Microinjection of Follicle-Enclosed Mouse Oocytes

NASA Astrophysics Data System (ADS)

Jaffe, Laurinda A.; Norris, Rachael P.; Freudzon, Marina; Ratzan, William J.; Mehlmann, Lisa M.

The mammalian oocyte develops within a complex of somatic cells known as a follicle, within which signals from the somatic cells regulate the oocyte, and signals from the oocyte regulate the somatic cells. Because isolation of the oocyte from the follicle disrupts these communication pathways, oocyte physiology is best studied within an intact follicle. Here we describe methods for quantitative microinjection of follicle-enclosed mouse oocytes, thus allowing the introduction of signaling molecules as well as optical probes into the oocyte within its physiological environment.

Study the Antinociceptive Effect of Intracerebroventricular Injection of Aqueous Extract of Origanum Vulgare Leaves in Rat: Possible Involvement of Opioid System

PubMed Central

Pahlavan, Yasaman; Sepehri, Gholamreza; Sheibani, Vahid; Afarinesh khaki, Mohammadreza; Gojazadeh, Morteza; Pahlavan, Bahare; Pahlavan, Fereshteh

2013-01-01

Objective(s): The aim of study was to investigate the antinociceptive effect of intracerebroventricular (ICV) microinjection of Origanum vulgare (ORG) extract and possible involvement of opioid receptors. Materials and Methods: Cannula was inserted into left ventricle of male rats. Five days after surgery Tail Flick Latency (TFL) was measured after ICV microinjection of, ORG (1, 3 and 6 µg / rat). Effective dose of ORG was injected ICV in concomitant with morphine (2 mg/kg, IP), naloxone (2 mg / kg, IP) and saline (0.5 µl/rat) and TFL was recorded. Results: The co- administration of ORG extract with morphine showed a significant increase in TFL and naloxone, pretreatment significantly inhibited the antinociceptive activity of ORG and morphine. Conclusion: The aqueous extract of ORG possesses antinociceptive activities in the tail-flick test in a dose dependent manner. ORG - induced antinociception may have been mediated by opioid systems. PMID:24379969

Targeting of loaded Sendai virus envelopes by covalently attached insulin molecules to virus receptor-depleted cells: fusion-mediated microinjection of ricin A and simian virus 40 DNA.

PubMed

Gitman, A G; Graessmann, A; Loyter, A

1985-11-01

Insulin molecules were covalently attached to detergent-solubilized Sendai virus envelope glycoproteins (HN and F polypeptides) by the use of the crosslinking reagent succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB). Reconstitution of modified viral glycoproteins (carrying covalently attached insulin) together with unmodified viral glycoproteins resulted in the formation of "fusogenic" viral envelopes bearing insulin molecules. Reconstitution of such fusogenic viral envelopes in the presence of ricin A or simian virus 40 (SV40) DNA resulted in the formation of viral envelopes bearing insulin molecules and loaded with ricin A or SV40 DNA. Such viral envelopes were able to bind to hepatoma tissue culture cells (HTCC) from which Sendai virus receptors were removed by treatment with neuraminidase. Incubation of viral envelopes loaded with ricin A with virus receptor-depleted HTCC resulted in fusion-mediated injection of the toxin, as inferred from inhibition of protein synthesis and decrease in cell viability of the microinjected cells. Fusion-mediated injection of SV40 DNA was inferred from the appearance of SV40 tumor antigen in microinjected cells. Binding and fusion of the loaded viral envelopes to neuraminidase-treated HTCC was mediated solely by the virus-associated insulin molecules. Addition of free insulin molecules inhibited binding of the viral envelopes and, consequently, the microinjection of ricin A and SV40 DNA.

Targeting of loaded Sendai virus envelopes by covalently attached insulin molecules to virus receptor-depleted cells: fusion-mediated microinjection of ricin A and simian virus 40 DNA.

PubMed Central

Gitman, A G; Graessmann, A; Loyter, A

1985-01-01

Insulin molecules were covalently attached to detergent-solubilized Sendai virus envelope glycoproteins (HN and F polypeptides) by the use of the crosslinking reagent succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB). Reconstitution of modified viral glycoproteins (carrying covalently attached insulin) together with unmodified viral glycoproteins resulted in the formation of "fusogenic" viral envelopes bearing insulin molecules. Reconstitution of such fusogenic viral envelopes in the presence of ricin A or simian virus 40 (SV40) DNA resulted in the formation of viral envelopes bearing insulin molecules and loaded with ricin A or SV40 DNA. Such viral envelopes were able to bind to hepatoma tissue culture cells (HTCC) from which Sendai virus receptors were removed by treatment with neuraminidase. Incubation of viral envelopes loaded with ricin A with virus receptor-depleted HTCC resulted in fusion-mediated injection of the toxin, as inferred from inhibition of protein synthesis and decrease in cell viability of the microinjected cells. Fusion-mediated injection of SV40 DNA was inferred from the appearance of SV40 tumor antigen in microinjected cells. Binding and fusion of the loaded viral envelopes to neuraminidase-treated HTCC was mediated solely by the virus-associated insulin molecules. Addition of free insulin molecules inhibited binding of the viral envelopes and, consequently, the microinjection of ricin A and SV40 DNA. PMID:2997783

Dynamics of Micropipette Vibration During Piezo-assisted Microinjection

NASA Astrophysics Data System (ADS)

Karzar-Jeddi, Mehdi; Olgac, Nejat; Fan, Tai-Hsi

Microinjection is a well-accepted method to introduce materials such as sperm, DNA materials, or nucleus into a living cell for biomedical applications. The conventional microinjection procedure consists of immobilizing the cell by applying suction through a holding pipette, and then an injecting micropipette penetrates through the cell membrane and introduces the materials into the cell. To assist the penetration process a piezo-generated pulse train is applied to the injecting pipette, which causes an undesirable lateral vibration at the injecting pipette tip. In this research we provide an analytical model to study the response of micropipette to the piezo-pulse train using the Duhamel integral method. Our results show that filling the micropipette tip with mercury causes a larger amplitude stroke vibration in micropipette tip than that of empty micropipette when it is submerged in the viscous medium surrounding the cell. The mercury introduced larger stroke vibration can cause a larger shear force and assist the penetration of micropipette through the cell membrane. This work is supported by NSF CBET-0828733 and NIH R24RR018934-01.

Generating chimeric mice from embryonic stem cells via vial coculturing or hypertonic microinjection.

PubMed

Lee, Kun-Hsiung

2014-01-01

The generation of a fertile embryonic stem cell (ESC)-derived or F0 (100 % coat color chimerism) mice is the final criterion in proving that the ESC is truly pluripotent. Many methods have been developed to produce chimeric mice. To date, the most popular methods for generating chimeric embryos is well sandwich aggregation between zona pellucida (ZP) removed (denuded) 2.5-day post-coitum (dpc) embryos and ESC clumps, or direct microinjection of ESCs into the cavity (blastocoel) of 3.5-dpc blastocysts. However, due to systemic limitations and the disadvantages of conventional microinjection, aggregation, and coculturing, two novel methods (vial coculturing and hypertonic microinjection) were developed in recent years at my laboratory.Coculturing 2.5-dpc denuded embryos with ESCs in 1.7-mL vials for ~3 h generates chimeras that have significantly high levels of chimerism (including 100 % coat color chimerism) and germline transmission. This method has significantly fewer instrumental and technological limitations than existing methods, and is an efficient, simple, inexpensive, and reproducible method for "mass production" of chimeric embryos. For laboratories without a microinjection system, this is the method of choice for generating chimeric embryos. Microinjecting ESCs into a subzonal space of 2.5-dpc embryos can generate germline-transmitted chimeras including 100 % coat color chimerism. However, this method is adopted rarely due to the very small and tight space between ZP and blastomeres. Using a laser pulse or Piezo-driven instrument/device to help introduce ESCs into the subzonal space of 2.5-dpc embryos demonstrates the superior efficiency in generating ESC-derived (F0) chimeras. Unfortunately, due to the need for an expensive instrument/device and extra fine skill, not many studies have used either method. Recently, ESCs injected into the large subzonal space of 2.5-dpc embryos in an injection medium containing 0.2-0.3 M sucrose very efficiently generated viable, healthy, and fertile chimeric mice with 100 % coat color chimerism.Both vial coculture and hypertonic microinjection methods are useful and effective alternatives for producing germline chimeric or F0 mice efficiently and reliably. Furthermore, both novel methods are also good for induced pluripotent stem cells (iPSCs) to generate chimeric embryos.

Differential expression of the major immediate early gene of human cytomegalovirus.

PubMed

Tsutsui, Y; Nogami-Satake, T

1990-01-01

We prepared a murine monoclonal antibody reactive to a human cytomegalovirus (HCMV)-induced nuclear protein with an Mr of 68,000. Expression of the 68K protein was compared with the major immediate early (IE) 72K protein in various cell types after infection with HCMV or microinjection of plasmid DNA containing the major IE gene. The 68K nuclear protein was detected 2 to 3 h after appearance of the 72K protein in human embryonal lung (HEL) cells infected with HCMV. The 68K protein was distributed throughout the cytoplasm in the late phase of infection, while the 72K protein remained chiefly in the nucleus. The 68K protein was barely detected in the cells under IE conditions by immunoprecipitation, but, together with the 72K protein, it was expressed after microinjection of cloned DNA, containing only the major IE region (region 1), into the nuclei of HEL cells. The 72K protein was expressed in nuclei 2 h after microinjection, whereas the 68K protein was detected 4 to 5 h after the injection. The 68K protein was expressed after microinjection in non-permissive rodent fibroblasts or non-permissive transformed human cells in which these proteins were not expressed after viral infection. Immunoprecipitations after chase-labelling from IE conditions or after partial digestions suggested that the 68K protein is neither a degradation nor a modification product of the major IE 72K protein.

Effects of tissue-preparation-induced callose synthesis on estimates of plasmodesma size exclusion limits.

PubMed

Radford, J E; White, R G

2001-01-01

Plasmodesmata are often characterised by their size exclusion limit (SEL), which is the molecular weight of the largest dye, introduced by microinjection, that will move from cell to cell. In this study, we investigated whether commonly used techniques for isolation and manipulation of tissues, and microinjection of fluorescent dyes, affected the SEL, and whether any such effects could be ameliorated by inhibiting callose deposition. We examined young root epidermal cells of Arabidopsis thaliana and staminal hair cells of Tradescantia virginiana, two tissues often used in experiments on symplastic transport. Transport in root tips dissected from the main plant body and in stamen hairs removed from the base of the stamen filament was compared with transport in undissected roots and stamen hairs attached to the base of the filament, respectively. Tissues were microinjected with fluorescent dyes (457 Da to > 3 kDa) with or without prior incubation in the callose deposition inhibitors 2-deoxy-D-glucose or aniline blue fluorochrome. In both tissues, dissection reduced the SEL, which was largely prevented by prior incubation in 2-deoxy-D-glucose but not by incubation in aniline blue fluorochrome. Thus, standard methods for tissue preparation can cause sufficient callose deposition to reduce cell-to-cell transport, and this needs to be considered in studies employing microinjection. Introduction of the dyes by pressure injection rather than iontophoresis decreased the SEL in A. thaliana but increased it in T. virginiana, showing that these two injection techniques do not necessarily give identical results and that plasmodesmata in different tissues may respond differently to similar experimental procedures.

A novel cell weighing method based on the minimum immobilization pressure for biological applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Qili; Institute of Robotics and Automatic Information System, Nankai University, Tianjin 300071; Shirinzadeh, Bijan

2015-07-28

A novel weighing method for cells with spherical and other regular shapes is proposed in this paper. In this method, the relationship between the cell mass and the minimum aspiration pressure to immobilize the cell (referred to as minimum immobilization pressure) is derived for the first time according to static theory. Based on this relationship, a robotic cell weighing process is established using a traditional micro-injection system. Experimental results on porcine oocytes demonstrate that the proposed method is able to weigh cells at an average speed of 16.3 s/cell and with a success rate of more than 90%. The derived cellmore » mass and density are in accordance with those reported in other published results. The experimental results also demonstrated that this method is able to detect less than 1% variation of the porcine oocyte mass quantitatively. It can be conducted by a pair of traditional micropipettes and a commercial pneumatic micro-injection system, and is expected to perform robotic operation on batch cells. At present, the minimum resolution of the proposed method for measuring the cell mass can be 1.25 × 10{sup −15 }kg. Above advantages make it very appropriate for quantifying the amount of the materials injected into or moved out of the cells in the biological applications, such as nuclear enucleations and embryo microinjections.« less

Preparation (pulling) of needles for gene delivery by microinjection.

PubMed

Dean, David A

2006-12-01

INTRODUCTIONThis protocol contains methods for pulling microinjection needles using two different models of pipette pullers. The advantage of pulling needles in the laboratory is that a variety of different needle types can be pulled, depending on the samples and cells being injected. An added advantage is cost; once a pipette puller has been purchased, boxes of glass capillaries are inexpensive compared to premade microinjection needles. The advantages to buying preformed and sterilized needles include increased uniformity of needles from one to another, ease of use, high quality, and not having to invest in a pipette puller. The pipette puller models described in this article are the Flaming/Brown Pipette Puller Model P-97 (Sutter) and the PUL-1 Micropipette Puller (World Precision Instruments). The PUL-1 instrument is the less expensive of the two, but it requires more user input, and it cannot be used to pull Femtotip-like microinjection pipettes.

Microinjection - a tool to study gravitropism

NASA Astrophysics Data System (ADS)

Scherp, P.; Hasenstein, K. H.

2003-05-01

Despite extensive studies on plant gravitropism this phenomenon is still poorly understood. The separation of gravity sensing, signal transduction and response is a common concept but especially the mechanism of gravisensing remains unclear. This paper focuses on microinjection as powerful tool to investigate gravisensing in plants. We describe the microinjection of magnetic beads in rhizoids of the green alga Chara and related subsequent manipulation of the gravisensing system. After injection, an external magnet can control the movement of the magnetic beads. We demonstrate successful injection of magnetic beads into rhizoids and describe a multitude of experiments that can be carried out to investigate gravitropism in Chara rhizoids. In addition to examining mechanical properties, bead microinjection is also useful for probing the function of the cytoskeleton by coating beads with drugs that interfere with the cytoskeleton. The injection of fluorescently labeled beads or probes may reveal the involvement of the cytoskeleton during gravistimulation and response in living cells.

Relationship of the Xeroderma Pigmentosum Group E DNA Repair Defect to the Chromatin and DNA Binding Proteins UV-DDB and Replication Protein A

PubMed Central

Rapić Otrin, Vesna; Kuraoka, Isao; Nardo, Tiziana; McLenigan, Mary; Eker, A. P. M.; Stefanini, Miria; Levine, Arthur S.; Wood, Richard D.

1998-01-01

Cells from complementation groups A through G of the heritable sun-sensitive disorder xeroderma pigmentosum (XP) show defects in nucleotide excision repair of damaged DNA. Proteins representing groups A, B, C, D, F, and G are subunits of the core recognition and incision machinery of repair. XP group E (XP-E) is the mildest form of the disorder, and cells generally show about 50% of the normal repair level. We investigated two protein factors previously implicated in the XP-E defect, UV-damaged DNA binding protein (UV-DDB) and replication protein A (RPA). Three newly identified XP-E cell lines (XP23PV, XP25PV, and a line formerly classified as an XP variant) were defective in UV-DDB binding activity but had levels of RPA in the normal range. The XP-E cell extracts did not display a significant nucleotide excision repair defect in vitro, with either UV-irradiated DNA or a uniquely placed cisplatin lesion used as a substrate. Purified UV-DDB protein did not stimulate repair of naked DNA by DDB− XP-E cell extracts, but microinjection of the protein into DDB− XP-E cells could partially correct the repair defect. RPA stimulated repair in normal, XP-E, or complemented extracts from other XP groups, and so the effect of RPA was not specific for XP-E cell extracts. These data strengthen the connection between XP-E and UV-DDB. Coupled with previous results, the findings suggest that UV-DDB has a role in the repair of DNA in chromatin. PMID:9584159

Activation of amphibian oocytes by sperm extracts.

PubMed

Bonilla, F; Ajmat, M T; Sánchez Toranzo, G; Zelarayán, L; Oterino, J; Bühler, M I

2008-11-01

In the fertilization of most animals, egg activation is accompanied by an increase in cytoplasmatic Ca2+; however, the mechanism through which the fertilizing sperm induce this phenomenon is still controversial. An increase in intracellular free Ca2+ is required to trigger egg activation events, a process that includes cortical granule exocytosis, resumption and completion of meiosis and DNA replication, and culminates in the first mitotic cleavage. In this work, we investigated the effect of microinjection and incubation of different fractions of homologous sperm extract on the activation of Bufo arenarum oocytes matured in vitro. Two heat treatment-sensitive fractions obtained by chromatography were able to induce oocyte activation. The sperm fraction, which contained a 24 kDa protein, induced 90% activation when it was microinjected into the oocytes. Whilst the sperm fraction, which contained a 36 kDa protein, was able to induce about 70% activation only when it was applied on the oocyte surface.

Nuclear envelope breakdown and mitosis in sand dollar embryos is inhibited by microinjection of calcium buffers in a calcium-reversible fashion, and by antagonists of intracellular Ca2+ channels.

PubMed

Silver, R B

1989-01-01

Transient elevations in intracellular free Ca2+ are believed to signal the initiation of mitosis. This model predicts that mitosis might be arrested prior to nuclear envelope breakdown (NEB) or anaphase onset if intracellular Ca2+ concentration is buffered or dampened. Microinjection of a discrete dose of Ca2+ into the cell might then release the cell to resume mitotic cycling. Experimentally, one blastomere of two cell sand dollar (Echinaracnius parma) embryos was microinjected with Ca2+ buffers, Ca2+ solutions, or Ca2+ channel antagonists; the uninjected blastomere was the control. Cells were loaded with 10 pl doses of the Ca2+ buffer antipyrylazo III (ApIII) at specific times in the cell cycle to attempt a competitive inhibition of Ca2+-dependent steps in NEB and initiation of mitosis. Injection of 50 microM ApIII 6 min prior to NEB blocked NEB and further cell cycling. Injections of solutions between 0 and 30 microM ApIII were without observable effect. Control injections had no observable effect on the injected cell. Cells injected with 50 microM ApIII 2 min prior to the onset of anaphase in control cells were blocked in metaphase. Cells were sensitive to Ca2+ buffer injections 6 min prior to NEB (with a 40- to 45-sec duration), and 2 min prior to anaphase onset (with a 10- to 20-sec duration). Vital staining of these cells with H33342 demonstrated that they contained only one nucleus that had the same fluorescence intensity as seen prior to microinjection, and thus did not undergo DNA synthesis following the imposition of the Ca2+ buffer block to mitosis. Cells arrested in this fashion did not spontaneously resume mitotic cycling. This Ca2+ buffer-induced mitotic arrest was, however, experimentally reversible. Cells arrested with 50 microM ApIII 6 min prior to NEB could be returned to mitotic activity by injecting 300 microM CaCl2 5 min after the ApIII injection. The double injected cells resumed cycling, NEB, and mitosis after a delay of one cell cycle period, and remained one cell cycle out of phase with the sister (control) cell. Microinjection of antagonists of endomembrane Ca2+ channels inhibited NEB and anaphase onset in a concentration- and time-dependent fashion. The effective doses of compounds tested were 7 micrograms/ml ryanodine and 500 micrograms/ml TMB-8. These results indicate that a transient elevation of intracellular Ca2+ from endomembrane stores is required to initiate mitotic events, namely NEB and anaphase onset.(ABSTRACT TRUNCATED AT 400 WORDS)

Aneuploidy screening of embryonic stem cell clones by metaphase karyotyping and droplet digital polymerase chain reaction.

PubMed

Codner, Gemma F; Lindner, Loic; Caulder, Adam; Wattenhofer-Donzé, Marie; Radage, Adam; Mertz, Annelyse; Eisenmann, Benjamin; Mianné, Joffrey; Evans, Edward P; Beechey, Colin V; Fray, Martin D; Birling, Marie-Christine; Hérault, Yann; Pavlovic, Guillaume; Teboul, Lydia

2016-08-05

Karyotypic integrity is essential for the successful germline transmission of alleles mutated in embryonic stem (ES) cells. Classical methods for the identification of aneuploidy involve cytological analyses that are both time consuming and require rare expertise to identify mouse chromosomes. As part of the International Mouse Phenotyping Consortium, we gathered data from over 1,500 ES cell clones and found that the germline transmission (GLT) efficiency of clones is compromised when over 50 % of cells harbour chromosome number abnormalities. In JM8 cells, chromosomes 1, 8, 11 or Y displayed copy number variation most frequently, whilst the remainder generally remain unchanged. We developed protocols employing droplet digital polymerase chain reaction (ddPCR) to accurately quantify the copy number of these four chromosomes, allowing efficient triage of ES clones prior to microinjection. We verified that assessments of aneuploidy, and thus decisions regarding the suitability of clones for microinjection, were concordant between classical cytological and ddPCR-based methods. Finally, we improved the method to include assay multiplexing so that two unstable chromosomes are counted simultaneously (and independently) in one reaction, to enhance throughput and further reduce the cost. We validated a PCR-based method as an alternative to classical karyotype analysis. This technique enables laboratories that are non-specialist, or work with large numbers of clones, to precisely screen ES cells for the most common aneuploidies prior to microinjection to ensure the highest level of germline transmission potential. The application of this method allows early exclusion of aneuploid ES cell clones in the ES cell to mouse conversion process, thus improving the chances of obtaining germline transmission and reducing the number of animals used in failed microinjection attempts. This method can be applied to any other experiments that require accurate analysis of the genome for copy number variation (CNV).

Microinjection of cytoplasm as a test of complementation in Paramecium

PubMed Central

1982-01-01

Mutants in Paramecium tetraurelia, unable to generate action potentials, have been isolated as cells which show no backward swimming in response to ionic stimulation. These "pawn" mutants belong to at least three complementation groups designated pwA, pwB, and pwC. We have found that microinjection of cytoplasm from a wild-type donor into a pawn recipient of any of the three complementation groups restores the ability of the pawn to generate action potentials and hence swim backward. In addition, the cytoplasm from a pawn cannot restore a recipient of the same complementation group, but that from a pawn of a different group can. Electrophysiological analysis had demonstrated that the restoration of backward swimming is not due to a simple addition of ions but represents a profound change in the excitable membrane of the recipient pawn cells. Using known pawn mutants and those which had previously been unclassified, we have been able to establish a perfect concordance of genetic complementation and complementation by cytoplasmic transfer through microinjection. This method has been used to classify pawn mutants that are sterile or hard- to-mate and to examine the ability of cytoplasms from different species of ciliated protozoa to restore the ability to swim backward in the pawn mutants of P. tetraurelia. A cell homogenate has also been fractionated by centrifugation to further purify the active components. These results demonstrate that transfer of cytoplasm between cells by microinjection can be a valid and systematic method to classify mutants. This test is simpler to perform than the genetic complementation test and can be used under favorable conditions in mutants that are sterile and in cells of different species. PMID:7061597

Optical micromanipulation methods for controlled rotation, transportation, and microinjection of biological objects.

PubMed

Mohanty, S K; Gupta, P K

2007-01-01

The use of laser microtools for rotation and controlled transport of microscopic biological objects and for microinjection of exogenous material in cells is discussed. We first provide a brief overview of the laser tweezers-based methods for rotation or orientation of microscopic objects. Particular emphasis is placed on the methods that are more suitable for the manipulation of biological objects, and the use of these for two-dimensional (2D) and 3D rotations/orientations of intracellular objects is discussed. We also discuss how a change in the shape of a red blood cell (RBC) suspended in hypertonic buffer leads to its rotation when it is optically tweezed. The potential use of this approach for the diagnosis of malaria is also illustrated. The use of a line tweezers having an asymmetric intensity distribution about the center of its major axis for simultaneous transport of microscopic objects, and the successful use of this approach for induction, enhancement, and guidance of neuronal growth cones is presented next. Finally, we describe laser microbeam-assisted microinjection of impermeable drugs into cells and also briefly discuss possible adverse effects of the laser trap or microbeams on cells.

Developing Xenopus Embryos Recover by Compacting and Expelling Single-Wall Carbon Nanotubes

PubMed Central

Holt, Brian D.; Shawky, Joseph H.; Dahl, Kris Noel; Davidson, Lance A.; Islam, Mohammad F.

2015-01-01

Single-wall carbon nanotubes are high aspect ratio nanomaterials that are being developed for use in materials, technological and biological applications due to their high mechanical stiffness, optical properties, and chemical inertness. Because of their prevalence, it is inevitable that biological systems will be exposed to nanotubes, yet studies of the effects of nanotubes on developing embryos have been inconclusive and are lacking for single-wall carbon nanotubes exposed to the widely studied model organism Xenopus laevis (African clawed frog). Microinjection of experimental substances into the Xenopus embryo is a standard technique for toxicology studies and cellular lineage tracing. Here we report the surprising finding that superficial (12.5 ± 7.5 μm below the membrane) microinjection of nanotubes dispersed with Pluronic F127 into one-to-two cell Xenopus embryos resulted in the formation and expulsion of compacted, nanotube-filled, punctate masses, at the blastula to mid-gastrula developmental stages, which we call “boluses”. Such expulsion of microinjected materials by Xenopus embryos has not been reported before and is dramatically different from the typical distribution of the materials throughout the progeny of the microinjected cells. Previous studies of microinjections of nanomaterials such as nanodiamonds, quantum dots or spherical nanoparticles report that nanomaterials often induce toxicity and remain localized within the embryos. In contrast, our results demonstrate an active recovery pathway for embryos after exposure to Pluronic F127-coated nanotubes, which we speculate is due to a combined effect of the membrane activity of the dispersing agent, Pluronic F127, and the large aspect ratio of nanotubes. PMID:26153061

Developing Xenopus embryos recover by compacting and expelling single wall carbon nanotubes.

PubMed

Holt, Brian D; Shawky, Joseph H; Dahl, Kris Noel; Davidson, Lance A; Islam, Mohammad F

2016-04-01

Single wall carbon nanotubes are high aspect ratio nanomaterials being developed for use in materials, technological and biological applications due to their high mechanical stiffness, optical properties and chemical inertness. Because of their prevalence, it is inevitable that biological systems will be exposed to nanotubes, yet studies of the effects of nanotubes on developing embryos have been inconclusive and are lacking for single wall carbon nanotubes exposed to the widely studied model organism Xenopus laevis (African clawed frog). Microinjection of experimental substances into the Xenopus embryo is a standard technique for toxicology studies and cellular lineage tracing. Here we report the surprising finding that superficial (12.5 ± 7.5 µm below the membrane) microinjection of nanotubes dispersed with Pluronic F127 into one- to two-cell Xenopus embryos resulted in the formation and expulsion of compacted, nanotube-filled, punctate masses, at the blastula to mid-gastrula developmental stages, which we call "boluses." Such expulsion of microinjected materials by Xenopus embryos has not been reported before and is dramatically different from the typical distribution of the materials throughout the progeny of the microinjected cells. Previous studies of microinjections of nanomaterials such as nanodiamonds, quantum dots or spherical nanoparticles report that nanomaterials often induce toxicity and remain localized within the embryos. In contrast, our results demonstrate an active recovery pathway for embryos after exposure to Pluronic F127-coated nanotubes, which we speculate is due to a combined effect of the membrane activity of the dispersing agent, Pluronic F127, and the large aspect ratio of nanotubes. Copyright © 2015 John Wiley & Sons, Ltd.

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing.

PubMed

Elaswad, Ahmed; Khalil, Karim; Cline, David; Page-McCaw, Patrick; Chen, Wenbiao; Michel, Maximilian; Cone, Roger; Dunham, Rex

2018-01-20

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

Microinjection of the SH2 domain of the 85-kilodalton subunit of phosphatidylinositol 3-kinase inhibits insulin-induced DNA synthesis and c-fos expression.

PubMed Central

Jhun, B H; Rose, D W; Seely, B L; Rameh, L; Cantley, L; Saltiel, A R; Olefsky, J M

1994-01-01

We have investigated the functional role of the SH2 domain of the 85-kDa subunit (p85) of the phosphatidylinositol 3-kinase in the insulin signal transduction pathway. Microinjection of a bacterial fusion protein containing the N-terminal SH2 domain of p85 inhibited insulin- and other growth factor-induced DNA synthesis by 90% and c-fos protein expression by 80% in insulin-responsive rat fibroblasts. The specificity of the fusion protein was examined by in vitro precipitation experiments, which showed that the SH2 domain of p85 can independently associate with both insulin receptor substrate 1 and the insulin receptor itself in the absence of detectable binding to other phosphoproteins. The microinjection results were confirmed through the use of an affinity-purified antibody directed against p85, which gave the same phenotype. Additional studies were carried out in another cell line expressing mutant insulin receptors which lack the cytoplasmic tyrosine residues with which p85 interacts. Microinjection of the SH2 domain fusion protein also inhibited insulin signaling in these cells, suggesting that association of p85 with insulin receptor substrate 1 is a key element in insulin-mediated cell cycle progression. In addition, coinjection of purified p21ras protein with the p85 fusion protein or the antibody restored DNA synthesis, suggesting that ras function is either downstream or independent of p85 SH2 domain interaction. Images PMID:7935461

Gene delivery by direct injection (microinjection) using a controlled-flow system.

PubMed

Dean, David A

2006-12-01

INTRODUCTIONThis protocol describes a method for constant-flow microinjection using the Pneumatic PicoPump (World Precision Instruments). This type of system is very simple and can be assembled on a relatively low budget. In this method, a constant flow of sample is delivered from the tip of the pipette, and the amount of sample injected into the cell is determined by how long the pipette remains in the cell. A typical system is composed of a pressure regulator that can be adjusted for two pressures (back pressure and injection pressure), a capillary holder, and a coarse and fine micromanipulator.

Microinjection--a tool to study gravitropism.

PubMed

Scherp, P; Hasenstein, K H

2003-01-01

Despite extensive studies on plant gravitropism this phenomenon is still poorly understood. The separation of gravity sensing, signal transduction and response is a common concept but especially the mechanism of gravisensing remains unclear. This paper focuses on microinjection as powerful tool to investigate gravisensing in plants. We describe the microinjection of magnetic beads in rhizoids of the green alga Chara and related subsequent manipulation of the gravisensing system. After injection, an external magnet can control the movement of the magnetic beads. We demonstrate successful injection of magnetic beads into rhizoids and describe a multitude of experiments that can be carried out to investigate gravitropism in Chara rhizoids. In addition to examining mechanical properties, bead microinjection is also useful for probing the function of the cytoskeleton by coating beads with drugs that interfere with the cytoskeleton. The injection of fluorescently labeled beads or probes may reveal the involvement of the cytoskeleton during gravistimulation and response in living cells. c2003 COSPAR. Published by Elsevier Ltd. All rights reserved.

Microinjection--a tool to study gravitropism

NASA Technical Reports Server (NTRS)

Scherp, P.; Hasenstein, K. H.

2003-01-01

Despite extensive studies on plant gravitropism this phenomenon is still poorly understood. The separation of gravity sensing, signal transduction and response is a common concept but especially the mechanism of gravisensing remains unclear. This paper focuses on microinjection as powerful tool to investigate gravisensing in plants. We describe the microinjection of magnetic beads in rhizoids of the green alga Chara and related subsequent manipulation of the gravisensing system. After injection, an external magnet can control the movement of the magnetic beads. We demonstrate successful injection of magnetic beads into rhizoids and describe a multitude of experiments that can be carried out to investigate gravitropism in Chara rhizoids. In addition to examining mechanical properties, bead microinjection is also useful for probing the function of the cytoskeleton by coating beads with drugs that interfere with the cytoskeleton. The injection of fluorescently labeled beads or probes may reveal the involvement of the cytoskeleton during gravistimulation and response in living cells. c2003 COSPAR. Published by Elsevier Ltd. All rights reserved.

Microinjection of recombinant O-GlcNAc transferase potentiates Xenopus oocytes M-phase entry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dehennaut, Vanessa; EA 4020, Laboratoire de Regulation des Signaux de Division, USTL, IFR147, Villeneuve d'Ascq; Hanoulle, Xavier

2008-05-02

In order to understand the importance of the cytosolic and nuclear-specific O-linked N-acetylglucosaminylation (O-GlcNAc) on cell cycle regulation, we recently reported that inhibition of O-GlcNAc transferase (OGT) delayed or blocked Xenopus laevis oocyte germinal vesicle breakdown (GVBD). Here, we show that increased levels of the long OGT isoform (ncOGT) accelerate X. laevis oocyte GVBD. A N-terminally truncated isoform (sOGT) with a similar in vitro catalytic activity towards a synthetic CKII-derived peptide had no effect, illustrating the important role played by the N-terminal tetratrico-peptide repeats. ncOGT microinjection in the oocytes increases both the speed and extent of O-GlcNAc addition, leads tomore » a quicker activation of the MPF and MAPK pathways and finally results in a faster GVBD. Microinjection of anti-OGT antibodies leads to a delay of the GVBD kinetics. Our results hence demonstrate that OGT is a key molecule for the timely progression of the cell cycle.« less

Intracellular Route of Canine Parvovirus Entry

PubMed Central

Vihinen-Ranta, Maija; Kalela, Anne; Mäkinen, Päivi; Kakkola, Laura; Marjomäki, Varpu; Vuento, Matti

1998-01-01

The present study was designed to investigate the endocytic pathway involved in canine parvovirus (CPV) infection. Reduced temperature (18°C) or the microtubule-depolymerizing drug nocodazole was found to inhibit productive infection of canine A72 cells by CPV and caused CPV to be retained in cytoplasmic vesicles as indicated by immunofluorescence microscopy. Consistent with previously published results, these data indicate that CPV enters a host cell via an endocytic route and further suggest that microtubule-dependent delivery of CPV to late endosomes is required for productive infection. Cytoplasmic microinjection of CPV particles was used to circumvent the endocytosis and membrane fusion steps in the entry process. Microinjection experiments showed that CPV particles which were injected directly into the cytoplasm, thus avoiding the endocytic pathway, were unable to initiate progeny virus production. CPV treated at pH 5.0 prior to microinjection was unable to initiate virus production, showing that factors of the endocytic route other than low pH are necessary for the initiation of infection by CPV. PMID:9420290

Pig cloning by microinjection of fetal fibroblast nuclei.

PubMed

Onishi, A; Iwamoto, M; Akita, T; Mikawa, S; Takeda, K; Awata, T; Hanada, H; Perry, A C

2000-08-18

Pig cloning will have a marked impact on the optimization of meat production and xenotransplantation. To clone pigs from differentiated cells, we microinjected the nuclei of porcine (Sus scrofa) fetal fibroblasts into enucleated oocytes, and development was induced by electroactivation. The transfer of 110 cloned embryos to four surrogate mothers produced an apparently normal female piglet. The clonal provenance of the piglet was indicated by her coat color and confirmed by DNA microsatellite analysis.

Direct observation of nucleocytoplasmic transport by microinjection of GFP-tagged proteins in living cells.

PubMed

Rosorius, O; Heger, P; Stelz, G; Hirschmann, N; Hauber, J; Stauber, R H

1999-08-01

We established a straightforward experimental system to investigate directly the requirements for nucleocytoplasmic transport in live cells. For this purpose, substrates were created containing nuclear localization signals (NLS) or nuclear export signals (NES) linked to a chimeric protein composed of the glutathione S-transferase (GST) fused to the green fluorescent protein (GFP). The combination of GST/GFP-tagging allowed us to control protein expression in bacteria and to monitor protein purification during chromatography. Following microinjection into somatic cells, nuclear export/import of the highly fluorescent substrates could be observed directly by fluorescence microscopy. This system sets the stage to quantitate, in real time, the kinetics of nuclear import/export in living cells and to evaluate qualitative differences in various NLS/NES signals and pathways.

Adenovirus-mediated heme oxygenase-1 gene transfer into rabbit ocular tissues.

PubMed

Abraham, N G; da Silva, J L; Lavrovsky, Y; Stoltz, R A; Kappas, A; Dunn, M W; Schwartzman, M L

1995-10-01

Heme oxygenase-1 (HO-1) is a stress protein induced up to 100-fold within a few hours after exposure to oxidative stress, and it has been shown to counteract oxidative injury induced by ultraviolet light or free radicals. The current study was undertaken to determine whether the HO-1 gene can be introduced into adult rabbit ocular tissues by microinjection of a recombinant replication-deficient adenovirus human HO-1 cDNA (Adv-HHO). Human HO-1 gene was used for transfection studies to differentiate endogenous from transfected HO. The purified Adv-HHO construct (10(8) pfu/ml) was mixed with lipofectamine and microinjected into the anterior chamber, vitreous cavity, and subretinal space of New Zealand rabbit eyes. After 2 weeks, total RNA was extracted from different ocular tissues, reverse transcription-polymerase chain reaction was performed using specific human HO-1 primers, and amplification products were subjected to Southern hybridization. Transfection with the Adv-HHO construct into rabbit corneal epithelial cells in culture resulted in a functional expression of the human HO-1 gene; the human HO-1 mRNA was detected, and enzyme activity increased threefold. Human HO-1 mRNA was detected in the retina after microinjection of the Adv-HHO construct into the subretinal space. Microinjection into the vitreous resulted in HO-1 mRNA expression in the corneal endothelium, iris, lens, and retina; after intracameral injection of the Adv-HHO construct, human HO-1 mRNA was detected in corneal epithelium and endothelium, ciliary body, lens, and iris. Regardless of the injection site, transfected human HO-1 mRNA was undetectable in tissues outside the eye, that is, brain, liver, and kidney. These results demonstrated a tissue-selective functional transfer of the human HO-1 gene into rabbit ocular tissues in vivo. This technique may be a promising means for delivering HO-1 gene in vivo as a protective mechanism against oxidative stress that contributes to the pathogenesis of ocular diseases such as cataract, light-induced injury, age-related macular degeneration, and diabetic retinopathy.

Comparative inhibition of rabbit globin mRNA translation by modified antisense oligodeoxynucleotides.

PubMed Central

Cazenave, C; Stein, C A; Loreau, N; Thuong, N T; Neckers, L M; Subasinghe, C; Hélène, C; Cohen, J S; Toulmé, J J

1989-01-01

We have studied the translation of rabbit globin mRNA in cell free systems (reticulocyte lysate and wheat germ extract) and in microinjected Xenopus oocytes in the presence of anti-sense oligodeoxynucleotides. Results obtained with the unmodified all-oxygen compounds were compared with those obtained when phosphorothioate or alpha-DNA was used. In the wheat germ system a 17-mer sequence targeted to the coding region of beta-globin mRNA was specifically inhibitory when either the unmodified phosphodiester oligonucleotide or its phosphorothioate analogue were used. In contrast no effect was observed with the alpha-oligomer. These results were ascribed to the fact that phosphorothioate oligomers elicit an RNase-H activity comparable to the all-oxygen congeners, while alpha-DNA/mRNA hybrids were a poor substrate. Microinjected Xenopus oocytes followed a similar pattern. The phosphorothioate oligomer was more efficient to prevent translation than the unmodified 17-mer. Inhibition of beta-globin synthesis was observed in the nanomolar concentration range. This result can be ascribed to the nuclease resistance of phosphorothioates as compared to natural phosphodiester linkages, alpha-oligomers were devoid of any inhibitory effect up to 30 microM. Phosphorothioate oligodeoxyribonucleotides were shown to be non-specific inhibitors of protein translation, at concentrations in the micromolar range, in both cell-free systems and oocytes. Non-specific inhibition of translation was dependent on the length of the phosphorothioate oligomer. These non-specific effects were not observed with the unmodified or the alpha-oligonucleotides. Images PMID:2472605

The antifreeze protein type I (AFP I) increases seabream (Sparus aurata) embryos tolerance to low temperatures.

PubMed

Robles, V; Barbosa, V; Herráez, M P; Martínez-Páramo, S; Cancela, M L

2007-07-15

To date, all attempts at fish embryo cryopreservation have failed. One of the main reasons for this to occur is the high chilling sensitivity reported in fish embryos thus emphasizing the need for further testing of different methods and alternative cryoprotective agents (CPAs) in order to improve our chances to succeed in this purpose. In this work we have used the antifreeze protein type I (AFP I) as a natural CPA. This protein is naturally expressed in sub-arctic fish species, and inhibits the growth of ice crystals as well as recrystallization during thawing. Embryos from Sparus aurata were microinjected with AFP I at different developmental stages, 2 cells and blastula, into the blastomere-yolk interface and into the yolk sac, respectively. Control, punctured and microinjected embryos were subjected to chilling at two different temperatures, 0 degrees C (1h) and -10 degrees C (15min) when embryos reached 5-somite stage. Embryos were subjected to -10 degrees C chilling in a 3M DMSO extender to avoid ice crystal formation in the external solution. Survival after chilling was established as the percentage of embryos that hatch. To study the AFP I distribution in the microinjected embryos, a confocal microscopy study was done. Results demonstrate that AFP I can significantly improve chilling resistance at 0 degrees C, particularly in 2-cell microinjected embryos, displaying nearly 100% hatching rates. This fact is in agreement with the confocal microscopy observations which confirmed the presence of the AFP protein in embryonic cells. These results support the hypothesis that AFP protect cellular structures by stabilizing cellular membranes.

Nucleolar changes after microinjection of antibodies to RNA polymerase I into the nucleus of mammalian cells.

PubMed

Benavente, R; Reimer, G; Rose, K M; Hügle-Dörr, B; Scheer, U

1988-01-01

After microinjection of antibodies against RNA polymerase I into the nuclei of cultured rat kangaroo (PtK2) and rat (RVF-SMC) cells alterations in nucleolar structure and composition were observed. These were detected by electron microscopy and double-label immunofluorescence microscopy using antibodies to proteins representative of the three major components of the nucleolus. The microinjected antibodies produced a progressive loss of the material of the dense fibrillar component (DFC) from the nucleoli which, at 4 h after injection, were transformed into bodies with purely granular component (GC) structure with attached fibrillar centers (FCs). Concomitantly, numerous extranucleolar aggregates appeared in the nucleoplasm which morphologically resembled fragments of the DFC and contained a protein (fibrillarin) diagnostic for this nucleolar structure. These observations indicate that the topological distribution of the material constituting the DFC can be experimentally influenced in interphase cells, apparently by modulating the transcriptional activity of the rRNA genes. These effects are different from nucleolar lesions induced by inhibitory drugs such as actinomycin D-dependent "nucleolar segregation". The structural alterations induced by antibodies to RNA polymerase I resemble, however, the initial events of nucleolar disintegration during mitotic prophase.

Survival rate of eukaryotic cells following electrophoretic nanoinjection.

PubMed

Simonis, Matthias; Hübner, Wolfgang; Wilking, Alice; Huser, Thomas; Hennig, Simon

2017-01-25

Insertion of foreign molecules such as functionalized fluorescent probes, antibodies, or plasmid DNA to living cells requires overcoming the plasma membrane barrier without harming the cell during the staining process. Many techniques such as electroporation, lipofection or microinjection have been developed to overcome the cellular plasma membrane, but they all result in reduced cell viability. A novel approach is the injection of cells with a nanopipette and using electrophoretic forces for the delivery of molecules. The tip size of these pipettes is approximately ten times smaller than typical microinjection pipettes and rather than pressure pulses as delivery method, moderate DC electric fields are used to drive charged molecules out of the tip. Here, we show that this approach leads to a significantly higher survival rate of nanoinjected cells and that injection with nanopipettes has a significantly lower impact on the proliferation behavior of injected cells. Thus, we propose that injection with nanopipettes using electrophoretic delivery is an excellent alternative when working with valuable and rare living cells, such as primary cells or stem cells.

Marshall Barber and the century of microinjection: from cloning of bacteria to cloning of everything.

PubMed

Korzh, Vladimir; Strähle, Uwe

2002-08-01

A hundred years ago, Dr. Marshall A. Barber proposed a new technique - the microinjection technique. He developed this method initially to clone bacteria and to confirm the germ theory of Koch and Pasteur. Later on, he refined his approach and was able to manipulate nuclei in protozoa and to implant bacteria into plant cells. Continuous improvement and adaptation of this method to new applications dramatically changed experimental embryology and cytology and led to the formation of several new scientific disciplines including animal cloning as one of its latest applications. Interestingly, microinjection originated as a method at the crossroad of bacteriology and plant biology, demonstrating once again the unforeseen impact that basic research in an unrelated field can have on the development of entirely different disciplines.

A Presumptive Developmental Role for a Sea Urchin Cyclin B Splice Variant

PubMed Central

Lozano, Jean-Claude; Schatt, Philippe; Marquès, François; Peaucellier, Gérard; Fort, Philippe; Féral, Jean-Pierre; Genevière, Anne-Marie; Picard, André

1998-01-01

We show that a splice variant–derived cyclin B is produced in sea urchin oocytes and embryos. This splice variant protein lacks highly conserved sequences in the COOH terminus of the protein. It is found strikingly abundant in growing oocytes and cells committed to differentiation during embryogenesis. Cyclin B splice variant (CBsv) protein associates weakly in the cell with Xenopus cdc2 and with budding yeast CDC28p. In contrast to classical cyclin B, CBsv very poorly complements a triple CLN deletion in budding yeast, and its microinjection prevents an initial step in MPF activation, leading to an important delay in oocyte meiosis reinitiation. CBsv microinjection in fertilized eggs induces cell cycle delay and abnormal development. We assume that CBsv is produced in growing oocytes to keep them in prophase, and during embryogenesis to slow down cell cycle in cells that will be committed to differentiation. PMID:9442104

Reversible inactivation and excitation of nucleus raphe magnus can modulate tail blood flow of male Wistar rats in response to hypothermia.

PubMed

Malakouti, Seyed Mansour; Kourosh Arami, Masoomeh; Sarihi, Abdorahman; Hajizadeh, Sohrab; Behzadi, Gila; Shahidi, Siamak; Komaki, Alireza; Heshmatian, Behnam; Vahabian, Mehrangiz

2008-10-01

The nucleus raphe magnus (NRM) is involved in thermoregulatory processing. There is a correlation between changes in the firing rates of the cells in the NRM and the application of the peripheral thermal stimulus. we examined the effect of reversible inactivation and excitation of NRM on mechanisms involved in tail blood flow (TBF) regulation in hypothermia. Hypothermia was induced in Male Wistar rats and cannula was implanted above the NRM. To evaluate the effect of nucleus inactivation on TBF, the amount of TBF was measured by Laser Doppler in hypothermic rats, before and after lidocaine microinjection into NRM. TBF was also measured after glutamate microinjection to assess the effect of nucleus excitation in hypothermic rats. Results indicated that after dropping TBF by hypothermia, microinjection of lidocaine into NRM significantly decreased TBF from 54.43 +- 5.7 to 46.81 +- 3.4, whereas glutamate microinjection caused a significant increase from 44.194 +- 0.6 to 98 +- 10.0 CONCLUSION: These data suggest that NRM have thermoregulatory effect in response to hypothermia.

Cloning of pigs from somatic cells and its prospects.

PubMed

Onishi, Akira

2002-01-01

The technology of somatic cell cloning in pigs is valuable for agricultural and therapeutic purposes. This paper will focus on the current methods of cloning pigs, including our successful microinjection#10; of somatic cell nuclei and its application. #10;

Microtubule dynamics in nerve cells: analysis using microinjection of biotinylated tubulin into PC12 cells

PubMed Central

1988-01-01

To study microtubule (MT) dynamics in nerve cells, we microinjected biotin-labeled tubulin into the cell body of chemically fused and differentiated PC12 cells and performed the immunofluorescence or immunogold procedure using an anti-biotin antibody followed by secondary antibodies coupled to fluorescent dye or colloidal gold. Incorporation of labeled subunits into the cytoskeleton of neurites was observed within minutes after microinjection. Serial electron microscopic reconstruction revealed that existing MTs in PC12 neurites incorporated labeled subunits mainly at their distal ends and the elongation rate of labeled segments was estimated to be less than 0.3 micron/min. Overall organization of MTs in the nerve cells was different from that in undifferentiated cells such as fibroblasts. Namely, we have not identified any MT-organizing centers from which labeled MTs are emanating in the cell bodies of the injected cells. Stereo electron microscopy revealed that some fully labeled segments seemed to start in the close vicinity of electron dense material within the neurites. This suggests new nucleation off some structures in the neurites. We have also studied the overall pattern of the incorporation of labeled subunits which extended progressively from the proximal part of the neurites toward their tips. To characterize the mechanism of tubulin incorporation, we have measured mean density of gold labeling per unit length of labeled segments at different parts of the neurites. The results indicate access of free tubulin subunits into the neurites and local incorporation into the neurite cytoskeleton. Our results lead to the conclusion that MTs are not static polymers but dynamic structures that continue to elongate even within the differentiated nerve cell processes. PMID:3047145

Pushing the envelope: microinjection of Minute virus of mice into Xenopus oocytes causes damage to the nuclear envelope.

PubMed

Cohen, Sarah; Panté, Nelly

2005-12-01

Parvoviruses are small DNA viruses that replicate in the nucleus of their host cells. It has been largely assumed that parvoviruses enter the nucleus through the nuclear pore complex (NPC). However, the details of this mechanism remain undefined. To study this problem, the parvovirus Minute virus of mice (MVM) was microinjected into the cytoplasm of Xenopus oocytes and a transmission electron microscope was used to visualize the effect of the virus on the host cell. It was found that MVM caused damage to the nuclear envelope (NE) in a time- and concentration-dependent manner. Damage was predominantly to the outer nuclear membrane and was often near the NPCs. However, microinjection experiments in which the NPCs were blocked showed that NE damage induced by MVM was independent of the NPC. To address the question of whether this effect of MVM is specific to the NE, purified organelles were incubated with MVM. Visualization by electron microscopy revealed that MVM did not affect all intracellular membranes. These data represent a novel form of virus-induced damage to host cell nuclear structure and suggest that MVM is imported into the nucleus using a unique mechanism that is independent of the NPC, and involves disruption of the NE and import through the resulting breaks.

Electron microscopic visualization of sites of nascent DNA synthesis by streptavidin-gold binding to biotinylated nucleotides incorporated in vivo

PubMed Central

1988-01-01

Biotinylated nucleotides (bio-11-dCTP, bio-11-dUTP, and bio-7-dATP) were microinjected into unfertilized and fertilized Xenopus laevis eggs. The amounts introduced were comparable to in vivo deoxy- nucleoside triphosphate pools. At various times after microinjection, DNA was extracted from eggs or embryos and subjected to electrophoresis on agarose gels. Newly synthesized biotinylated DNA was analyzed by Southern transfer and visualized using either the BluGENE or Detek-hrp streptavidin-based nucleic acid detection systems. Quantitation of the amount of biotinylated DNA observed at various times showed that the microinjected biotinylated nucleotides were efficiently incorporated in vivo, both into replicating endogenous chromosomal DNA and into replicating microinjected exogenous plasmid DNA. At least one biotinylated nucleotide could be incorporated in vivo for every eight nucleotides of DNA synthesized. Control experiments also showed that heavily biotinylated DNA was not subjected to detectable DNA repair during early embryogenesis (for at least 5 h after activation of the eggs). The incorporated biotinylated nucleotides were visualized by electron microscopy by using streptavidin-colloidal gold or streptavidin-ferritin conjugates to bind specifically to the biotin groups projecting from the newly replicated DNA. The incorporated biotinylated nucleotides were thus made visible as electron-dense spots on the underlying DNA molecules. Biotinylated nucleotides separated by 20-50 bases could be resolved. We conclude that nascent DNA synthesized in vivo in Xenopus laevis eggs can be visualized efficiently and specifically using the techniques described. PMID:3392102

Dynamics of hybrid amoeba proteus containing zoochlorellae studied using fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Liu, C.-H.; Fong, B. A.; Alfano, S. A., Jr.; Rakhlin, I.; Wang, W. B.; Ni, X. H.; Yang, Y. L.; Zhou, F.; Zuzolo, R. C.; Alfano, R. R.

2011-03-01

The microinjection of organelles, plants, particles or chemical solutions into Amoeba proteus coupled with spectroscopic analysis and observed for a period of time provides a unique new model for cancer treatment and studies. The amoeba is a eukaryote having many similar features of mammalian cells. The amoeba biochemical functions monitored spectroscopically can provide time sequence in vivo information about many metabolic transitions and metabolic exchanges between cellar organelles and substances microinjected into the amoeba. It is possible to microinject algae, plant mitochondria, drugs or carcinogenic solutions followed by recording the native fluorescence spectra of these composites. This model can be used to spectroscopically monitor the pre-metabolic transitions in developing diseased cells such as a cancer. Knowing specific metabolic transitions could offer solutions to inhibit cancer or reverse it as well as many other diseases. In the present study a simple experiment was designed to test the feasibility of this unique new model by injecting algae and chloroplasts into amoeba. The nonradiative dynamics found from these composites are evidence in terms of the emission ratios between the intensities at 337nm and 419nm; and 684nm bands. There were reductions in the metabolic and photosynthetic processes in amoebae that were microinjected with chloroplasts and zoochlorellae as well of those amoebae that ingested the algae and chloroplasts. The changes in the intensity of the emissions of the peaks indicate that the zoochlorellae lived in the amoebae for ten days. Spectral changes in intensity under the UV and 633nm wavelength excitation are from the energy transfer of DNA and RNA, protein-bound chromophores and chlorophylls present in zoochlorellae undergoing photosynthesis. The fluorescence spectroscopic probes established the biochemical interplay between the cell organelles and the algae present in the cell cytoplasm. This hybrid state is indicative that a symbiotic system is in place and the results definitely support the potential use of this unique new model. This model many help in plant / animal and cancer processes.

Survival rate of eukaryotic cells following electrophoretic nanoinjection

PubMed Central

Simonis, Matthias; Hübner, Wolfgang; Wilking, Alice; Huser, Thomas; Hennig, Simon

2017-01-01

Insertion of foreign molecules such as functionalized fluorescent probes, antibodies, or plasmid DNA to living cells requires overcoming the plasma membrane barrier without harming the cell during the staining process. Many techniques such as electroporation, lipofection or microinjection have been developed to overcome the cellular plasma membrane, but they all result in reduced cell viability. A novel approach is the injection of cells with a nanopipette and using electrophoretic forces for the delivery of molecules. The tip size of these pipettes is approximately ten times smaller than typical microinjection pipettes and rather than pressure pulses as delivery method, moderate DC electric fields are used to drive charged molecules out of the tip. Here, we show that this approach leads to a significantly higher survival rate of nanoinjected cells and that injection with nanopipettes has a significantly lower impact on the proliferation behavior of injected cells. Thus, we propose that injection with nanopipettes using electrophoretic delivery is an excellent alternative when working with valuable and rare living cells, such as primary cells or stem cells. PMID:28120926

Optical coherence tomography guided microinjections in live mouse embryos: high-resolution targeted manipulation for mouse embryonic research.

PubMed

Syed, Saba H; Coughlin, Andrew J; Garcia, Monica D; Wang, Shang; West, Jennifer L; Larin, Kirill V; Larina, Irina V

2015-05-01

The ability to conduct highly localized delivery of contrast agents, viral vectors, therapeutic or pharmacological agents, and signaling molecules or dyes to live mammalian embryos is greatly desired to enable a variety of studies in the field of developmental biology, such as investigating the molecular regulation of cardiovascular morphogenesis. To meet such a demand, we introduce, for the first time, the concept of employing optical coherence tomography (OCT)-guide microinjections in live mouse embryos, which provides precisely targeted manipulation with spatial resolution at the micrometer scale. The feasibility demonstration is performed with experimental studies on cultured live mouse embryos at E8.5 and E9.5. Additionally, we investigate the OCT-guided microinjection of gold–silica nanoshells to the yolk sac vasculature of live cultured mouse embryos at the stage when the heart just starts to beat, as a potential approach for dynamic assessment of cardiovascular form and function before the onset of blood cell circulation. Also, the capability of OCT to quantitatively monitor and measure injection volume is presented. Our results indicate that OCT-guided microinjection could be a useful tool for mouse embryonic research.

Optical coherence tomography guided microinjections in live mouse embryos: high-resolution targeted manipulation for mouse embryonic research

PubMed Central

Syed, Saba H.; Coughlin, Andrew J.; Garcia, Monica D.; Wang, Shang; West, Jennifer L.; Larin, Kirill V.; Larina, Irina V.

2015-01-01

Abstract. The ability to conduct highly localized delivery of contrast agents, viral vectors, therapeutic or pharmacological agents, and signaling molecules or dyes to live mammalian embryos is greatly desired to enable a variety of studies in the field of developmental biology, such as investigating the molecular regulation of cardiovascular morphogenesis. To meet such a demand, we introduce, for the first time, the concept of employing optical coherence tomography (OCT)-guide microinjections in live mouse embryos, which provides precisely targeted manipulation with spatial resolution at the micrometer scale. The feasibility demonstration is performed with experimental studies on cultured live mouse embryos at E8.5 and E9.5. Additionally, we investigate the OCT-guided microinjection of gold–silica nanoshells to the yolk sac vasculature of live cultured mouse embryos at the stage when the heart just starts to beat, as a potential approach for dynamic assessment of cardiovascular form and function before the onset of blood cell circulation. Also, the capability of OCT to quantitatively monitor and measure injection volume is presented. Our results indicate that OCT-guided microinjection could be a useful tool for mouse embryonic research. PMID:25581495

Analysis of 14-3-3 Family Member Function in Xenopus Embryos by Microinjection of Antisense Morpholino Oligos

NASA Astrophysics Data System (ADS)

Lau, Jeffrey M. C.; Muslin, Anthony J.

The 14-3-3 intracellular phosphoserine/threonine-binding proteins are adapter molecules that regulate signal transduction, cell cycle, nutrient sensing, apoptotic, and cytoskeletal pathways. There are seven 14-3-3 family members, encoded by separate genes, in vertebrate organisms. To evaluate the role of individual 14-3-3 proteins in vertebrate embryonic development, we utilized an antisense morpholino oligo microinjection technique in Xenopus laevis embryos. By use of this method, we showed that embryos lacking specific 14-3-3 proteins displayed unique phenotypic abnormalities. Specifically, embryos lacking 14-3-3 τ exhibited gastrulation and axial patterning defects, but embryos lacking 14-3-3 γ exhibited eye defects without other abnormalities, and embryos lacking 14-3-3 ζ appeared completely normal. These and other results demonstrate the power and specificity of the morpholino antisense oligo microinjection technique.

Diffusion tensor driven contour closing for cell microinjection targeting.

PubMed

Becattini, Gabriele; Mattos, Leonardo S; Caldwell, Darwin G

2010-01-01

This article introduces a novel approach to robust automatic detection of unstained living cells in bright-field (BF) microscope images with the goal of producing a target list for an automated microinjection system. The overall image analysis process is described and includes: preprocessing, ridge enhancement, image segmentation, shape analysis and injection point definition. The developed algorithm implements a new version of anisotropic contour completion (ACC) based on the partial differential equation (PDE) for heat diffusion which improves the cell segmentation process by elongating the edges only along their tangent direction. The developed ACC algorithm is equivalent to a dilation of the binary edge image with a continuous elliptic structural element that takes into account local orientation of the contours preventing extension towards normal direction. Experiments carried out on real images of 10 to 50 microm CHO-K1 adherent cells show a remarkable reliability in the algorithm along with up to 85% success for cell detection and injection point definition.

Effect of diadenosine tetraphosphate microinjection on heat shock protein synthesis in Xenopus laevis oocytes.

PubMed Central

Guedon, G; Sovia, D; Ebel, J P; Befort, N; Remy, P

1985-01-01

Bisnucleosides polyphosphates are thought to be chemical messengers signalling to the cell the onset of various stresses. Diadenosine tri- and tetraphosphates (respectively, Ap3A and Ap4A) accumulate in prokaryotic and eukaryotic cells under heat shock conditions, suggesting they could trigger the synthesis of heat shock proteins (hsps). In this study, Ap4A, Ap3A and, as a control, Ap4 (adenosine tetraphosphate) were injected into Xenopus oocytes. Whereas none of these compounds is able to trigger the synthesis of hsps in the absence of hyperthermic treatment, nuclear microinjection of Ap4A after a mild heat shock specifically enhances the synthesis of the 70-kd hsp, which is involved in the regulation and possibly the termination of the heat shock response. The microinjection of Ap4A prior to the hyperthermic treatment results in a strong inhibition of hsps synthesis (with the exception of the 70-kd hsp) suggesting that Ap4A is involved in the regulation and/or termination of the heat shock response. Ap3A and Ap4 do not induce any detectable modification of hsps expression. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. PMID:4092696

Casein kinase II induces c-fos expression via the serum response element pathway and p67SRF phosphorylation in living fibroblasts.

PubMed Central

Gauthier-Rouvière, C; Basset, M; Blanchard, J M; Cavadore, J C; Fernandez, A; Lamb, N J

1991-01-01

Elevation of intracellular casein kinase II (CKII) levels through microinjection of purified CKII results in the rapid and transient induction of c-fos in quiescent rat embryo fibroblasts, and activation of quiescent cells by serum is accompanied by the nuclear relocation of endogenous CKII. The induction of c-fos by CKII is inhibited by coinjection of oligonucleotides corresponding to the sequence of the serum response element (SRE) present in the c-fos promoter, indicating that competitive displacement of positive factors from the endogenous c-fos SRE prevents c-fos induction by CKII. Furthermore, the expression of c-fos induced by either CKII injection or serum activation is also inhibited by microinjection of antibodies against the 67 kDa serum response factor (p67SRF) indicating the absolute requirement of p67SRF in this process. Finally, we show the specific phosphorylation of p67SRF in vivo following microinjection of CKII into quiescent cells. Together, these data strongly support that CKII induces c-fos expression through binding/activation of the phosphorylated p67SRF at the SRE sequence. Images PMID:1915270

Mitosis in sand dollar embryos is inhibited by antibodies directed against the calcium transport enzyme of muscle.

PubMed

Silver, R B

1986-06-01

Monospecific antibodies to the calcium transport enzyme (alpha-Ca pump) inhibit mitosis when microinjected into sand dollar embryos. Immunoglobulins were raised against the calcium transport enzyme (Ca pump) of sarcoplasmic reticulum (SR) from rat skeletal muscle and guinea pig ileum smooth muscle. Specific antibodies were further isolated from IgG fractions by using electrophoretically purified SR Ca-pump protein as the immobilized ligand for immunoaffinity chromatography. ELISA demonstrated that common antigenic determinants are shared by SR, SR Ca pump (of rat skeletal and guinea pig ileum smooth muscle), and isolated membrane containing "native" mitotic apparatus (MA). Preimmune sera gave negative results in identical control assays. Triton X-100 extraction of MA removes the Ca-pump antigen. SR Ca pump and the MA Ca pump have nearly identical molecular masses as determined by NaDodSO4/PAGE. These alpha-SR Ca-pump IgGs inhibit ATP-dependent Ca2+ sequestration by purified SR and MA membranes. Indirect immunofluorescence of isolated native MA demonstrated coincident localization of the MA Ca pump, sequestered calcium, and membrane vesicles. Fluorescent foci were regionally concentrated within the volumes of the asters and spindle. Microinjection of the anti-Ca-pump IgGs into one of two sister blastomeres at second metaphase resulted in mitotic arrest of the injected cell accompanied by a rapid loss of spindle birefringence. Karyomeres formed and fused to form nuclei either at the site of the metaphase plate or at the position the chromosomes occupied during anaphase A. The cleavage furrow did not develop in the injected cell, while the sister and neighbor cells continued normal mitotic cycling. Injection later in mitosis yielded cells with two nuclei whose cleavage furrow relaxed completely. Routine control injections of boiled immune IgG, preimmune IgG, Wesson oil, buffer, or goat anti-rabbit IgG did not affect mitosis, birefringence of the MA, or cleavage furrow activity.

Mitosis in sand dollar embryos is inhibited by antibodies directed against the calcium transport enzyme of muscle.

PubMed Central

Silver, R B

1986-01-01

Monospecific antibodies to the calcium transport enzyme (alpha-Ca pump) inhibit mitosis when microinjected into sand dollar embryos. Immunoglobulins were raised against the calcium transport enzyme (Ca pump) of sarcoplasmic reticulum (SR) from rat skeletal muscle and guinea pig ileum smooth muscle. Specific antibodies were further isolated from IgG fractions by using electrophoretically purified SR Ca-pump protein as the immobilized ligand for immunoaffinity chromatography. ELISA demonstrated that common antigenic determinants are shared by SR, SR Ca pump (of rat skeletal and guinea pig ileum smooth muscle), and isolated membrane containing "native" mitotic apparatus (MA). Preimmune sera gave negative results in identical control assays. Triton X-100 extraction of MA removes the Ca-pump antigen. SR Ca pump and the MA Ca pump have nearly identical molecular masses as determined by NaDodSO4/PAGE. These alpha-SR Ca-pump IgGs inhibit ATP-dependent Ca2+ sequestration by purified SR and MA membranes. Indirect immunofluorescence of isolated native MA demonstrated coincident localization of the MA Ca pump, sequestered calcium, and membrane vesicles. Fluorescent foci were regionally concentrated within the volumes of the asters and spindle. Microinjection of the anti-Ca-pump IgGs into one of two sister blastomeres at second metaphase resulted in mitotic arrest of the injected cell accompanied by a rapid loss of spindle birefringence. Karyomeres formed and fused to form nuclei either at the site of the metaphase plate or at the position the chromosomes occupied during anaphase A. The cleavage furrow did not develop in the injected cell, while the sister and neighbor cells continued normal mitotic cycling. Injection later in mitosis yielded cells with two nuclei whose cleavage furrow relaxed completely. Routine control injections of boiled immune IgG, preimmune IgG, Wesson oil, buffer, or goat anti-rabbit IgG did not affect mitosis, birefringence of the MA, or cleavage furrow activity. Images PMID:2940599

Effects of the Particulate Matter₂.₅ (PM₂.₅) on Lipoprotein Metabolism, Uptake and Degradation, and Embryo Toxicity.

PubMed

Kim, Jae-Yong; Lee, Eun-Young; Choi, Inho; Kim, Jihoe; Cho, Kyung-Hyun

2015-12-01

Particulate matter2.5 (PM2.5) is notorious for its strong toxic effects on the cardiovascular, skin, nervous, and reproduction systems. However, the molecular mechanism by which PM2.5 aggravates disease progression is poorly understood, especially in a water-soluble state. In the current study, we investigated the putative physiological effects of aqueous PM2.5 solution on lipoprotein metabolism. Collected PM2.5 from Seoul, Korea was dissolved in water, and the water extract (final 3 and 30 ppm) was treated to human serum lipoproteins, macrophages, and dermal cells. PM2.5 extract resulted in degradation and aggregation of high-density lipoprotein (HDL) as well as low-density lipoprotein (LDL); apoA-I in HDL aggregated and apo-B in LDL disappeared. PM2.5 treatment (final 30 ppm) also induced cellular uptake of oxidized LDL (oxLDL) into macrophages, especially in the presence of fructose (final 50 mM). Uptake of oxLDL along with production of reactive oxygen species was accelerated by PM2.5 solution in a dose-dependent manner. Further, PM2.5 solution caused cellular senescence in human dermal fibroblast cells. Microinjection of PM2.5 solution into zebrafish embryos induced severe mortality accompanied by impairment of skeletal development. In conclusion, water extract of PM2.5 induced oxidative stress as a precursor to cardiovascular toxicity, skin cell senescence, and embryonic toxicity via aggregation and proteolytic degradation of serum lipoproteins.

Effects of the Particulate Matter2.5 (PM2.5) on Lipoprotein Metabolism, Uptake and Degradation, and Embryo Toxicity

PubMed Central

Kim, Jae-Yong; Lee, Eun-Young; Choi, Inho; Kim, Jihoe; Cho, Kyung-Hyun

2015-01-01

Particulate matter2.5 (PM2.5) is notorious for its strong toxic effects on the cardiovascular, skin, nervous, and reproduction systems. However, the molecular mechanism by which PM2.5 aggravates disease progression is poorly understood, especially in a water-soluble state. In the current study, we investigated the putative physiological effects of aqueous PM2.5 solution on lipoprotein metabolism. Collected PM2.5 from Seoul, Korea was dissolved in water, and the water extract (final 3 and 30 ppm) was treated to human serum lipoproteins, macrophages, and dermal cells. PM2.5 extract resulted in degradation and aggregation of high-density lipoprotein (HDL) as well as low-density lipoprotein (LDL); apoA-I in HDL aggregated and apo-B in LDL disappeared. PM2.5 treatment (final 30 ppm) also induced cellular uptake of oxidized LDL (oxLDL) into macrophages, especially in the presence of fructose (final 50 mM). Uptake of oxLDL along with production of reactive oxygen species was accelerated by PM2.5 solution in a dose-dependent manner. Further, PM2.5 solution caused cellular senescence in human dermal fibroblast cells. Microinjection of PM2.5 solution into zebrafish embryos induced severe mortality accompanied by impairment of skeletal development. In conclusion, water extract of PM2.5 induced oxidative stress as a precursor to cardiovascular toxicity, skin cell senescence, and embryonic toxicity via aggregation and proteolytic degradation of serum lipoproteins. PMID:26615830

Intracerebral dendritic cells critically modulate encephalitogenic versus regulatory immune responses in the CNS

PubMed Central

Zozulya, Alla L.; Ortler, Sonja; Lee, JangEun; Weidenfeller, Christian; Sandor, Matyas; Wiendl, Heinz; Fabry, Zsuzsanna

2010-01-01

Dendritic cells (DCs) appear in higher numbers within the CNS as a consequence of inflammation associated with autoimmune disorders, such as multiple sclerosis (MS), but the contribution of these cells to the outcome of disease is not yet clear. Here we show that stimulatory or tolerogenic functional states of intracerebral DCs regulate the systemic activation of neuroantigen-specific T cells, the recruitment of these cells into the CNS and the onset and progression of experimental autoimmune encephalomyelitis (EAE). Intracerebral microinjection of stimulatory DCs exacerbated the onset and clinical course of EAE, accompanied with an early T-cell infiltration and a decreased proportion of regulatory FoxP3-expressing cells in the brain. In contrast, the intracerebral microinjection of DCs modified by tumor necrosis factor alpha (TNF-α) induced their tolerogenic functional state and delayed or prevented EAE onset. This triggered the generation of interleukin 10 (IL-10)-producing neuroantigen-specific lymphocytes in the periphery and restricted IL-17 production in the CNS. Our findings suggest that DCs are a rate-limiting factor for neuroinflammation. PMID:19129392

Human immunodeficiency virus type 1 Tat does not transactivate mature trans-acting responsive region RNA species in the nucleus or cytoplasm of primate cells.

PubMed Central

Chin, D J; Selby, M J; Peterlin, B M

1991-01-01

Human immunodeficiency virus (HIV)-encoded transactivator Tat is essential for viral gene expression and replication. By interacting with a nascent RNA stem-loop called the trans-acting responsive region (TAR). Tat increases rates of initiation and/or elongation of HIV transcription. Several reports have also suggested that Tat has additional effects on mature HIV RNA species including modification of primary transcripts in the nucleus and their increased translation in the cytoplasm. These posttranscriptional effects are most pronounced in the Xenopus oocyte. To investigate directly whether Tat has similar effects on viral transcripts in cells that are permissive for HIV replication, we cotransfected and microinjected human and monkey cells with Tat and TAR in the form of DNA or RNA. Whereas Tat transactivated TAR DNA targets, it did not transactivate TAR RNA targets in the nucleus of microinjected cells or in the cytoplasm of transfected cells. We conclude that in cells permissive for viral replication, Tat exerts its effect primarily at the level of HIV transcription. Images PMID:1900539

Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides

PubMed Central

Wefers, Benedikt; Meyer, Melanie; Ortiz, Oskar; Hrabé de Angelis, Martin; Hansen, Jens; Wurst, Wolfgang; Kühn, Ralf

2013-01-01

The study of genetic disease mechanisms relies mostly on targeted mouse mutants that are derived from engineered embryonic stem (ES) cells. Nevertheless, the establishment of mutant ES cells is laborious and time-consuming, restricting the study of the increasing number of human disease mutations discovered by high-throughput genomic analysis. Here, we present an advanced approach for the production of mouse disease models by microinjection of transcription activator-like effector nucleases (TALENs) and synthetic oligodeoxynucleotides into one-cell embryos. Within 2 d of embryo injection, we created and corrected chocolate missense mutations in the small GTPase RAB38; a regulator of intracellular vesicle trafficking and phenotypic model of Hermansky-Pudlak syndrome. Because ES cell cultures and targeting vectors are not required, this technology enables instant germline modifications, making heterozygous mutants available within 18 wk. The key features of direct mutagenesis by TALENs and oligodeoxynucleotides, minimal effort and high speed, catalyze the generation of future in vivo models for the study of human disease mechanisms and interventions. PMID:23426636

Intracellular distribution of nontargeted quantum dots after natural uptake and microinjection

PubMed Central

Damalakiene, Leona; Karabanovas, Vitalijus; Bagdonas, Saulius; Valius, Mindaugas; Rotomskis, Ricardas

2013-01-01

Background: The purpose of this study was to elucidate the mechanism of natural uptake of nonfunctionalized quantum dots in comparison with microinjected quantum dots by focusing on their time-dependent accumulation and intracellular localization in different cell lines. Methods: The accumulation dynamics of nontargeted CdSe/ZnS carboxyl-coated quantum dots (emission peak 625 nm) was analyzed in NIH3T3, MCF-7, and HepG2 cells by applying the methods of confocal and steady-state fluorescence spectroscopy. Intracellular colocalization of the quantum dots was investigated by staining with Lysotracker®. Results: The uptake of quantum dots into cells was dramatically reduced at a low temperature (4°C), indicating that the process is energy-dependent. The uptake kinetics and imaging of intracellular localization of quantum dots revealed three accumulation stages of carboxyl-coated quantum dots at 37°C, ie, a plateau stage, growth stage, and a saturation stage, which comprised four morphological phases: adherence to the cell membrane; formation of granulated clusters spread throughout the cytoplasm; localization of granulated clusters in the perinuclear region; and formation of multivesicular body-like structures and their redistribution in the cytoplasm. Diverse quantum dots containing intracellular vesicles in the range of approximately 0.5–8 μm in diameter were observed in the cytoplasm, but none were found in the nucleus. Vesicles containing quantum dots formed multivesicular body-like structures in NIH3T3 cells after 24 hours of incubation, which were Lysotracker-negative in serum-free medium and Lysotracker-positive in complete medium. The microinjected quantum dots remained uniformly distributed in the cytosol for at least 24 hours. Conclusion: Natural uptake of quantum dots in cells occurs through three accumulation stages via a mechanism requiring energy. The sharp contrast of the intracellular distribution after microinjection of quantum dots in comparison with incubation as well as the limited transfer of quantum dots from vesicles into the cytosol and vice versa support the endocytotic origin of the natural uptake of quantum dots. Quantum dots with proteins adsorbed from the culture medium had a different fate in the final stage of accumulation from that of the protein-free quantum dots, implying different internalization pathways. PMID:23429995

Functions of AT1 and AT2 angiotensin receptors in the paraventricular nucleus of the rat, correlating single-unit and cardiovascular responses.

PubMed

Khanmoradi, Mehrangiz; Nasimi, Ali

2017-06-01

The paraventricular hypothalamic nucleus (PVN) is a complex structure with both neuroendocrine and autonomic functions including cardiovascular control. The PVN contains angiotensin II (AngII) immunoreactive cells, fibers, as well as AT1 and AT2 receptors of AngII. We microinjected AngII into the PVN of normotensive anesthetized rats and simultaneously recorded blood pressure, heart rate (HR) and single-unit responses. The roles of AT1 and AT2 receptors in these responses were also evaluated. Microinjection of AngII into the PVN produced a short excitatory single-unit response and two types of pressor responses: short duration with a decrease in HR and long with an increase in HR. Microinjection of losartan, an AT1 antagonist, into the PVN produced two response types, attenuation and augmentation of the pressor and firing rate responses to AngII. Microinjection of PD123319, an AT2 antagonist, into the PVN greatly attenuated pressor and single-unit response to AngII, indicating that the pressor response was mediated through AT2 receptors too. In conclusion, microinjection of AngII into the PVN stimulates neurons resulting in an increase in firing rate and consequently produces a short or long pressor response. These responses were mediated through AT1 and AT2 receptors; however, AT1 receptor may produce inhibition too. The results suggest that AngII of the PVN may be a neurotransmitter playing a role in arterial pressure regulation. Copyright © 2017 Elsevier Inc. All rights reserved.

Acute pancreatitis decreases the sensitivity of pancreas-projecting dorsal motor nucleus of the vagus neurones to group II metabotropic glutamate receptor agonists in rats

PubMed Central

Babic, Tanja; Travagli, R Alberto

2014-01-01

Recent studies have shown that pancreatic exocrine secretions (PES) are modulated by dorsal motor nucleus of the vagus (DMV) neurones, whose activity is finely tuned by GABAergic and glutamatergic synaptic inputs. Group II metabotropic glutamate receptors (mGluR) decrease synaptic transmission to pancreas-projecting DMV neurones and increase PES. In the present study, we used a combination of in vivo and in vitro approaches aimed at characterising the effects of caerulein-induced acute pancreatitis (AP) on the vagal neurocircuitry modulating pancreatic functions. In control rats, microinjection of bicuculline into the DMV increased PES, whereas microinjections of kynurenic acid had no effect. Conversely, in AP rats, microinjection of bicuculline had no effect, whereas kynurenic acid decreased PES. DMV microinjections of the group II mGluR agonist APDC and whole cell recordings of excitatory currents in identified pancreas-projecting DMV neurones showed a reduced functional response in AP rats compared to controls. Moreover, these changes persisted up to 3 weeks following the induction of AP. These data demonstrate that AP increases the excitatory input to pancreas-projecting DMV neurones by decreasing the response of excitatory synaptic terminals to group II mGluR agonist. PMID:24445314

Hypothalamic supraoptic but not paraventricular nucleus is involved in cardiovascular responses to carbachol microinjected into the bed nucleus of stria terminalis of unanesthetized rats.

PubMed

Alves, Fernando H F; Crestani, Carlos C; Busnardo, Cristiane; Antunes-Rodrigues, José; Gomes, Felipe V; Resstel, Leonardo B M; Corrêa, Fernando M A

2011-06-01

Microinjection of the cholinergic agonist carbachol into the bed nucleus of the stria terminalis (BST) has been reported to cause pressor response in unanesthetized rats, which was shown to be mediated by an acute release of vasopressin into the systemic circulation and followed by baroreflex-mediated bradycardia. In the present study, we tested the possible involvement of the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei in the pressor response evoked by carbachol microinjection into the BST of unanesthetized rats. For this, cardiovascular responses following carbachol (1 nmol/100 nL) microinjection into the BST were studied before and after PVN or SON pretreatment, either ipsilateral or contralateral in relation to BST microinjection site, with the nonselective neurotransmission blocker cobalt chloride (CoCl₂, 1 mM/100 nL). Carbachol microinjection into the BST evoked pressor response. Moreover, BST treatment with carbachol significantly increased plasma vasopressin levels, thus confirming previous evidences that carbachol microinjection into the BST evokes pressor response due to vasopressin release into the circulation. SON pretreatment with CoCl₂, either ipsilateral or contralateral in relation to BST microinjection site, inhibited the pressor response to carbachol microinjection into the BST. However, CoCl₂ microinjection into the ipsilateral or contralateral PVN did not affect carbachol-evoked pressor response. In conclusion, our results suggest that pressor response to carbachol microinjection into the BST is mediated by SON magnocellular neurons, without significant involvement of those in the PVN. The results also indicate that responses to carbachol microinjection into the BST are mediated by a neural pathway that depends on the activation of both ipsilateral and contralateral SON. Copyright © 2011 Elsevier B.V. All rights reserved.

Effective intracellular inhibition of the cAMP-dependent protein kinase by microinjection of a modified form of the specific inhibitor peptide PKi in living fibroblasts.

PubMed

Fernandez, A; Mery, J; Vandromme, M; Basset, M; Cavadore, J C; Lamb, N J

1991-08-01

In order to obtain a peptide retaining its biological activity following microinjection into living cells, we have modified a synthetic peptide [PKi(m)(6-24)], derived from the specific inhibitor protein of the cAMP-dependent protein kinase (A-kinase) in two ways: (1) substitution of the arginine at position 18 for a D-arginine; (2) blockade of the side chain on the C-terminal aspartic acid by a cyclohexyl ester group. In an in vitro assay, PKi(m) has retained a specific inhibitory activity against A-kinase as assessed against six other kinases, with similar efficiency to that of the unmodified PKi(5-24) peptide. Microinjection of PKi(m) into living fibroblasts reveals its capacity to prevent the changes in cell morphology and cytoskeleton induced by drugs which activate endogenous A-kinase, whereas the original PKi peptide failed to do so. This inhibition of A-kinase in vivo by PKi(m) lasts between 4 and 6 h after injection. In light of its effective half-life, this modified peptide opens a route for the use of biologically active peptides in vivo, an approach which has been hampered until now by the exceedingly short half-life of peptides inside living cells. By providing a direct means of inhibiting A-kinase activity for sufficiently long periods to observe effects on cellular functions in living cells, PKi(m) represents a powerful tool in studying the potential role of cAMP-dependent phosphorylation in vivo.

High throughput, detailed, cell-specific neuroanatomy of dendritic spines using microinjection and confocal microscopy

PubMed Central

Dumitriu, Dani; Rodriguez, Alfredo; Morrison, John H.

2012-01-01

Morphological features such as size, shape and density of dendritic spines have been shown to reflect important synaptic functional attributes and potential for plasticity. Here we describe in detail a protocol for obtaining detailed morphometric analysis of spines using microinjection of fluorescent dyes, high resolution confocal microscopy, deconvolution and image analysis using NeuronStudio. Recent technical advancements include better preservation of tissue resulting in prolonged ability to microinject, and algorithmic improvements that compensate for the residual Z-smear inherent in all optical imaging. Confocal imaging parameters were probed systematically for the identification of both optimal resolution as well as highest efficiency. When combined, our methods yield size and density measurements comparable to serial section transmission electron microscopy in a fraction of the time. An experiment containing 3 experimental groups with 8 subjects in each can take as little as one month if optimized for speed, or approximately 4 to 5 months if the highest resolution and morphometric detail is sought. PMID:21886104

Substance P analogues potentiate the pressor response to microinjection of L-glutamate into laminas I and II of the cat dorsal horn.

PubMed

Beyaert, C A; Hill, J M; Kaufman, M P

1997-06-06

Microinjection of a substance P analogue (1 mM; 7 or 10 nl) into laminae I and II of the L7 dorsal horn of decerebrate cats significantly potentiated (P < 0.05) the increase in arterial pressure evoked by microinjection of L-glutamate (109 mM; 7 or 10 nl) into these spinal sites. Microinjection of the substance P analogues (i.e., GR73638 and [Sar9,Met(O2)11]-substance P) which were selective NK-1 receptor agonists, had no impact on the cardioacceleration evoked by microinjection of L-glutamate (P > 0.05). In addition, microinjection of these analogues had no effect on the modest and non-significant increase in phrenic nerve discharge evoked by L-glutamate. We conclude that stimulation of NK-1 receptors in the superficial laminae of the dorsal horn potentiates the pressor responses to microinjection of L-glutamate.

Correction of xeroderma pigmentosum repair defect by basal transcription factor BTF2 (TFIIH).

PubMed Central

van Vuuren, A J; Vermeulen, W; Ma, L; Weeda, G; Appeldoorn, E; Jaspers, N G; van der Eb, A J; Bootsma, D; Hoeijmakers, J H; Humbert, S

1994-01-01

ERCC3 was initially identified as a gene correcting the nucleotide excision repair (NER) defect of xeroderma pigmentosum complementation group B (XP-B). The recent finding that its gene product is identical to the p89 subunit of basal transcription factor BTF2(TFIIH), opened the possibility that it is not directly involved in NER but that it regulates the transcription of one or more NER genes. Using an in vivo microinjection repair assay and an in vitro NER system based on cell-free extracts we demonstrate that ERCC3 in BTF2 is directly implicated in excision repair. Antibody depletion experiments support the idea that the p62 BTF2 subunit and perhaps the entire transcription factor function in NER. Microinjection experiments suggest that exogenous ERCC3 can exchange with ERCC3 subunits in the complex. Expression of a dominant negative K436-->R ERCC3 mutant, expected to have lost all helicase activity, completely abrogates NER and transcription and concomitantly induces a dramatic chromatin collapse. These findings establish the role of ERCC3 and probably the entire BTF2 complex in transcription in vivo which was hitherto only demonstrated in vitro. The results strongly suggest that transcription itself is a critical component for maintenance of chromatin structure. The remarkable dual role of ERCC3 in NER and transcription provides a clue in understanding the complex clinical features of some inherited repair syndromes. Images PMID:8157004

Opioid microinjection into raphe magnus modulates cardiorespiratory function in mice and rats.

PubMed

Hellman, Kevin M; Mendelson, Scott J; Mendez-Duarte, Marco A; Russell, James L; Mason, Peggy

2009-11-01

The raphe magnus (RM) participates in opioid analgesia and contains pain-modulatory neurons with respiration-related discharge. Here, we asked whether RM contributes to respiratory depression, the most prevalent lethal effect of opioids. To investigate whether opioidergic transmission in RM produces respiratory depression, we microinjected a mu-opioid receptor agonist, DAMGO, or morphine into the RM of awake rodents. In mice, opioid microinjection produced sustained decreases in respiratory rate (170 to 120 breaths/min), as well as heart rate (520 to 400 beats/min). Respiratory sinus arrhythmia, indicative of enhanced parasympathetic activity, was prevalent in mice receiving DAMGO microinjection. We performed similar experiments in rats but observed no changes in breathing rate or heart rate. Both rats and mice experienced significantly more episodes of bradypnea, indicative of impaired respiratory drive, after opioid microinjection. During spontaneous arousals, rats showed less tachycardia after opioid microinjection than before microinjection, suggestive of an attenuated sympathetic tone. Thus, activation of opioidergic signaling within RM produces effects beyond analgesia, including the unwanted destabilization of cardiorespiratory function. These adverse effects on homeostasis consequent to opioid microinjection imply a role for RM in regulating the balance of sympathetic and parasympathetic tone.

SPATIAL MEMORY IMPAIRMENT AND HIPPOCAMPAL CELL LOSS INDUCED BY OKADAIC ACID (EXPERIMENTAL STUDY).

PubMed

Chighladze, M; Dashniani, M; Beselia, G; Kruashvili, L; Naneishvili, T

2016-01-01

In the present study, we evaluated and compared effect of intracerebroventricular (ICV) and intrahippocampal bilateral microinjection of okadaic acid (OA) on spatial memory function assessed in one day water maze paradigm and hippocampal structure in rats. Rats were divided in following groups: Control(icv) - rats injected with ICV and aCSF; Control(hipp) - rats injected intrahippocampally with aCSF; OAicv - rats injected with ICV and OA; OAhipp - rats injected intrahippocampally with OA. Nissl staining of hippocampal sections showed that the pyramidal cell loss in OAhipp group is significantly higher than that in the OAicv. The results of behavioral experiments showed that ICV or intrahippocampal bilateral microinjection of OA did not affect learning process and short-term spatial memory but induced impairment in spatial long-term memory assessed in probe test performance 24 h after training. OA-induced spatial memory impairment may be attributed to the hippocampal cell death. Based on these results OA induced memory deficit and hippocampal cell loss in rat may be considered as a potential animal model for preclinical evaluation of antidementic drug activity.

The behavioural importance of dynamically activated descending inhibition from the nucleus reticularis gigantocellularis pars alpha.

PubMed

Azami, J; Green, D L; Roberts, M H; Monhemius, R

2001-05-01

We have recently demonstrated (J Physiol 506 (1998) 459) that the dynamic activation of descending inhibition of the nociceptive response of spinal multireceptive cells occurs in the nucleus reticularis gigantocellularis pars alpha (GiA). In the same paper we have shown that Lamina I dorsal horn cells are responsible for activating this inhibition via a pathway which runs in the contralateral dorsolateral funiculus. The effects of dynamically activating this system by noxious stimulation on behavioural responses to noxious stimuli have not been established. Here we demonstrate the effects of GiA on the behavioural response during application of standardized noxious stimuli. As this system is activated in response to noxious stimulation (J Physiol 506 (1998) 459), it is possible that chronic pain states may also activate GiA. We have therefore investigated this possibility in animals following partial sciatic nerve ligation (an animal model of chronic pain; Pain 43 (1990) 205). Male Wistar rats (280-310 g) were anaesthetized with halothane (0.5-2% in O(2)). Guide cannulae for microinjections were stereotaxically placed above GiA. In one group of animals the sciatic nerve was partially ligated. Animals were allowed to recover for 4-6 days. The responses of each animal during the formalin test (Pain 4 (1977) 161) and the tail flick test (Pain 12 (1982) 229) were recorded on different days. Microinjections (0.5 microl) of either gamma-aminobutyric acid (GABA, 200 mM), D-L homocysteic acid (DLH, 25 mM) or 0.9% saline (as control) into GiA were performed during these tests in a randomized, blind manner. In animals without sciatic nerve ligation, microinjection of GABA to GiA did not significantly affect the animal's response during the tail flick test. However microinjection of DLH significantly increased the latency of tail flick from 6.2 +/- 0.8 to 8.4 +/- 0.5 s for up to 15 min (n = 7, P < 0.01, Mann-Whitney U-test). Microinjection of GABA to GiA increased the behavioural response to formalin between 10 and 20 min post-injection, while microinjection of DLH reduced this response at all time points except 10 min post-injection (n = 8, P < 0.05, Mann-Whitney U-test). In animals with sciatic nerve ligation, microinjections (0.5 microl) of either GABA (200 mM), or saline (as control) into GiA contralateral to the partial sciatic ligation were performed during these tests in a randomized, blind manner. Partial sciatic ligation significantly reduced the behavioural response to contralaterally applied formalin from 15 min post-injection onwards, compared to controls without sciatic nerve ligation. Microinjection of GABA to GiA significantly increased the behavioural response to formalin from 20 to 50 min post-injection. The inactivation of GiA only causes behavioural effects in nociceptive tests of a long enough duration to activate the system (i.e. the formalin test but not the tail flick test). Chemical activation of the system affects both tests. These data strongly support the concept of an important analgesic system which is activated in response to noxious stimulation, and subsequently acts to reduce behavioural responses to noxious stimuli.

High resolution ultrasound-guided microinjection for interventional studies of early embryonic and placental development in vivo in mice

PubMed Central

Slevin, John C; Byers, Lois; Gertsenstein, Marina; Qu, Dawei; Mu, Junwu; Sunn, Nana; Kingdom, John CP; Rossant, Janet; Adamson, S Lee

2006-01-01

Background In utero microinjection has proven valuable for exploring the developmental consequences of altering gene expression, and for studying cell lineage or migration during the latter half of embryonic mouse development (from embryonic day 9.5 of gestation (E9.5)). In the current study, we use ultrasound guidance to accurately target microinjections in the conceptus at E6.5–E7.5, which is prior to cardiovascular or placental dependence. This method may be useful for determining the developmental effects of targeted genetic or cellular interventions at critical stages of placentation, gastrulation, axis formation, and neural tube closure. Results In 40 MHz ultrasound images at E6.5, the ectoplacental cone region and proamniotic cavity could be visualized. The ectoplacental cone region was successfully targeted with 13.8 nL of a fluorescent bead suspension with few or no beads off-target in 51% of concepti microinjected at E6.5 (28/55 injected). Seventy eight percent of the embryos survived 2 to 12 days post injection (93/119), 73% (41/56) survived to term of which 68% (38/56) survived and appeared normal one week after birth. At E7.5, the amniotic and exocoelomic cavities, and ectoplacental cone region were discernable. Our success at targeting with few or no beads off-target was 90% (36/40) for the ectoplacental cone region and 81% (35/43) for the exocoelomic cavity but tended to be less, 68% (34/50), for the smaller amniotic cavity. At E11.5, beads microinjected at E7.5 into the ectoplacental cone region were found in the placental spongiotrophoblast layer, those injected into the exocoelomic cavity were found on the surface or within the placental labyrinth, and those injected into the amniotic cavity were found on the surface or within the embryo. Following microinjection at E7.5, survival one week after birth was 60% (26/43) when the amniotic cavity was the target and 66% (19/29) when the target was the ectoplacental cone region. The survival rate was similar in sham experiments, 54% (33/61), for which procedures were identical but no microinjection was performed, suggesting that surgery and manipulation of the uterus were the main causes of embryonic death. Conclusion Ultrasound-guided microinjection into the ectoplacental cone region at E6.5 or E7.5 and the amniotic cavity at E7.5 was achieved with a 7 day postnatal survival of ≥60%. Target accuracy of these sites and of the exocoelomic cavity at E7.5 was ≥51%. We suggest that this approach may be useful for exploring gene function during early placental and embryonic development. PMID:16504164

Potassium salt microinjection into Xenopus oocytes mimics gonadotropin treatment.

PubMed

Lau, Y T; Yassin, R R; Horowitz, S B

1988-06-03

Gonadotropin stimulates protein synthesis and growth in ovarian oocytes. The hormone is also known to modify transfollicular K+ fluxes and is now shown to cause increased intraoocytic K+ activity (aK). The hormone's effect on aK was duplicated by microinjecting K+ salts into oocytes which were incubated in paraffin oil. This treatment mimicked the influence of gonadotropin on both the rate of protein synthesis and the synthesis of specific polypeptides. These findings suggest that gonadotropin-stimulated oocyte growth is attributable largely to the hormone's influence on transfollicular K+ fluxes. They support the hypothesis that the K+ flux and aK changes observed during cell activation are critical in causing subsequent increases in protein synthesis and growth.

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids.

PubMed

Hill, David R; Huang, Sha; Tsai, Yu-Hwai; Spence, Jason R; Young, Vincent B

2017-12-18

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

Brain regions involved in the development of acute phase responses accompanying fever in rabbits.

PubMed Central

Morimoto, A; Murakami, N; Nakamori, T; Sakata, Y; Watanabe, T

1989-01-01

1. The effects of microinjection of rabbit endogenous pyrogen and human recombinant interleukin-1 alpha on rectal temperature and acute phase responses were extensively examined in forty different brain regions of rabbits. The acute phase responses that were investigated were the changes in plasma levels of iron, zinc and copper concentration and the changes in circulating leucocyte count. 2. The rostral hypothalamic regions, such as nucleus broca ventralis, preoptic area and anterior hypothalamic region, responded to the microinjection of endogenous pyrogen or interleukin-1 by producing both fever and acute phase responses. 3. The microinjection of endogenous pyrogen or interleukin-1 into the rostral hypothalamic regions significantly decreased the plasma levels of iron and zinc concentration 8 and 24 h after injection. The circulating leucocyte count increased 8 h after injection. However, neither the injections of endogenous pyrogen nor interleukin-1 affected the number of red blood cells. 4. The present results show that the rostral hypothalamic regions respond directly to endogenous pyrogen or interleukin-1 with the consequent development of fever and acute phase responses. PMID:2514261

p67SRF is a constitutive nuclear protein implicated in the modulation of genes required throughout the G1 period.

PubMed Central

Gauthier-Rouvière, C; Cavadore, J C; Blanchard, J M; Lamb, N J; Fernandez, A

1991-01-01

Indirect immunofluorescence analysis, using antibodies directed against peptide sequences outside the DNA-binding domain of the 67-kDa serum response factor (p67SRF), revealed a punctuated nuclear staining, constant throughout the cell cycle and in all different cell lines tested. p67SRF was also tightly associated with chromatin through all stages of mitosis. Inhibition of p67SRF activity in vivo, through microinjection of anti-p67SRF antibodies, specifically suppressed DNA synthesis induced after serum addition or ras microinjection, suggesting that these antibodies were effective in preventing expression of serum response element (SRE)-regulated genes. A similar inhibition was also obtained in cells injected with oligonucleotides corresponding to the DNA binding sequence for p67SRF protein, SRE. Moreover, this inhibition of DNA synthesis by anti-p67SRF or SRE injection was still observed in cells injected during late G1, well after c-fos induction. These data imply that genes regulated by p67SRF are continuously involved in the proliferation pathway throughout G1 and that p67SRF forms an integral component of mammalian cell transcriptional control. Images PMID:1782216

A5 region modulation of the cardiorespiratory responses evoked from parabrachial cell bodies in the anaesthetised rat.

PubMed

Dawid Milner, M S; Lara, J P; López de Miguel, M P; López-González, M V; Spyer, K M; González-Barón, S

2003-08-22

We have examined the importance of the A5 region modulating cardiorespiratory responses evoked from the parabrachial complex (PB) in spontaneously breathing rats. Cardiorespiratory changes were analyzed in response to electrical stimulation and glutamate microinjections into the PB (10-20 nl, 1-2 nmol) before and after ipsilateral microinjection of muscimol (50 nl, 0.25 nmol) or lidocaine (50 nl, 0.5 nmol) within the A5 region. Stimulation of medial parabrachial and Kölliker-Fuse nuclei (mPB-KF) evoked a decrease in respiratory rate (P<0.001) with a rise in blood pressure (P<0.001) and heart rate (P<0.05). After muscimol or lidocaine microinjections within the A5 region, the pressor and heart rate responses to mPB-KF stimulation were reduced (P<0.05, both cases). Muscimol within the A5 region altered the respiratory response to glutamate stimulation of mPB-KF, evoking an increase in respiratory rate (P<0.05). Lidocaine abolished the respiratory response to mPB-KF stimulation. Stimulation of the lateral parabrachial nuclei (lPB) caused an increase in respiratory rate (P<0.001) with a rise in blood pressure (P<0.001) and heart rate (P<0.05). Muscimol or lidocaine microinjections within A5 region decreased heart rate (P<0.05) and pressor responses (P<0.05) evoked from lPB. The increase of respiratory rate persisted unchanged. To confirm functional interactions between A5 and PB, extracellular recordings of putative A5 neurones were obtained during PB stimulation. Eighty-three A5 cells were recorded, 35 were activated from the mPB-KF (42%). The results indicate that neurones of the A5 region participate in the cardiorespiratory response evoked from the different regions of the PB complex. The possible mechanisms involved in these interactions are discussed.

Functionally Selective Signaling for Morphine and Fentanyl Antinociception and Tolerance Mediated by the Rat Periaqueductal Gray

PubMed Central

Morgan, Michael M.; Reid, Rachel A.; Saville, Kimber A.

2014-01-01

Functionally selective signaling appears to contribute to the variability in mechanisms that underlie tolerance to the antinociceptive effects of opioids. The present study tested this hypothesis by examining the contribution of G protein-coupled receptor kinase (GRK)/Protein kinase C (PKC) and C-Jun N-terminal kinase (JNK) activation on both the expression and development of tolerance to morphine and fentanyl microinjected into the ventrolateral periaqueductal gray of the rat. Microinjection of morphine or fentanyl into the periaqueductal gray produced a dose-dependent increase in hot plate latency. Microinjection of the non-specific GRK/PKC inhibitor Ro 32-0432 into the periaqueductal gray to block mu-opioid receptor phosphorylation enhanced the antinociceptive effect of morphine but had no effect on fentanyl antinociception. Microinjection of the JNK inhibitor SP600125 had no effect on morphine or fentanyl antinociception, but blocked the expression of tolerance to repeated morphine microinjections. In contrast, a microinjection of Ro 32-0432 blocked the expression of fentanyl, but not morphine tolerance. Repeated microinjections of Ro 32-0432 blocked the development of morphine tolerance and inhibited fentanyl antinociception whether rats were tolerant or not. Repeated microinjections of SP600125 into the periaqueductal gray blocked the development of tolerance to both morphine and fentanyl microinjections. These data demonstrate that the signaling molecules that contribute to tolerance vary depending on the opioid and methodology used to assess tolerance (expression vs. development of tolerance). This signaling difference is especially clear for the expression of tolerance in which JNK contributes to morphine tolerance and GRK/PKC contributes to fentanyl tolerance. PMID:25503060

Comparison of male chimeric mice generated from microinjection of JM8.N4 embryonic stem cells into C57BL/6J and C57BL/6NTac blastocysts.

PubMed

Fielder, Thomas J; Yi, Charles S; Masumi, Juliet; Waymire, Katrina G; Chen, Hsiao-Wen; Wang, Shuling; Shi, Kai-Xuan; Wallace, Douglas C; MacGregor, Grant R

2012-12-01

To identify ways to improve the efficiency of generating chimeric mice via microinjection of blastocysts with ES cells, we compared production and performance of ES-cell derived chimeric mice using blastocysts from two closely related and commonly used sub-strains of C57BL/6. Chimeras were produced by injection of the same JM8.N4 (C57BL/6NTac) derived ES cell line into blastocysts of mixed sex from either C57BL/6J (B6J) or C57BL/6NTac (B6NTac) mice. Similar efficiency of production and sex-conversion of chimeric animals was observed with each strain of blastocyst. However, B6J chimeric males had fewer developmental abnormalities involving urogenital and reproductive tissues (1/12, 8%) compared with B6NTac chimeric males (7/9, 78%). The low sample size did not permit determination of statistical significance for many parameters. However, in each category analyzed the B6J-derived chimeric males performed as well, or better, than their B6NTac counterparts. Twelve of 14 (86%) B6J male chimeras were fertile compared with 6 of 11 (55%) B6NTac male chimeras. Ten of 12 (83%) B6J chimeric males sired more than 1 litter compared with only 3 of 6 (50%) B6NTac chimeras. B6J male chimeras produced more litters per productive mating (3.42 ± 1.73, n = 12) compared to B6NTac chimeras (2.17 ± 1.33, n = 6). Finally, a greater ratio of germline transmitting chimeric males was obtained using B6J blastocysts (9/14; 64%) compared with chimeras produced using B6NTac blastocysts (4/11; 36%). Use of B6J host blastocysts for microinjection of ES cells may offer improvements over blastocysts from B6NTac and possibly other sub-strains of C57BL/6 mice.

RNA imaging: tracking in real-time RNA transport in neurons using molecular beacons and confocal microscopy.

PubMed

Zepeda, Angélica; Arias, Clorinda; Flores-Jasso, Fabian; Vaca, Luis

2013-01-01

RNAs are present within eukaryotic cells and are involved in several biological processes. RNA transport within cell compartments is important for proper cell function. To understand in depth the cellular processes in which RNA is involved requires a method that reveals RNA localization in real time in a sub-cellular context in living cells. In this protocol we describe a method for imaging RNA in living cells and in particular in neuronal cultures based on cell microinjection of molecular beacons in conjunction with confocal microscopy. This methodology overcomes some of the main obstacles for imaging RNA in live cells since microinjection allows the delivery of the probe to a desired cellular compartment and MBs bind with high specificity to its target RNA without inhibiting its function. The proper design of the MBs is essential to obtain RNA-MB association at the temperature of the cell cytosol. MBs design with other purposes in mind (such as PCR experiments) have a design that facilitates association to its target at high temperatures, rendering them unsuitable for live cell imaging. Using the methodology described in this chapter allows the study of RNA transport to different regions of neurons and may be combined with the tagging of proteins of interest to measure co-transport of the protein and the RNA to different cellular regions. Copyright © 2013 Elsevier Inc. All rights reserved.

Muscarinic type 2 receptors in the lateral dorsal tegmental area modulate cocaine and food seeking behavior in rats.

PubMed

Shabani, S; Foster, R; Gubner, N; Phillips, T J; Mark, G P

2010-10-13

The cholinergic input from the lateral dorsal tegmental area (LDTg) modulates the dopamine cells of the ventral tegmental area (VTA) and plays an important role in cocaine taking. Specific pharmacological agents that block or stimulate muscarinic receptors in the LDTg change acetylcholine (ACh) levels in the VTA. Furthermore, manipulations of cholinergic input in the VTA can change cocaine taking. In the current study, the ACh output from the LDTg was attenuated by treatment with the selective muscarinic type 2 (M2) autoreceptor agonist oxotremorine.sesquifumarate (OxoSQ). We hypothesized that OxoSQ would reduce the motivation of rats to self-administer both natural and drug rewards. Animals were tested on progressive ratio (PR) schedules of reinforcement for food pellets and cocaine. On test days, animals on food and on cocaine schedules were bilaterally microinjected prior to the test. Rats received either LDTg OxoSQ infusions or LDTg artificial cerebrospinal fluid (aCSF) infusions in a within-subjects design. In addition, infusions were delivered into a dorsal brain area above the LDTg as an anatomical control region. OxoSQ microinjection in the LDTg, compared to aCSF, significantly reduced both the number of self-administered pellets and cocaine infusions during the initial half of the session; this reduction was dose-dependent. OxoSQ microinjections into the area just dorsal to the LDTg had no significant effect on self-administration of food pellets or cocaine. Animals were also tested in locomotor activity chambers for motor effects following the above microinjections. Locomotor activity was mildly increased by OxoSQ microinjection into the LDTg during the initial half of the session. Overall, these data suggest that LDTg cholinergic neurons play an important role in modifying the reinforcing value of natural and drug rewards. These effects cannot be attributed to significant alterations of locomotor behavior and are likely accomplished through LDTg muscarinic autoreceptors. Published by Elsevier Ltd.

Generation of transgenic goats by pronuclear microinjection: a retrospective analysis of a commercial operation (1995-2012).

PubMed

Gavin, W; Blash, S; Buzzell, N; Pollock, D; Chen, L; Hawkins, N; Howe, J; Miner, K; Pollock, J; Porter, C; Schofield, M; Echelard, Y; Meade, H

2018-02-01

Production of transgenic founder goats involves introducing and stably integrating an engineered piece of DNA into the genome of the animal. At LFB USA, the ultimate use of these transgenic goats is for the production of recombinant human protein therapeutics in the milk of these dairy animals. The transgene or construct typically links a milk protein specific promoter sequence, the coding sequence for the gene of interest, and the necessary downstream regulatory sequences thereby directing expression of the recombinant protein in the milk during the lactation period. Over the time period indicated (1995-2012), pronuclear microinjection was used in a number of programs to insert transgenes into 18,120, 1- or 2- cell stage fertilized embryos. These embryos were transferred into 4180 synchronized recipient females with 1934 (47%) recipients becoming pregnant, 2594 offspring generated, and a 109 (4.2%) of those offspring determined to be transgenic. Even with new and improving genome editing tools now available, pronuclear microinjection is still the predominant and proven technology used in this commercial setting supporting regulatory filings and market authorizations when producing founder transgenic animals with large transgenes (> 10 kb) such as those necessary for directing monoclonal antibody production in milk.

Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection

PubMed Central

Michlewski, Gracjan; Finnegan, David J.; Elfick, Alistair; Rosser, Susan J.

2017-01-01

Abstract Delivery of DNA to cells and its subsequent integration into the host genome is a fundamental task in molecular biology, biotechnology and gene therapy. Here we describe an IP-free one-step method that enables stable genome integration into either prokaryotic or eukaryotic cells. A synthetic mariner transposon is generated by flanking a DNA sequence with short inverted repeats. When purified recombinant Mos1 or Mboumar-9 transposase is co-transfected with transposon-containing plasmid DNA, it penetrates prokaryotic or eukaryotic cells and integrates the target DNA into the genome. In vivo integrations by purified transposase can be achieved by electroporation, chemical transfection or Lipofection of the transposase:DNA mixture, in contrast to other published transposon-based protocols which require electroporation or microinjection. As in other transposome systems, no helper plasmids are required since transposases are not expressed inside the host cells, thus leading to generation of stable cell lines. Since it does not require electroporation or microinjection, this tool has the potential to be applied for automated high-throughput creation of libraries of random integrants for purposes including gene knock-out libraries, screening for optimal integration positions or safe genome locations in different organisms, selection of the highest production of valuable compounds for biotechnology, and sequencing. PMID:28204586

Symplastic Transport of Carboxyfluorescein in Staminal Hairs of Setcreasea purpurea Is Diffusive and Includes Loss to the Vacuole.

PubMed

Tucker, J E; Mauzerall, D; Tucker, E B

1989-07-01

The kinetics of symplastic transport in staminal hairs of Setcreasea purpurea was studied. The tip cell of a staminal hair was microinjected with carboxyfluorescein (CF) and the symplastic transport of this CF was videotaped and the digital data analyzed to produce kinetic curves. Using a finite difference equation for diffusion between cells and for loss of dye into the vacuole, kinetic curves were calculated and fitted to the observed data. These curves were matched with data from actual microinjection experiments by adjusting K (the coefficient of intercellular junction diffusion) and L (the coefficient of intracellular loss) until a minimum in the least squares difference between the curves was obtained. (a) Symplastic transport of CF was governed by diffusion through intercellular pores (plasmodesmata) and intracellular loss. Diffusion within the cell cytoplasm was never limiting. (b) Each cell and its plasmodesmata must be considered as its own diffusion system. Therefore, a diffusion coefficient cannot be calculated for an entire chain of cells. (c) The movement through plasmodesmata in either direction was the same since the data are fit by a diffusion equation. (d) Diffusion through the intercellular pores was estimated to be slower than diffusion through similar pores filled with water.

Symplastic Transport of Carboxyfluorescein in Staminal Hairs of Setcreasea purpurea Is Diffusive and Includes Loss to the Vacuole 1

PubMed Central

Tucker, Joseph E.; Mauzerall, David; Tucker, Edward B.

1989-01-01

The kinetics of symplastic transport in staminal hairs of Setcreasea purpurea was studied. The tip cell of a staminal hair was microinjected with carboxyfluorescein (CF) and the symplastic transport of this CF was videotaped and the digital data analyzed to produce kinetic curves. Using a finite difference equation for diffusion between cells and for loss of dye into the vacuole, kinetic curves were calculated and fitted to the observed data. These curves were matched with data from actual microinjection experiments by adjusting K (the coefficient of intercellular junction diffusion) and L (the coefficient of intracellular loss) until a minimum in the least squares difference between the curves was obtained. (a) Symplastic transport of CF was governed by diffusion through intercellular pores (plasmodesmata) and intracellular loss. Diffusion within the cell cytoplasm was never limiting. (b) Each cell and its plasmodesmata must be considered as its own diffusion system. Therefore, a diffusion coefficient cannot be calculated for an entire chain of cells. (c) The movement through plasmodesmata in either direction was the same since the data are fit by a diffusion equation. (d) Diffusion through the intercellular pores was estimated to be slower than diffusion through similar pores filled with water. PMID:16666864

Inhibitory effect of the nucleus reticularis pontis oralis on the pontine micturition center and pontine urine storage center in decerebrate cats.

PubMed

Sugaya, Kimio; Nishijima, Saori; Miyazato, Minoru; Oda, Masami; Ogawa, Yoshihide

2006-10-01

The influence of the nucleus reticularis pontis oralis (PoO) on the pontine micturition center (PMC) and pontine urine storage center (PUSC) was examined in decerebrate cats by electrical and chemical stimulations of the PMC, PUSC or PoO. Microinjection of carbachol into the rostral and dorsolateral part of the PoO rapidly inhibited reflex micturition and external urethral sphincter (EUS) activity. After confirming the inhibition of reflex micturition and EUS activity by microinjection of carbachol into the PoO, intravenous injection of atropine sulfate or its microinjection into the PoO recovered both reflex micturition and EUS activity. Microinjection of carbachol into the PMC evoked micturition and then inhibited reflex micturition, but intravenous injection of atropine or its microinjection into the PoO recovered reflex micturition. After confi rming the inhibition of reflex micturition and EUS activity by microinjection of carbachol into the PoO, electrical stimulation of the PUSC enhanced EUS activity, but electrical stimulation of the PMC failed to evoke micturition. However, electrical stimulation of the PMC evoked micturition after microinjection of atropine into the PoO. These results suggest that the PoO strongly inhibits the PMC and less strongly inhibits the PUSC. Therefore, the PoO seems to be the pontine micturition inhibitory area.

Spatio-temporal propagation of Ca2+ signals by cyclic ADP-ribose in 3T3 cells stimulated via purinergic P2Y receptors

PubMed Central

Bruzzone, Santina; Kunerth, Svenja; Zocchi, Elena; De Flora, Antonio; Guse, Andreas H.

2003-01-01

The role of cyclic ADP-ribose in the amplification of subcellular and global Ca2+ signaling upon stimulation of P2Y purinergic receptors was studied in 3T3 fibroblasts. Either (1) 3T3 fibroblasts (CD38− cells), (2) 3T3 fibroblasts preloaded by incubation with extracellular cyclic ADP-ribose (cADPR), (3) 3T3 fibroblasts microinjected with ryanodine, or (4) 3T3 fibroblasts transfected to express the ADP-ribosyl cyclase CD38 (CD38+ cells) were used. Both preincubation with cADPR and CD38 expression resulted in comparable intracellular amounts of cyclic ADP-ribose (42.3 ± 5.2 and 50.5 ± 8.0 pmol/mg protein). P2Y receptor stimulation of CD38− cells yielded a small increase of intracellular Ca2+ concentration and a much higher Ca2+ signal in CD38-transfected cells, in cADPR-preloaded cells, or in cells microinjected with ryanodine. Confocal Ca2+ imaging revealed that stimulation of ryanodine receptors by cADPR or ryanodine amplified localized pacemaker Ca2+ signals with properties resembling Ca2+ quarks and triggered the propagation of such localized signals from the plasma membrane toward the internal environment, thereby initiating a global Ca2+ wave. PMID:14623867

Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases.

PubMed

Remy, Séverine; Tesson, Laurent; Menoret, Séverine; Usal, Claire; De Cian, Anne; Thepenier, Virginie; Thinard, Reynald; Baron, Daniel; Charpentier, Marine; Renaud, Jean-Baptiste; Buelow, Roland; Cost, Gregory J; Giovannangeli, Carine; Fraichard, Alexandre; Concordet, Jean-Paul; Anegon, Ignacio

2014-08-01

The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner. © 2014 Remy et al.; Published by Cold Spring Harbor Laboratory Press.

Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases

PubMed Central

Remy, Séverine; Tesson, Laurent; Menoret, Séverine; Usal, Claire; De Cian, Anne; Thepenier, Virginie; Thinard, Reynald; Baron, Daniel; Charpentier, Marine; Renaud, Jean-Baptiste; Buelow, Roland; Cost, Gregory J.; Giovannangeli, Carine; Fraichard, Alexandre; Concordet, Jean-Paul; Anegon, Ignacio

2014-01-01

The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%–5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner. PMID:24989021

Ricinus communis cyclophilin: functional characterisation of a sieve tube protein involved in protein folding.

PubMed

Gottschalk, Maren; Dolgener, Elmar; Xoconostle-Cázares, Beatriz; Lucas, William J; Komor, Ewald; Schobert, Christian

2008-09-01

The phloem translocation stream of the angiosperms contains a special population of proteins and RNA molecules which appear to be produced in the companion cells prior to being transported into the sieve tube system through the interconnecting plasmodesmata. During this process, these non-cell-autonomous proteins are thought to undergo partial unfolding. Recent mass spectroscopy studies identified peptidyl-prolyl cis-trans isomerase (PPIases) as potential molecular chaperones functioning in the phloem translocation stream (Giavalisco et al. 2006). In the present study, we describe the cloning and characterisation of a castor bean phloem cyclophilin, RcCYP1 that has high peptidyl-prolyl cis-trans isomerase activity. Equivalent enzymatic activity was detected with phloem sap or purified recombinant (His)(6)-tagged RcCYP1. Mass spectrometry analysis of proteolytic peptides, derived from a 22 kDa band in HPLC-fractionated phloem sap, immunolocalisation studies and Western analysis of proteins extracted from castor bean tissues/organs indicated that RcCYP1 is an abundant protein in the companion cell-sieve element complex. Microinjection experiments established that purified recombinant (His)(6)-RcCYP1 can interact with plasmodesmata to both induce an increase in size exclusion limit and mediate its own cell-to-cell trafficking. Collectively, these findings support the hypothesis that RcCYP1 plays a role in the refolding of non-cell-autonomous proteins after their entry into the phloem translocation stream.

In vivo operation of the pentose phosphate pathway in frog oocytes is limited by NADP+ availability.

PubMed

Preller, A; Guixé, V; Ureta, T

1999-03-05

Evolution of CO2 from labelled glucose microinjected into frog oocytes in vivo may be ascribed to the pentose-P pathway, as measured by radioactive CO2 production from [1-(14)C] and [6-(14)C]glucose. Coinjection of NADP+ and [14C]glucose significantly stimulated 14CO2 production. The effect depends on the amount of NADP+ injected, half maximal stimulation being obtained at 0.13 mM. The increase in CO2 production was also observed with microinjected glucose-1-P, glucose-6-P or fructose-6-P used as substrates. Phenazine methosulfate, mimicked the effects of NADP+. A high NADPH/NADP+ ratio of 4.3 was found in the cells, the intracellular concentration of NADP+ being 19 microM.

Investigation of micro-injection molding based on longitudinal ultrasonic vibration core.

PubMed

Qiu, Zhongjun; Yang, Xue; Zheng, Hui; Gao, Shan; Fang, Fengzhou

2015-10-01

An ultrasound-assisted micro-injection molding method is proposed to improve the rheological behavior of the polymer melt radically, and a micro-injection molding system based on a longitudinal ultrasonic vibration core is developed and employed in the micro-injection molding process of Fresnel lenses. The verification experiments show that the filling mold area of the polymer melt is increased by 6.08% to 19.12%, and the symmetric deviation of the Fresnel lens is improved 15.62% on average. This method improved the filling performance and replication quality of the polymer melt in the injection molding process effectively.

DNA (DEOXYRIBONUCLEIC ACID) SYNTHESIS FOLLOWING MICROINJECTION OF HETEROLOGOUS SPERM AND SOMATIC CELL NUCLEI INTO HAMSTER OOCYTES

EPA Science Inventory

The authors have investigated the ability of the hamster oocyte to initiate DNA synthesis in nuclei differing in basic protein content. DNA synthesis was studied by autoradiography in oocytes that had been incubated in 3H-thymidine after being parthenogenetically activated by sha...

The posterior ventral tegmental area mediates alcohol-seeking behavior in alcohol-preferring rats.

PubMed

Hauser, Sheketha R; Ding, Zheng-Ming; Getachew, Bruk; Toalston, Jamie E; Oster, Scott M; McBride, William J; Rodd, Zachary A

2011-03-01

The mesolimbic dopamine (DA) system is involved in the rewarding process of drugs of abuse and is activated during the anticipation of drug availability. However, the neurocircuitry that regulates ethanol (EtOH)-seeking has not been adequately investigated. The objectives of the present study were to determine 1) whether the posterior ventral tegmental area (p-VTA) mediates EtOH-seeking, 2) whether microinjections of EtOH into the p-VTA could stimulate EtOH-seeking, and (3) the involvement of p-VTA DA neurons in EtOH-seeking. Alcohol-preferring rats were trained to self-administer 15% EtOH and water. After 10 weeks, rats underwent extinction training, followed by 2 weeks in their home cages. During the home-cage period, rats were then bilaterally implanted with guide cannulae aimed at the p-VTA or anterior ventral tegmental area (a-VTA). EtOH-seeking was assessed by the Pavlovian spontaneous recovery model. Separate experiments examined the effects of: 1) microinjection of quinpirole into the p-VTA, 2) EtOH microinjected into the p-VTA, 3) coadministration of EtOH and quinpirole into the p-VTA, 4) microinjection of quinpirole into the a-VTA, and 5) microinjection of EtOH into the a-VTA. Quinpirole microinjected into the p-VTA reduced EtOH-seeking. Microinjections of EtOH into the p-VTA increased EtOH-seeking. Pretreatment with both quinpirole and EtOH into the p-VTA reduced EtOH-seeking. Microinjections of quinpirole or EtOH into the a-VTA did not alter EtOH-seeking. Overall, the results suggest that the p-VTA is a neuroanatomical substrate mediating alcohol-seeking behavior and that activation of local DA neurons is involved.

Modulation of fear/anxiety responses, but not food intake, following α-adrenoceptor agonist microinjections in the nucleus accumbens shell of free-feeding rats.

PubMed

Kochenborger, Larissa; Zanatta, Débora; Berretta, Luigi Marins; Lopes, Ana Paula Fraga; Wunderlich, Bruna Luiza; Januário, Ana Cláudia; Neto, José Marino; Terenzi, Mariana Graciela; Paschoalini, Marta Aparecida; Faria, Moacir Serralvo

2012-01-01

This study investigated the effect of α-adrenoceptor agonists microinjected into the shell region of the accumbens nucleus (AcbSh) on feeding and anxiety-related behaviors in free-feeding rats. Male Wistar rats with a chronically implanted cannula into the AcbSh were unilaterally microinjected with either clonidine (CLON, α(2)-adrenoceptor agonist) or phenylephrine (PHEN, α(1)-adrenoceptor agonist) at the doses of 6 and 20 nmol and submitted to the elevated plus-maze (EPM), a pre-clinical test of anxiety. Immediately after the EPM test, the animals underwent food intake evaluation for 30 min. The data showed that rats microinjected with CLON (20 nmol/0.2 μl) into the AcbSh exhibited increased %Open arm time, which is compatible with an anxiolytic-like effect. The CLON-induced anxiolysis was corroborated by increased head-dipping and decreased stretched-attend posture, two ethologically derived behaviors which are fear/anxiety-motivated. The animal's locomotor activity was not changed by 20 nmol CLON microinjection into the AcbSh. However, neither dose of PHEN microinjected into the AcbSh was able to alter either the spatial-temporal or ethological variables representative of fear/anxiety and locomotion. Food intake was not altered by any dose of CLON and PHEN microinjected into the AcbSh, but the 20 nmol CLON microinjection induced increased motor activity in the feeding test. The data suggests that noradrenergic projections to the AcbSh may underlie fear/anxiety modulation through α(2)-adrenoceptor in the AcbSh, while feeding behavior was unaffected by noradrenergic modulation in the AcbSh of free-feeding rats. This article is part of a Special Issue entitled 'Anxiety and Depression'. Copyright © 2011 Elsevier Ltd. All rights reserved.

Protective effect of histamine microinjected into cerebellar fastigial nucleus on stress gastric mucosal damage in rats.

PubMed

Qiao, Xiao; Yang, Jun; Fei, Su-Juan; Zhu, Jin-Zhou; Zhu, Sheng-Ping; Liu, Zhang-Bo; Li, Ting-Ting; Zhang, Jian-Fu

2015-12-10

In the study, we investigated the effect of histamine microinjected into cerebellar fastigial nucleus (FN) on stress gastric mucosal damage (SGMD), and its mechanisms in rats. The model of SGMD was established by restraining and water (21±1°C)-immersion for 3h. The gastric mucosal damage index (GMDI) indicated the severity of gastric mucosal damage. Histamine or receptor antagonist was microinjected into the FN. The decussation of superior cerebellar peduncle (DSCP) and the lateral hypothalamic area (LHA) were destroyed, respectively. The pathological changes of gastric mucosa were evaluated using biological signal acquisition system, Laser-Doppler flowmeter, and western blotting. We found that the microinjection of histamine (0.05, 0.5, and 5μg) into FN significantly attenuated the SGMD, in a dose-dependent manner, whereas, the microinjection of histamine H2 receptor antagonist, ranitidine, and glutamic acid decarboxylase antagonist, 3-mercaptopropionic acid (3-MPA) exacerbated the SGMD. The protective effect of histamine on SGMD was abolished by electrical lesion of DSCP or chemical ablation of LHA. The microinjection of histamine decreased the discharge frequency of the greater splanchnic nerve, and the gastric mucosal blood flow was increased. In addition, the cellular proliferation was enhanced, but the cellular apoptosis was reduced in the gastric mucosa. Also the pro-apoptosis protein, Bax, and caspase-3 were down-regulated, and the anti-apoptosis protein, Bcl-2 was up-regulated following microinjection of histamine. In conclusion, the FN participated in the regulation of SGMD after histamine microinjected into FN, and cerebellar-hypothalamic circuits (include: DSCP, LHA) contribute to the process, which may provide a new therapeutic strategy for SGMD. Copyright © 2015 Elsevier B.V. All rights reserved.

Genetic engineering including superseding microinjection: new ways to make GM pigs.

PubMed

Galli, Cesare; Perota, Andrea; Brunetti, Dario; Lagutina, Irina; Lazzari, Giovanna; Lucchini, Franco

2010-01-01

Techniques for genetic engineering of swine are providing genetically modified animals of importance for the field of xenotransplantation, animal models for human diseases and for a variety of research applications. Many of these modifications have been directed toward avoiding naturally existing cellular and antibody responses to species-specific antigens. A number of techniques are today available to engineering the genome of mammals, these range from the well established less efficient method of DNA microinjection into the zygote, the use of viral vectors, to the more recent use of somatic cell nuclear transfer. The use of enzymatic engineering that are being developed now will refine the precision of the genetic modification combined with the use of new vectors like transposons. The use of somatic cell nuclear transfer is currently the most efficient way to generate genetically modified pigs. The development of enzymatic engineering with zinc-finger nucleases, recombinases and transposons will revolutionize the field. Nevertheless, genetic engineering in large domesticated animals will remain a challenging task. Recent improvements in several fields of cell and molecular biology offer new promises and opportunities toward an easier, cost-effective and efficient generation of transgenic pigs. © 2010 John Wiley & Sons A/S.

Live-cell imaging of Salmonella Typhimurium interaction with zebrafish larvae after injection and immersion delivery methods.

PubMed

Varas, Macarena; Fariña, Alonso; Díaz-Pascual, Francisco; Ortíz-Severín, Javiera; Marcoleta, Andrés E; Allende, Miguel L; Santiviago, Carlos A; Chávez, Francisco P

2017-04-01

The zebrafish model has been used to determine the role of vertebrate innate immunity during bacterial infections. Here, we compare the in vivo immune response induced by GFP-tagged Salmonella Typhimurium inoculated by immersion and microinjection in transgenic zebrafish larvae. Our novel infection protocols in zebrafish allow live-cell imaging of Salmonella colonization. Copyright © 2017 Elsevier B.V. All rights reserved.

The Role of TSC Proteins in Regulating Cell Adhesion and Motility

DTIC Science & Technology

2006-09-01

Pennsylvania Philadelphia, Pennsylvania 19104-6270 9 . SPONSORING...from seizures, mental retardation, and autism . Thus, TSC represents a major cause of developmental disorders and epilepsy in the pediatric...images are from 138 microinjected cells 2. 0 20 40 60 80 100 * * GF P C el l m ig ra tio n, % Pɘ.001 TS C2 TS C2 -C TS C2 -N Figure 9 . N-terminal

Electrical detection of cellular penetration during microinjection with carbon nanopipettes.

PubMed

Anderson, Sean E; Bau, Haim H

2014-06-20

The carbon nanopipette (CNP) is comprised of a pulled-glass pipette terminating with a nanoscale (tens to hundreds of nm) diameter carbon pipe. The entire inner glass surface of the CNP is coated with a carbon film, providing an electrically conductive path from the carbon tip to the distal, macroscopic end of the pipette. The CNP can double as a nanoelectrode, enabling electrical measurements through its carbon lining, and as a nanoinjector, facilitating reagent injection through its hollow bore. With the aid of a lock-in amplifier, we measured, in real time and with millisecond resolution, variations in impedance and interfacial capacitance as the CNP penetrated into the cytoplasm and nucleus of adherent human osteosarcoma (U20S) cells during microinjection. The capacitance change associated with nucleus penetration was, on average, 1.5 times greater than the one associated with cell membrane penetration. The experimental data was compared and favorably agreed with theoretical predictions based on a simple electrical network model. As a proof of concept, the cytoplasm and nucleus were transfected with fluorescent tRNA, enabling real-time monitoring of tRNA trafficking across the nuclear membrane. The CNP provides a robust and reliable means to detect cell and nucleus penetration, and trigger injection, thereby enabling the automation of cell injection.

Electrical detection of cellular penetration during microinjection with carbon nanopipettes

NASA Astrophysics Data System (ADS)

Anderson, Sean E.; Bau, Haim H.

2014-06-01

The carbon nanopipette (CNP) is comprised of a pulled-glass pipette terminating with a nanoscale (tens to hundreds of nm) diameter carbon pipe. The entire inner glass surface of the CNP is coated with a carbon film, providing an electrically conductive path from the carbon tip to the distal, macroscopic end of the pipette. The CNP can double as a nanoelectrode, enabling electrical measurements through its carbon lining, and as a nanoinjector, facilitating reagent injection through its hollow bore. With the aid of a lock-in amplifier, we measured, in real time and with millisecond resolution, variations in impedance and interfacial capacitance as the CNP penetrated into the cytoplasm and nucleus of adherent human osteosarcoma (U20S) cells during microinjection. The capacitance change associated with nucleus penetration was, on average, 1.5 times greater than the one associated with cell membrane penetration. The experimental data was compared and favorably agreed with theoretical predictions based on a simple electrical network model. As a proof of concept, the cytoplasm and nucleus were transfected with fluorescent tRNA, enabling real-time monitoring of tRNA trafficking across the nuclear membrane. The CNP provides a robust and reliable means to detect cell and nucleus penetration, and trigger injection, thereby enabling the automation of cell injection.

Poor embryo development in post-ovulatory in vivo-aged mouse oocytes is associated with mitochondrial dysfunction, but mitochondrial transfer from somatic cells is not sufficient for rejuvenation.

PubMed

Igarashi, Hideki; Takahashi, Toshifumi; Abe, Hiroyuki; Nakano, Hiroshi; Nakajima, Osamu; Nagase, Satoru

2016-10-01

Does in vivo aging of mouse oocytes affect mitochondrial function? Mitochondrial function was impaired in post-ovulatory in vivo-aged mouse oocytes and microinjection of somatic cell mitochondria did not rescue poor fertilization and embryonic development rates. The mechanisms underlying the decline in oocyte quality associated with oocyte aging remain unknown, although studies have suggested that the decline is regulated by mitochondrial dysfunction. However, only a limited number of studies have provided direct evidence implicating mitochondrial dysfunction in oocyte quality during the aging of oocytes. We used post-ovulatory, in vivo-aged mouse oocytes as a model for studying low-quality oocytes in oocyte aging. Superovulated oocytes released from the oviduct at 14 h and 20-24 h post-hCG injection were designated as 'fresh' and 'aged' oocytes, respectively. Membrane potentials and oxygen consumption in single oocytes were evaluated as measures of mitochondrial function in fresh and aged oocytes. Mitochondrial transcriptional factor A (TFAM) expression levels were examined by western blotting, and colocalization of mitochondria and TFAM was analyzed by measuring immunofluorescence in fresh and aged oocytes. IVF and blastocyst formation rates were calculated after oocyte microinjection with mitochondria derived from liver cells. The average mitochondrial membrane potential in fresh oocytes was significantly higher than that in aged oocytes (P < 0.05). The average oxygen consumption rate in aged oocytes was significantly lower than that in fresh oocytes (P < 0.05). Although total TFAM expression was unchanged, its colocalization with mitochondria decreased in aged oocytes. IVF and blastocyst formation rates for mitochondrion-injected aged oocytes were not significantly different from those for buffer-injected aged oocytes. Not applicable. A limitation of this study is that we did not examine the effects of microinjecting mitochondria from other somatic cell types into aged oocytes on their fertilization and embryonic development rates. The results from the present study showed that poor embryonic development was associated with impairment of mitochondrial functions in in vivo-aged oocytes. However, the microinjection of mitochondria from liver cells did not improve the low fertilization and embryonic development rates of aged oocytes. It remains to be demonstrated whether oocyte quality can be rescued by the transfer of cytosolic factors or cellular organelles, such as the endoplasmic reticulum or mitochondria, from specific cell types. This study was supported by Grants-in-Aid for General Science Research to Toshifumi Takahashi (No. 25462550) and Hideki Igarashi (No. 26462474). The funding source played no role in study design in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. The authors have no conflict of interest to disclose. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Transcription-dependent induction of G1 phase during the zebra fish midblastula transition.

PubMed

Zamir, E; Kam, Z; Yarden, A

1997-02-01

The early development of the zebra fish (Danio rerio) embryo is characterized by a series of rapid and synchronous cell cycles with no detectable transcription. This period is followed by the midblastula transition (MBT), during which the cell cycle gradually lengthens, cell synchrony is lost, and zygotic transcription is initially detected. In this work, we examined the changes in the pattern of the cell cycle during MBT in zebra fish and whether these changes are dependent on the initiation of zygotic transcription. To characterize the pattern of the early zebra fish cell cycles, the embryonic DNA content was determined by flow cytometric analysis. We found that G1 phase is below detection levels during the first 10 cleavages and can be initially detected at the onset of MBT. Inhibition of zygotic transcription, by microinjection of actinomycin D, abolished the appearance of G1 phase at MBT. Premature activation of zygotic transcription, by microinjection of nonspecific DNA, induced G1 phase before the onset of MBT, while coinjection of actinomycin D and nonspecific DNA abolished this early appearance of G1 phase. We therefore suggest that during the early development of the zebra fish embryo, G1 phase appears at the onset of MBT and that the activation of transcription at MBT is essential and sufficient for the G1-phase induction.

Potato virus X TGBp1 induces plasmodesmata gating and moves between cells in several host species whereas CP moves only in N. benthamiana leaves

DOE Office of Scientific and Technical Information (OSTI.GOV)

Howard, Amanda R.; Heppler, Marty L.; Ju, Ho-Jong

Experiments were conducted to compare the plasmodesmal transport activities of Potato virus X (PVX) TGBp1 and coat protein (CP) in several plant species. Microinjection experiments indicated that TGBp1 gates plasmodesmata in Nicotiana tabacum leaves. These results support previous microinjection studies indicating that TGBp1 gates plasmodesmata in Nicotiana benthamiana and Nicotiana clevelandii leaves. To study protein movement, plasmids expressing the green fluorescent protein (GFP) gene fused to the PVX TGBp1 or CP genes were biolistically bombarded to leaves taken from four different PVX host species. GFP/TGBp1 moved between adjacent cells in N. tabacum, N. clevelandii, N. benthamiana, and Lycopersicon esculentum, whereasmore » GFP/CP moved only in N. benthamiana leaves. Mutations m12 and m13 were introduced into the TGBp1 gene and both mutations eliminated TGBp1 ATPase active site motifs, inhibited PVX movement, reduced GFP/TGBp1 cell-to-cell movement in N. benthamiana leaves, and eliminated GFP/TGBp1 movement in N. tabacum, N. clevelandii, and L. esculentum leaves. GFP/TGBp1m13 formed aggregates in tobacco cells. The ability of GFP/CP and mutant GFP/TGBp1 fusion proteins to move in N. benthamiana and not in the other PVX host species suggests that N. benthamiana plants have a unique ability to promote protein intercellular movement.« less

The role of spinal GABAergic circuits in the control of phrenic nerve motor output

PubMed Central

Ghali, Michael G. Z.; Rogers, Robert F.

2015-01-01

While supraspinal mechanisms underlying respiratory pattern formation are well characterized, the contribution of spinal circuitry to the same remains poorly understood. In this study, we tested the hypothesis that intraspinal GABAergic circuits are involved in shaping phrenic motor output. To this end, we performed bilateral phrenic nerve recordings in anesthetized adult rats and observed neurogram changes in response to knocking down expression of both isoforms (65 and 67 kDa) of glutamate decarboxylase (GAD65/67) using microinjections of anti-GAD65/67 short-interference RNA (siRNA) in the phrenic nucleus. The number of GAD65/67-positive cells was drastically reduced on the side of siRNA microinjections, especially in the lateral aspects of Rexed's laminae VII and IX in the ventral horn of cervical segment C4, but not contralateral to microinjections. We hypothesize that intraspinal GABAergic control of phrenic output is primarily phasic, but also plays an important role in tonic regulation of phrenic discharge. Also, we identified respiration-modulated GABAergic interneurons (both inspiratory and expiratory) located slightly dorsal to the phrenic nucleus. Our data provide the first direct evidence for the existence of intraspinal GABAergic circuits contributing to the formation of phrenic output. The physiological role of local intraspinal inhibition, independent of descending direct bulbospinal control, is discussed. PMID:25833937

Distribution of Single-Wall Carbon Nanotubes in the Xenopus laevis Embryo after Microinjection

PubMed Central

Holt, Brian D.; Shawky, Joseph H.; Dahl, Kris Noel; Davidson, Lance A.; Islam, Mohammad F.

2016-01-01

Single-wall carbon nanotubes (SWCNTs) are advanced materials with the potential for a myriad of diverse applications, including biological technologies and largescale usage with the potential for environmental impacts. SWCNTs have been exposed to developing organisms to determine their effects on embryogenesis, and results have been inconsistent arising, in part, from differing material quality, dispersion status, material size, impurity from catalysts, and stability. For this study, we utilized highly purified SWCNT samples with short, uniform lengths (145 ± 17 nm) well dispersed in solution. To test high exposure doses, we microinjected > 500 μg mL-1 SWCNT concentrations into the well-established embryogenesis model, Xenopus laevis, and determined embryo compatibility and sub-cellular localization during development. SWCNTs localized within cellular progeny of the microinjected cells, but heterogeneously distributed throughout the target-injected tissue. Co-registering unique Raman spectral intensity of SWCNTs with images of fluorescently labelled sub-cellular compartments demonstrated that even at the regions of highest SWCNT concentration, there were no gross alterations to sub-cellular microstructures, including filamentous actin, endoplasmic reticulum and vesicles. Furthermore, SWCNTs did not aggregate or localize to the perinuclear sub-cellular region. Combined, these results suggest that purified and dispersed SWCNTs are not toxic to X. laevis animal cap ectoderm and may be suitable candidate materials for biological applications. PMID:26510384

The nuclear membrane-associated honeycomb structure of the unicellular organism Amoeba proteus: on the search for homologies with the nuclear lamina of metazoa.

PubMed

Schmidt, M; Grossmann, U; Krohne, G

1995-07-01

In the protozoon Amoeba proteus, a complex and highly organized structure with the morphology of a honeycomb is associated with the nucleoplasmic surface of the nuclear membrane. We have tested whether this structure exhibits similarity to the nuclear lamina of metazoic organisms. First, we have shown that the honeycomb layer is composed of 3 to 5 nm thick protein fibrils resistant to treatment with detergent, high salt, and digestion with nucleases, thus possessing properties typical for karyoskeletal elements. However, in contrast to the meshwork of lamin filaments in somatic cells of metazoic organisms, the honeycomb layer is not tightly anchored to the nucleoplasmic side of pore complexes, or to the inner nuclear membrane. Second, in microinjection experiments we investigated whether fluorescently labeled lamins of Xenopus laevis (lamins A and LI) and Drosophila melanogaster (lamin Dmo) were able to associate in vivo with the Amoeba proteus honeycomb structure. In microinjected amoeba these three lamins were efficiently transported into the nucleus, but did not associate with the nuclear envelope. Our results suggest that the Amoeba proteus nuclear envelope, including the honeycomb layer, does not contain proteins exhibiting high homologies to lamins of metazoan species thus preventing the localized assembly of microinjected lamins along the nuclear periphery.

Dissecting non-coding RNA mechanisms in cellulo by single-molecule high-resolution localization and counting

PubMed Central

Pitchiaya, Sethuramasundaram; Krishnan, Vishalakshi; Custer, Thomas C.; Walter, Nils G.

2013-01-01

Non-coding RNAs (ncRNAs) recently were discovered to outnumber their protein-coding counterparts, yet their diverse functions are still poorly understood. Here we report on a method for the intracellular Single-molecule High Resolution Localization and Counting (iSHiRLoC) of microRNAs (miRNAs), a conserved, ubiquitous class of regulatory ncRNAs that controls the expression of over 60% of all mammalian protein coding genes post-transcriptionally, by a mechanism shrouded by seemingly contradictory observations. We present protocols to execute single particle tracking (SPT) and single-molecule counting of functional microinjected, fluorophore-labeled miRNAs and thereby extract diffusion coefficients and molecular stoichiometries of micro-ribonucleoprotein (miRNP) complexes from living and fixed cells, respectively. This probing of miRNAs at the single molecule level sheds new light on the intracellular assembly/disassembly of miRNPs, thus beginning to unravel the dynamic nature of this important gene regulatory pathway and facilitating the development of a parsimonious model for their obscured mechanism of action. PMID:23820309

A universal piezo-driven ultrasonic cell microinjection system.

PubMed

Huang, Haibo; Mills, James K; Lu, Cong; Sun, Dong

2011-08-01

Over the past decade, the rapid development of biotechnologies such as gene injection, in-vitro fertilization, intracytoplasmic sperm injection (ICSI) and drug development have led to great demand for highly automated, high precision equipment for microinjection. Recently a new cell injection technology using piezo-driven pipettes with a very small mercury column was proposed and successfully applied in ICSI to a variety of mammal species. Although this technique significantly improves the survival rates of the ICSI process, shortcomings due to the toxicity of mercury and damage to the cell membrane due to large lateral tip oscillations of the injector pipette may limit its application. In this paper, a new cell injection system for automatic batch injection of suspended cells is developed. A new design of the piezo-driven cell injector is proposed for automated suspended cell injection. This new piezo-driven cell injector design relocates the piezo oscillation actuator to the injector pipette which eliminates the vibration effect on other parts of the micromanipulator. A small piezo stack is sufficient to perform the cell injection process. Harmful lateral tip oscillations of the injector pipette are reduced substantially without the use of a mercury column. Furthermore, ultrasonic vibration micro-dissection (UVM) theory is utilized to analyze the piezo-driven cell injection process, and the source of the lateral oscillations of the injector pipette is investigated. From preliminary experiments of cell injection of a large number of zebrafish embryos (n = 200), the injector pipette can easily pierce through the cell membrane at a low injection speed and almost no deformation of the cell wall, and with a high success rate(96%) and survival rate(80.7%) This new injection approach shows good potential for precision injection with less damage to the injected cells.

Direct Tumor Microinjection and FDG-PET in Testing Drug Sensitivity in Patients With Relapsed or Refractory Non-Hodgkin Lymphoma, Hodgkin Lymphoma, or Stage IV Breast Cancer

ClinicalTrials.gov

2018-03-28

Breast Adenocarcinoma; Recurrent Breast Carcinoma; Recurrent Hodgkin Lymphoma; Recurrent Mycosis Fungoides; Recurrent Non-Hodgkin Lymphoma; Recurrent Primary Cutaneous T-Cell Non-Hodgkin Lymphoma; Refractory Hodgkin Lymphoma; Refractory Mycosis Fungoides; Refractory Nodal Marginal Zone Lymphoma; Refractory Non-Hodgkin Lymphoma; Refractory Primary Cutaneous T-Cell Non-Hodgkin Lymphoma; Stage IV Breast Cancer AJCC v6 and v7

Hippocampal asymmetry in exploratory behavior to vasoactive intestinal polypeptide.

PubMed

Ivanova, Margarita; Ternianov, Alexandar; Belcheva, Stiliana; Tashev, Roman; Negrev, Negrin; Belcheva, Iren

2008-06-01

The effects of vasoactive intestinal polypeptide (VIP) microinjected uni- or bilaterally into the CA1 hippocampal area of male Wistar rats at a dose of 10, 50 and 100 ng on exploratory behavior were examined. VIP microinjected bilaterally at a high dose (100 ng) significantly decreased the horizontal movements, while at low doses (10 and 50 ng) had no effect on the exploratory activity. Microinjections of VIP into the left hippocampal CA1 area at doses 50 and 100 ng suppressed the exploratory activity, while right-side VIP administration at a dose 100 ng significantly increased horizontal movements compared to the respective controls. Vertical activity was stimulated only by VIP administered into the right hippocampal CA1 area at the three doses used. Neither bilateral nor left injections of VIP induced changes in the vertical movements. The main finding was the presence of hippocampal asymmetry in exploratory behavior to unilateral microinjections of VIP depending on the dose and the microinjected hemisphere.

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

PubMed Central

Poland, Ryan S.; Bull, Cecilia; Syed, Wahab A.; Bowers, M. Scott

2015-01-01

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as appropriate models, partly because the response to behaviorally relevant stimuli and the circuitry parsing stimulus-action outcomes is astonishingly similar between humans and rodents. In studying molecular substrates of complex behaviors, the microinjection of reagents that modify, augment, or silence specific systems is an invaluable technique. However, it is crucial that the microinjection site is precisely targeted in order to aid interpretation of the results. We present a method for the manufacture of surgical implements and microinjection needles that enables accurate microinjection and unlimited customizability with minimal cost. Importantly, this technique can be successfully completed in awake rodents if conducted in conjunction with other JoVE articles that covered requisite surgical procedures. Additionally, there are many behavioral paradigms that are well suited for measuring motivation. The progressive ratio is a commonly used method that quantifies the efficacy of a reinforcer to maintain responding despite an (often exponentially) increasing work requirement. This assay is sensitive to reinforcer magnitude and pharmacological manipulations, which allows reinforcing efficacy and/ or motivation to be determined. We also present a straightforward approach to program operant software to accommodate a progressive ratio reinforcement schedule. PMID:26437131

Actin dynamics at the living cell submembrane imaged by total internal reflection fluorescence photobleaching.

PubMed Central

Sund, S E; Axelrod, D

2000-01-01

Although reversible chemistry is crucial to dynamical processes in living cells, relatively little is known about relevant chemical kinetic rates in vivo. Total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP), an established technique previously demonstrated to measure reversible biomolecular kinetic rates at surfaces in vitro, is extended here to measure reversible biomolecular kinetic rates of actin at the cytofacial (subplasma membrane) surface of living cells. For the first time, spatial imaging (with a charge-coupled device camera) is used in conjunction with TIR/FRAP. TIR/FRAP imaging produces both spatial maps of kinetic parameters (off-rates and mobile fractions) and estimates of kinetic correlation distances, cell-wide kinetic gradients, and dependences of kinetic parameters on initial fluorescence intensity. For microinjected rhodamine actin in living cultured smooth muscle (BC3H1) cells, the unbinding rate at or near the cytofacial surface of the plasma membrane (averaged over the entire cell) is measured at 0.032 +/- 0.007 s(-1). The corresponding rate for actin marked by microinjected rhodamine phalloidin is very similar, 0.033 +/- 0.013 s(-1), suggesting that TIR/FRAP is reporting the dynamics of entire filaments or protofilaments. For submembrane fluorescence-marked actin, the intensity, off-rate, and mobile fraction show a positive correlation over a characteristic distance of 1-3 microm and a negative correlation over larger distances greater than approximately 7-14 microm. Furthermore, the kinetic parameters display a statistically significant cell-wide gradient, with the cell having a "fast" and "slow" end with respect to actin kinetics. PMID:10969025

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences.

PubMed

Dudczig, Stefanie; Currie, Peter D; Poggi, Lucia; Jusuf, Patricia R

2017-03-22

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments. In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

Embryo development, fetal growth and postnatal phenotype of eGFP lambs generated by lentiviral transgenesis.

PubMed

Crispo, M; Vilariño, M; dos Santos-Neto, P C; Núñez-Olivera, R; Cuadro, F; Barrera, N; Mulet, A P; Nguyen, T H; Anegón, I; Menchaca, A

2015-02-01

Lentiviral technology has been recently proposed to generate transgenic farm animals more efficiently and easier than traditional techniques. The objective was to evaluate several parameters of lambs obtained by lentiviral transgenesis in comparison with non-transgenic counterparts. In vitro produced embryos were microinjected (TG group) at two-cell stage with a lentiviral construct containing enhanced green fluorescent protein (eGFP) gene, while embryos produced by in vitro fertilization (IVF group) or intrauterine insemination (IUI group) were not microinjected. Microinjection technique efficiently generated eight-cell transgenic embryos (97.4%; 114/117). Development rate on day 5 after fertilization was similar for TG (39.3%, 46/117) and IVF embryos (39.6%, 44/111). Pregnancy rate was detected in 50.0% (6/12) of recipient ewes with TG embryos, in 46.7% (7/15) with IVF embryos, and in 65.0% (13/20) of IUI ewes (P = NS). Nine lambs were born in TG group, six lambs in IVF group, and 16 lambs in IUI group. All TG lambs (9/9) were GFP positive to real-time PCR and eight (88.9%) showed a strong and evident GFP expression in mucosae, eyes and keratin tissues. Fetal growth monitored every 15 day by ultrasonography did not show significant differences. Transgenic lambs neither differ in morphometric variables in comparison with non transgenic IVF lambs within 3 months after birth. Transmission of the transgene to the progeny was observed in green fluorescent embryos produced by IVF using semen from the TG founder lambs. In conclusion, this study demonstrates the high efficiency of lentiviral technology to produce transgenic sheep, with no clinic differences in comparison with non transgenic lambs.

Somatic cell nuclear transfer followed by CRIPSR/CAS9 microinjection results in highly efficient genome editing in cloned pigs

USDA-ARS?s Scientific Manuscript database

The domestic pig is an ideal “dual purpose” animal model for agricultural and biomedical research. With the availability of genome editing tools [e.g. clustered regularly interspersed short palindromic repeat (CRISPR) and associated nuclease Cas9 (CRISPR/Cas9)] it is now possible to perform site-sp...

Electrolytic lesion of the nucleus raphe magnus reduced the antinociceptive effects of bilateral morphine microinjected into the nucleus cuneiformis in rats.

PubMed

Haghparast, Abbas; Ordikhani-Seyedlar, Mehdi; Ziaei, Maryam

2008-06-27

Several lines of investigation show that the rostral ventromedial medulla is a critical relay for midbrain regions, including the nucleus cuneiformis (CnF), which control nociception at the spinal cord. There is some evidence that local stimulation or morphine administration into the CnF produces the effective analgesia through the nucleus raphe magnus (NRM). The present study tries to determine the effect of morphine-induced analgesia following microinjection into the CnF in the absence of NRM. Seven days after the cannulae implantation, morphine was microinjected bilaterally into the CnF at the doses of 0.25, 1, 2.5, 5, 7.5 and 10 microg/0.3 microl saline per side. The morphine-induced antinociceptive effect measured by tail-flick test at 30, 60, 90 and 120 min after microinjection. The results showed that bilateral microinjection of morphine into the CnF dose-dependently causes increase in tail-flick latency (TFL). The 50% effective dose of morphine was determined and microinjected into the CnF (2.5 microg/0.3 microl saline per side) in rats after NRM electrolytic lesion (1 mA, 30 s). Lesion of the NRM significantly decreased TFLs, 30 (P<0.01) and 60 (P<0.05) but not 90-120 min after morphine microinjection into the CnF, compared with sham-lesion group. We concluded that morphine induces the analgesic effects through the opioid receptors in the CnF. It is also appeared that morphine-induced antinociception decreases following the NRM lesion but it seems that there are some other descending pain modulatory pathways that activate in the absence of NRM.

Role of glutamatergic receptors located in the nucleus raphe magnus on antinociceptive effect of morphine microinjected into the nucleus cuneiformis of rat.

PubMed

Haghparast, Abbas; Soltani-Hekmat, Ava; Khani, Abbas; Komaki, Alireza

2007-10-29

Neurons in the nucleus cuneiformis (CnF), located just ventrolateral to the periaqueductal gray, project to medullary nucleus raphe magnus (NRM), which is a key medullary relay for descending pain modulation and is critically involved in opioid-induced analgesia. Previous studies have shown that antinociceptive response of CnF-microinjected morphine can be modulated by the specific subtypes of glutamatergic receptors within the CnF. In this study, we evaluated the role of NMDA and kainate/AMPA receptors that are widely distributed within the NRM on morphine-induced antinociception elicited from the CnF. Hundred and five male Wistar rats weighing 250-300 g were used. Morphine (10, 20 and 40 microg) and NMDA receptor antagonist, MK-801 (10 microg) or kainate/AMPA receptor antagonist, DNQX (0.5 microg) in 0.5 microl saline were stereotaxically microinjected into the CnF and NRM, respectively. The latency of tail-flick response was measured at set intervals (2, 7, 12, 17, 22, 27 min after microinjection) by using an automated tail-flick analgesiometer. The results showed that morphine microinjection into the CnF dose-dependently causes increase in tail-flick latency (TFL). MK-801 microinjected into the NRM, just 1 min before morphine injection into the CnF, significantly attenuated antinociceptive effects of morphine. On the other hand, DNQX microinjected into the NRM, significantly increased TFL after local application of morphine into the CnF. We suggest that morphine related antinociceptive effect elicited from the CnF is mediated, in part, by NMDA receptor at the level of the NRM whereas kainite/AMPA receptor has a net inhibitory influence at the same pathway.

Microinjector for blasocysts

DOEpatents

Remenyik, Carl J.; Woychik, Richard P.; Patek, David R.; Hawk, James A.; Turner, John C.

1999-01-01

An electromechanical device for driving the tip of a microinjection cannula, or needle, through the outer barrier of a blastocyst, cell, or cell nucleus for the injection of cells or other bioactive materials. Either a flexible frame or a ram moving within a base member is employed. Cannula motion is achieved by means of a piezoelectric stack and spring return system. The thrust motion over a predetermined microscopic distance is achieved without cannula setback prior to the thrust movement. Instead of specially prepared beveled and tipped needles, standard unimproved cannulas or needles can be used.

Microinjector for blasocysts

DOEpatents

Remenyik, C.J.; Woychik, R.P.; Patek, D.R.; Hawk, J.A.; Turner, J.C.

1999-03-02

An electromechanical device is disclosed for driving the tip of a microinjection cannula, or needle, through the outer barrier of a blastocyst, cell, or cell nucleus for the injection of cells or other bioactive materials. Either a flexible frame or a ram moving within a base member is employed. Cannula motion is achieved by means of a piezoelectric stack and spring return system. The thrust motion over a predetermined microscopic distance is achieved without cannula setback prior to the thrust movement. Instead of specially prepared beveled and tipped needles, standard unimproved cannulas or needles can be used. 6 figs.

Intracytoplasmic sperm injection for treatment of the infertile male.

PubMed

Kim, E D; Lamb, D J; Lipshultz, L I

1997-07-01

Intracytoplasmic sperm injection (ICSI) with in vitro fertilization represents one of the most significant advances in fertility technology. In this relatively new procedure, a single viable sperm is microinjected into an oocyte that has been extracted transvaginally. After fertilization occurs, the embryo is transferred into the uterus. This procedure now affords men who were previously thought to be irreversibly infertile the chance to initiate their own biologic pregnancy. However, because of the procedure's significant costs and its potential risk to the mother, careful selection of couples following a thorough male factor evaluation is mandatory.

Culture time of vitrified/warmed zygotes before microinjection affects the production efficiency of CRISPR-Cas9-mediated knock-in mice.

PubMed

Nakagawa, Yoshiko; Sakuma, Tetsushi; Nishimichi, Norihisa; Yokosaki, Yasuyuki; Takeo, Toru; Nakagata, Naomi; Yamamoto, Takashi

2017-05-15

Robust reproductive engineering techniques are required for the efficient and rapid production of genetically modified mice. We have reported the efficient production of genome-edited mice using reproductive engineering techniques, such as ultra-superovulation, in vitro fertilization (IVF) and vitrification/warming of zygotes. We usually use vitrified/warmed fertilized oocytes created by IVF for microinjection because of work efficiency and flexible scheduling. Here, we investigated whether the culture time of zygotes before microinjection influences the efficiency of producing knock-in mice. Knock-in mice were generated using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system and single-stranded oligodeoxynucleotide (ssODN) or PITCh (Precise Integration into Target Chromosome) system, a method of integrating a donor vector assisted by microhomology-mediated end-joining. The cryopreserved fertilized oocytes were warmed, cultured for several hours and microinjected at different timings. Microinjection was performed with Cas9 protein, guide RNA(s), and an ssODN or PITCh donor plasmid for the ssODN knock-in and the PITCh knock-in, respectively. Different production efficiencies of knock-in mice were observed by changing the timing of microinjection. Our study provides useful information for the CRISPR-Cas9-based generation of knock-in mice. © 2017. Published by The Company of Biologists Ltd.

Correlations between conceptal concentrations of all-trans-retinoic acid and dysmorphogenesis after microinjections of all-trans-retinoic acid, 13-cis-retinoic acid, all-trans-retinoyl-beta-glucuronide, or retinol in cultured whole rat embryos.

PubMed

Kraft, J C; Juchau, M R

1992-01-01

Retinol (4,000 ng/ml), all-trans-retinoyl-beta-glucuronide (4,000 ng/ml), and 13-cis-retinoic acid (1,500 ng/ml) each produced dysmorphogenic effects qualitatively similar to those elicited by 250 ng/ml of all-trans-retinoic acid after microinjections of the respective individual retinoids into the amniotic cavities of cultured whole rat embryos. Subsequent HPLC analyses of the cultured whole conceptuses, embryos proper, yolk sacs, and culture media (24 hr after microinjections) indicated that conceptal biotransformation of each of the retinoids had occurred during the culture period. All-trans-retinoic acid was present in the embryos proper at quantitatively similar concentrations (20-100 nM) after microinjections of the selected quantities of each of the microinjected retinoids: retinol, all-trans-retinoyl-beta-glucuronide, 13-cis-retinoic acid, or all-trans-retinoic acid. The results suggested that all-trans-retinoic acid acted as an ultimate dysmorphogen for the retinoids tested with respect to the anomalies monitored in the embryo culture system.

The role of spinal GABAergic circuits in the control of phrenic nerve motor output.

PubMed

Marchenko, Vitaliy; Ghali, Michael G Z; Rogers, Robert F

2015-06-01

While supraspinal mechanisms underlying respiratory pattern formation are well characterized, the contribution of spinal circuitry to the same remains poorly understood. In this study, we tested the hypothesis that intraspinal GABAergic circuits are involved in shaping phrenic motor output. To this end, we performed bilateral phrenic nerve recordings in anesthetized adult rats and observed neurogram changes in response to knocking down expression of both isoforms (65 and 67 kDa) of glutamate decarboxylase (GAD65/67) using microinjections of anti-GAD65/67 short-interference RNA (siRNA) in the phrenic nucleus. The number of GAD65/67-positive cells was drastically reduced on the side of siRNA microinjections, especially in the lateral aspects of Rexed's laminae VII and IX in the ventral horn of cervical segment C4, but not contralateral to microinjections. We hypothesize that intraspinal GABAergic control of phrenic output is primarily phasic, but also plays an important role in tonic regulation of phrenic discharge. Also, we identified respiration-modulated GABAergic interneurons (both inspiratory and expiratory) located slightly dorsal to the phrenic nucleus. Our data provide the first direct evidence for the existence of intraspinal GABAergic circuits contributing to the formation of phrenic output. The physiological role of local intraspinal inhibition, independent of descending direct bulbospinal control, is discussed. Copyright © 2015 the American Physiological Society.

Efficient transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection.

PubMed

Masani, Mat Yunus Abdul; Noll, Gundula A; Parveez, Ghulam Kadir Ahmad; Sambanthamurthi, Ravigadevi; Prüfer, Dirk

2014-01-01

Genetic engineering remains a major challenge in oil palm (Elaeis guineensis) because particle bombardment and Agrobacterium-mediated transformation are laborious and/or inefficient in this species, often producing chimeric plants and escapes. Protoplasts are beneficial as a starting material for genetic engineering because they are totipotent, and chimeras are avoided by regenerating transgenic plants from single cells. Novel approaches for the transformation of oil palm protoplasts could therefore offer a new and efficient strategy for the development of transgenic oil palm plants. We recently achieved the regeneration of healthy and fertile oil palms from protoplasts. Therefore, we focused on the development of a reliable PEG-mediated transformation protocol for oil palm protoplasts by establishing and validating optimal heat shock conditions, concentrations of DNA, PEG and magnesium chloride, and the transfection procedure. We also investigated the transformation of oil palm protoplasts by DNA microinjection and successfully regenerated transgenic microcalli expressing green fluorescent protein as a visible marker to determine the efficiency of transformation. We have established the first successful protocols for the transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. These novel protocols allow the rapid and efficient generation of non-chimeric transgenic callus and represent a significant milestone in the use of protoplasts as a starting material for the development of genetically-engineered oil palm plants.

Efficient Transformation of Oil Palm Protoplasts by PEG-Mediated Transfection and DNA Microinjection

PubMed Central

Masani, Mat Yunus Abdul; Noll, Gundula A.; Parveez, Ghulam Kadir Ahmad; Sambanthamurthi, Ravigadevi; Prüfer, Dirk

2014-01-01

Background Genetic engineering remains a major challenge in oil palm (Elaeis guineensis) because particle bombardment and Agrobacterium-mediated transformation are laborious and/or inefficient in this species, often producing chimeric plants and escapes. Protoplasts are beneficial as a starting material for genetic engineering because they are totipotent, and chimeras are avoided by regenerating transgenic plants from single cells. Novel approaches for the transformation of oil palm protoplasts could therefore offer a new and efficient strategy for the development of transgenic oil palm plants. Methodology/Principal Findings We recently achieved the regeneration of healthy and fertile oil palms from protoplasts. Therefore, we focused on the development of a reliable PEG-mediated transformation protocol for oil palm protoplasts by establishing and validating optimal heat shock conditions, concentrations of DNA, PEG and magnesium chloride, and the transfection procedure. We also investigated the transformation of oil palm protoplasts by DNA microinjection and successfully regenerated transgenic microcalli expressing green fluorescent protein as a visible marker to determine the efficiency of transformation. Conclusions/Significance We have established the first successful protocols for the transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. These novel protocols allow the rapid and efficient generation of non-chimeric transgenic callus and represent a significant milestone in the use of protoplasts as a starting material for the development of genetically-engineered oil palm plants. PMID:24821306

Cross state-dependency of learning between tramadol and MK-801 in the mouse dorsal hippocampus: involvement of nitric oxide (NO) signaling pathway.

PubMed

Jafari-Sabet, Majid; Amiri, Shiva; Ataee, Ramin

2018-04-21

Tramadol, an atypical μ-opioid receptor agonist, as a psychoactive drug, is frequently abused by human beings. Understanding the neurobiological mechanisms of drug-associated learning and memory formation may help prevent drug addiction and relapse. Previous study revealed that dorsal hippocampus (CA1) plays a crucial role in the retrieval of tramadol-associated memory and that its role depends on the expression of CA1 N-methyl-D-aspartate (NMDA) receptors (Jafari-Sabet et al. Can J Physiol Pharmacol 96:45-50, 2018). To clarify the exact mechanisms involved, the activation of CA1 nitric oxide (NO) signaling pathway by L-arginine (a nitric oxide precursor) on the interaction between tramadol and MK-801 in memory retrieval was examined. The dorsal hippocampal CA1 regions of adult male NMRI mice were bilaterally cannulated and a single-trial step-down inhibitory avoidance apparatus was used for the assessment of memory retrieval. Post-training and/or pre-test microinjection of tramadol (0.5 and 1 μg/mouse) and/or a non-competitive NMDA receptor antagonist, MK-801 (0.25 and 0.5 μg/mouse), induced amnesia which were reversed when the same doses of the drugs were administered 24 h later in a pre-test session, suggesting tramadol state-dependent learning (SDL) and MK-801 SDL. The amnesia induced by post-training microinjection of tramadol (1 μg/mouse) was reversed by pre-test microinjection of MK-801 (0.25 and 0.5 μg/mouse). Pre-test microinjection of MK-801 (0.125 and 0.25 μg/mouse) with an ineffective dose of tramadol (0.25 μg/mouse) potentiated tramadol SDL. The amnesia induced by post-training microinjection of MK-801 (0.5 μg/mouse) was reversed by pre-test microinjection of tramadol (0.5 and 1 μg/mouse). Pre-test microinjection of tramadol (0.25 and 0.5 μg/mouse) with an ineffective dose of MK-801 (0.125 μg/mouse) potentiated MK-801 SDL. Pre-test microinjection of ineffective doses of L-arginine (0.125, 025, and 0.5 μg/mouse) improved amnesia induced by the co-administration of tramadol and MK-801. Pre-test microinjection of L-arginine (0.125, 025, and 0.5 μg/mouse) could not reverse amnesia induced by post-training microinjection of tramadol while same doses of L-arginine improved MK-801 response on tramadol SDL. The results strongly propose that activation of CA1 NO signaling pathway has a pivotal role in cross SDL among tramadol and MK-801.

Identification of phospholipase activity in Rhinella arenarum sperm extract capable of inducing oocyte activation.

PubMed

Bonilla, Federico; Minahk, Carlos; Ajmat, María Teresa; Toranzo, Graciela Sánchez; Bühler, Marta Inés

2014-11-01

Egg activation, which includes cortical granule exocytosis, resumption and completion of meiosis and pronuclear formation culminates in the first mitotic cleavage. However, the mechanism through which the fertilizing sperm induces this phenomenon is still controversial. We investigated the effect of the microinjection of homologous sperm soluble fractions obtained by fast protein liquid chromatography (FPLC) from reacted sperm (without acrosome) and non-reacted sperm on the activation of Rhinella arenarum oocytes matured in vitro. The FPLC-purified sperm fraction obtained from reacted or non-reacted sperm is able to induce oocyte activation when it is microinjected. This fraction has a 24 kDa protein and showed phospholipase C (PLC) activity in vitro, which was inhibited by D-609 but not by n-butanol or neomycin, suggesting that it is a PLC that is specific for phosphatidylcholine (PC-PLC). The assays conducted using inhibitors of inositol triphosphate (IP3) and ryanodine receptors (RyRs) indicate that the fraction with biological activity would act mainly through the cADPr (cyclic ADP ribose) pathway. Moreover, protein kinase C (PKC) inhibition blocks the activation produced by the same fraction. Immunocytochemical studies indicate that this PC-PLC can be found throughout the sperm head.

Innate immune system still works at diapause, a physiological state of dormancy in insects

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakamura, Akihiro; Miyado, Kenji, E-mail: kmiyado@nch.go.jp; Takezawa, Youki

Highlights: {yields} Two major types of cells are present in the body fluid isolated from the thoracic region of a diapausing pupa. {yields} Phagocytosis and encapsulation by these cells were observed when latex beads as foreign targets were microinjected into a pupa. {yields} Such behavior by these cells was still observed even when pupae were continuously chilled at 4 {sup o}C. {yields} Innate cellular reactions can work in diapausing insects in a dormant state. -- Abstract: Diapause is most often observed in insects and is a physiologically dormant state different from other types of dormancy, such as hibernation. It allowsmore » insects to survive in harsh environments or extend longevity. In general, larval, pupal, or adult non-diapausing insects possess an innate immune system preventing the invasion of microorganisms into their bodies; however, it is unclear whether this system works under the dormant condition of diapause. We here report the occurrence of innate cellular reactions during diapause using pupae of a giant silkmoth, Samia cynthia pryeri. Scanning electron microscopic analysis demonstrated the presence of two major types of cells in the body fluid isolated from the thoracic region of a pupa. Phagocytosis and encapsulation, characteristics of innate cellular reactions, by these cells were observed when latex beads as foreign targets were microinjected into the internal portion of a pupa. Such behavior by these cells was still observed even when pupae were continuously chilled at 4 {sup o}C. Our results indicate that innate cellular reactions can work in diapausing insects in a dormant state.« less

Ventrolateral orbital cortex oxytocin attenuates neuropathic pain through periaqueductal gray opioid receptor.

PubMed

Taati, Mina; Tamaddonfard, Esmaeal

2018-06-01

Oxytocin plays an important role in supraspinal modulation of pain. In the present study, we investigated the effects of ventrolateral orbital cortex (VLOC) microinjection of oxytocin on neuropathic pain after blockade of opioid receptors in this area and ventrolateral periaqueductal gray (vlPAG). Neuropathic pain was induced by complete transcection of preoneal and tibial branches of sciatic nerve. The VLOC and vlPAG were unilaterally (contralateral to the sciatic nerve-injured side) and bilaterally implanted with guide cannulas, respectively. Mechanical paw withdrawal threshold (PWT) was measured using von Frey filaments. Area under curve (AUC) was also calculated. Microinjection of oxytocin (5, 10 and 20 ng/site) into the VLOC increased PWT. Antiallodynia induced by oxytocin (20 ng/site) was inhibited by prior intra-VLOC administration of atosiban (an oxytocin receptor antagonist, 100 ng/site) and naloxone (an opioid receptor antagonist, 500 ng/site). Prior microinjection of naloxone (500 ng/site) into the vlPAG also inhibited antiallodynia induced by intra-VLOC microinjection of oxytocin (20 ng/site). All the VLOC and vlPAG microinjected drugs did not alter locomotor activity. It is concluded that oxytocin and its receptor may be involved in modulation of neuropathic pain at the VLOC level. Opioid receptors of VLOC and vlPAG might be involved in the antiallodynic effect of the VLOC-microinjected oxytocin. Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.

Polygalacturonase Causes Lygus-like Damage on Plants: Cloning and Identification of Western Tarnished Plant Bug (Lygus hesperus) Polygalacturonases Secreted During Feeding

USDA-ARS?s Scientific Manuscript database

Abstract: Polygalacturonase (PG), an enzyme that degrades pectin within the plant tissue cell wall, has been postulated as the chemical cause of damage to plants by the mirid Lygus hesperus. Micro-injection of two pure recombinant Aspergillus niger PG II protein forms, the wild type enzymically acti...

Microinjection of the monoclonal anti-tubulin antibody YL1/2 inhibits cleavage of sand dollar eggs.

PubMed

Oka, M T; Arai, T; Hamaguchi, Y

1990-12-01

Two monoclonal antibodies against alpha-tubulin (YL1/2 and D2D6) were microinjected into the egg of the sand dollar Clypeaster japonicus, and their effects on cleavage of the egg were investigated. They had already been shown by immunoblotting to react specifically with egg tubulin and by immunofluorescence to stain the mitotic apparatus [OKA et al., (1990). Cell Motil. Cytoskel. 16:239-250]. Injection of YL1/2 prevented chromosome movement and cleavage, although the cleavage furrow developed in some cases. In all eggs injected at prometaphase, metaphase, or anaphase, the birefringence of the mitotic apparatus disappeared immediately after injection. Injection of D2D6 had no significant effect on mitosis or cleavage of whole eggs injected after nuclear disappearance, although it prevented the disappearance of the nuclear envelope in 54% of the eggs injected before the disappearance. FITC-conjugated D2D6 did not accumulate in the spindle when injected into the dividing sand dollar egg. These results indicate that YL1/2 disassembled microtubules, whereas D2D6 did not bind to microtubules in the living cell.

Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes.

PubMed

Crispo, M; Mulet, A P; Tesson, L; Barrera, N; Cuadro, F; dos Santos-Neto, P C; Nguyen, T H; Crénéguy, A; Brusselle, L; Anegón, I; Menchaca, A

2015-01-01

While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and livestock.

The nucleus raphe magnus OFF-cells are involved in diffuse noxious inhibitory controls.

PubMed

Chebbi, R; Boyer, N; Monconduit, L; Artola, A; Luccarini, P; Dallel, R

2014-06-01

Diffuse noxious inhibitory controls (DNIC) are very powerful long-lasting descending inhibitory controls which are pivotal in modulating the activity of spinal and trigeminal nociceptive neurons. DNIC are subserved by a loop involving supraspinal structures such as the lateral parabrachial nucleus and the subnucleus reticularis dorsalis. Surprisingly, though, whether the nucleus raphe magnus (NRM), another supraspinal area which is long known to be important in pain modulation, is involved in DNIC is still a matter of discussion. Here, we reassessed the role of the NRM neurons in DNIC by electrophysiologically recording from wide dynamic range (WDR) neurons in the trigeminal subnucleus oralis and pharmacologically manipulating the NRM OFF- and ON-cells. In control conditions, C-fiber-evoked responses in trigeminal WDR neurons are inhibited by a conditioning noxious heat stimulation applied to the hindpaw. We show that inactivating the NRM by microinjecting the GABAA receptor agonist, muscimol, both facilitates C-fiber-evoked responses of trigeminal WDR neurons and strongly attenuates their inhibition by heat applied to the hindpaw. Interestingly, selective blockade of ON-cells by microinjecting the broad-spectrum excitatory amino acid antagonist, kynurenate, into the NRM neither affects C-fiber-evoked responses nor attenuates DNIC of trigeminal WDR neurons. These results indicate that the NRM tonically inhibits trigeminal nociceptive inputs and is involved in the neuronal network underlying DNIC. Moreover, within NRM, OFF-cells might be more specifically involved in both the tonic and phasic descending inhibitory controls of trigeminal nociception. Copyright © 2014 Elsevier Inc. All rights reserved.

Characterisation of the Rac/PAK pathway in Amoeba proteus.

PubMed

Kłopocka, W; Moraczewska, J; Redowicz, M J

2005-04-01

Molecular mechanisms underlying the unique locomotion of the highly motile Amoeba proteus still remain poorly understood. Recently, we have shown that blocking the endogenous amoebal Rac-like protein(s) leads to distinct and irreversible changes in the appearance of these large migrating cells as well as to a significant inhibition of their locomotion. To elucidate the mechanism of the Rac pathway in Amoeba proteus, we tested the effects of blocking the endogenous myosin I heavy chain kinase (MIHCK), one of the Rac effectors in Acanthamoeba castellanii and Dictyostelium discoideum, with anti-MIHCK antibodies in migrating amoebae, as well as the effect of inhibiting Rac and MIHCK on the actin polymerisation process. Antibodies against A. castellanii MIHCK detected an A. proteus protein with a molecular mass (ca. 95 kDa) similar to the A. castellanii kinase. The cellular distribution of MIHCK in A. proteus was very similar to those of Rac-like protein in amoebae and MIHCK in A. castellanii. Amoebae microinjected with anti-MIHCK antibodies moved slower and protruded fewer wide pseudopodia (5-6) than the control cells (9-10), resembling to some extent the phenotype of cells microinjected with anti-Rac antibodies. The in vitro studies indicate that the A. proteus Rac-like protein, but not the MIHCK isoform, is engaged in the regulation of the nucleation step of the actin polymerisation process. These observations suggest that MIHCK may be one of the effectors for Rac in these extremely large cells.

Probing the role of nonmuscle tropomyosin isoforms in intracellular granule movement by microinjection of monoclonal antibodies

PubMed Central

1989-01-01

Chicken embryo fibroblast (CEF) cells were microinjected with several different monoclonal antibodies that recognize certain nonmuscle isoforms of tropomyosin. Immediately after injection, cells were recorded with a time-lapse video imaging system; later analysis of the tapes revealed that particles in cells injected with one of these antibodies (CG1, specific for CEF tropomyosin isoforms 1 and 3) showed a dramatic decrease in instantaneous speed while moving, distance moved per saltation, and proportion of time spent in motion. Injection of Fab fragments of CG1 resulted in similar changes in the pattern of granule movement. This inhibition of granule movement by CG1 antibody was reversible; at 2.5 h after injection, granules in injected cells had already reached three-fourths of normal speed. The speed of granule movement in cells injected either with antibody specific for tropomyosin isoforms not present in CEF cells, or with CG1 antibody preabsorbed with tropomyosin, was not significantly different from the speed of granules in uninjected cells. When cells were injected with CG1 or Fab fragments of CG1, fixed, and counter-stained with rabbit antibodies to reveal the microtubule, microfilament, and intermediate filament systems, no obvious differences from the patterns normally seen in uninjected cells were observed. Examination of the ultrastructure of injected cells by EM confirmed the presence of apparently intact and normal microtubule, actin, and intermediate filament networks. These experiments suggest that tropomyosin may play an important role in the movement of vesicles and organelles in the cell cytoplasm. Also, we have shown previously that the CG1 determinant can undergo a motility-dependent change in reactivity, that may be important for the regulatory function of nonmuscle tropomyosin (Hegmann, T. E., J. L.-C. Lin, and J. J.-C. Lin. 1988. J. Cell Biol. 106:385-393). Therefore, in addition to postulated microtubule-based motors, microfilaments may play a critical role in regulating granule movement in nonmuscle cells. PMID:2670955

Neuropeptide Y in the rostral ventromedial medulla reverses inflammatory and nerve injury hyperalgesia in rats via non-selective excitation of local neurons

PubMed Central

Cleary, Daniel R.; Roeder, Zachary; Elkhatib, Rania; Heinricher, Mary M.

2014-01-01

Chronic pain reflects not only sensitization of the ascending nociceptive pathways, but also changes in descending modulation. The rostral ventromedial medulla (RVM) is a key structure in a well-studied descending pathway, and contains two classes of modulatory neurons, the ON-cells and the OFF-cells. Disinhibition of OFF-cells depresses nociception; increased ON-cell activity facilitates nociception. Multiple lines of evidence show that sensitization of ON-cells contributes to chronic pain, and reversing or blocking this sensitization is of interest as a treatment of persistent pain. Neuropeptide Y (NPY) acting via the Y1 receptor has been shown to attenuate hypersensitivity in nerve-injured animals without affecting normal nociception when microinjected into the RVM, but the neural basis for this effect was unknown. We hypothesized that behavioral anti-hyperalgesia was due to selective inhibition of ON-cells by NPY at the Y1 receptor. To explore the possibility of Y1 selectivity on ON-cells, we stained for the NPY-Y1 receptor in the RVM, and found it broadly expressed on both serotonergic and non-serotonergic neurons. In subsequent behavioral experiments, NPY microinjected into the RVM in lightly anesthetized animals reversed signs of mechanical hyperalgesia following either nerve injury or chronic hindpaw inflammation. Unexpectedly, rather than decreasing ON-cell activity, NPY increased spontaneous activity of both ON- and OFF-cells without altering noxious-evoked changes in firing. Based on these results, we conclude that the anti-hyperalgesic effects of NPY in the RVM are not explained by selective inhibition of ON-cells, but rather by increased spontaneous activity of OFF-cells. Although ON-cells undoubtedly facilitate nociception and contribute to hypersensitivity, the present results highlight the importance of parallel OFF-cell mediated descending inhibition in limiting the expression of chronic pain. PMID:24792711

Cerebroventricular Microinjection (CVMI) into Adult Zebrafish Brain Is an Efficient Misexpression Method for Forebrain Ventricular Cells

PubMed Central

Kizil, Caghan; Brand, Michael

2011-01-01

The teleost fish Danio rerio (zebrafish) has a remarkable ability to generate newborn neurons in its brain at adult stages of its lifespan-a process called adult neurogenesis. This ability relies on proliferating ventricular progenitors and is in striking contrast to mammalian brains that have rather restricted capacity for adult neurogenesis. Therefore, investigating the zebrafish brain can help not only to elucidate the molecular mechanisms of widespread adult neurogenesis in a vertebrate species, but also to design therapies in humans with what we learn from this teleost. Yet, understanding the cellular behavior and molecular programs underlying different biological processes in the adult zebrafish brain requires techniques that allow manipulation of gene function. As a complementary method to the currently used misexpression techniques in zebrafish, such as transgenic approaches or electroporation-based delivery of DNA, we devised a cerebroventricular microinjection (CVMI)-assisted knockdown protocol that relies on vivo morpholino oligonucleotides, which do not require electroporation for cellular uptake. This rapid method allows uniform and efficient knockdown of genes in the ventricular cells of the zebrafish brain, which contain the neurogenic progenitors. We also provide data on the use of CVMI for growth factor administration to the brain – in our case FGF8, which modulates the proliferation rate of the ventricular cells. In this paper, we describe the CVMI method and discuss its potential uses in zebrafish. PMID:22076157

ING2 (inhibitor of growth protein-2) plays a crucial role in preimplantation development.

PubMed

Zhou, Lin; Wang, Pei; Zhang, Juanjuan; Heng, Boon Chin; Tong, Guo Qing

2016-02-01

ING2 (inhibitor of growth protein-2) is a member of the ING-gene family and participates in diverse cellular processes involving tumor suppression, DNA repair, cell cycle regulation, and cellular senescence. As a subunit of the Sin3 histone deacetylase complex co-repressor complex, ING2 binds to H3K4me3 to regulate chromatin modification and gene expression. Additionally, ING2 recruits histone methyltransferase (HMT) activity for gene repression, which is independent of the HDAC class I or II pathway. However, the physiological function of ING2 in mouse preimplantation embryo development has not yet been characterized previously. The expression, localization and function of ING2 during preimplantation development were investigated in this study. We showed increasing expression of ING2 within the nucleus from the 4-cell embryo stage onwards; and that down-regulation of ING2 expression by endoribonuclease-prepared small interfering RNA (esiRNA) microinjection results in developmental arrest during the morula to blastocyst transition. Embryonic cells microinjected with ING2-specific esiRNA exhibited decreased blastulation rate compared to the negative control. Further investigation of the underlying mechanism indicated that down-regulation of ING2 significantly increased expression of p21, whilst decreasing expression of HDAC1. These results suggest that ING2 may play a crucial role in the process of preimplantation embryo development through chromatin regulation.

A microfluidic device for automated, high-speed microinjection of Caenorhabditis elegans

PubMed Central

Song, Pengfei; Dong, Xianke; Liu, Xinyu

2016-01-01

The nematode worm Caenorhabditis elegans has been widely used as a model organism in biological studies because of its short and prolific life cycle, relatively simple body structure, significant genetic overlap with human, and facile/inexpensive cultivation. Microinjection, as an established and versatile tool for delivering liquid substances into cellular/organismal objects, plays an important role in C. elegans research. However, the conventional manual procedure of C. elegans microinjection is labor-intensive and time-consuming and thus hinders large-scale C. elegans studies involving microinjection of a large number of C. elegans on a daily basis. In this paper, we report a novel microfluidic device that enables, for the first time, fully automated, high-speed microinjection of C. elegans. The device is automatically regulated by on-chip pneumatic valves and allows rapid loading, immobilization, injection, and downstream sorting of single C. elegans. For demonstration, we performed microinjection experiments on 200 C. elegans worms and demonstrated an average injection speed of 6.6 worm/min (average worm handling time: 9.45 s/worm) and a success rate of 77.5% (post-sorting success rate: 100%), both much higher than the performance of manual operation (speed: 1 worm/4 min and success rate: 30%). We conducted typical viability tests on the injected C. elegans and confirmed that the automated injection system does not impose significant adverse effect on the physiological condition of the injected C. elegans. We believe that the developed microfluidic device holds great potential to become a useful tool for facilitating high-throughput, large-scale worm biology research. PMID:26958099

Microinjection of muscimol into caudal periaqueductal gray lowers body temperature and attenuates increases in temperature and activity evoked from the dorsomedial hypothalamus.

PubMed

de Menezes, Rodrigo C A; Zaretsky, Dmitry V; Fontes, Marco A P; DiMicco, Joseph A

2006-05-30

Microinjection of the neuronal inhibitor muscimol into the midbrain lateral/dorsolateral periaqueductal gray (l/dlPAG) suppresses increases in heart rate (HR) and mean arterial pressure (MAP) evoked by microinjection of the GABA(A) receptor antagonist bicuculline methiodide (BMI) into the dorsomedial hypothalamus (DMH) in rats. Injection of BMI into the DMH also increases body temperature (Tco) and motor activity. Here, our goal was to extend previous findings by examining the effect of microinjection of muscimol into the PAG on these thermogenic and behavioral responses in conscious freely moving rats. Microinjection of muscimol (300 pmol and 1 nmol) alone into the l/dlPAG reduced baseline Tco without affecting activity, HR, or MAP. Similar injection of a dose that failed to alter baseline Tco (100 pmol) suppressed the increases in Tco evoked from the DMH and significantly attenuated DMH-induced increases in locomotor activity. Whereas microinjection of 1 nmol muscimol into the ldlPAG abolished the increases in Tco evoked from the DMH and in fact lowered body temperature to a degree similar to that seen after this dose of muscimol alone, 1 nmol muscimol at adjacent sites outside the targeted region of the PAG had no significant effect on DMH-induced increases in Tco or any other parameter. These results indicate a role for neuronal activity in the l/dlPAG in (1) the temperature and behavioral responses to disinhibition of neurons in the DMH, and (2) the maintenance of basal body temperature in conscious freely moving rats.

A strain of Serratia marcescens pathogenic for larvae of Lymantria dispar: Infectivity and mechanisms of pathogenicity

Treesearch

J.D. Podgwaite; B.J. Cosenza

1976-01-01

The ED50 of a strain of Serratia marcescens for microinjected instar III and IV gypsy moth larvae was 7.5 and 14.5 viable cells, respectively. Percentage and rate of mortality were found to be highly variable among replicates of the same instar and between instars in free-feeding bioassays. Mortality in second instar larvae...

Mastoparan-Induced Intracellular Ca2+ Fluxes May Regulate Cell-to-Cell Communication in Plants.

PubMed

Tucker, E. B.; Boss, W. F.

1996-06-01

The relationship of Ca2+ and plasmodesmatal closure was examined in staminal hairs of Setcreasea purpurea by microinjecting cells with active mastoparan (Mas-7), inactive mastoparan (Mas-17), active inositol-1,4,5-trisphosphate (IP3), or inactive IP3. Calcium green dextran 10,000 was used to study cellular free Ca2+, and carboxyfluorescein was used to monitor plasmodesmatal closure. When Mas-7 was microinjected into the cytoplasm of cell 1 (the tip cell of a chain of cells), a rapid increase in calcium green dextran-10,000 fluorescence was observed in the cytoplasmic areas on both sides of the plasmodesmata connecting cells 1 and 2 during the same time that the diffusion of carboxyfluorescein through them was blocked. The inhibition of cell-to-cell diffusion was transient, and the closed plasmodesmata reopened within 30 s. The elevated Ca2+ level near plasmodesmata was also transient and returned to base level in about 1.5 min. The transient increase in Ca2+, once initiated in cell 1, repeated with an oscillatory period of 3 min. Elevated Ca2+ and oscillations of Ca2+ were also observed near interconnecting cell walls throughout the chain of cells, indicating that the signal had been transmitted. Previously, we reported that IP3 closed plasmodesmata; now we report that it stimulated Ca2+ and oscillations similar to Mas-7. The effect was specific for similar concentrations of Mas-7 over Mas-17 and active IP3 over inactive IP3. It is important that the Ca2+ channel blocker La3+ eliminated the responses from Mas-7 and IP3, indicating that an influx of Ca2+ was required. These results support the contention that plasmodesmata functioning is regulated via Ca2+ and that IP3 may be an intermediary between the stimulus and Ca2+ elevations.

Mastoparan-Induced Intracellular Ca2+ Fluxes May Regulate Cell-to-Cell Communication in Plants.

PubMed Central

Tucker, E. B.; Boss, W. F.

1996-01-01

The relationship of Ca2+ and plasmodesmatal closure was examined in staminal hairs of Setcreasea purpurea by microinjecting cells with active mastoparan (Mas-7), inactive mastoparan (Mas-17), active inositol-1,4,5-trisphosphate (IP3), or inactive IP3. Calcium green dextran 10,000 was used to study cellular free Ca2+, and carboxyfluorescein was used to monitor plasmodesmatal closure. When Mas-7 was microinjected into the cytoplasm of cell 1 (the tip cell of a chain of cells), a rapid increase in calcium green dextran-10,000 fluorescence was observed in the cytoplasmic areas on both sides of the plasmodesmata connecting cells 1 and 2 during the same time that the diffusion of carboxyfluorescein through them was blocked. The inhibition of cell-to-cell diffusion was transient, and the closed plasmodesmata reopened within 30 s. The elevated Ca2+ level near plasmodesmata was also transient and returned to base level in about 1.5 min. The transient increase in Ca2+, once initiated in cell 1, repeated with an oscillatory period of 3 min. Elevated Ca2+ and oscillations of Ca2+ were also observed near interconnecting cell walls throughout the chain of cells, indicating that the signal had been transmitted. Previously, we reported that IP3 closed plasmodesmata; now we report that it stimulated Ca2+ and oscillations similar to Mas-7. The effect was specific for similar concentrations of Mas-7 over Mas-17 and active IP3 over inactive IP3. It is important that the Ca2+ channel blocker La3+ eliminated the responses from Mas-7 and IP3, indicating that an influx of Ca2+ was required. These results support the contention that plasmodesmata functioning is regulated via Ca2+ and that IP3 may be an intermediary between the stimulus and Ca2+ elevations. PMID:12226302

Blockade of mGluR5 in the nucleus accumbens shell but not core attenuates heroin seeking behavior in rats

PubMed Central

Lou, Zhong-ze; Chen, Ling-hong; Liu, Hui-feng; Ruan, Lie-min; Zhou, Wen-hua

2014-01-01

Aim: Glutamatergic neurotransmission in the nucleus accumbens (NAc) is crucial for the relapse to heroin seeking. The aim of this study was to determine whether mGluR5 in the NAc core or shell involved in heroin seeking behavior in rats. Methods: Male SD rats were self-administered heroin under a fixed-ratio 1 (FR1) reinforcement schedule for 14 d, and subsequently withdrawn for 2 weeks. The selective mGluR5 antagonist 2-methyl-6-phenylethynyl-pyridine (MPEP, 5, 15 and 50 nmol per side) was then microinjected into the NAc core or shell 10 min before a heroin-seeking test induced by context, cues or heroin priming. Results: Microinjection of MPEP into the NAc shell dose-dependently decreased the heroin seeking induced by context, cues or heroin priming. In contrast, microinjection of MPEP into the NAc core did not alter the heroin seeking induced by cues or heroin priming. In addition, microinjection with MPEP (15 nmol per side) in the NAc shell reversed both the percentage of open arms entries (OE%) and the percentage of time spent in open arms (OT%) after heroin withdrawal. Microinjection of MPEP (50 nmol per side) in the striatum as a control location did not affect the heroin seeking behavior. Microinjection of MPEP in the 3 locations did not change the locomotion activities. Conclusion: Blockade of mGluR5 in NAc shell in rats specifically suppresses the relapse to heroin-seeking and anxiety-like behavior, suggesting that mGluR5 antagonists may be a potential candidate for the therapy of heroin addiction. PMID:25399651

Practical Guide for Ascidian Microinjection: Phallusia mammillata.

PubMed

Yasuo, Hitoyoshi; McDougall, Alex

2018-01-01

Phallusia mammillata has recently emerged as a new ascidian model. Its unique characteristics, including the optical transparency of eggs and embryos and efficient translation of exogenously introduced mRNA in eggs, make the Phallusia system suitable for fluorescent protein (FP)-based imaging approaches. In addition, genomic and transcriptomic resources are readily available for this ascidian species, facilitating functional gene studies. Microinjection is probably the most versatile technique for introducing exogenous molecules such as plasmids, mRNAs, and proteins into ascidian eggs/embryos. However, it is not practiced widely within the community; presumably, because the system is rather laborious to set up and it requires practice. Here, we describe in as much detail as possible two microinjection methods that we use daily in the laboratory: one based on an inverted microscope and the other on a stereomicroscope. Along the stepwise description of system setup and injection procedure, we provide practical tips in the hope that this chapter might be a useful guide for introducing or improving a microinjection setup.

Isolation of chicken embryonic stem cell and preparation of chicken chimeric model.

PubMed

Zhang, Yani; Yang, Haiyan; Zhang, Zhentao; Shi, Qingqing; Wang, Dan; Zheng, Mengmeng; Li, Bichun; Song, Jiuzhou

2013-03-01

Chicken embryonic stem cells (ESCs) were separated from blastoderms at stage-X and cultured in vitro. Alkaline phosphatase activity and stage-specific embryonic antigen-1 staining was conducted to detect ESCs. Then, chicken ESCs were transfected with linearized plasmid pEGFP-N1 in order to produce chimeric chicken. Firstly, the optimal electrotransfection condition was compared; the results showed the highest transfection efficiency was obtained when the field strength and pulse duration was 280 V and 75 μs, respectively. Secondly, the hatchability of shedding methods, drilling a window at the blunt end of egg and drilling a window at the lateral shell of egg was compared, the results showed that the hatchability was the highest for drilling a window at the lateral shell of egg. Thirdly, the hatchability of microinjection (ESCs was microinjected into chick embryo cavity) was compared too, the results showed there were significant difference between the injection group transfected with ESCs and that of other two groups. In addition, five chimeric chickens were obtained in this study and EGFP gene was expressed in some organs, but only two chimeric chicken expressed EGFP gene in the gonad, indicating that the chimeric chicken could be obtained through chick embryo cavity injection by drilling a window at the lateral shell of egg.

Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes

PubMed Central

Crispo, M.; Mulet, A. P.; Tesson, L.; Barrera, N.; Cuadro, F.; dos Santos-Neto, P. C.; Nguyen, T. H.; Crénéguy, A.; Brusselle, L.; Anegón, I.; Menchaca, A.

2015-01-01

While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and livestock. PMID:26305800

Microinjection of kynurenic acid in the rostral nucleus of the tractus solitarius disrupts spatiotemporal aspects of mechanically induced tracheobronchial cough.

PubMed

Poliacek, Ivan; Pitts, Teresa; Rose, Melanie J; Davenport, Paul W; Simera, Michal; Veternik, Marcel; Kotmanova, Zuzana; Bolser, Donald C

2017-06-01

The importance of neurons in the nucleus of the solitary tract (NTS) in the production of coughing was tested by microinjections of the nonspecific glutamate receptor antagonist kynurenic acid (kyn; 100 mM in artificial cerebrospinal fluid) in 15 adult spontaneously breathing anesthetized cats. Repetitive coughing was elicited by mechanical stimulation of the intrathoracic airway. Electromyograms (EMG) were recorded from inspiratory parasternal and expiratory transversus abdominis (ABD) muscles. Bilateral microinjections of kyn into the NTS rostral to obex [55 ± 4 nl total in 2 locations ( n = 6) or 110 ± 4 nl total in 4 locations ( n = 5)], primarily the ventrolateral subnucleus, reduced cough number and expiratory cough efforts (amplitudes of ABD EMG and maxima of esophageal pressure) compared with control. These microinjections also markedly prolonged the inspiratory phase, all cough-related EMG activation, and the total cough cycle duration as well as some other cough-related time intervals. In response to microinjections of kyn into the NTS rostral to the obex respiratory rate decreased, and there were increases in the durations of the inspiratory and postinspiratory phases and mean blood pressure. However, bilateral microinjections of kyn into the NTS caudal to obex as well as control vehicle microinjections in the NTS location rostral to obex had no effect on coughing or cardiorespiratory variables. These results are consistent with the existence of a critical component of the cough rhythmogenic circuit located in the rostral ventral and lateral NTS. Neuronal structures of the rostral NTS are significantly involved specifically in the regulation of cough magnitude and phase timing. NEW & NOTEWORTHY The nucleus of the solitary tract contains significant neuronal structures responsible for control of 1 ) cough excitability, 2 ) motor drive during cough, 3 ) cough phase timing, and 4 ) cough rhythmicity. Significant elimination of neurons in the solitary tract nucleus results in cough apraxia (incomplete and/or disordered cough pattern). The mechanism of the cough impairment is different from that for the concomitant changes in breathing. Copyright © 2017 the American Physiological Society.

The glass micropipette electrode: A history of its inventors and users to 1950

PubMed Central

2017-01-01

Soon after the glass micropipette was invented as a micro-tool for manipulation of single bacteria and the microinjection and microsurgery of living cells, it was seen to hold promise as a microelectrode to stimulate individual cells electrically and to study electrical potentials in them. Initial successes and accurate mechanistic explanations of the results were achieved in giant plant cells in the 1920s. Long known surface electrical activity in nerves and muscles was only resolved at a similar cellular level in the 1930s and 1940s after the discovery of giant nerve fibers and the development of finer tipped microelectrodes for normal-sized cells. PMID:28298356

Germline modification of domestic animals

PubMed Central

Tang, L.; González, R.; Dobrinski, I.

2016-01-01

Genetically-modified domestic animal models are of increasing significance in biomedical research and agriculture. As authentic ES cells derived from domestic animals are not yet available, the prevailing approaches for engineering genetic modifications in those animals are pronuclear microinjection and somatic cell nuclear transfer (SCNT, also known as cloning). Both pronuclear microinjection and SCNT are inefficient, costly, and time-consuming. In animals produced by pronuclear microinjection, the exogenous transgene is usually inserted randomly into the genome, which results in highly variable expression patterns and levels in different founders. Therefore, significant efforts are required to generate and screen multiple founders to obtain animals with optimal transgene expression. For SCNT, specific genetic modifications (both gain-of-function and loss-of-function) can be engineered and carefully selected in the somatic cell nucleus before nuclear transfer. SCNT has been used to generate a variety of genetically modified animals such as goats, pigs, sheep and cattle; however, animals resulting from SCNT frequently suffer from developmental abnormalities associated with incomplete nuclear reprogramming. Other strategies to generate genetically-modified animals rely on the use of the spermatozoon as a natural vector to introduce genetic material into the female gamete. This sperm mediated DNA transfer (SMGT) combined with intracytoplasmatic sperm injection (ICSI) has relatively high efficiency and allows the insertion of large DNA fragments, which, in turn, enhance proper gene expression. An approach currently being developed to complement SCNT for producing genetically modified animals is germ cell transplantation using genetically modified male germline stem cells (GSCs). This approach relies on the ability of GSCs that are genetically modified in vitro to colonize the recipient testis and produce donor derived sperm upon transplantation. As the genetic change is introduced into the male germ line just before the onset of spermatogenesis, the time required for the production of genetically modified sperm is significantly shorter using germ cell transplantation compared to cloning or embryonic stem (ES) cell based technology. Moreover, the GSC-mediated germline modification circumvents problems associated with embryo manipulation and nuclear reprogramming. Currently, engineering targeted mutations in domestic animals using GSCs remains a challenge as GSCs from those animals are difficult to maintain in vitro for an extended period of time. Recent advances in genome editing techniques such as Zinc-Finger Nucleases (ZFNs), Transcription Activator-like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) greatly enhance the efficiency of engineering targeted genetic change in domestic animals as demonstrated by the generation of several gene knock-out pig and cattle models using those techniques. The potential of GSC-mediated germline modification in making targeted genetic modifications in domestic animal models will be maximized if those genome editing techniques can be applied in GSCs. PMID:27390591

Thermo-rheological behaviour of polymer melts in microinjection moulding

NASA Astrophysics Data System (ADS)

Vasco, J. C.; Maia, J. M.; Pouzada, A. S.

2009-10-01

Microinjection has proven to be one of the most efficient replication methods for microcomponents and microsystems in various domains of microengineering. The use of available commercial microinjection equipment to evaluate the polymeric flow in microchannels would surely contribute to enhancing knowledge on polymeric flow at the microscale under industrial conditions. This approach is appropriate since rheological phenomena such as wall slip, surface tension, melt pressure drop and polymer flow length can be studied. These aspects are not fully dealt with in current commercial simulation software packages. In this study a micromould was designed to assess and characterize the flow in microchannels under realistic industrial conditions.

Harmane produces hypotension following microinjection into the RVLM: possible role of I1-imidazoline receptors

PubMed Central

Musgrave, I F; Badoer, E

2000-01-01

The β-carboline, harmane (0.1–1.0 nmol) produces dose dependent hypotension when microinjected unilaterally into the rostral ventrolateral medulla (RVLM) of the anaesthetized rat. The potency of harmane on blood pressure is similar to that of the imidazoline, clonidine. The hypotensive effects of both clonidine and harmane are reversed by microinjection of the relatively I1-receptor selective antagonist efaroxan (20 nmol). These results are consistent with harmane acting at an I1-receptor in the RVLM. This is the first report of an endogenous ligand for I1-receptors that has central effects on blood pressure. PMID:10725251

Harmane produces hypotension following microinjection into the RVLM: possible role of I(1)-imidazoline receptors.

PubMed

Musgrave, I F; Badoer, E

2000-03-01

The beta-carboline, harmane (0.1 - 1.0 nmol) produces dose dependent hypotension when microinjected unilaterally into the rostral ventrolateral medulla (RVLM) of the anaesthetized rat. The potency of harmane on blood pressure is similar to that of the imidazoline, clonidine. The hypotensive effects of both clonidine and harmane are reversed by microinjection of the relatively I(1)-receptor selective antagonist efaroxan (20 nmol). These results are consistent with harmane acting at an I(1)-receptor in the RVLM. This is the first report of an endogenous ligand for I(1)-receptors that has central effects on blood pressure.

Three-dimensional imaging of nucleolin trafficking in normal cells, transfectants, and heterokaryons

NASA Astrophysics Data System (ADS)

Ballou, Byron T.; Fisher, Gregory W.; Deng, Jau-Shyong; Hakala, Thomas R.; Srivastava, Meera; Farkas, Daniel L.

1996-04-01

The study of intracellular trafficking using labeled molecules has been aided by the development of the cyanine fluorochromes, which are easily coupled, very soluble, resist photobleaching, and fluoresce at far-red wavelengths where background fluorescence is minimal. We have used Cy3-, Cy5-, and Cy5.5-labeled antibodies, antigen-binding fragments, and specifically binding single-stranded oligonucleotides to follow expression and trafficking of nucleolin, the most abundant protein of the nucleolus. Nucleolin shuttles between the nucleolus and the cytoplasm, and is also expressed on the cell surface, allowing us to test our techniques at all three cellular sites. Differentially cyanine-labeled non-specific antibodies were used to control for non-specific binding. Similarly, the differentially labeled non-binding strand of the cloned oligonucleotide served as a control. The multimode microscope allowed us to follow both rapid and slow redistributions of labeled ligands in the same study. We also performed 3-D reconstructions of nucleolin distribution in cells using rapid acquisition and deconvolution. Microinjection of labeled ligands was used to follow intracellular distribution, while incubation of whole cells with antibody and antigen-binding fragments was used to study uptake. To unambiguously define trafficking, and eliminate the possibility of interference by cross-reactive proteins, we transfected mouse renal cell carcinoma cells that express cell surface nucleolin with human nucleolin. We used microinjection and cell surface staining with Cy3- or Cy5- labeled monoclonal antibody D3 (specific for human nucleolin) to assess the cellular distribution of the human protein. Several clones expressed human nucleolin on their surfaces and showed high levels of transport of the human protein into the mouse nucleus and nucleolus. This distribution roughly parallels that of mouse nucleolin as determined by labeled polyclonal antibody. We have used these engineered transfectants to determine whether the cell surface-expressed xenogeneic nucleolin can serve as a target for antibodies in vivo.

Thermostability of sperm nuclei assessed by microinjection into hamster oocytes

EPA Science Inventory

Nuclei isolated from spermatozoa of various species (golden hamster, mouse, human, rooster, and the fish tilapia) were heated at 60 degrees-125 degrees C for 20-120 min and then microinjected into hamster oocytes to determine whether they could decondense and develop into pronucl...

Dorsomedial pontine neurons with descending projections to the medullary reticular formation express orexin-1 and adrenergic alpha2A receptor mRNA.

PubMed

Volgin, Denys V; Malinowska, Monika; Kubin, Leszek

2009-08-14

Neurons located in the dorsomedial pontine rapid eye movement (REM) sleep-triggering region send axons to the medial medullary reticular formation (mMRF). This pathway is believed to be important for the generation of REM sleep motor atonia, but other than that they are glutamatergic little is known about neurochemical signatures of these pontine neurons important for REM sleep. We used single-cell reverse transcription and polymerase chain reaction (RT-PCR) to determine whether dorsomedial pontine cells with projections to the mMRF express mRNA for selected membrane receptors that mediate modulatory influences on REM sleep. Fluorescein (FITC)-labeled latex microspheres were microinjected into the mMRF of 26-34-day-old rats under pentobarbital anesthesia. After 5-6 days, rats were sacrificed, pontine slices were obtained and neurons were dissociated from 400 to 600 microm micropunches extracted from dorsomedial pontine reticular formation. We found that 32 out of 51 FITC-labeled cells tested (63+/-7% (SE)) contained the orexin type 1 receptor (ORX1r) mRNA, 27 out of 73 (37+/-6%) contained the adrenergic alpha(2A) receptor (alpha(2A)r) RNA, and 6 out of 31 (19+/-7%) contained both mRNAs. The percentage of cells positive for the ORX1r mRNA was significantly lower (p<0.04) for the dorsomedial pontine cells that were not retrogradely labeled from the mMRF (32+/-11%), whereas alpha(2A)r mRNA was present in a similar percentage of FITC-labeled and unlabeled neurons. Our data suggest that ORX and adrenergic pathways converge on a subpopulation of cells of the pontine REM sleep-triggering region that have descending projections to the medullary region important for the motor control during REM sleep.

Kinesin is the motor for microtubule-mediated Golgi-to-ER membrane traffic [published errata appear in J Cell Biol 1995 Mar;128(5):following 988 and 1995 May;129(3):893

PubMed Central

1995-01-01

The distribution and dynamics of both the ER and Golgi complex in animal cells are known to be dependent on microtubules; in many cell types the ER extends toward the plus ends of microtubules at the cell periphery and the Golgi clusters at the minus ends of microtubules near the centrosome. In this study we provide evidence that the microtubule motor, kinesin, is present on membranes cycling between the ER and Golgi and powers peripherally directed movements of membrane within this system. Immunolocalization of kinesin at both the light and electron microscopy levels in NRK cells using the H1 monoclonal antibody to kinesin heavy chain, revealed kinesin to be associated with all membranes of the ER/Golgi system. At steady-state at 37 degrees C, however, kinesin was most concentrated on peripherally distributed, pre- Golgi structures containing beta COP and vesicular stomatitis virus glycoprotein newly released from the ER. Upon temperature reduction or nocodazole treatment, kinesin's distribution shifted onto the Golgi, while with brefeldin A (BFA)-treatment, kinesin could be found in both Golgi-derived tubules and in the ER. This suggested that kinesin associates with membranes that constitutively cycle between the ER and Golgi. Kinesin's role on these membranes was examined by microinjecting kinesin antibody. Golgi-to-ER but not ER-to-Golgi membrane transport was found to be inhibited by the microinjected anti-kinesin, suggesting kinesin powers the microtubule plus end-directed recycling of membrane to the ER, and remains inactive on pre-Golgi intermediates that move toward the Golgi complex. PMID:7844144

A hybrid microfluidic device for on-demand orientation and multidirectional imaging of C. elegans organs and neurons

PubMed Central

Ardeshiri, Ramtin; Mulcahy, Ben; Zhen, Mei; Rezai, Pouya

2016-01-01

C. elegans is a well-known model organism in biology and neuroscience with a simple cellular (959 cells) and nervous (302 neurons) system and a relatively homologous (40%) genome to humans. Lateral and longitudinal manipulation of C. elegans to a favorable orientation is important in many applications such as neural and cellular imaging, laser ablation, microinjection, and electrophysiology. In this paper, we describe a micro-electro-fluidic device for on-demand manipulation of C. elegans and demonstrate its application in imaging of organs and neurons that cannot be visualized efficiently under natural orientation. To achieve this, we have used the electrotaxis technique to longitudinally orient the worm in a microchannel and then insert it into an orientation and imaging channel in which we integrated a rotatable glass capillary for orientation of the worm in any desired direction. The success rates of longitudinal and lateral orientations were 76% and 100%, respectively. We have demonstrated the application of our device in optical and fluorescent imaging of vulva, uterine-vulval cell (uv1), vulB1\\2 (adult vulval toroid cells), and ventral nerve cord of wild-type and mutant worms. In comparison to existing methods, the developed technique is capable of orienting the worm at any desired angle and maintaining the orientation while providing access to the worm for potential post-manipulation assays. This versatile tool can be potentially used in various applications such as neurobehavioral imaging, neuronal ablation, microinjection, and electrophysiology. PMID:27990213

Expression of mammalian beta-adrenergic receptors in Xenopus laevis oocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bahouth, S.W.; Malbon, C.C.

1987-05-01

Xenopus laevis oocytes are a useful transcription and expression system for DNA and RNA, respectively. Total cellular RNA was extracted from mouse lymphoma S49 cells and poly(A)/sup +/mRNA prepared by affinity chromatography of RNA on oligo(dT) cellulose. The membranes of S49 cells contain beta-adrenergic receptors that display pharmacological characteristics of beta/sub 2/-subtype. Xenopus laevis oocytes were injected with 50 ng of mRNA/oocyte. Expression of beta-adrenergic receptors in oocytes incubated for 30 hr after microinjection was assessed in membranes by radioligand binding using (/sup 3/H) dihydroalprenolol. The injected oocytes displayed 0.34 fmol receptor/oocyte as compared to 0.02 fmol receptor/oocyte in themore » control oocytes. The affinity of beta-adrenergic receptors in injected oocytes for this radioligand was 2 nM, a value similar to the affinity of beta-adrenergic receptors for DHA in S49 cell membranes. The potency of beta-adrenergic agonists in competing for DHA binding to oocytes membranes was isoproterenol > epinephrine > norepineprine, indicating that the expressed beta-adrenergic receptors were of the beta/sub 2/-subtype. The K/sub I/ of these agonists for the beta-adrenergic receptor in oocyte membranes was 0.03, 0.15 and 1.2 ..mu..M, respectively. The role of post-translational modification in dictating receptor subtype is analyzed using mRNA of beta/sub 1/- as well as beta/sub 2/-adrenergic receptors.« less

Knock-in of Enhanced Green Fluorescent Protein or/and Human Fibroblast Growth Factor 2 Gene into β-Casein Gene Locus in the Porcine Fibroblasts to Produce Therapeutic Protein.

PubMed

Lee, Sang Mi; Kim, Ji Woo; Jeong, Young-Hee; Kim, Se Eun; Kim, Yeong Ji; Moon, Seung Ju; Lee, Ji-Hye; Kim, Keun-Jung; Kim, Min-Kyu; Kang, Man-Jong

2014-11-01

Transgenic animals have become important tools for the production of therapeutic proteins in the domestic animal. Production efficiencies of transgenic animals by conventional methods as microinjection and retrovirus vector methods are low, and the foreign gene expression levels are also low because of their random integration in the host genome. In this study, we investigated the homologous recombination on the porcine β-casein gene locus using a knock-in vector for the β-casein gene locus. We developed the knock-in vector on the porcine β-casein gene locus and isolated knock-in fibroblast for nuclear transfer. The knock-in vector consisted of the neomycin resistance gene (neo) as a positive selectable marker gene, diphtheria toxin-A gene as negative selection marker, and 5' arm and 3' arm from the porcine β-casein gene. The secretion of enhanced green fluorescent protein (EGFP) was more easily detected in the cell culture media than it was by western blot analysis of cell extract of the HC11 mouse mammary epithelial cells transfected with EGFP knock-in vector. These results indicated that a knock-in system using β-casein gene induced high expression of transgene by the gene regulatory sequence of endogenous β-casein gene. These fibroblasts may be used to produce transgenic pigs for the production of therapeutic proteins via the mammary glands.

Knock-in of Enhanced Green Fluorescent Protein or/and Human Fibroblast Growth Factor 2 Gene into β-Casein Gene Locus in the Porcine Fibroblasts to Produce Therapeutic Protein

PubMed Central

Lee, Sang Mi; Kim, Ji Woo; Jeong, Young-Hee; Kim, Se Eun; Kim, Yeong Ji; Moon, Seung Ju; Lee, Ji-Hye; Kim, Keun-Jung; Kim, Min-Kyu; Kang, Man-Jong

2014-01-01

Transgenic animals have become important tools for the production of therapeutic proteins in the domestic animal. Production efficiencies of transgenic animals by conventional methods as microinjection and retrovirus vector methods are low, and the foreign gene expression levels are also low because of their random integration in the host genome. In this study, we investigated the homologous recombination on the porcine β-casein gene locus using a knock-in vector for the β-casein gene locus. We developed the knock-in vector on the porcine β-casein gene locus and isolated knock-in fibroblast for nuclear transfer. The knock-in vector consisted of the neomycin resistance gene (neo) as a positive selectable marker gene, diphtheria toxin-A gene as negative selection marker, and 5′ arm and 3′ arm from the porcine β-casein gene. The secretion of enhanced green fluorescent protein (EGFP) was more easily detected in the cell culture media than it was by western blot analysis of cell extract of the HC11 mouse mammary epithelial cells transfected with EGFP knock-in vector. These results indicated that a knock-in system using β-casein gene induced high expression of transgene by the gene regulatory sequence of endogenous β-casein gene. These fibroblasts may be used to produce transgenic pigs for the production of therapeutic proteins via the mammary glands. PMID:25358326

[Methods of substances and organelles introduction in living cell for cell engineering technologies].

PubMed

Nikitin, V A

2007-01-01

We have presented the classification of more than 40 methods of genetic material, substances and organelles introduction into a living cell. Each of them has its characteristic advantages, disadvantages and limitations with respect to cell viability, transfer efficiency, general applicability, and technical requirements. It this article we have enlarged on the description of our developments of several new and improved approaches, methods and devices of the direct microinjection into a single cell and cell microsurgery with the help of glass micropipettes. The problem of low efficiency of mammalian cloning is discussed with emphasis on the necessity of expertizing of each step of single cell reconstruction to begin with microsurgical manipulations and necessity of the development of such methods of single cell resonstruction that could minimize the possible damage of the cell.

A rho-like protein is involved in the organisation of the contractile ring in dividing sand dollar eggs.

PubMed

Mabuchi, I; Hamaguchi, Y; Fujimoto, H; Morii, N; Mishima, M; Narumiya, S

1993-11-01

Sand dollar eggs were microinjected with botulinum C3 exoenzyme, an ADP-ribosyltransferase from Clostridium botulinum that specifically ADP-ribosylates and inactivates rho proteins. C3 exoenzyme microinjected during nuclear division interfered with subsequent cleavage furrow formation. No actin filaments were detected in the equatorial cortical layer of these eggs by rhodamine-phalloidin staining. When microinjected into furrowing eggs, C3 exoenzyme rapidly disrupted the contractile ring actin filaments and caused regression of the cleavage furrows. C3 exoenzyme had no apparent effect on nuclear division, however, and multinucleated embryos developed from the microinjected eggs. By contrast, C3 exoenzyme did not affect the organisation of cortical actin filaments immediately after fertilisation. Only one protein (molecular weight 22,000) was ADP-ribosylated by C3 exoenzyme in the isolated cleavage furrow. This protein co-migrated with ADP-ribosylated rhoA derived from human platelets when analysed by two-dimensional gel electrophoresis. These results strongly suggest that a rho-like, small GTP-binding protein is selectively involved in the organisation and maintenance of the contractile ring.

Human sperm chromosome analysis after subzonal sperm insemination of hamster oocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cozzi, J.

1994-09-01

Sperm microinjection techniques, subzonal sperm insemination (SUZI) and intracytoplasmic sperm injection (ICSI), have achieved a wide spread clinical application for the treatment of male infertility. To date, only one study has focused on sperm karyotypes after microinjection. Martin et al. reported a very high incidence of abnormal human sperm complements after ICSI into hamster oocytes. In the present study, are reported the first human sperm karyotypes after SUZI of hamster oocytes. Spermatozoa from two control donors were treated by calcium ionophore A23187 and injected under the zona of hamster eggs. The microinjected eggs were then cultured for cytogenetic analysis ofmore » the pronuclei. Out of 47 analyzed sperm chromosome metaphases, 5 (10.6%) were abnormal, 4 (8.5%) were hypohaploid and 1 (2.1%) had a structural abnormality. The sex ratio was not significantly different from the expected 1:1 ratio. Rates of chromosomal abnormalities in microinjected spermatozoa were similar to those observed in spermatozoa inseminated with zona free eggs, suggesting that SUZI procedure per se does not increase sperm chromosomal abnormalities.« less

Conceptual biotransformation of 4-oxo-all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid and all-trans-retinoyl-beta-glucuronide in rat whole embryo culture.

PubMed

Creech Kraft, J; Juchau, M R

1992-05-28

In cultured rat conceptuses, intraamniotic microinjections of 2500 ng/mL of 4-oxo-13-cis-retinoic acid, 600 ng/mL 4-oxo-all-trans-retinoic acid or 4000 ng/mL all-trans-retinoyl-beta-glucuronide, produce qualitatively and quantitatively similar patterns of dysmorphogenesis as those reported after the intraamniotic microinjection of 250 ng/mL all-trans-retinoic acid [Lee et al., Teratology 44: 313-323, 1991; Creech Kraft et al., Teratology 45: 259-270, 1992]. In the present study, we utilized HPLC techniques to analyze retinoid levels in cultured rat conceptuses, 1.5 hr after intraamniotic microinjections of 4-oxo-13-cis-retinoic acid (2500 ng/mL), 4-oxo-all-trans-retinoic acid (600 ng/mL) or all-trans-retinoyl-beta-glucuronide (4000 ng/mL). Our findings show that, after the microinjections of 4-oxo-all-trans-retinoic acid or 4-oxo-13-cis-retinoic acid (at these selected concentrations), 4-oxo-all-trans-retinoic acid was predominant in the embryos proper at concentrations of about 200 nM. This was roughly equivalent to the levels of all-trans-retinoic acid assayed after microinjections of all-trans-retinoyl-beta-glucuronide (4000 ng/mL). We conclude from these studies that both 4-oxo-all-trans-retinoic acid and all-trans-retinoic acid behave as ultimate or proximate dysmorphogens.

Basolateral Amygdala and the Regulation of Fear-Conditioned Changes in Sleep: Role of Corticotropin-Releasing Factor

PubMed Central

Wellman, Laurie L.; Yang, Linghui; Ambrozewicz, Marta A.; Machida, Mayumi; Sanford, Larry D.

2013-01-01

Study Objective: To determine whether corticotropin-releasing factor (CRF) in the basolateral amygdala (BLA) modulated sleep and fear-conditioned alterations in sleep. Design: After 2 days of habituation to recording procedures, baseline sleep recordings were obtained. The animals were then habituated to the handling procedure necessary for microinjections over 2 consecutive days. In experiment 1, rats received microinjections of 0.5 μL antalarmin (1.61 or 4.82 mM), a CRF receptor 1 antagonist, or distilled water once a week for 3 wk. In experiment 2, rats received a microinjection of either antalarmin or vehicle prior to inescapable shock training (ST; 20 shocks; 0.8 mA, 0.5 sec; 1 min interstimulus interval). The animals were placed back in the context 7 days later for 30 min without shock (CR; context re-exposure). Sleep was recorded for 8 h after each manipulation. Setting: NA. Subjects: Outbred Wistar rats. Interventions: The rats were surgically implanted with electrodes for recording the electroencephalogram and electromyogram for determining arousal state and with bilateral guide cannulae directed at BLA. Measurements and Results: Antalarmin microinjected into BLA did not significantly alter sleep under undisturbed conditions. However, antalarmin microinjected bilaterally into BLA prior to ST blocked reductions in rapid eye movement sleep that ST normally produces. Further, the single microinjection prior to ST blocked the reduction in rapid eye movement typically seen after subsequent CR. Behavioral freezing, an indicator of fear memory, was not altered. Conclusions: CRF in BLA is involved in regulating stress-induced alterations in sleep and it plays a role in modulating how stressful memories influence sleep. Citation: Wellman LL; Yang L; Ambrozewicz MA; Machida M; Sanford LD. Basolateral amygdala and the regulation of fear-conditioned changes in sleep: role of corticotropin-releasing factor. SLEEP 2013;36(4):471-480. PMID:23564994

Parabrachial complex glutamate receptors modulate the cardiorespiratory response evoked from hypothalamic defense area.

PubMed

Díaz-Casares, A; López-González, M V; Peinado-Aragonés, C A; González-Barón, S; Dawid-Milner, M S

2012-08-16

To characterize the possible role of glutamate in the interaction between Hypothalamic Defense Area (HDA) and Parabrachial complex (PBc) nuclei, cardiorespiratory changes were analyzed in response to electrical stimulation of the HDA (1 ms pulses, 30-50 μA given at 100 Hz for 5s) before and after the microinjection of the nonspecific glutamate receptor antagonist kynurenic acid (50 nl, 5 nmol), NMDA receptor antagonist MK-801 (50 nl, 50 nmol), non-NMDA receptor antagonist CNQX (50 nl, 50 nmol) or metabotropic glutamate receptor antagonist MCPG (50 nl, 5 nmol) within the PBc. HDA stimulation evoked an inspiratory facilitatory response, consisting of an increase in respiratory rate (p<0.001) due to a decrease in expiratory time (p<0.01). The respiratory response was accompanied by a pressor (p<0.001) and a tachycardic response (p<0.001). Kynurenic acid within the lateral parabrachial region (lPB) abolished the tachycardia (p<0.001) and decreased the magnitude of blood pressure response (p<0.001) to HDA stimulation. Similarly, the magnitude of the tachycardia and the pressor response was decreased after the microinjection of MK-801 (p<0.01 and p<0.001, respectively) and CNQX (p<0.05 in both cases) into the lPB. Kynurenic acid microinjection in this region produced an inhibition of the tachypnea (p<0.001) to HDA stimulation but the respiratory response persisted unchanged after MK-801 or CNQX microinjection into the lPB. Kynurenic acid within the medial parabrachial region (mPB) abolished the tachycardia (p<0.01) and decreased the magnitude of the pressor response (p<0.001) to HDA stimulation. MK-801 and CNQX microinjection in this region decreased the magnitude of the tachycardia (p<0.05, in both cases) and pressor response (p<0.05, in both cases). The respiratory response evoked by HDA stimulation was not changed after the microinjection of kynurenic acid, MK-801 or CNQX within the mPB. No changes were observed in the cardiorespiratory response evoked to HDA stimulation after MCPG microinjection within lPB and mPB. These results indicate that glutamate PBc receptors are involved in the cardiorespiratory response evoked from the HDA. The possible mechanisms involved in these interactions are discussed. Copyright © 2012 Elsevier B.V. All rights reserved.

Interallelic complementation of mutations in propionic acidemia by microinjection of mutant cDNAs into fibroblasts of affected patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Loyer, M.; Leclerc, D.; Gravel, R.A.

1994-09-01

Propionic acidemia is a rare autosomal recessive disorder resulting from defects of the {alpha} or {beta} subunit of biotin-dependent propionyl-CoA carboxylase (PCC). Mutations are assigned to defects of the PCCA ({alpha} subunit) or PCCB ({beta} subunit) gene through complementation studies after somatic fusion of patient cell lines. About two-thirds of patients with {beta} subunit defects (complementation group pccBC) show interallelic complementation in cell fusion experiments (subgroups pccB and pccC), monitored by the PCC-dependent metabolisms of {sup 14}C-propionate. Most patient cell lines are heteroallelic for two different mutations, leaving ambiguous the identity of the mutation participating in interallelic complementation. To identifymore » the complementing mutations, we have expressed {beta}-subunit cDNAs containing individual mutations by microinjection of the cDNAs in recipient cells from patients with {beta} subunit defects. Correction of the PCC defect was monitored by autoradiography of {sup 14}C-propionate incorporation. In some experiments, cDNAs were co-injected with a plasmid expressing the E. coli lacZ gene as a positive control for successful injection. Two mutations from the pccB subgroup showed complementation when injected into pccC cells; dupKICK140-143 and Pro228Leu. Similarly, two mutations from the pccC subgroup complemented after injection into pccB cells; {Delta}Ile408 and Arg410Trp. No mutation complemented with mutation of the pccBC group which are classified as non-complementing in cell fusion experiments. The results show that the complementing pccB mutations are found in the N-terminal half of the {beta} subunit, while the complementing pccC mutations cluxter at a site in the C-terminal half. The latter site is a candidate for the propionyl-CoA binding site based on sequence identity with a region of transcarboxylase from Propionibacterium shermanii.« less

Observing the conformation of individual SNARE proteins inside live cells

NASA Astrophysics Data System (ADS)

Weninger, Keith

2010-10-01

Protein conformational dynamics are directly linked to function in many instances. Within living cells, protein dynamics are rarely synchronized so observing ensemble-averaged behaviors can hide details of signaling pathways. Here we present an approach using single molecule fluorescence resonance energy transfer (FRET) to observe the conformation of individual SNARE proteins as they fold to enter the SNARE complex in living cells. Proteins were recombinantly expressed, labeled with small-molecule fluorescent dyes and microinjected for in vivo imaging and tracking using total internal reflection microscopy. Observing single molecules avoids the difficulties of averaging over unsynchronized ensembles. Our approach is easily generalized to a wide variety of proteins in many cellular signaling pathways.

Novel Treatments for Botulism: Development of Antagonists for Identified Steps in the Action of Botulinum Neurotoxins

DTIC Science & Technology

1989-11-07

dialyzed against the appropriate medium prior to dilution into the bath. The buccal ganglion in Aplysia contain two large, identified cholinergic neurons...transmitters. Micro-injection of nanomolar final concentrations of BoNT A (Fig. 2A) or B (Fig. 2B) into pre-synaptic cholinergic neurons in the buccal ...already been made to use rat pituitary cells together with patch pipette techniques in this study; this system has facilitated intracellular

Mesodermal and axial determinants contribute to mesoderm regionalization in Bufo arenarum embryos.

PubMed

Manes, Mario E; Campos Casal, Fernando H

2002-09-01

The existence of mesodermal determinants in the equator of Bufo arenarum embryos has been previously demonstrated. In this work, their role in dorso-ventral regionalization of mesoderm was studied by transferring the determinants to animal blastomeres. The transfer was performed by cleavage reorientation and cytoplasmic microinjection. Forced inclination during early cleavage caused deviation of the third cleavage plane and annexation of equatorial cytoplasm into animal quartets. Animal blastomeres from embryos oriented with the dorsal side up, incorporated ventro-equatorial cytoplasm and formed blood cells, mesenchyme, and coelomic epithelium. In contrast, animal blastomeres from embryos oriented with the ventral side up, acquired dorso-equatorial cytoplasm and developed notochord, somites, mesenchyme, coelomic epithelium and nervous tissue. In order to investigate if this dorso-ventral differentiation pattern responds to an interaction of mesodermal and axial factors, isolated 8-cell-stage animal quartets were microinjected with subcortical cytoplasm from: (a) the ventro-equatorial region of synchronous embryos; (b) the vegetal pole of uncleaved eggs; (c) a combination of both cytoplasms. As expected, the implanted ventro-equatorial cytoplasm promoted ventral mesoderm differentiation. Conversely, the joint transfer of ventro-equatorial cytoplasm and vegetal pole cytoplasm behaved as the dorso-equatorial cytoplasm, promoting dorso-lateral mesoderm and neural formation. Thus, mesoderm regionalization in B. arenarum embryos seems to be caused by a concurrent action of both mesodermal and axial determinants.

Interactive roles of Ras, insulin receptor substrate-1, and proteins with Src homology-2 domains in insulin signaling in Xenopus oocytes.

PubMed

Chuang, L M; Hausdorff, S F; Myers, M G; White, M F; Birnbaum, M J; Kahn, C R

1994-11-04

Insulin receptor substrate-1 (IRS-1) serves as the major immediate substrate of insulin/insulin-like growth factor (IGF)-1 receptors and following tyrosine phosphorylation binds to specific Src homology-2 (SH2) domain-containing proteins including the p85 subunit of phosphatidylinositol (PI) 3-kinase and GRB2, a molecule believed to link IRS-1 to the Ras pathway. To investigate how these SH2-containing signaling molecules interact to regulate insulin/IGF-1 action, IRS-1, glutathione S-transferase (GST)-SH2 domain fusion proteins and Ras proteins were microinjected into Xenopus oocytes. We found that pleiotropic insulin actions are mediated by IRS-1 through two independent, but convergent, pathways involving PI 3-kinase and GRB2. Thus, microinjection of GST-fusion proteins of either p85 or GRB2 inhibited IRS-1-dependent activation of mitogen-activated protein (MAP) and S6 kinases and oocyte maturation, although only the GST-SH2 of p85 reduced insulin-stimulated PI 3-kinase activation. Co-injection of a dominant negative Ras (S17N) with IRS-1 inhibited insulin-stimulated MAP and S6 kinase activation. Micro-injection of activated [Arg12,Thr59]Ras increased basal MAP and S6 kinase activities and sensitized the oocytes to insulin-stimulated maturation without altering insulin-stimulated PI 3-kinase. The Ras-enhanced oocyte maturation response, but not the elevated basal level of MAP and S6 kinase, was partially blocked by the SH2-p85, but not SH2-GRB2. These data strongly suggest that IRS-1 can mediate many of insulin's actions on cellular enzyme activation and cell cycle progression requires binding and activation of multiple different SH2-domain proteins.

Protective effects of agmatine on lipopolysaccharide-injured microglia and inducible nitric oxide synthase activity.

PubMed

Ahn, Soo Kyung; Hong, Samin; Park, Yu Mi; Choi, Ja Yong; Lee, Won Taek; Park, Kyung Ah; Lee, Jong Eun

2012-12-17

Proinflammatory factors released from activated microglia contribute to maintaining homeostasis against various noxious stimuli in the central nervous system. If excessive, however, they may initiate a pathologic neuroinflammatory process. In this investigation, we evaluated whether agmatine, a primary polyamine known to protect neurons, reduces lipopolysaccharide (LPS)-induced damage to microglia in vitro and in vivo. For in vitro study, BV2-immortalized murine microglia were exposed to LPS with agmatine treatment. After 24hours, cell viability and the amount of nitrite generated were determined. For in vivo study, LPS was microinjected into the corpus callosum of adult male albino mice. Agmatine was intraperitoneally administered at the time of injury. Brains were evaluated 24hours after LPS microinjection to check for immunoreactivity with a microglial marker of ionized calcium binding adaptor molecule 1 (Iba1) and inducible nitric oxide synthase (iNOS). Using western blot analysis, protein expression of iNOS as well as that of the proinflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1β, was determined. Agmatine significantly reduced the LPS-induced BV2 microglial cytotoxicity from over 80% to less than 60% (p<0.001), as determined by lactate dehydrogenase assay. It suppressed the nitrite production from 16.4±3.14μM to 5.5±1.27μM (p<0.001), as measured using the Griess reaction. Agmatine also decreased the activities of microglia and iNOS induced by LPS microinjection into corpus callosum. Our findings reveal that agmatine attenuates LPS-induced microglial damage and suggest that agmatine may serve as a novel therapeutic strategy for neuroinflammatory diseases. Copyright © 2012 Elsevier Inc. All rights reserved.

Ultrasound-guided microinjection into the mouse forebrain in utero at E9.5.

PubMed

Pierfelice, Tarran J; Gaiano, Nicholas

2010-11-13

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Differential development of antinociceptive tolerance to morphine and fentanyl is not linked to efficacy in the ventrolateral periaqueductal gray of the rat

PubMed Central

Bobeck, Erin N.; Haseman, Rachel A.; Hong, Dana; Ingram, Susan L.; Morgan, Michael M.

2012-01-01

Systemic administration of morphine typically produces greater tolerance than higher efficacy mu-opioid receptor (MOPr) agonists, such as fentanyl. The objective of the present study was to test this relationship by measuring antinociceptive efficacy and tolerance to morphine and fentanyl microinjected into the ventrolateral periaqueductal gray (vlPAG). MOPr agonist efficacy was evaluated by microinjecting the irreversible opioid receptor antagonist β-funaltrexamine hydrochloride (β-FNA) into the vlPAG prior to a dose-response analysis of morphine and fentanyl antinociception. In contrast to systemic administration of morphine and fentanyl, microinjection of these drugs into the vlPAG had similar efficacy as measured by similar reductions in maximal antinociception following β-FNA administration. Analysis of tolerance revealed a rightward shift in the dose-response curve to a single pretreatment with morphine, but not fentanyl. Moreover, the magnitude of tolerance to morphine was comparable following one, four, or eight pretreatments. Tolerance to fentanyl also was evident following four or eight microinjections. These data are surprising in that antinociceptive efficacy appears to vary depending on the site of administration. Moreover, the similar efficacy following microinjection of morphine and fentanyl into the vlPAG was associated with comparable tolerance, with the one exception of no tolerance to acute administration of fentanyl. Perspective These data reveal that antinociceptive tolerance following vlPAG administration of opioids develops rapidly, is evident with both morphine and fentanyl, and the magnitude is relatively consistent regardless of the number of pretreatments. PMID:22766006

REM sleep-like episodes of motoneuronal depression and respiratory rate increase are triggered by pontine carbachol microinjections in in situ perfused rat brainstem preparation.

PubMed

Brandes, Ivo F; Stettner, Georg M; Mörschel, Michael; Kubin, Leszek; Dutschmann, Mathias

2011-05-01

Hypoglossal nerve activity (HNA) controls the position and movements of the tongue. In persons with compromised upper airway anatomy, sleep-related hypotonia of the tongue and other pharyngeal muscles causes increased upper airway resistance, or total upper airway obstructions, thus disrupting both sleep and breathing. Hypoglossal nerve activity reaches its nadir, and obstructive episodes are longest and most severe, during rapid eye movement stage of sleep (REMS). Microinjections of a cholinergic agonist, carbachol, into the pons have been used in vivo to investigate the mechanisms of respiratory control during REMS. Here, we recorded inspiratory-modulated phrenic nerve activity and HNA and microinjected carbachol (25-50 nl, 10 mm) into the pons in an in situ perfused working heart-brainstem rat preparation (WHBP), an ex vivo model previously validated for studies of the chemical and reflex control of breathing. Carbachol microinjections were made into 40 sites in 33 juvenile rat preparations and, at 24 sites, they triggered depression of HNA with increased respiratory rate and little change of phrenic nerve activity, a pattern akin to that during natural REMS in vivo. The REMS-like episodes started 151 ± 73 s (SD) following microinjections, lasted 20.3 ± 4.5 min, were elicited most effectively from the dorsal part of the rostral nucleus pontis oralis, and were prevented by perfusion of the preparation with atropine. The WHBP offers a novel model with which to investigate cellular and neurochemical mechanisms of REMS-related upper airway hypotonia in situ without anaesthesia and with full control over the cellular environment.

Effects of NMDA and non-NMDA ionotropic glutamate receptors in the medial preoptic area on body temperature in awake rats.

PubMed

Sengupta, Trina; Jaryal, Ashok Kumar; Mallick, Hruda Nanda

2016-10-01

Glutamate when microinjected at the medial preoptic area (mPOA) influences brain temperature (T br ) and body temperature (T b ) in rats. Glutamate and its various receptors are present at the mPOA. The aim of this study was to identify the contribution of each of the ionotropic glutamatergic receptors at the mPOA on changes in T br and T b in freely moving rats. Adult male Wistar rats (n=40) were implanted with bilateral guide cannula with indwelling styli above the mPOA. A telemetric transmitter was implanted at the peritoneum to record T b and locomotor activity (LMA). A precalibrated thermocouple wire implanted near the hypothalamus was used to assess T br . Specific agonist for each ionotropic glutamate receptor was microinjected into the mPOA and its effects on temperature and LMA were measured in the rats. The rats were also microinjected with the respective ionotropic receptor antagonists, 15min prior to the microinjection of each agonist. Amongst amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-d-aspartate (NMDA) and kainic acid, AMPA increased T b and LMA when injected at the mPOA. Specific antagonists for AMPA receptors was able to attenuate this increase (p<0.005). Pharmacological blockade of NMDA was able to lower T br only. Microinjection of kainic acid and its antagonist had no effect on the variables. The finding of the study suggests that activation of the AMPA receptors at the mPOA, leads to the rise in body temperature. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification of a nuclear localization sequence in the polyomavirus capsid protein VP2

NASA Technical Reports Server (NTRS)

Chang, D.; Haynes, J. I. 2nd; Brady, J. N.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1992-01-01

A nuclear localization signal (NLS) has been identified in the C-terminal (Glu307-Glu-Asp-Gly-Pro-Gln-Lys-Lys-Lys-Arg-Arg-Leu318) amino acid sequence of the polyomavirus minor capsid protein VP2. The importance of this amino acid sequence for nuclear transport of newly synthesized VP2 was demonstrated by a genetic "subtractive" study using the constructs pSG5VP2 (expressing full-length VP2) and pSG5 delta 3VP2 (expressing truncated VP2, lacking amino acids Glu307-Leu318). These constructs were transfected into COS-7 cells, and the intracellular localization of the VP2 protein was determined by indirect immunofluorescence. These studies revealed that the full-length VP2 was localized in the nucleus, while the truncated VP2 protein was localized in the cytoplasm and not transported to the nucleus. A biochemical "additive" approach was also used to determine whether this sequence could target nonnuclear proteins to the nucleus. A synthetic peptide identical to VP2 amino acids Glu307-Leu318 was cross-linked to the nonnuclear proteins bovine serum albumin (BSA) or immunoglobulin G (IgG). The conjugates were then labeled with fluorescein isothiocyanate and microinjected into the cytoplasm of NIH 3T6 cells. Both conjugates localized in the nucleus of the microinjected cells, whereas unconjugated BSA and IgG remained in the cytoplasm. Taken together, these genetic subtractive and biochemical additive approaches have identified the C-terminal sequence of polyoma-virus VP2 (containing amino acids Glu307-Leu318) as the NLS of this protein.

Glial cell line-derived neurotrophic factor mediates the desirable actions of the anti-addiction drug ibogaine against alcohol consumption.

PubMed

He, Dao-Yao; McGough, Nancy N H; Ravindranathan, Ajay; Jeanblanc, Jerome; Logrip, Marian L; Phamluong, Khanhky; Janak, Patricia H; Ron, Dorit

2005-01-19

Alcohol addiction manifests as uncontrolled drinking despite negative consequences. Few medications are available to treat the disorder. Anecdotal reports suggest that ibogaine, a natural alkaloid, reverses behaviors associated with addiction including alcoholism; however, because of side effects, ibogaine is not used clinically. In this study, we first characterized the actions of ibogaine on ethanol self-administration in rodents. Ibogaine decreased ethanol intake by rats in two-bottle choice and operant self-administration paradigms. Ibogaine also reduced operant self-administration of ethanol in a relapse model. Next, we identified a molecular mechanism that mediates the desirable activities of ibogaine on ethanol intake. Microinjection of ibogaine into the ventral tegmental area (VTA), but not the substantia nigra, reduced self-administration of ethanol, and systemic administration of ibogaine increased the expression of glial cell line-derived neurotrophic factor (GDNF) in a midbrain region that includes the VTA. In dopaminergic neuron-like SHSY5Y cells, ibogaine treatment upregulated the GDNF pathway as indicated by increases in phosphorylation of the GDNF receptor, Ret, and the downstream kinase, ERK1 (extracellular signal-regulated kinase 1). Finally, the ibogaine-mediated decrease in ethanol self-administration was mimicked by intra-VTA microinjection of GDNF and was reduced by intra-VTA delivery of anti-GDNF neutralizing antibodies. Together, these results suggest that GDNF in the VTA mediates the action of ibogaine on ethanol consumption. These findings highlight the importance of GDNF as a new target for drug development for alcoholism that may mimic the effect of ibogaine against alcohol consumption but avoid the negative side effects.

Glial Cell Line-Derived Neurotrophic Factor Mediates the Desirable Actions of the Anti-Addiction Drug Ibogaine against Alcohol Consumption

PubMed Central

He, Dao-Yao; McGough, Nancy N. H.; Ravindranathan, Ajay; Jeanblanc, Jerome; Logrip, Marian L.; Phamluong, Khanhky; Janak, Patricia H.; Ron, Dorit

2005-01-01

Alcohol addiction manifests as uncontrolled drinking despite negative consequences. Few medications are available to treat the disorder. Anecdotal reports suggest that ibogaine, a natural alkaloid, reverses behaviors associated with addiction including alcoholism; however, because of side effects, ibogaine is not used clinically. In this study, we first characterized the actions of ibogaine on ethanol self-administration in rodents. Ibogaine decreased ethanol intake by rats in two-bottle choice and operant self-administration paradigms. Ibogaine also reduced operant self-administration of ethanol in a relapse model. Next, we identified a molecular mechanism that mediates the desirable activities of ibogaine on ethanol intake. Microinjection of ibogaine into the ventral tegmental area (VTA), but not the substantia nigra, reduced self-administration of ethanol, and systemic administration of ibogaine increased the expression of glial cell line-derived neurotrophic factor (GDNF) in a midbrain region that includes the VTA. In dopaminergic neuron-like SHSY5Y cells, ibogaine treatment upregulated the GDNF pathway as indicated by increases in phosphorylation of the GDNF receptor, Ret, and the downstream kinase, ERK1 (extracellular signal-regulated kinase 1). Finally, the ibogaine-mediated decrease in ethanol self-administration was mimicked by intra-VTA microinjection of GDNF and was reduced by intra-VTA delivery of anti-GDNF neutralizing antibodies. Together, these results suggest that GDNF in the VTA mediates the action of ibogaine on ethanol consumption. These findings highlight the importance of GDNF as a new target for drug development for alcoholism that may mimic the effect of ibogaine against alcohol consumption but avoid the negative side effects. PMID:15659598

Microinjection of muscimol into the dorsomedial hypothalamus suppresses MDMA-evoked sympathetic and behavioral responses

PubMed Central

Rusyniak, Daniel E.; Zaretskaia, Maria V.; Zaretsky, Dmitry V.; DiMicco, Joseph A.

2008-01-01

When given systemically to rats and humans, the drug of abuse 3–4 methylenedioxymethamphetamine (ecstasy, MDMA) elicits hyperthermia, hyperactivity, tachycardia, and hypertension. Chemically stimulating the dorsomedial hypothalamus (DMH), a brain region known to be involved in thermoregulation and in stress responses, causes similar effects. We therefore tested the hypothesis that neuronal activity in the DMH plays a role in MDMA-evoked sympathetic and behavioral responses by microinjecting artificial CSF or muscimol, a neuronal inhibitor, into the DMH prior to intravenous infusion of saline or MDMA in conscious rats. Core temperature, heart rate, mean arterial pressure and locomotor activity were recorded by telemetry every minute for 120 minutes. In rats previously microinjected with CSF, MDMA elicited significant increases from baseline in core temperature (+1.3 ± 0.3°C), locomotion (+50 ± 6 counts/min), heart rate (+142 ± 16 beats/min), and mean arterial pressure (+26 ±3 mmHg). Microinjecting muscimol into the DMH prior to MDMA prevented increases in core temperature and locomotion and attenuated increases in heart rate and mean arterial pressure. These results indicate that neuronal activity in the DMH is necessary for the sympathetic and behavioral responses evoked by MDMA. PMID:18586013

Electrical stimulation or MK-801 in the inferior colliculus improve motor deficits in MPTP-treated mice.

PubMed

Melo-Thomas, L; Gil-Martínez, A L; Cuenca, L; Estrada, C; Gonzalez-Cuello, A; Schwarting, R K; Herrero, M T

2018-03-01

The inferior colliculus (IC) is an important midbrain relay station for the integration of descending and ascending auditory information. Additionally, the IC has been implicated in processing sensorimotor responses. Glutamatergic and GABAergic manipulations in the IC can improve motor deficits as demonstrated by the animal model of haloperidol-induced catalepsy. However, how the IC influences motor function remains unclear. We investigated the effects of either intracollicular deep brain stimulation (DBS) or microinjection of the glutamatergic antagonist MK-801 or the agonist NMDA in C57BL/6J mice chronically treated with saline or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). After DBS or microinjections, the mice were submitted to rotarod and open field tests, respectively. DBS in the IC was effective to increase the time spent on the rotarod in MPTP-treated mice. After unilateral microinjection of MK-801, but not NMDA, MPTP-treated mice increased the distance travelled in the open field (p < 0.05). In conclusion, intracollicular DBS or MK-801 microinjection can improve motor performance in parkinsonian mice suggesting the IC as a new and non-conventional therapeutic target in motor impairment. Copyright © 2018 Elsevier B.V. All rights reserved.

Mediation by neurotensin-receptors of effects of neurotensin on self-stimulation of the medial prefrontal cortex.

PubMed Central

Fernández, R.; Sabater, R.; Sáez, J. A.; Montes, R.; Alba, F.; Ferrer, J. M.

1996-01-01

1 Intracortical microinjections of neurotensin (NT) selectively decreased intracranial self-stimulation (ICSS) of the medial prefrontal cortex in the rat. 2 To elucidate whether this effect is mediated by NT receptors or by the formation of NT-dopamine complexes, we investigated the effects on ICSS of intracortical microinjections of neurotensin (1-11), an NT fragment that forms extracellular complexes with dopamine but does not bind to NT receptors. 3 We also studied the effects of the peripheral administration of SR 48692, a selective antagonist of NT receptors, on the inhibition of ICSS produced by the intracortical administration of NT. 4 Unilateral microinjections of neurotensin (1-11) at doses of 10, 20 and 40 nmol into the medial prefrontal cortex did not change the basal ICSS rate of this area. 5 The intraperitoneal administration of SR 48692 at doses of 0.08 and 0.16 mg kg-1 30 min before microinjection of 10 nmol of NT into the medial prefrontal cortex, antagonized the inhibition of ICSS produced by the neuropeptide. 6 These results demonstrate that the inhibitory effect of NT on ICSS is mediated by NT receptors. PMID:8886412

Increase in sensitivity of the baroreceptor reflex following microinjection of carbachol into the posterior hypothalamic nucleus of awake rats.

PubMed

Newey, C R; Martin, J R

2016-01-01

In a rat model, the baroreceptor reflex can be assessed by graded infusions of either phenylephrine or sodium nitroprusside with continuous hemodynamic monitoring. Microinjection of the cholinergic agonist carbachol (CCh) into the posterior hypothalamic nucleus (PHN) evokes an increase in mean arterial pressure and a change in heart rate. Lower doses of CCh evoke only tachycardia, whereas middle and higher doses evoke a biphasic change in heart rate of tachycardia followed by bradycardia. The bradycardia following the microinjection of CCh into the PHN can be attenuated by the previous administration of the vasopressin V1 receptor antagonist [d(CH2 )5 Tyr(Me)] arginine vasopressin (AVPX). Circulating arginine vasopressin (AVP) has been shown to increase the sensitivity of the baroreceptor reflex by stimulating vasopressin V1 receptors in the area postrema. The attenuation by AVPX of the bradycardia that results following the high doses of CCh suggests that AVP is released into the circulation following stimulation of cholinergic systems within the PHN. Thus, microinjection of a high dose of CCh (11 nmol) into the PHN alters the sensitivity of the baroreceptor reflex by increasing peripheral levels of AVP. © 2016 John Wiley & Sons Ltd.

Cannabinoid system of dorsomedial telencephalon modulates behavioral responses to noxious stimulation in the fish Leporinus macrocephalus.

PubMed

Wolkers, Carla Patricia Bejo; Menescal-de-Oliveira, Leda; Hoffmann, Anette

2017-10-01

Fish dorsomedial telencephalon has been considered a pallial region homologous to mammals amygdala, being considered a possible substrate for nociception modulation in this animal group. The present study aimed to evaluate the participation of the cannabinoid system of Dm telencephalon on nociception modulation in the fish Leporinus macrocephalus. We demonstrated that cannabidiol microinjection in Dm telecephalon inhibits the behavioral nociceptive response to the subcutaneous injection of 3% formaldehyde, and this antinociception is blocked by previous treatment with AM251 microinjection. Furthermore, AM251 microinjection in Dm prior to restraint stress also blockades the stress-induced antinociception. These results reinforce the hypothesis that this pallial telencephalic structure has a pivotal role in nociception modulation in fish. Copyright © 2017 Elsevier Inc. All rights reserved.

Distinct effect of orphanin FQ in nucleus raphe magnus and nucleus reticularis gigantocellularis on the rat tail flick reflex.

PubMed

Yang, Z; Zhang, Y; Wu, G

2001-06-22

The aim of the present study is to investigate the effects of orphanin FQ (OFQ) microinjected into the nucleus raphe magnus (NRM) and the nucleus reticularis gigantocellularis (NGC) on pain modulation. The tail-flick latency (TFL) was used as a behavioral index of nociceptive responsiveness. The result showed microinjection of OFQ into the NRM significantly increased the TFL, whereas microinjection of OFQ into the NGC decreased the TFL, suggesting the analgesic effect of OFQ in the NRM and the hyperalgesic effect of OFQ in the NGC. As there are three classes of putative pain modulating neurons in the rostral ventromedial medulla (RVM), the hyperalgesic or analgesic effect of OFQ in the RVM might depend upon the different class of the neurons being acted.

Genome Editing in Mice Using TALE Nucleases.

PubMed

Wefers, Benedikt; Brandl, Christina; Ortiz, Oskar; Wurst, Wolfgang; Kühn, Ralf

2016-01-01

Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as sequence-specific nucleases to induce targeted double-strand breaks, the mouse genome can be directly modified in zygotes in a single step without the need for embryonic stem cells. By embryo microinjection of TALEN mRNAs and targeting vectors, knockout and knock-in alleles can be generated fast and efficiently. In this chapter we provide protocols for the application of TALENs in mouse zygotes.

Hippocampal nicotinic receptors have a modulatory role for ethanol and MDMA interaction in memory retrieval.

PubMed

Rostami, Maryam; Rezayof, Ameneh; Alijanpour, Sakineh; Sharifi, Khadijeh Alsadat

2017-08-15

The aim of the current study was to examine the effect of dorsal hippocampal nicotinic acetylcholine receptors (nAChRs) activation on the functional interaction between ethanol and 3,4-methylenedioxy-N-methylamphetamine (MDMA or ecstasy) in memory retrieval. The dorsal hippocampal CA1 regions of adult male NMRI mice were bilaterally cannulated and memory retrieval was measured in a step-down type passive avoidance apparatus. Post-training or pre-test systemic administration of ethanol (1g/kg, i.p.) induced amnesia. Pre-test administration of ethanol reversed pre-training ethanol-induced amnesia, suggesting ethanol state-dependent learning. Pre-test intra-CA1 microinjection of different doses of MDMA (0.25-1µg/mouse) with an ineffective dose of ethanol (0.25g/kg, i.p.) also induced amnesia. Interestingly, pre-test intra-CA1 microinjection of MDMA (0.25-1µg/mouse) potentiated ethanol state-dependent learning. On the other hand, the activation of the dorsal hippocampal nAChRs by pre-test microinjection of nicotine (0.1-1µg/mouse, intra-CA1) improved amnesia induced by the co-administration of MDMD and ethanol. It is important to note that intra-CA1 microinjection of the same doses of MDMA or nicotine could not affect memory formation by itself. Pre-test intra-CA1 microinjection of nicotine (0.3-0.9µg/mouse) could not reverse amnesia induced by pre-training administration of ethanol while this treatment enhanced MDMA response on ethanol state-dependent learning. Thus, it can be concluded that there may be functional interactions among ethanol, MDMA and nicotine via the dorsal hippocampal nicotinic acetylcholine receptor mechanism in memory retrieval and drug state-dependent learning. Copyright © 2017 Elsevier B.V. All rights reserved.

Orexin in Rostral Hotspot of Nucleus Accumbens Enhances Sucrose ‘Liking' and Intake but Scopolamine in Caudal Shell Shifts ‘Liking' Toward ‘Disgust' and ‘Fear'

PubMed Central

Castro, Daniel C; Terry, Rachel A; Berridge, Kent C

2016-01-01

The nucleus accumbens (NAc) contains a hedonic hotspot in the rostral half of medial shell, where opioid agonist microinjections are known to enhance positive hedonic orofacial reactions to the taste of sucrose (‘liking' reactions). Within NAc shell, orexin/hypocretin also has been reported to stimulate food intake and is implicated in reward, whereas blockade of muscarinic acetylcholine receptors by scopolamine suppresses intake and may have anti-reward effects. Here, we show that NAc microinjection of orexin-A in medial shell amplifies the hedonic impact of sucrose taste, but only within the same anatomically rostral site, identical to the opioid hotspot. By comparison, at all sites throughout medial shell, orexin microinjections stimulated ‘wanting' to eat, as reflected by increases in intake of palatable sweet chocolates. At NAc shell sites outside the hotspot, orexin selectively enhanced ‘wanting' to eat without enhancing sweetness ‘liking' reactions. In contrast, microinjections of the antagonist scopolamine at all sites in NAc shell suppressed sucrose ‘liking' reactions as well as suppressing intake of palatable food. Conversely, scopolamine increased aversive ‘disgust' reactions elicited by bitter quinine at all NAc shell sites. Finally, scopolamine microinjections localized to the caudal half of medial shell additionally generated a fear-related anti-predator reaction of defensive treading and burying directed toward the corners of the transparent chamber. Together, these results confirm a rostral hotspot in NAc medial shell as a unique site for orexin induction of hedonic ‘liking' enhancement, similar to opioid enhancement. They also reveal distinct roles for orexin and acetylcholine signals in NAc shell for hedonic reactions and motivated behaviors. PMID:26787120

REM sleep-like episodes of motoneuronal depression and respiratory rate increase are triggered by pontine carbachol microinjections in in situ perfused rat brainstem preparation

PubMed Central

Brandes, Ivo F.; Stettner, Georg M.; Mörschel, Michael; Kubin, Leszek; Dutschmann, Mathias

2015-01-01

Hypoglossal nerve activity (HNA) controls the position and movements of the tongue. In persons with compromised upper airway anatomy, sleep-related hypotonia of the tongue and other pharyngeal muscles causes increased upper airway resistance, or total upper airway obstructions, thus disrupting both sleep and breathing. Hypoglossal nerve activity reaches its nadir, and obstructive episodes are longest and most severe, during rapid eye movement stage of sleep (REMS). Microinjections of a cholinergic agonist, carbachol, into the pons have been used in vivo to investigate the mechanisms of respiratory control during REMS. Here, we recorded inspiratory-modulated phrenic nerve activity and HNA and microinjected carbachol (25–50 nl, 10 mm) into the pons in an in situ perfused working heart–brainstem rat preparation (WHBP), an ex vivo model previously validated for studies of the chemical and reflex control of breathing. Carbachol microinjections were made into 40 sites in 33 juvenile rat preparations and, at 24 sites, they triggered depression of HNA with increased respiratory rate and little change of phrenic nerve activity, a pattern akin to that during natural REMS in vivo. The REMS-like episodes started 151±73 s (SD) following microinjections, lasted 20.3±4.5 min, were elicited most effectively from the dorsal part of the rostral nucleus pontis oralis, and were prevented by perfusion of the preparation with atropine. The WHBP offers a novel model with which to investigate cellular and neurochemical mechanisms of REMS-related upper airway hypotonia in situ without anaesthesia and with full control over the cellular environment. PMID:21335420

Dorsal hippocampal NMDA receptors mediate the interactive effects of arachidonylcyclopropylamide and MDMA/ecstasy on memory retrieval in rats.

PubMed

Ghaderi, Marzieh; Rezayof, Ameneh; Vousooghi, Nasim; Zarrindast, Mohammad-Reza

2016-04-03

A combination of cannabis and ecstasy may change the cognitive functions more than either drug alone. The present study was designed to investigate the possible involvement of dorsal hippocampal NMDA receptors in the interactive effects of arachidonylcyclopropylamide (ACPA) and ecstasy/MDMA on memory retrieval. Adult male Wistar rats were cannulated into the CA1 regions of the dorsal hippocampus (intra-CA1) and memory retrieval was examined using the step-through type of passive avoidance task. Intra-CA1 microinjection of a selective CB1 receptor agonist, ACPA (0.5-4ng/rat) immediately before the testing phase (pre-test), but not after the training phase (post-training), impaired memory retrieval. In addition, pre-test intra-CA1 microinjection of MDMA (0.5-1μg/rat) dose-dependently decreased step-through latency, indicating an amnesic effect of the drug by itself. Interestingly, pre-test microinjection of a higher dose of MDMA into the CA1 regions significantly improved ACPA-induced memory impairment. Moreover, pre-test intra-CA1 microinjection of a selective NMDA receptor antagonist, D-AP5 (1 and 2μg/rat) inhibited the reversal effect of MDMA on the impairment of memory retrieval induced by ACPA. Pre-test intra-CA1 microinjection of the same doses of D-AP5 had no effect on memory retrieval alone. These findings suggest that ACPA or MDMA consumption can induce memory retrieval impairment, while their co-administration improves this amnesic effect through interacting with hippocampal glutamatergic-NMDA receptor mechanism. Thus, it seems that the tendency to abuse cannabis with ecstasy may be for avoiding cognitive dysfunction. Copyright © 2015. Published by Elsevier Inc.

Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering.

PubMed

Horii, Takuro; Arai, Yuji; Yamazaki, Miho; Morita, Sumiyo; Kimura, Mika; Itoh, Masahiro; Abe, Yumiko; Hatada, Izuho

2014-03-28

The CRISPR/Cas system, in which the Cas9 endonuclease and a guide RNA complementary to the target are sufficient for RNA-guided cleavage of the target DNA, is a powerful new approach recently developed for targeted gene disruption in various animal models. However, there is little verification of microinjection methods for generating knockout mice using this approach. Here, we report the verification of microinjection methods of the CRISPR/Cas system. We compared three methods for injection: (1) injection of DNA into the pronucleus, (2) injection of RNA into the pronucleus, and (3) injection of RNA into the cytoplasm. We found that injection of RNA into the cytoplasm was the most efficient method in terms of the numbers of viable blastocyst stage embryos and full-term pups generated. This method also showed the best overall knockout efficiency.

Overexpression of Robo2 causes defects in the recruitment of metanephric mesenchymal cells and ureteric bud branching morphogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ji, Jiayao; Medical College of NanKai University, Tianjin; Li, Qinggang

2012-05-11

Highlights: Black-Right-Pointing-Pointer Overexpression of Robo2 caused reduced UB branching and glomerular number. Black-Right-Pointing-Pointer Fewer MM cells surrounding the UB after overexpression of Robo2 in vitro. Black-Right-Pointing-Pointer No abnormal Epithelial Morphology of UB or apoptosis of mm cells in the kidney. Black-Right-Pointing-Pointer Overexpression of Robo2 affected MM cells migration and caused UB deficit. Black-Right-Pointing-Pointer The reduced glomerular number can also be caused by fewer MM cells. -- Abstract: Roundabout 2 (Robo2) is a member of the membrane protein receptor family. The chemorepulsive effect of Slit2-Robo2 signaling plays vital roles in nervous system development and neuron migration. Slit2-Robo2 signaling is also importantmore » for maintaining the normal morphogenesis of the kidney and urinary collecting system, especially for the branching of the ureteric bud (UB) at the proper site. Slit2 or Robo2 mouse mutants exhibit multilobular kidneys, multiple ureters, and dilatation of the ureter, renal pelvis, and collecting duct system, which lead to vesicoureteral reflux. To understand the effect of Robo2 on kidney development, we used microinjection and electroporation to overexpress GFP-Robo2 in an in vitro embryonic kidney model. Our results show reduced UB branching and decreased glomerular number after in vitro Robo2 overexpression in the embryonic kidneys. We found fewer metanephric mesenchymal (MM) cells surrounding the UB but no abnormal morphology in the branching epithelial UB. Meanwhile, no significant change in MM proliferation or apoptosis was observed. These findings indicate that Robo2 is involved in the development of embryonic kidneys and that the normal expression of Robo2 can help maintain proper UB branching and glomerular morphogenesis. Overexpression of Robo2 leads to reduced UB branching caused by fewer surrounding MM cells, but MM cell apoptosis is not involved in this effect. Our study demonstrates that overexpression of Robo2 by microinjection in embryonic kidneys is an effective approach to study the function of Robo2.« less

A method of microinjection: delivering monoclonal antibody 1223 into sea urchin embryos.

PubMed

Cho, J W

1999-08-31

In this paper, a simpler method of microinjecting sea urchin embryos without using the conventional microinjection chamber designed by Kiehart is reported. A trough was made on a surface of 0.6% agarose gel dissolved in artificial sea water. Approximately fifty hatched embryos could be loaded in the trough and, consequently, swimming embryos were trapped in the trough. Monoclonal antibody (mAB) 1223 which blocks spiculogenesis in vitro was delivered into the blastocoels of sea urchin embryos to test whether this antibody inhibits spiculogenesis in vivo and also, whether this new technique is effective for the microinjection of the sea urchin embryos. The embryos were injected with mAB1223 at the hatched blastula, early mesenchyme blastula and early gastrula stages, and 63%, 90% and 97% of the embryos did not form spicules at the late gastrula stage, respectively. Therefore, mAB1223 was shown to also block spiculogenesis in vivo. From the fact that spiculogenesis occurred at a lower rate when mAB1223 was injected at the hatched blastula stage than at later stages, it may be speculated that endogenous proteases degraded the injected antibodies. Using this technique, extracellular events in the blastocoel or the function of certain molecules expressed in blastocoel can be easily investigated in vivo.

In vivo imaging and toxicity assessments of fluorescent nanodiamonds in Caenorhabditis elegans.

PubMed

Mohan, Nitin; Chen, Chao-Sheng; Hsieh, Hsiao-Han; Wu, Yi-Chun; Chang, Huan-Cheng

2010-09-08

Nanoscale carbon materials hold great promise for biotechnological and biomedical applications. Fluorescent nanodiamond (FND) is a recent new addition to members of the nanocarbon family. Here, we report long-term in vivo imaging of FNDs in Caenorhabditis elegans (C. elegans) and explore the nano-biointeractions between this novel nanomaterial and the model organism. FNDs are introduced into wild-type C. elegans by either feeding them with colloidal FND solution or microinjecting FND suspension into the gonads of the worms. On feeding, bare FNDs stay in the intestinal lumen, while FNDs conjugated with biomolecules (such as dextran and bovine serum albumin) are absorbed into the intestinal cells. On microinjection, FNDs are dispersed in the gonad and delivered to the embryos and eventually into the hatched larvae in the next generation. The toxicity assessments, performed by employing longevity and reproductive potential as physiological indicators and measuring stress responses with use of reporter genes, show that FNDs are stable and nontoxic and do not cause any detectable stress to the worms. The high brightness, excellent photostability, and nontoxic nature of the nanomaterial have enabled continuous imaging of the whole digestive system and tracking of the cellular and developmental processes of the living organism for several days.

Recognition of the CDEI motif GTCACATG by mouse nuclear proteins and interference with the early development of the mouse embryo.

PubMed Central

Blangy, A; Léopold, P; Vidal, F; Rassoulzadegan, M; Cuzin, F

1991-01-01

We have reported previously (1) two unexpected consequences of the microinjection into fertilized mouse eggs of a recombinant plasmid designated p12B1, carrying a 343 bp insert of non-repetitive mouse DNA. Injected at very low concentrations, this plasmid could be established as an extrachromosomal genetic element. When injected in greater concentration, an early arrest of embryonic development resulted. In the present work, we have studied this toxic effect in more detail by microinjecting short synthetic oligonucleotides with sequences from the mouse insert. Lethality was associated with the nucleotide sequence GTCACATG, identical with the CDEl element of yeast centromeres. Development of injected embryos was arrested between the one-cell and the early morula stages, with abnormal structures and DNA contents. Electrophoretic mobility shift and DNAse foot-printing assays demonstrated the binding of mouse nuclear protein(s) to the CDEl-like box. Base changes within the CDEl sequence prevented both the toxic effects in embryos and the formation of protein complex in vitro, suggesting that protein binding at such sites in chromosomal DNA plays an important role in early development. Images PMID:1766880

Role of the major antigenic membrane protein in phytoplasma transmission by two insect vector species.

PubMed

Rashidi, Mahnaz; Galetto, Luciana; Bosco, Domenico; Bulgarelli, Andrea; Vallino, Marta; Veratti, Flavio; Marzachì, Cristina

2015-09-30

Phytoplasmas are bacterial plant pathogens (class Mollicutes), transmitted by phloem feeding leafhoppers, planthoppers and psyllids in a persistent/propagative manner. Transmission of phytoplasmas is under the control of behavioral, environmental and geographical factors, but molecular interactions between membrane proteins of phytoplasma and vectors may also be involved. The aim of the work was to provide experimental evidence that in vivo interaction between phytoplasma antigenic membrane protein (Amp) and vector proteins has a role in the transmission process. In doing so, we also investigated the topology of the interaction at the gut epithelium and at the salivary glands, the two barriers encountered by the phytoplasma during vector colonization. Experiments were performed on the 'Candidatus Phytoplasma asteris' chrysanthemum yellows strain (CYP), and the two leafhopper vectors Macrosteles quadripunctulatus Kirschbaum and Euscelidius variegatus Kirschbaum. To specifically address the interaction of CYP Amp at the gut epithelium barrier, insects were artificially fed with media containing either the recombinant phytoplasma protein Amp, or the antibody (A416) or both, and transmission, acquisition and inoculation efficiencies were measured. An abdominal microinjection protocol was employed to specifically address the interaction of CYP Amp at the salivary gland barrier. Phytoplasma suspension was added with Amp or A416 or both, injected into healthy E. variegatus adults and then infection and inoculation efficiencies were measured. An internalization assay was developed, consisting of dissected salivary glands from healthy E. variegatus exposed to phytoplasma suspension alone or together with A416 antibody. The organs were then either observed in confocal microscopy or subjected to DNA extraction and phytoplasma quantification by qPCR, to visualize and quantify possible differences among treatments in localization/presence/number of CYP cells. Artificial feeding and abdominal microinjection protocols were developed to address the two barriers separately. The in vivo interactions between Amp of 'Candidatus Phytoplasma asteris' Chrysanthemum yellows strain (CYP) and vector proteins were studied by evaluating their effects on phytoplasma transmission by Euscelidius variegatus and Macrosteles quadripunctulatus leafhoppers. An internalization assay was developed, consisting of dissected salivary glands from healthy E. variegatus exposed to phytoplasma suspension alone or together with anti-Amp antibody. To visualize possible differences among treatments in localization/presence of CYP cells, the organs were observed in confocal microscopy. Pre-feeding of E. variegatus and M. quadripunctulatus on anti-Amp antibody resulted in a significant decrease of acquisition efficiencies in both species. Inoculation efficiency of microinjected E. variegatus with CYP suspension and anti-Amp antibody was significantly reduced compared to that of the control with phytoplasma suspension only. The possibility that this was due to reduced infection efficiency or antibody-mediated inhibition of phytoplasma multiplication was ruled out. These results provided the first indirect proof of the role of Amp in the transmission process. Protocols were developed to assess the in vivo role of the phytoplasma native major antigenic membrane protein in two phases of the vector transmission process: movement through the midgut epithelium and colonization of the salivary glands. These methods will be useful also to characterize other phytoplasma-vector combinations. Results indicated for the first time that native CYP Amp is involved in vivo in specific crossing of the gut epithelium and salivary gland colonization during early phases of vector infection.

In Situ Activation of Antigen-Specific CD8+ T Cells in the Presence of Antigen in Organotypic Brain Slices1

PubMed Central

Ling, Changying; Verbny, Yakov I.; Banks, Matthew I.; Sandor, Matyas; Fabry, Zsuzsanna

2012-01-01

The activation of Ag-specific T cells locally in the CNS could potentially contribute to the development of immune-mediated brain diseases. We addressed whether Ag-specific T cells could be stimulated in the CNS in the absence of peripheral lymphoid tissues by analyzing Ag-specific T cell responses in organotypic brain slice cultures. Organotypic brain slice cultures were established 1 h after intracerebral OVA Ag microinjection. We showed that when OVA-specific CD8+ T cells were added to Ag-containing brain slices, these cells became activated and migrated into the brain to the sites of their specific Ags. This activation of OVA-specific T cells was abrogated by the deletion of CD11c+ cells from the brain slices of the donor mice. These data suggest that brain-resident CD11c+ cells stimulate Ag-specific naive CD8+ T cells locally in the CNS and may contribute to immune responses in the brain. PMID:18523307

Rho is Required for the Initiation of Calcium Signaling and Phagocytosis by Fcγ Receptors in Macrophages

PubMed Central

Hackam, David J.; Rotstein, Ori D.; Schreiber, Alan; Zhang, Wei-jian; Grinstein, Sergio

1997-01-01

Phagocytosis of bacteria by macrophages and neutrophils is an essential component of host defense against infection. The mechanism whereby the interaction of opsonized particles with Fcγ receptors triggers the engulfment of opsonized particles remains incompletely understood, although activation of tyrosine kinases has been recognized as an early step. Recent studies in other systems have demonstrated that tyrosine kinases can in turn signal the activation of small GTPases of the ras superfamily. We therefore investigated the possible role of Rho in Fc receptor–mediated phagocytosis. To this end we microinjected J774 macrophages with C3 exotoxin from Clostridium botulinum, which ADP-ribosylates and inactivates Rho. C3 exotoxin induced the retraction of filopodia, the disappearance of focal complexes, and a global decrease in the F-actin content of J774 cells. In addition, these cells exhibited increased spreading and the formation of vacuolar structures. Importantly, inactivation of Rho resulted in the complete abrogation of phagocytosis. Inhibition of Fcγ receptor–mediated phagocytosis by C3 exotoxin was confirmed in COS cells, which become phagocytic upon transfection of the FcγRIIA receptor. Rho was found to be essential for the accumulation of phosphotyrosine and of F-actin around phagocytic cups and for Fcγ receptor–mediated Ca2+ signaling. The clustering of receptors in response to opsonin, an essential step in Fcγ-induced signaling, was the earliest event shown to be inhibited by C3 exotoxin. The effect of the toxin was specific, since clustering and internalization of transferrin receptors were unaffected by microinjection of C3. These data identify a role for small GTPases in Fcγ receptor–mediated phagocytosis by leukocytes. PMID:9294149

Distribution and dynamics of the cytoskeleton in graviresponding protonemata and rhizoids of characean algae: exclusion of microtubules and a convergence of actin filaments in the apex suggest an actin-mediated gravitropism.

PubMed

Braun, M; Wasteneys, G O

1998-05-01

The organization of the microtubule (MT) and actin microfilament (MF) cytoskeleton of tip-growing rhizoids and protonemata of characean green algae was examined by confocal laser scanning microscopy. This analysis included microinjection of fluorescent tubulin and phallotoxins into living cells, as well as immunofluorescence labeling of fixed material and fluorescent phallotoxin labeling of unfixed material. Although the morphologically very similar positively gravitropic (downward growing) rhizoids and negatively gravitropic (upward growing) protonemata show opposite gravitropic responses, no differences were detected in the extensive three-dimensional distribution of actin MFs and MTs in both cell types. Tubulin microinjection revealed that in contrast to internodal cells, fluorescent tubulin incorporated very slowly into the MT arrays of rhizoids, suggesting that MT dynamics are very different in tip-growing and diffusely expanding cells. Microtubules assembled from multiple sites at the plasma membrane in the basal zone, and a dense subapical array emerged from a diffuse nucleation centre on the basal side of the nuclear envelope. Immunofluorescence confirmed these distribution patterns but revealed more extensive MT arrays. In the basal zone, short branching clusters of MTs form two cortical hemicylinders. Subapical, axially oriented MTs are distributed in equal density throughout the peripheral and inner cytoplasm and are closely associated with subapical organelles. Microtubules, however, are completely absent from the apical zones of rhizoids and protonemata. Actin MFs were found in all zones of rhizoids and protonemata including the apex. Two files of axially oriented bundles of subcortical actin MFs and ring-like actin structures in the streaming endoplasm of rhizoids were detected in the basal zones by microinjection or rhodamine-phalloidin labeling. The subapical zone contains a dense array of mainly axially oriented actin MFs that co-distribute with the subapical MT array. In the apex, actin MFs form thicker bundles that converge into a remarkably distinct actin patch in the apical dome, whose position coincides with the position of the endoplasmic reticulum aggregate in the centre of the Spitzenkörper. Actin MFs radiate from the actin patch towards the apical membrane. Together with results from previous inhibitor studies (Braun and Sievers, 1994, Eur J Cell Biol 63: 289-298), these results suggest that MTs have a stabilizing function in maintaining the polar cytoplasmic and cytoskeletal organization. The motile processes, however, are mediated by actin. In particular, the actin cytoskeleton appears to be involved in the structural and functional organization of the Spitzenkörper and thus is responsible for controlling cell shape and growth direction. Despite the similar structural arrangements of the actin cytoskeleton, major differences in the function of actin MFs have been observed in rhizoids and protonemata. Since actin MFs are more directly involved in the gravitropic response of protonemata than of rhizoids, the opposite gravitropsim in the two cell types seems to be based mainly on different properties and activities of the actin cytoskeleton.

Intact vinculin protein is required for control of cell shape, cell mechanics, and rac-dependent lamellipodia formation

NASA Technical Reports Server (NTRS)

Goldmann, Wolfgang H.; Ingber, Donald E.

2002-01-01

Studies were carried out using vinculin-deficient F9 embryonic carcinoma (gamma229) cells to analyze the relationship between structure and function within the focal adhesion protein vinculin, in the context of control of cell shape, cell mechanics, and movement. Atomic force microscopy studies revealed that transfection of the head (aa 1-821) or tail (aa 811-1066) domain of vinculin, alone or together, was unable to fully reverse the decrease in cell stiffness, spreading, and lamellipodia formation caused by vinculin deficiency. In contrast, replacement with intact vinculin completely restored normal cell mechanics and spreading regardless of whether its tyrosine phosphorylation site was deleted. Constitutively active rac also only induced extension of lamellipodia when microinjected into cells that expressed intact vinculin protein. These data indicate that vinculin's ability to physically couple integrins to the cytoskeleton, to mechanically stabilize cell shape, and to support rac-dependent lamellipodia formation all appear to depend on its intact three-dimensional structure.

Micromanipulation by laser microbeam and optical tweezers: from plant cells to single molecules.

PubMed

Greulich, K O; Pilarczyk, G; Hoffmann, A; Meyer Zu Hörste, G; Schäfer, B; Uhl, V; Monajembashi, S

2000-06-01

Complete manipulation by laser light allows precise and gentle treatment of plant cells, subcellular structures, and even individual DNA molecules. Recently, affordable lasers have become available for the construction of microbeams as well as for optical tweezers. This may generate new interest in these tools for plant biologists. Early experiments, reviewed in this journal, showed that laser supported microinjection of material into plant cells or tissues circumvents mechanical problems encountered in microinjection by fragile glass capillaries. Plant protoplasts could be fused with each other when under microscopical observation, and it was no major problem to generate a triple or quadruple fusion product. In the present paper we review experiments where membrane material was prepared from root hair tips and microgravity was simulated in algae. As many plant cells are transparent, it is possible to work inside living, intact cells. New experiments show that it is possible to release by optical micromanipulation, with high spatial resolutions, intracellular calcium from caged compounds and to study calcium oscillations. An example for avian cardiac tissue is given, but the technique is also suitable for plant cell research. As a more technical tool, optical tweezers can be used to spatially fix subcellular structures otherwise moving inside a cell and thus make them available for investigation with a confocal microscope even when the time for image formation is extended (for example at low fluorescence emission). A molecular biological example is the handling of chromosomes and isolated individual DNA molecules by laser microtools. For example, chromosomes can be cut along complex trajectories, not only perpendicular to their long axis. Single DNA molecules are cut by the laser microbeam and, after coupling such a molecule to a polystrene microbead, are handled in complex geometries. Here, the individual DNA molecules are made visible with a conventional fluorescence microscope by fluorescent dyes such as SYBRGreen. The cutting of a single DNA molecule by molecules of the restriction endonuclease EcoRI can be observed directly, i.e. a type of single molecule restriction analysis is possible. Finally, mechanical properties of individual DNA molecules can be observed directly.

Dorsomedial pontine neurons with descending projections to the medullary reticular formation express orexin-1 and adrenergic α2A receptor mRNA

PubMed Central

Volgin, Denys V.; Malinowska, Monika; Kubin, Leszek

2009-01-01

Neurons located in the dorsomedial pontine rapid eye movement (REM) sleep-triggering region send axons to the medial medullary reticular formation (mMRF). This pathway is believed to be important for the generation of REM sleep motor atonia, but other than that they are glutamatergic little is known about neurochemical signatures of these pontine neurons important for REM sleep. We used single-cell reverse transcription and polymerase chain reaction (RT-PCR) to determine whether dorsomedial pontine cells with projections to the mMRF express mRNA for selected membrane receptors that mediate modulatory influences on REM sleep. Fluorescein (FITC)-labeled latex microspheres were microinjected into the mMRF of 26–34 day-old rats under pentobarbital anesthesia. After 5–6 days, rats were sacrificed, pontine slices were obtained and neurons were dissociated from 400–600 μm micropunches extracted from dorsomedial pontine reticular formation. We found that 32 out of 51 FITC-labeled cells tested (63%±7(SE)) contained the orexin type 1 receptor (ORX1r) mRNA, 27 out of 73 (37%±6) contained the adrenergic α2A receptor (α2Ar) RNA, and 6 out of 31 (19%±7) contained both mRNAs. The percentage of cells positive for the ORX1r mRNA was significantly lower (p<0.04) for the dorsomedial pontine cells that were not retrogradely labeled from the mMRF (32%±11), whereas α2Ar mRNA was present in a similar percentage of FITC-labeled and unlabeled neurons. Our data suggest that ORX and adrenergic pathways converge on a subpopulation of cells of the pontine REM sleep-triggering region that have descending projections to the medullary region important for the motor control during REM sleep. PMID:19427365

Parvoviral nuclear import: bypassing the host nuclear-transport machinery.

PubMed

Cohen, Sarah; Behzad, Ali R; Carroll, Jeffrey B; Panté, Nelly

2006-11-01

The parvovirus Minute virus of mice (MVM) is a small DNA virus that replicates in the nucleus of its host cells. However, very little is known about the mechanisms underlying parvovirus' nuclear import. Recently, it was found that microinjection of MVM into the cytoplasm of Xenopus oocytes causes damage to the nuclear envelope (NE), suggesting that the nuclear-import mechanism of MVM involves disruption of the NE and import through the resulting breaks. Here, fluorescence microscopy and electron microscopy were used to examine the effect of MVM on host-cell nuclear structure during infection of mouse fibroblast cells. It was found that MVM caused dramatic changes in nuclear shape and morphology, alterations of nuclear lamin immunostaining and breaks in the NE of infected cells. Thus, it seems that the unusual nuclear-import mechanism observed in Xenopus oocytes is in fact used by MVM during infection of host cells.

Involvement of myosin VI immunoanalog in pinocytosis and phagocytosis in Amoeba proteus.

PubMed

Sobczak, Magdalena; Wasik, Anna; Kłopocka, Wanda; Redowicz, Maria Jolanta

2008-12-01

Recently, we found a 130-kDa myosin VI immunoanalog in amoeba, which bound to actin in an ATP-sensitive manner and in migrating amoebae colocalized to filamentous actin and dynamin II-containing vesicular structures. To further characterize this protein, we assessed its involvement in amoeba pinocytosis and phagocytosis. Confocal immunofluorescence microscopy and electron microscopy of immunogold-stained cells revealed that, in pinocytotic and phagocytotic amoebae, the myosin VI immunoanalog was visible throughout the cells, including pinocytotic channels and pinocytotic vesicles as well as phagosomes and emerging phagocytic cups. Blocking endogenous protein with anti-porcine myosin VI antibody (introduced into cells by means of microinjection) caused severe defects in pinocytosis and phagocytosis. In comparison with control cells, the treated amoebae formed ~75% less pinocytotic channels and phagocytosed ~65% less Tetrahymena cells. These data indicate that the myosin VI immunoanalog has an important role in pinocytosis and phagocytosis in Amoeba proteus (Pal.).

Cloning of the short-tailed Gyeongju Donggyeong dog via SCNT: conserving phenotypic inheritance.

PubMed

Choi, Yoo Bin; Kim, Geon A; Oh, Hyun Ju; Kim, Min Jung; Jo, Young Kwang; Setyawan, Erif Maha Nugraha; Lee, Seok Hee; Lee, Byeong Chun

2016-02-01

Somatic cell nuclear transfer is a useful tool to maintain genetic information of animals. The Gyeongju Donggyeong dog is a breed registered as natural monument in Korea. The unique feature of the Donggyeong dog is its tail, as the Donggyeong dog can be classified as either short tailed or tailless. The aim of this study was to preserve the Donggyeong dog's unique feature by cloning. Fibroblasts were obtained from a short-tailed Donggyeong dog. In vivo matured oocytes were enucleated, microinjected with a donor cell and fused electrically. Reconstructed embryos were transferred to six recipient dogs. One surrogate became pregnant, and one short-tailed Donggyeong dog was delivered. This study demonstrated that the phenotype of the Donggyeong dog could be conserved by somatic cell nuclear transfer.

[Advances of transgenic breeding in livestock].

PubMed

Yu, Da-Wei; Zhu, Hua-Bin; DU, Wei-Hua

2011-05-01

Transgenic technology represents a revolutionary way to produce elite livestock breeds, allowing introduction of alien gene into livestock genome. Currently, pronuclear microinjection of DNA and somatic cell nuclear transfer are two popular methods used to make transgenic farm animals. Transgenic technology can be used in livestock breeding for improving disease resistance, carcass composition, lactational performance, wool production, growth rate, and reproductive performance, as well as reducing negative environmental impact. In addition to introduction of animal transgenic technologies, this review described the status and the future perspective of transgenic breeding in livestock.

In vitro reestablishment of cell-cell contacts in adult rat cardiomyocytes. Functional role of transmembrane components in the formation of new intercalated disk-like cell contacts.

PubMed

Eppenberger, H M; Zuppinger, C

1999-01-01

Primary adult rat cardiomyocytes (ARC)in culture are shown to be a model system for cardiac cell hypertrophy in vitro. ARC undergo a process of morphological transformation and grow only by increase in cell size, however, without loss of the cardiac phenotype. The isolated cells spread and establish new cell-cell contacts, eventually forming a two-dimensional heart tissue-like synchronously beating cell sheet. The reformation of specific cell contacts (intercalated disks) is shown also between ventricular and atrial cardiomyocytes by using antibodies against the gap junction protein connexin-43 and after microinjection into ARC of N-cadherin cDNA fused to reporter green fluorescent protein (GFP) cDNA. The expressed fusion protein allowed the study of live cell cultures and of the dynamics of the adherens junction protein N-cadherin during the formation of new cell-cell contacts. The possible use of the formed ARC cell-sheet cells under microgravity conditions as a test system for the reformation of the cytoskeleton of heart muscle cells is proposed.

GABAA-benzodiazepine receptors in the dorsomedial (Dm) telencephalon modulate restraint-induced antinociception in the fish Leporinus macrocephalus.

PubMed

Wolkers, Carla Patricia Bejo; Barbosa Junior, Augusto; Menescal-de-Oliveira, Leda; Hoffmann, Anette

2015-08-01

The possibility that fish experience pain has been denied based on the absence of the neural substrates to support this "experience". In this context, the identification of brain regions involved in nociception modulation could provide important insights regarding the processing of nociceptive information in fish. Our study evaluated the participation of the GABAA-benzodiazepine receptor in the dorsomedial (Dm) telencephalon in restraint-induced antinociception in the fish Leporinus macrocephalus through the microinjection of the anxiolytic drug midazolam. The microinjection of midazolam in the Dm did not alter the nocifensive response; however, this drug did block the inhibition of the nocifensive response to formaldehyde promoted by restraint stress. The fish that received midazolam (40nmol) microinjection prior to restraint (3 or 5min), followed by subcutaneous injection with formaldehyde presented a higher distance traveled than the fish that received saline microinjection. This effect might reflect the specific action of midazolam on benzodiazepine receptors in the Dm telencephalon, as pre-treatment with flumazenil, a benzodiazepine receptor antagonist, inhibited the effects of this drug. In the present study, we present the first evidence demonstrating a role for the dorsomedial telencephalic region in the modulation of stress-induced antinociception in fish, revealing new perspectives in the understanding of nociceptive information processing in this group. Copyright © 2015 Elsevier Inc. All rights reserved.

Mechanical properties of micro-injected HDPE composites

NASA Astrophysics Data System (ADS)

Bongiorno, A.; Pagano, C.; Agnelli, S.; Baldi, F.; Fassi, I.

2016-03-01

Micro-injection moulding is one of the key manufacturing technologies for the mass production of high value polymeric miniaturized-components. However, this process is not just a straightforward down scaling of the conventional injection moulding technique. Indeed, during the micro-injection the polymer melt is forced to flow at high strain rates through very small channels in non-isothermal conditions, and this can lead to complex microstructures and to parts with unexpected performances. In this work, the relationships among the processing conditions, the mechanical properties and the microstructural characteristics of miniaturized specimens obtained by injection moulding were investigated. Two model systems were considered with the same filler content of 15% wt. (HDPE-talc and HDPE-glass beads), representative of two different types of micro-composites: containing lamellar and spherical micro-particles, respectively. The attention was focused on the influence of the filler type and the process conditions on the mechanical behaviour, examined by uniaxial tensile tests and dynamic-mechanical analyses, and on the morphological characteristics of the specimens, examined by microscopy analyses. The results highlight that mechanical response of the miniaturized specimens is significantly affected by both the filler and the process conditions that can have an influence on the polymer microstructure. Lamellar composites showed the best performance due to the orientation of the talc particles during the micro-injection process, while, different morphologies of the skin/core transition region in dependence on the process temperatures were observable.

A role of nucleus accumbens dopamine receptors in the nucleus accumbens core, but not shell, in fear prediction error.

PubMed

Li, Susan S Y; McNally, Gavan P

2015-08-01

Two experiments used an associative blocking design to study the role of dopamine receptors in the nucleus accumbens shell (AcbSh) and core (AcbC) in fear prediction error. Rats in the experimental groups were trained to a visual fear-conditioned stimulus (conditional stimulus [CS]) A in Stage I, whereas rats in the control groups were not. In Stage II, all rats received compound fear conditioning of the visual CSA and an auditory CSB. Rats were later tested for their fear responses to CSB. All rats received microinjections of saline or the D1-D2 receptor antagonist cis-(z)-flupenthixol prior to Stage II. These microinjections targeted either the AcbSh (Experiment 1) or the AcbC (Experiment 2). In each experiment, Stage I fear conditioning of CSA blocked fear learning to CSB. Microinjection of cis-(z)-flupenthixol (10 or 20 μg) into the AcbSh (Experiment 1) had no effect on fear learning or associative blocking. In contrast, microinjection of cis-(z)-flupenthixol (10 or 20 μg) into the AcbC (Experiment 2) attenuated blocking and so enabled fear learning to CSB. These results identify the AcbC as the critical locus for dopamine receptor contributions to fear prediction error and the associative blocking of fear learning. (c) 2015 APA, all rights reserved).

Hypothalamic GABAergic influences on treadmill exercise responses in rats.

PubMed

Overton, J M; Redding, M W; Yancey, S L; Stremel, R W

1994-01-01

Microinjection of GABAergic antagonists in the posterior hypothalamus (PH) produces exercise-like adjustments in cardiovascular function. To test the hypothesis that a hypothalamic GABAergic mechanism within the PH modulates the cardiovascular adjustments to dynamic exercise in conscious animals, Sprague-Dawley rats (n = 10) were instrumented with bilateral guide cannula directed at the pH, an arterial cannula, and Doppler flow probes on the iliac and mesenteric arteries. Saline (100 nl) or the GABAA receptor agonist muscimol (125 ng.100 nl-1) was bilaterally injected into the PH during treadmill exercise (20 m.min-1). Microinjection of saline had no effect on mean arterial pressure (MAP), heart rate (HR), mesenteric vascular resistance (MR), or iliac vascular resistance (IR) during exercise. Microinjection of muscimol during exercise produced no significant changes in MAP (mean change +/- SE; +0 +/- 1 mmHg), HR (+17 +/- 12 b.min-1), or MR (+7 +/- 13%). However, microinjection of muscimol produced a significant increase in IR during exercise (16 +/- 6%). In addition, muscimol significantly decreased treadmill run time (saline = 19.6 +/- 0.4 min; muscimol = 17.8 +/- 0.6 min) and produced behavioral effects (including mild sedation) that were most evident after exercise. The results of these experiments suggest that while the posterior hypothalamic GABAergic system may modulate iliac blood flow during exercise in rats, this system does not modulate HR and MR responses to dynamic exercise.

Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice.

PubMed

Nakagawa, Yoshiko; Sakuma, Tetsushi; Nishimichi, Norihisa; Yokosaki, Yasuyuki; Yanaka, Noriyuki; Takeo, Toru; Nakagata, Naomi; Yamamoto, Takashi

2016-08-15

Current advances in producing genetically modified mice using genome-editing technologies have indicated the need for improvement of limiting factors including zygote collection for microinjection and their cryopreservation. Recently, we developed a novel superovulation technique using inhibin antiserum and equine chorionic gonadotropin to promote follicle growth. This method enabled the increased production of fertilized oocytes via in vitro fertilization compared with the conventional superovulation method. Here, we verify that the ultra-superovulation technique can be used for the efficient generation of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated knockout mice by microinjection of plasmid vector or ribonucleoprotein into zygotes. We also investigated whether single-amino-acid-substituted mice and conditional knockout mice could be generated. Founder mice bearing base substitutions were generated more efficiently by co-microinjection of Cas9 protein, a guide RNA and single-stranded oligodeoxynucleotide (ssODN) than by plasmid microinjection with ssODN. The conditional allele was successfully introduced by the one-step insertion of an ssODN designed to carry an exon flanked by two loxP sequences and homology arms using a double-cut CRISPR-Cas9 strategy. Our study presents a useful method for the CRISPR-Cas9-based generation of genetically modified mice from the viewpoints of animal welfare and work efficiency. © 2016. Published by The Company of Biologists Ltd.

A zebrafish embryo behaves both as a "cortical shell-liquid core" structure and a homogeneous solid when experiencing mechanical forces.

PubMed

Liu, Fei; Wu, Dan; Chen, Ken

2014-12-01

Mechanical properties are vital for living cells, and various models have been developed to study the mechanical behavior of cells. However, there is debate regarding whether a cell behaves more similarly to a "cortical shell-liquid core" structure (membrane-like) or a homogeneous solid (cytoskeleton-like) when experiencing stress by mechanical forces. Unlike most experimental methods, which concern the small-strain deformation of a cell, we focused on the mechanical behavior of a cell undergoing small to large strain by conducting microinjection experiments on zebrafish embryo cells. The power law with order of 1.5 between the injection force and the injection distance indicates that the cell behaves as a homogenous solid at small-strain deformation. The linear relation between the rupture force and the microinjector radius suggests that the embryo behaves as membrane-like when subjected to large-strain deformation. We also discuss the possible reasons causing the debate by analyzing the mechanical properties of F-actin filaments.

CB1 receptor mediated analgesia from the Nucleus Reticularis Gigantocellularis pars alpha is activated in an animal model of neuropathic pain.

PubMed

Monhemius, R; Azami, J; Green, D L; Roberts, M H

2001-07-20

Cannabinoids are known to suppress responses to noxious stimulation in animals and man. Recent research has suggested a role for endogenous cannabinoids in the descending inhibition of dorsal horn cells via a supraspinal site of action. We have recently demonstrated [J. Physiol. 506(2) (1998) 459] that the nucleus reticularis gigantocellularis pars alpha (GiA) is a major source of such descending modulation, and importantly, that this system is activated in response to noxious stimulation. We have therefore investigated the role of CB1 receptor activation in mediating the antinociceptive effects of activation of GiA in models of acute and chronic pain. Microinjections (0.5 microl 60% DMSO) of either WIN 55,212-2 (5 microg, selective CB1 agonist), SR141716A (50 microg, competitive CB1 antagonist), both compounds together, or vehicle alone into GiA were performed prior to these tests in a randomised, blind manner. In control animals, WIN 55,212-2 markedly increased withdrawal latencies in the tail flick test and reduced responses to subcutaneous formalin. These effects were blocked by co-administration of SR141716A. These data suggest that activation of cannabinoid CB1 receptor subtypes in GiA leads to behavioural analgesia. In animals with partial sciatic nerve ligation, microinjection of drugs and injection of formalin were performed contralaterally to the site of ligation. Partial sciatic nerve ligation significantly reduced behavioural responses to contralaterally applied formalin. Microinjection of SR141716A to GiA reversed this inhibition of responses to formalin in animals with partial sciatic nerve ligation. These data provide evidence that endogenous CB1 receptor ligands are involved in GiA mediated antinociception, and that this system is important for the modulation of nociceptive transmission in an animal model of chronic neuropathic pain.

Studies on the in vivo sensitivity of spindle microtubules to calcium ions and evidence for a vesicular calcium-sequestering system

PubMed Central

1981-01-01

I microinjected calcium ions into echinoderm eggs during mitosis to determine the calcium sensitivity of microtubules (Mts) in vivo. Spindle birefringence (BR), a measure of the number of aligned Mts in the spindle, is locally, rapidly, and reversibly abolished by small volumes of microinjected CaCl2 (1 mM). Rapid return of BR is followed by anaphase, and subsequent divisions are normal. Similar doses of MgCl2, BaCl2, KCl, NaCl, pH buffers, distilled water, or vegetable oil have no effect on spindle BR, whereas large doses of such agents sometimes cause slow, uniform loss in BR over the course of a minute or more. Of the ions tested, only Sr++ causes effects comparable to Ca++. Ca-EGTA buffers, containing greater than micromolar free Ca++, abolishes BR in a manner similar to millimolar concentrations of injected CaCl2. Caffeine, a potent uncoupler of the Ca++-pump/ATPase of sarcoplasmic reticulum, causes a local, transient depression in spindle BR in the injected region. Finally, injection of potassium oxalate results in the formation of small, highly BR crystals, presumably CA- oxalate, in Triton-sensitive compartments in the cytoplasm. Taken together, these findings demonstrate that spindle Mts are sensitive to levels of free Ca++ in the physiological range, provide evidence for the existence of a strong cytoplasmic Ca++-sequestering system, and support the notion that Mt assembly and disassembly in local regions of the spindle may be orchestrated by local changes in the cytoplasmic free Ca++ concentration during mitosis. An appendix offers the design of a new chamber for immobilizing echinoderm eggs for injection, a new method for determining the volume of the injected solution, and a description of the microinjection technique, which was designed, but never fully described, by Hiramoto (Y. Hiramoto, Exp. Cell. Res., 1962, 27:416-426.). PMID:7194345

Egr1 gene knockdown affects embryonic ocular development in zebrafish.

PubMed

Hu, Chao-Yu; Yang, Chang-Hao; Chen, Wei-Yu; Huang, Chiu-Ju; Huang, Hsing-Yen; Chen, Muh-Shy; Tsai, Huai-Jen

2006-10-26

To identify the changes in zebrafish embryonic ocular development after early growth response factor 1 (Egr1) gene knockdown by Egr1-specific translation inhibitor, morpholino oligonucleotides (MO). Two kinds of Egr1-MO were microinjected separately with various dosages into one to four celled zebrafish embryos to find an optimal dose generating an acceptable mortality rate and high frequency of specific phenotype. Chordin-MO served as the positive control; a 5 mismatch MO of Egr1-MO1 and a nonspecific MO served as negative controls. We graded the Egr1 morphants according to their gross abnormalities, and measured their ocular dimensions accordingly. Western blot analysis and synthetic Egr1 mRNA rescue experiments confirmed whether the deformities were caused by Egr1 gene knockdown. Histological examination and three kinds of immunohistochemical staining were applied to identify glutamate receptor one expression in retinal ganglion cells and amacrine cells, to recognize acetylated alpha-tubulin expression which indicated axonogenesis, and to label photoreceptor cells with zpr-1 antibody. After microinjection of 8 ng Egr1-MO1 or 2 ng Egr1-MO2, 81.8% and 97.3% of larvae at 72 h postfertilization had specific defects, respectively. The gross phenotype included string-like heart, flat head, and deformed tail. The more severely deformed larvae had smaller eyes and pupils. Co-injection of 8 ng Egr1-MO1 and supplementary 12 pg synthetic Egr1 mRNA reduced the gross abnormality rate from 84.4% to 29.7%, and decreased the severity of deformities. Egr1 protein appeared in the wildtype and rescued morphants, but was lacking in the Egr1 morphants with specific deformities. Lenses of Egr1 morphants were smaller and had some residual nucleated lens fiber cells. Morphants' retinal cells arranged disorderly and compactly with thin plexiform layers. Immunohistochemical studies showed that morphants had a markedly decreased number of mature retinal ganglion cells, amacrine cells, and photoreceptor cells. Retinal axonogenesis was prominently reduced in morphants. The Egr1 gene plays an important role in zebrafish embryonic oculogenesis. Ocular structures including lens and retina were primitive and lacked appropriate differentiation. Such arrested retinal and lenticular development in Egr1 morphants resulted in microphthalmos.

Detection of biotin in individual sea urchin oocytes using a bioluminescence binding assay.

PubMed

Feltus, A; Grosvenor, A L; Conover, R C; Anderson, K W; Daunert, S

2001-04-01

The ability to detect biomolecules in single cells is important in order to fully understand the processes by which many biochemical events occur. To that end, we have developed a bioluminescence binding assay capable of measuring the intracellular biotin content of individual cells. The assay depends on competition between an aequorin-biotin conjugate (AEQ-biotin) and free biotin within the oocytes for binding sites on the protein avidin. The assay is performed by microinjecting each component into the oocytes and following the resulting bioluminescence within the oocyte upon triggering of aequorin. Results obtained using sea urchin oocytes show that the assay performed within the cells behaves in a manner consistent with assay theory. Using the assay, the individual biotin content of the oocytes is an average of approximately 20 amol. To our knowledge, this is the first reported multicomponent binding assay to be performed inside an intact single cell.

[The effect of tachykinins microinjections into the solitary tract nucleus on respiration and blood circulation in rats].

PubMed

Chepurnov, S A; Iniushkin, A N

1997-04-01

Administration of substance P and kassinin into the solitary tract nucleus of anesthetized rats induced a dose-dependent increase in ventilation, tidal volume, inspiratory muscle activity, and a decrease in the mean blood pressure and heart rate. Microinjections of peptides caused a decrease in ventilatory response to hypoxia and an inhibition of the Breuer-Hering reflex. The data obtained suggest involvement of tachykinins in the respiratory and circulatory control via the solitary tract nucleus.

Identification of the Sensory Neuron Specific Regulatory Region for the Mouse Gene Encoding the Voltage Gated Sodium Channel Nav1.8

PubMed Central

Puhl, Henry L.; Ikeda, Stephen R.

2008-01-01

Voltage-gated sodium channels (VGSC) are critical membrane components that participate in the electrical activity of excitable cells. The type one VGSC family includes the tetrodotoxin insensitive sodium channel, Nav1.8, encoded by the Scn10a gene. Nav1.8 expression is restricted to small and medium diameter nociceptive sensory neurons of the dorsal root (DRG) and cranial sensory ganglia. In order to understand the stringent transcriptional regulation of the Scn10a gene, the sensory neuron specific promoter was functionally identified. While identifying the mRNA 5’ end, alternative splicing within the 5’ UTR was observed to create heterogeneity in the RNA transcript. Four kilobases of upstream genomic DNA was cloned and the presence of tissue specific promoter activity was tested by microinjection and adenoviral infection of fluorescent protein reporter constructs into primary mouse and rat neurons, and cell lines. The region contained many putative transcription factor binding sites and strong homology with the predicted rat ortholog. Homology to the predicted human ortholog was limited to the proximal end and several conserved cis elements were noted. Two regulatory modules were identified by microinjection of reporter constructs into DRG and superior cervical ganglia neurons: a neuron specific proximal promoter region between −1.6 and −0.2kb of the transcription start site cluster, and a distal sensory neuron switch region beyond −1.6kb that restricted fluorescent protein expression to a subset of primary sensory neurons. PMID:18466327

Microinjected magnetic beads induce curvature in Chara rhizoids

NASA Astrophysics Data System (ADS)

Scherp, P.; Hasenstein, K.

The gravitropic response of the Chara rhizoid is based on the interaction between the statoliths and the actin network located in the rhizoid apex. The rhizoid represents a model system for the study of gravitropism, because its apical cell contains the gravity sensing and response mechanism. In order to study the function of the statoliths and the cytoskeleton, we supplemented the naturally occurring statoliths with magnetic beads. These beads can be moved by an external magnetic field and they can be coated to interact with the cytoskeleton. The magnetic beads (1μm diameter) were injected close to the tip of the rhizoid in the presence of an external osmoticum to offset turgor pressure. The injection caused the formation of a noticeable plug of dense material at the site of impalement. After a recovery period of ca. 2 - 4 hours, the whole plant was mounted on the rotatable stage of a custom- built horizontal microscope, equipped with a long-working distance objective and a video camera. This stage is designed to reorientate the cell and/or the injected beads. In order to study the effect of the displacement of magnetic beads, an external magnetic field was applied. This external field was capable of displacing the magnetic particles but did not affect the natural statoliths. Work is in progress to quantify the response, to study the effect of microinjection on wall formation, and utilize coating of the beads to investigate their possible interaction with the original statoliths and with the microfilament network. Supported by NASA grant NAG 2- 1423.

Modeling of Fluid-Membrane Interaction in Cellular Microinjection Process

NASA Astrophysics Data System (ADS)

Karzar-Jeddi, Mehdi; Diaz, Jhon; Olgac, Nejat; Fan, Tai-Hsi

2009-11-01

Cellular microinjection is a well-accepted method to deliver matters such as sperm, nucleus, or macromolecules into biological cells. To improve the success rate of in vitro fertilization and to establish the ideal operating conditions for a novel computer controlled rotationally oscillating intracytoplasmic sperm injection (ICSI) technology, we investigate the fluid-membrane interactions in the ICSI procedure. The procedure consists of anchoring the oocyte (a developing egg) using a holding pipette, penetrating oocyte's zona pellucida (the outer membrane) and the oolemma (the plasma or inner membrane) using an injection micropipette, and finally to deliver sperm into the oocyte for fertilization. To predict the large deformation of the oocyte membranes up to the piercing of the oolemma and the motion of fluids across both membranes, the dynamic fluid-pipette-membrane interactions are formulated by the coupled Stokes' equations and the continuum membrane model based on Helfrich's energy theory. A boundary integral model is developed to simulate the transient membrane deformation and the local membrane stress induced by the longitudinal motion of the injection pipette. The model captures the essential features of the membranes shown on optical images of ICSI experiments, and is capable of suggesting the optimal deformation level of the oolemma to start the rotational oscillations for piercing into the oolemma.

MEMANTINE ATTENUATES THE OKADAIC ACID INDUCED SHORT-TERM SPATIAL MEMORY IMPAIRMENT AND HIPPOCAMPAL CELL LOSS IN RATS.

PubMed

Dashniani, M; Chighladze, M; Burjanadze, M; Beselia, G; Kruashvili, L

2016-03-01

In the present study, the possible beneficial effect of memantine on the Okadaic Acid (OA) induced spatial short-term memory impairment was examined in spatial alternation task, and the neuroprotective potential of memantine on OA-induced structural changes in the hippocampus was evaluated by Nissl staining. OA was dissolved in artificial cerebrospinal fluid (aCSF) and injected intracerebroventriculary (ICV) 200 ng in a volume of 10 μl bilaterally. Vehicle control received aCSF ICV bilaterally. Control and OA injected rats were divided into 2 subgroups injected i.p. with saline or memantine (5 mg/kg). Memantine or saline were given daily for 13 days starting from the day of OA injection. Behavioral study showed that bilateral ICV microinjection of OA induced impairment in spatial short-term memory. Nissl staining in the present study showed that the ICV microinjection of OA significantly decreased the number of surviving pyramidal neurons in the CA1 region of the hippocampus. Chronic administration of memantine effectively attenuated OA induced spatial short-term memory impairment and the OA-induced neuropathological changes in the hippocampus. Therefore, ICV injection of OA can be used as an experimental model to study mechanisms of neurodegeneration and define novel therapeutics targets for AD pathology.

Optoporation of impermeable molecules and genes for visualization and activation of cells

NASA Astrophysics Data System (ADS)

Dhakal, Kamal; Batbyal, Subrata; Kim, Young-Tae; Mohanty, Samarendra

2015-03-01

Visualization, activation, and detection of the cell(s) and their electrical activity require delivery of exogenous impermeable molecules and targeted expression of genes encoding labeling proteins, ion-channels and voltage indicators. While genes can be delivered by viral vector to cells, delivery of other impermeable molecules into the cytoplasm of targeted cells requires microinjection by mechanical needle or microelectrodes, which pose significant challenge to the viability of the cells. Further, it will be useful to localize the expression of the targeted molecules not only in specific cell types, but to specific cells in restricted spatial regions. Here, we report use of focused near-infrared (NIR) femtosecond laser beam to transiently perforate targeted cell membrane to insert genes encoding blue light activatable channelrhodopsin-2 (ChR2) and red-shifted opsin (ReachR). Optoporation of nanomolar concentrations of rhodamine phalloidin (an impermeable dye molecule for staining filamentous actin) into targeted living mammalian cells (both HEK and primary cortical neurons) is also achieved allowing imaging of dynamics and intact morphology of cellular structures without requiring fixation.

Inhibiting Myosin Light Chain Kinase Induces Apoptosis In Vitro and In Vivo

PubMed Central

Fazal, Fabeha; Gu, Lianzhi; Ihnatovych, Ivanna; Han, YooJeong; Hu, WenYang; Antic, Nenad; Carreira, Fernando; Blomquist, James F.; Hope, Thomas J.; Ucker, David S.; de Lanerolle, Primal

2005-01-01

Previous short-term studies have correlated an increase in the phosphorylation of the 20-kDa light chain of myosin II (MLC20) with blebbing in apoptotic cells. We have found that this increase in MLC20 phosphorylation is rapidly followed by MLC20 dephosphorylation when cells are stimulated with various apoptotic agents. MLC20 dephosphorylation is not a consequence of apoptosis because MLC20 dephosphorylation precedes caspase activation when cells are stimulated with a proapoptotic agent or when myosin light chain kinase (MLCK) is inhibited pharmacologically or by microinjecting an inhibitory antibody to MLCK. Moreover, blocking caspase activation increased cell survival when MLCK is inhibited or when cells are treated with tumor necrosis factor alpha. Depolymerizing actin filaments or detaching cells, processes that destabilize the cytoskeleton, or inhibiting myosin ATPase activity also resulted in MLC20 dephosphorylation and cell death. In vivo experiments showed that inhibiting MLCK increased the number of apoptotic cells and retarded the growth of mammary cancer cells in mice. Thus, MLC20 dephosphorylation occurs during physiological cell death and prolonged MLC20 dephosphorylation can trigger apoptosis. PMID:15988034

Maternal Supply of Cas9 to Zygotes Facilitates the Efficient Generation of Site-Specific Mutant Mouse Models

PubMed Central

Cebrian-Serrano, Alberto; Zha, Shijun; Hanssen, Lars; Biggs, Daniel; Preece, Christopher

2017-01-01

Genome manipulation in the mouse via microinjection of CRISPR/Cas9 site-specific nucleases has allowed the production time for genetically modified mouse models to be significantly reduced. Successful genome manipulation in the mouse has already been reported using Cas9 supplied by microinjection of a DNA construct, in vitro transcribed mRNA and recombinant protein. Recently the use of transgenic strains of mice overexpressing Cas9 has been shown to facilitate site-specific mutagenesis via maternal supply to zygotes and this route may provide an alternative to exogenous supply. We have investigated the feasibility of supplying Cas9 genetically in more detail and for this purpose we report the generation of a transgenic mice which overexpress Cas9 ubiquitously, via a CAG-Cas9 transgene targeted to the Gt(ROSA26)Sor locus. We show that zygotes prepared from female mice harbouring this transgene are sufficiently loaded with maternally contributed Cas9 for efficient production of embryos and mice harbouring indel, genomic deletion and knock-in alleles by microinjection of guide RNAs and templates alone. We compare the mutagenesis rates and efficacy of mutagenesis using this genetic supply with exogenous Cas9 supply by either mRNA or protein microinjection. In general, we report increased generation rates of knock-in alleles and show that the levels of mutagenesis at certain genome target sites are significantly higher and more consistent when Cas9 is supplied genetically relative to exogenous supply. PMID:28081254

GABAergic regulation of REM sleep in reticularis pontis oralis and caudalis in rats.

PubMed

Sanford, Larry D; Tang, Xiangdong; Xiao, Jihua; Ross, Richard J; Morrison, Adrian R

2003-08-01

The nucleus reticularis pontis oralis (RPO) and nucleus reticularis pontis caudalis (RPC) are implicated in the generation of rapid eye movement sleep (REM). Work in cats has indicated that GABA in RPO plays a role in the regulation of REM. We assessed REM after local microinjections into RPO and RPC of the gamma-aminobutyric acid-A (GABA(A)) agonist, muscimol (MUS), and the GABA(A) antagonist, bicuculline (BIC). Rats (90-day-old male Sprague-Dawley) were implanted with electrodes for recording electroencephalographs (EEG) and electromyographs (EMG). Guide cannulae were aimed into RPO (n = 9) and RPC (n = 8) for microinjecting MUS (200, 1,000.0 microM) and BIC (0.056, 0.333, 1.0, 1,000.0, and 10,000.0 microM). Animals received bilateral microinjections of saline, MUS, and BIC (0.2 microl microinjected at 0.1 microl/min) into each region followed by 6-h sleep recordings. In RPO, MUS (1,000.0 microM) suppressed REM and BIC (1,000.0 microM) enhanced REM. In RPC, MUS (200, 1,000.0 microM) suppressed REM, but BIC (1,000.0 microM and less) did not significantly affect REM. Higher concentrations of BIC (10,000.0 microM) injected into RPO (n = 9) and RPC (n = 4) produced wakefulness and escape behavior. The results indicate that GABA in RPO/RPC is involved in the regulation of REM and suggest site-specific differences in this regulation.

Optimization of factors influencing microinjection method for Agrobacterium tumefaciens-mediated transformation of tomato.

PubMed

Vinoth, S; Gurusaravanan, P; Jayabalan, N

2013-02-01

A simple and efficient protocol for Agrobacterium-mediated genetic transformation of tomato was developed using combination of non-tissue culture and micropropagation systems. Initially, ESAM region of 1-day-old germinated tomato seeds were microinjected for one to five times with Agrobacterium inoculums (OD(600) = 0.2-1.0). The germinated seeds were cocultivated in the MS medium fortified with (0-200 mM) acetosyringone and minimal concentrations of (0-20 mg L(-1)) kanamycin, and the antibiotic concentration was doubled during the second round of selection. Bacterial concentration of OD(600) = 0.6 served as an optimal concentration for infection and the transformation efficiency was significantly higher of about 46.28 %. In another set of experiment, an improved and stable regeneration system was adapted for the explants from the selection medium. Four-day-old double cotyledonary nodal explants were excised from the microinjected seedlings and cultured onto the MS medium supplemented with 1.5 mg L(-1) thidiazuron, 1.5 mg L(-1) indole-3-butyric acid, 30 mg L(-1) kanamycin, and 0-1.5 mg L(-1) adenine sulphate. Maximum of 9 out of 13 micropropagated shoots were shown positive to GUS assay. By this technique, the transformation efficiency was increased from 46.28 to 65.90 %. Thus, this paper reports the successful protocol for the mass production of transformants using microinjection and micropropagation techniques.

Cell migration without a lamellipodium: translation of actin dynamics into cell movement mediated by tropomyosin.

PubMed

Gupton, Stephanie L; Anderson, Karen L; Kole, Thomas P; Fischer, Robert S; Ponti, Aaron; Hitchcock-DeGregori, Sarah E; Danuser, Gaudenz; Fowler, Velia M; Wirtz, Denis; Hanein, Dorit; Waterman-Storer, Clare M

2005-02-14

The actin cytoskeleton is locally regulated for functional specializations for cell motility. Using quantitative fluorescent speckle microscopy (qFSM) of migrating epithelial cells, we previously defined two distinct F-actin networks based on their F-actin-binding proteins and distinct patterns of F-actin turnover and movement. The lamellipodium consists of a treadmilling F-actin array with rapid polymerization-dependent retrograde flow and contains high concentrations of Arp2/3 and ADF/cofilin, whereas the lamella exhibits spatially random punctae of F-actin assembly and disassembly with slow myosin-mediated retrograde flow and contains myosin II and tropomyosin (TM). In this paper, we microinjected skeletal muscle alphaTM into epithelial cells, and using qFSM, electron microscopy, and immunolocalization show that this inhibits functional lamellipodium formation. Cells with inhibited lamellipodia exhibit persistent leading edge protrusion and rapid cell migration. Inhibition of endogenous long TM isoforms alters protrusion persistence. Thus, cells can migrate with inhibited lamellipodia, and we suggest that TM is a major regulator of F-actin functional specialization in migrating cells.

Peroxisome proliferator-activated receptors γ/mitochondrial uncoupling protein 2 signaling protects against seizure-induced neuronal cell death in the hippocampus following experimental status epilepticus

PubMed Central

2012-01-01

Background Status epilepticus induces subcellular changes that may lead to neuronal cell death in the hippocampus. However, the mechanism of seizure-induced neuronal cell death remains unclear. The mitochondrial uncoupling protein 2 (UCP2) is expressed in selected regions of the brain and is emerged as an endogenous neuroprotective molecule in many neurological disorders. We evaluated the neuroprotective role of UCP2 against seizure-induced hippocampal neuronal cell death under experimental status epilepticus. Methods In Sprague–Dawley rats, kainic acid (KA) was microinjected unilaterally into the hippocampal CA3 subfield to induce prolonged bilateral seizure activity. Oxidized protein level, translocation of Bcl-2, Bax and cytochrome c between cytosol and mitochondria, and expression of peroxisome proliferator-activated receptors γ (PPARγ) and UCP2 were examined in the hippocampal CA3 subfield following KA-induced status epilepticus. The effects of microinjection bilaterally into CA3 area of a PPARγ agonist, rosiglitazone or a PPARγ antagonist, GW9662 on UCP2 expression, induced superoxide anion (O2· -) production, oxidized protein level, mitochondrial respiratory chain enzyme activities, translocation of Bcl-2, Bax and cytochrome c, and DNA fragmentation in bilateral CA3 subfields were examined. Results Increased oxidized proteins and mitochondrial or cytosol translocation of Bax or cytochrome c in the hippocampal CA3 subfield was observed 3–48 h after experimental status epilepticus. Expression of PPARγ and UCP2 increased 12–48 h after KA-induced status epilepticus. Pretreatment with rosiglitazone increased UCP2 expression, reduced protein oxidation, O2· - overproduction and dysfunction of mitochondrial Complex I, hindered the translocation of Bax and cytochrome c, and reduced DNA fragmentation in the CA3 subfield. Pretreatment with GW9662 produced opposite effects. Conclusions Activation of PPARγ upregulated mitochondrial UCP2 expression, which decreased overproduction of reactive oxygen species, improved mitochondrial Complex I dysfunction, inhibited mitochondrial translocation of Bax and prevented cytosolic release of cytochrome c by stabilizing the mitochondrial transmembrane potential, leading to amelioration of apoptotic neuronal cell death in the hippocampus following status epilepticus. PMID:22849356

Cdc6 is regulated by E2F and is essential for DNA replication in mammalian cells.

PubMed

Yan, Z; DeGregori, J; Shohet, R; Leone, G; Stillman, B; Nevins, J R; Williams, R S

1998-03-31

Cdc6 has a critical regulatory role in the initiation of DNA replication in yeasts, but its function in mammalian cells has not been characterized. We show here that Cdc6 is expressed selectively in proliferating but not quiescent mammalian cells, both in culture and within tissues of intact animals. During the transition from a growth-arrested to a proliferative state, transcription of mammalian Cdc6 is regulated by E2F proteins, as revealed by a functional analysis of the human Cdc6 promoter and by the ability of exogenously expressed E2F proteins to stimulate the endogenous Cdc6 gene. Immunodepletion of Cdc6 by microinjection of anti-Cdc6 antibody blocks initiation of DNA replication in a human tumor cell line. We conclude that expression of human Cdc6 is regulated in response to mitogenic signals though transcriptional control mechanisms involving E2F proteins, and that Cdc6 is required for initiation of DNA replication in mammalian cells.

Flexibility contra Stiffness: The Phragmoplast as a Physical Barrier for Beads But Not for Vesicles[OA

PubMed Central

Esseling-Ozdoba, Agnieszka; Kik, Richard A.; van Lammeren, André A.M.; Kleijn, J. Mieke; Emons, Anne Mie C.

2010-01-01

In plant cells, Golgi vesicles are transported to the division plane to fuse with each other, forming the cell plate, the initial membrane-bordered cell wall separating daughter cells. Vesicles, but not organelles, move through the phragmoplast, which consists of two opposing cylinders of microtubules and actin filaments, interlaced with endoplasmic reticulum membrane. To study physical aspects of this transport/inhibition process, we microinjected fluorescent synthetic 1,2-dioleoyl-sn-glycero-3-phospho-rac-1-glycerol (DOPG) vesicles and polystyrene beads into Tradescantia virginiana stamen hair cells. The phragmoplast was nonselective for DOPG vesicles of a size up to 150 nm in diameter but was a physical barrier for polystyrene beads having a diameter of 20 and 40 nm and also when beads were coated with the same DOPG membrane. We conclude that stiffness is a parameter for vesicle transit through the phragmoplast and discuss that cytoskeleton configurations can physically block such transit. PMID:19939943

Parvovirus particles and movement in the cellular cytoplasm and effects of the cytoskeleton

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lyi, Sangbom Michael; Tan, Min Jie Alvin, E-mail: tanmja@gis.a-star.edu.sg; Parrish, Colin R., E-mail: crp3@cornell.edu

2014-05-15

Cell infection by parvoviruses requires that capsids be delivered from outside the cell to the cytoplasm, followed by genome trafficking to the nucleus. Here we microinject capsids into cells that lack receptors and followed their movements within the cell over time. In general the capsids remained close to the positions where they were injected, and most particles did not move to the vicinity of or enter the nucleus. When 70 kDa-dextran was injected along with the capsids that did not enter the nucleus in significant amounts. Capsids conjugated to peptides containing the SV40 large T-antigen nuclear localization signal remained inmore » the cytoplasm, although bovine serum albumen conjugated to the same peptide entered the nucleus rapidly. No effects of disruption of microfilaments, intermediate filaments, or microtubules on the distribution of the capsids were observed. These results suggest that movement of intact capsids within cells is primarily associated with passive processes.« less

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

PubMed Central

Lee, Aih Cheun; Decourt, Boris; Suter, Daniel

2008-01-01

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones. PMID:19066568

Integrated sequencing of exome and mRNA of large-sized single cells.

PubMed

Wang, Lily Yan; Guo, Jiajie; Cao, Wei; Zhang, Meng; He, Jiankui; Li, Zhoufang

2018-01-10

Current approaches of single cell DNA-RNA integrated sequencing are difficult to call SNPs, because a large amount of DNA and RNA is lost during DNA-RNA separation. Here, we performed simultaneous single-cell exome and transcriptome sequencing on individual mouse oocytes. Using microinjection, we kept the nuclei intact to avoid DNA loss, while retaining the cytoplasm inside the cell membrane, to maximize the amount of DNA and RNA captured from the single cell. We then conducted exome-sequencing on the isolated nuclei and mRNA-sequencing on the enucleated cytoplasm. For single oocytes, exome-seq can cover up to 92% of exome region with an average sequencing depth of 10+, while mRNA-sequencing reveals more than 10,000 expressed genes in enucleated cytoplasm, with similar performance for intact oocytes. This approach provides unprecedented opportunities to study DNA-RNA regulation, such as RNA editing at single nucleotide level in oocytes. In future, this method can also be applied to other large cells, including neurons, large dendritic cells and large tumour cells for integrated exome and transcriptome sequencing.

Alteration of Epithelial Structure and Function Associated with PtdIns(4,5)P2 Degradation by a Bacterial Phosphatase

PubMed Central

Mason, David; Mallo, Gustavo V.; Terebiznik, Mauricio R.; Payrastre, Bernard; Finlay, B. Brett; Brumell, John H.; Rameh, Lucia; Grinstein, Sergio

2007-01-01

Elucidation of the role of PtdIns(4,5)P2 in epithelial function has been hampered by the inability to selectively manipulate the cellular content of this phosphoinositide. Here we report that SigD, a phosphatase derived from Salmonella, can effectively hydrolyze PtdIns(4,5)P2, generating PtdIns(5)P. When expressed by microinjecting cDNA into epithelial cells forming confluent monolayers, wild-type SigD induced striking morphological and functional changes that were not mimicked by a phosphatase-deficient SigD mutant (C462S). Depletion of PtdIns(4,5)P2 in intact SigD-injected cells was verified by detachment from the membrane of the pleckstrin homology domain of phospholipase Cδ, used as a probe for the phosphoinositide by conjugation to green fluorescent protein. Single-cell measurements of cytosolic pH indicated that the Na+/H+ exchange activity of epithelia was markedly inhibited by depletion of PtdIns(4,5)P2. Similarly, anion permeability, measured using two different halide-sensitive probes, was depressed in cells expressing SigD. Depletion of PtdIns(4,5)P2 was associated with marked alterations in the actin cytoskeleton and its association with the plasma membrane. The junctional complexes surrounding the injected cells gradually opened and the PtdIns(4,5)P2-depleted cells eventually detached from the monolayer, which underwent rapid restitution. Similar observations were made in intestinal and renal epithelial cultures. In addition to its effects on phosphoinositides, SigD has been shown to convert inositol 1,3,4,5,6-pentakisphosphate (IP5) into inositol 1,4,5,6-tetrakisphosphate (IP4), and the latter has been postulated to mediate the diarrhea caused by Salmonella. However, the effects of SigD on epithelial cells were not mimicked by microinjection of IP4. In contrast, the cytoskeletal and ion transport effects were replicated by hydrolyzing PtdIns(4,5)P2 with a membrane-targeted 5-phosphatase or by occluding the inositide using high-avidity tandem PH domain constructs. We therefore suggest that opening of the tight junctions and inhibition of Na+/H+ exchange caused by PtdIns(4,5)P2 hydrolysis combine to account, at least in part, for the fluid loss observed during Salmonella-induced diarrhea. PMID:17389247

Role of ventrolateral orbital cortex muscarinic and nicotinic receptors in modulation of capsaicin-induced orofacial pain-related behaviors in rats.

PubMed

Tamaddonfard, Esmaeal; Erfanparast, Amir; Abbas Farshid, Amir; Delkhosh-Kasmaie, Fatmeh

2017-11-15

Acetylcholine, as a major neurotransmitter, mediates many brain functions such as pain. This study was aimed to investigate the effects of microinjection of muscarinic and nicotinic acetylcholine receptor antagonists and agonists into the ventrolateral orbital cortex (VLOC) on capsaicin-induced orofacial nociception and subsequent hyperalgesia. The right side of VLOC was surgically implanted with a guide cannula in anaesthetized rats. Orofacial pain-related behaviors were induced by subcutaneous injection of a capsaicin solution (1.5µg/20µl) into the left vibrissa pad. The time spent face rubbing with ipsilateral forepaw and general behavior were recorded for 10min, and then mechanical hyperalgesia was determined using von Frey filaments at 15, 30, 45 and 60min post-capsaicin injection. Alone intra-VLOC microinjection of atropine (a muscarinic acetylcholine receptor antagonist) and mecamylamine (a nicotinic acetylcholine receptor antagonist) at a similar dose of 200ng/site did not alter nocifensive behavior and hyperalgesia. Microinjection of oxotremorine (a muscarinic acetylcholine receptor agonist) at doses of 50 and 100ng/site and epibatidine (a nicotinic acetylcholine receptor agonist) at doses of 12.5, 25, 50 and 100ng/site into the VLOC suppressed pain-related behaviors. Prior microinjections of 200ng/site atropine and mecamylamine (200ng/site) prevented oxotremorine (100ng/site)-, and epibatidine (100ng/site)-induced antinociception, respectively. None of the above-mentioned chemicals changed general behavior. These results showed that the VLOC muscarinic and nicotinic acetylcholine receptors might be involved in modulation of orofacial nociception and hypersensitivity. Copyright © 2017 Elsevier B.V. All rights reserved.

Uncovering Wolbachia Diversity upon Artificial Host Transfer

PubMed Central

Schneider, Daniela I.; Riegler, Markus; Arthofer, Wolfgang; Merçot, Hervé; Stauffer, Christian; Miller, Wolfgang J.

2013-01-01

The common endosymbiotic Wolbachia bacteria influence arthropod hosts in multiple ways. They are mostly recognized for their manipulations of host reproduction, yet, more recent studies demonstrate that Wolbachia also impact host behavior, metabolic pathways and immunity. Besides their biological and evolutionary roles, Wolbachia are new potential biological control agents for pest and vector management. Importantly, Wolbachia-based control strategies require controlled symbiont transfer between host species and predictable outcomes of novel Wolbachia-host associations. Theoretically, this artificial horizontal transfer could inflict genetic changes within transferred Wolbachia populations. This could be facilitated through de novo mutations in the novel recipient host or changes of haplotype frequencies of polymorphic Wolbachia populations when transferred from donor to recipient hosts. Here we show that Wolbachia resident in the European cherry fruit fly, Rhagoletis cerasi, exhibit ancestral and cryptic sequence polymorphism in three symbiont genes, which are exposed upon microinjection into the new hosts Drosophila simulans and Ceratitis capitata. Our analyses of Wolbachia in microinjected D. simulans over 150 generations after microinjection uncovered infections with multiple Wolbachia strains in trans-infected lines that had previously been typed as single infections. This confirms the persistence of low-titer Wolbachia strains in microinjection experiments that had previously escaped standard detection techniques. Our study demonstrates that infections by multiple Wolbachia strains can shift in prevalence after artificial host transfer driven by either stochastic or selective processes. Trans-infection of Wolbachia can claim fitness costs in new hosts and we speculate that these costs may have driven the shifts of Wolbachia strains that we saw in our model system. PMID:24376534

Uncovering Wolbachia diversity upon artificial host transfer.

PubMed

Schneider, Daniela I; Riegler, Markus; Arthofer, Wolfgang; Merçot, Hervé; Stauffer, Christian; Miller, Wolfgang J

2013-01-01

The common endosymbiotic Wolbachia bacteria influence arthropod hosts in multiple ways. They are mostly recognized for their manipulations of host reproduction, yet, more recent studies demonstrate that Wolbachia also impact host behavior, metabolic pathways and immunity. Besides their biological and evolutionary roles, Wolbachia are new potential biological control agents for pest and vector management. Importantly, Wolbachia-based control strategies require controlled symbiont transfer between host species and predictable outcomes of novel Wolbachia-host associations. Theoretically, this artificial horizontal transfer could inflict genetic changes within transferred Wolbachia populations. This could be facilitated through de novo mutations in the novel recipient host or changes of haplotype frequencies of polymorphic Wolbachia populations when transferred from donor to recipient hosts. Here we show that Wolbachia resident in the European cherry fruit fly, Rhagoletis cerasi, exhibit ancestral and cryptic sequence polymorphism in three symbiont genes, which are exposed upon microinjection into the new hosts Drosophila simulans and Ceratitis capitata. Our analyses of Wolbachia in microinjected D. simulans over 150 generations after microinjection uncovered infections with multiple Wolbachia strains in trans-infected lines that had previously been typed as single infections. This confirms the persistence of low-titer Wolbachia strains in microinjection experiments that had previously escaped standard detection techniques. Our study demonstrates that infections by multiple Wolbachia strains can shift in prevalence after artificial host transfer driven by either stochastic or selective processes. Trans-infection of Wolbachia can claim fitness costs in new hosts and we speculate that these costs may have driven the shifts of Wolbachia strains that we saw in our model system.

Excitatory amino acid receptors in the dorsomedial hypothalamus mediate prostaglandin-evoked thermogenesis in brown adipose tissue.

PubMed

Madden, C J; Morrison, S F

2004-02-01

We determined whether the dorsomedial hypothalamus (DMH) plays a role in the thermogenic, metabolic, and cardiovascular effects evoked by centrally administered PGE2. Microinjection of PGE2 (170 pmol/60 nl) into the medial preoptic area of the hypothalamus in urethane-chloralose-anesthetized, artificially ventilated rats increased brown adipose tissue (BAT) sympathetic nerve activity (SNA; +207 +/- 18% of control), BAT temperature (1.5 +/- 0.2 degrees C), expired CO2 (0.9 +/- 0.1%), heart rate (HR; 106 +/- 12 beats/min), and mean arterial pressure (22 +/- 4 mmHg). Within 5 min of subsequent bilateral microinjections of the GABAA receptor agonist muscimol (120 pmol.60 nl-1.side-1) or the ionotropic excitatory amino acid antagonist kynurenate (6 nmol.60 nl-1.side-1) into the DMH, the PGE2-evoked increases were, respectively, attenuated by 91 +/- 3% and 108 +/- 7% for BAT SNA, by 73 +/- 12% and 102 +/- 28% for BAT temperature, by 100 +/- 4% and 125 +/- 21% for expired CO2, by 72 +/- 11% and 70 +/- 16% for HR, and by 84 +/- 19% and 113 +/- 16% for mean arterial pressure. Microinjections outside the DMH within the dorsal hypothalamic area adjacent to the mamillothalamic tracts or within the ventromedial hypothalamus were less effective for attenuating the PGE2-evoked thermogenic, metabolic, and cardiovascular responses. These results demonstrate that activation of excitatory amino acid receptors within the DMH is necessary for the thermogenic, metabolic, and cardiovascular responses evoked by microinjection of PGE2 into the medial preoptic area.

[Microinjections of heat shock protein 70 kDa into the nucleus reticularis pontis oralis induce inhibition of rapid eye movement sleep in pigeons].

PubMed

Gusel'nikova, E A; Pastukhov, Iu F

2008-03-01

Recently it was indicated that microinjections of heat shock proteins 70 kDa (Hsp70) into the third ventricle of brain in pigeons results in an increase in the duration of slow wave sleep and a decrease in somato-visceral indices. It is suggested that Hsp70 effect may be related to GABA(A) receptors activation in the preoptic area of the hypothalamus. However, what transmitter mechanisms of activation are related to the removal effect (in 2-3 hrs) of rapid eye movement sleep inhibition still remains poorly understood. To solve this problem in the present study, microinjections of Hsp70 into the Nucleus reticularis pontis oralis (NRPO) were done. It is well known that cholinergic neurons of the NRPO are crucial for rapid eye movement sleep generation. The data show that Hsp70 produces more early (for first two hrs) a decrease in number of episodes and total time of rapid eye movement sleep, a diminution of electroencephalogram (EEG) power spectra in the 9-14 Hz band, a decrease in contractile muscle activity and brain temperature. It is suggested that Hsp70 effects are realized due to activation of GABA(A) receptors in the NRPO and induced inhibition of cholinergic mechanisms of rapid eye movement sleep triggering. The microinjections of Hsp70 into the NRPO increase the slow wave sleep total time with long latency (for 8-12 hrs). This effect may be related to influence of Hsp70 on neurons population, which are responsible for slow wave sleep maintenance outside the NRPO.

Exploring cytoplasmic dynamics in zebrafish yolk cells by single particle tracking of fluorescent nanodiamonds

NASA Astrophysics Data System (ADS)

Chang, Cheng-Chun; Zhang, Bailin; Li, Che-Yu; Hsieh, Chih-Chien; Duclos, Guillaume; Treussart, François; Chang, Huan-Cheng

2012-02-01

Fluorescent nanodiamonds (FNDs) have recently developed into an exciting new tool for bioimaging applications. The material possesses several unique features including high biocompatibility, easy bioconjugation, and perfect photostability, making it a promising optical nanoprobe in vitro as well as in vivo. This work explores the potential application of this novel nanomaterial as a photostable, nontoxic tracer in vivo using zebrafish as a model organism. We introduced FNDs into the yolk of a zebrafish embryo by microinjection at the 1-cell stage. Movements of the injected particles were investigated by using single particle tracking techniques. We observed unidirectional and stop-and-go traffic as part of the intricate cytoplasmic movements in the yolk cell. We determined a velocity in the range of 0.19 - 0.40 μm/s for 40 particles moving along with the axial streaming in the early developmental stage (1 to 2 hours post fertilization) of the zebrafish embryos.

Structural and functional identification of vasculogenic mimicry in vitro.

PubMed

Racordon, Dusan; Valdivia, Andrés; Mingo, Gabriel; Erices, Rafaela; Aravena, Raúl; Santoro, Felice; Bravo, Maria Loreto; Ramirez, Carolina; Gonzalez, Pamela; Sandoval, Alejandra; González, Alfonso; Retamal, Claudio; Kogan, Marcelo J; Kato, Sumie; Cuello, Mauricio A; Osorio, German; Nualart, Francisco; Alvares, Pedro; Gago-Arias, Araceli; Fabri, Daniella; Espinoza, Ignacio; Sanchez, Beatriz; Corvalán, Alejandro H; Pinto, Mauricio P; Owen, Gareth I

2017-08-01

Vasculogenic mimicry (VM) describes a process by which cancer cells establish an alternative perfusion pathway in an endothelial cell-free manner. Despite its strong correlation with reduced patient survival, controversy still surrounds the existence of an in vitro model of VM. Furthermore, many studies that claim to demonstrate VM fail to provide solid evidence of true hollow channels, raising concerns as to whether actual VM is actually being examined. Herein, we provide a standardized in vitro assay that recreates the formation of functional hollow channels using ovarian cancer cell lines, cancer spheres and primary cultures derived from ovarian cancer ascites. X-ray microtomography 3D-reconstruction, fluorescence confocal microscopy and dye microinjection conclusively confirm the existence of functional glycoprotein-rich lined tubular structures in vitro and demonstrate that many of structures reported in the literature may not represent VM. This assay may be useful to design and test future VM-blocking anticancer therapies.

Successful development of viable blastocysts from enhanced green fluorescent protein transgene-microinjected mouse embryos: comparison of culture media.

PubMed

Devgan, Vikram; Seshagiri, Polani B

2003-07-01

To improve efficiency of transgenesis, we compared M16 and CZB embryo culture media, supporting development to blastocysts of FVB/N mouse pronuclear-eggs, microinjected with enhanced green fluorescent protein (EGFP) transgene. When EGFP-injected-eggs were cultured (120 hr), blastocyst development was significantly (P < 0.03) higher in M16 medium (72.5 +/- 2.4%) than that in CZB (13.2 +/- 4.3%) or CZBG (CZB with 5.6 mM glucose at 48 hr culture) (62.1 +/- 3.7%) media. Blastocyst development of noninjected embryos was higher in M16 (92.0 +/- 2.6%) and CZBG (83.9 +/- 3.9%) media than in CZB (31.9 +/- 2.8%) medium (P < 0.0001). However, percentages of morulae at 72 hr were comparable in all treatments. Developed blastocysts were better in M16 than in CZB or CZBG media. Consistent with this, mean cell number per blastocyst, developed from injected embryos, was significantly (P < 0.002) higher in M16 medium (79.6), than those in CZB (31.3) or CZBG media (60.7); similar with noninjected embryos. Cell allocation to trophectoderm (TE) and inner cell mass (ICM), i.e., TE:ICM ratio, for injected blastocysts in M16 (3.0) was less than (P < 0.05) those in CZB (4.2) and CZBG (4.4) media; similar with noninjected blastocysts. Moreover, blastocysts, developed in M16 and CZBG media, hatched, attached, and exhibited trophoblast outgrowth; 18% of them showed EGFP-expression. Importantly, blastocysts from M16 medium produced live transgenic "green" pups (11%) following embryo transfer. Taken together, our results indicate that supplementation of glucose, at 48 hr of culture (CZBG), is required for morula to blastocyst transition; M16 medium, containing glucose from the beginning of culture, is superior to CZB or CZBG for supporting development of biologically viable blastocysts from EGFP-transgene-injected mouse embryos. Copyright 2003 Wiley-Liss, Inc.

MiR-285 targets P450 (CYP6N23) to regulate pyrethroid resistance in Culex pipiens pallens.

PubMed

Tian, Mengmeng; Liu, Bingqian; Hu, Hongxia; Li, Xixi; Guo, Qin; Zou, Feifei; Liu, Xianmiao; Hu, Mengxue; Guo, Juxin; Ma, Lei; Zhou, Dan; Sun, Yan; Shen, Bo; Zhu, Changliang

2016-12-01

MicroRNAs play critical roles in post-transcriptional regulation of gene expression, which participate in the modulation of almost all of the cellular processes. Although emerging evidence indicates that microRNAs are related with antineoplastic drugs resistance, whether microRNAs are responsible for insecticide resistance in mosquitos is poorly understood. In this paper, we found that miR-285 was significantly upregulated in the deltamethrin-resistant strain of Culex pipiens pallens, and overexpression miR-285 through microinjection increased mosquito survival rate against deltamethrin treatement. Using bioinformatic software, quantitative reverse transcription PCR, luciferase reporter assay and microinjection approaches, we conformed that CYP6N23 was the target of miR-285. Lower expression of CYP6N23 was observed in the deltamethrin-resistant strain. While, mosquito mortality rate was decreased after downregulating expression of CYP6N23 by dsRNA against CYP6N23 or miR-285 mimic microinjection. These findings revealed that miR-285 could target CYP6N23 to regulate pyrethroid resistance, providing new insights into mosquito insecticide resistance surveillance and control.

Leptin in the nucleus accumbens core disrupts acute cocaine effects: Implications for GSK3β connections.

PubMed

Lee, Jung Won; Kim, Wha Young; Cho, Bo Ram; Vezina, Paul; Kim, Jeong-Hoon

2018-01-30

An adipose-derived peptide hormone, leptin, has a regulatory role in reward-related behaviors produced by drugs of abuse. Although it is known that leptin modulates mesolimbic dopaminergic pathways, little is known about its direct role in the nucleus accumbens (NAcc). In the present study, we measured acute cocaine-induced locomotor activity in the rat and the phosphorylation levels of GSK3β after bilateral microinjections of leptin into the NAcc core. Interestingly, leptin in the NAcc core significantly disrupts acute cocaine's effects on both locomotor activity and signaling molecules. In order to further confirm the role of GSK3β in these processes, we microinjected S9 peptide, a small synthetic peptide acting as a competitive inhibitor against phosphorylation site of GSK3β, followed by leptin co-microinjection, and found that leptin's effects on cocaine were all nullified. These results indicate that leptin in the NAcc core has a negative regulatory role in acute cocaine' effects, and suggest that GSK3β may play a major role in mediating these processes. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of morphine-induced antinociception is altered by AF64A-induced lesions on cholinergic neurons in rat nucleus raphe magnus.

PubMed

Abe, Kenji; Ishida, Kota; Kato, Masatoshi; Shigenaga, Toshiro; Taguchi, Kyoji; Miyatake, Tadashi

2002-11-01

To examine the role of cholinergic neurons in the nucleus raphe magnus (NRM) in noxious heat stimulation and in the effects of morphine-induced antinociception by rats. After the cholinergic neuron selective toxin, AF64A, was microinjected into the NRM, we examined changes in the antinociceptive threshold and effects of morphine (5 mg/kg, ip) using the hot-plate (HP) and tail-flick (TF) tests. Systemic administration of morphine inhibited HP and TF responses in control rats. Microinjection of AF64A (2 nmol/site) into the NRM significantly decreased the threshold of HP response after 14 d, whereas the TF response was not affected. Morphine-induced antinociception was significantly attenuated in rats administered AF64A. Extracellular acetylcholine was attenuated after 14 d to below detectable levels in rats given AF64A. Naloxone (1 microg/site) microinjected into control rat NRM also antagonized the antinociceptive effect of systemic morphine. These findings suggest that cholinergic neuron activation in the NRM modulates the antinociceptive effect of morphine simultaneously with the opiate system.

Efficient mouse genome engineering by CRISPR-EZ technology.

PubMed

Modzelewski, Andrew J; Chen, Sean; Willis, Brandon J; Lloyd, K C Kent; Wood, Joshua A; He, Lin

2018-06-01

CRISPR/Cas9 technology has transformed mouse genome editing with unprecedented precision, efficiency, and ease; however, the current practice of microinjecting CRISPR reagents into pronuclear-stage embryos remains rate-limiting. We thus developed CRISPR ribonucleoprotein (RNP) electroporation of zygotes (CRISPR-EZ), an electroporation-based technology that outperforms pronuclear and cytoplasmic microinjection in efficiency, simplicity, cost, and throughput. In C57BL/6J and C57BL/6N mouse strains, CRISPR-EZ achieves 100% delivery of Cas9/single-guide RNA (sgRNA) RNPs, facilitating indel mutations (insertions or deletions), exon deletions, point mutations, and small insertions. In a side-by-side comparison in the high-throughput KnockOut Mouse Project (KOMP) pipeline, CRISPR-EZ consistently outperformed microinjection. Here, we provide an optimized protocol covering sgRNA synthesis, embryo collection, RNP electroporation, mouse generation, and genotyping strategies. Using CRISPR-EZ, a graduate-level researcher with basic embryo-manipulation skills can obtain genetically modified mice in 6 weeks. Altogether, CRISPR-EZ is a simple, economic, efficient, and high-throughput technology that is potentially applicable to other mammalian species.

L-glutamate microinjection in the preoptic area increases brain and body temperature in freely moving rats.

PubMed

Sengupta, Trina; Jaryal, Ashok K; Kumar, Velayudhan M; Mallick, Hruda N

2014-01-08

The role of the preoptic area (POA) in thermoregulation is well documented. Microinjection of various neurotransmitters into the POA in rats has been shown to influence body temperature. Alhough there are reports showing changes in temperature on administration of L-glutamate into the POA, the role of this excitatory amino acid in thermoregulation has not been studied in unanaesthetized rats. In the present study, brain and body temperatures were recorded in freely moving adult male Wistar rats with K-type thermocouple implanted near the hypothalamus and temperature transmitter implanted inside the peritoneum. Recordings were performed 2 h preinjection and 4 h postinjection. L-glutamate (0.14 nM) microinjection into the POA induced long-lasting hyperthermia and reduced locomotor activity. The rats remained curled up and showed piloerection. L-glutamate-induced hyperthermia was attenuated by previous injection of the ionotropic L-glutamate receptor antagonist, kynurenate (0.11 nM). We propose that L-glutamate in the POA participates not only in heat production and conservation but also plays a role in interlinking sleep and thermoregulation.

The use of additive and subtractive approaches to examine the nuclear localization sequence of the polyomavirus major capsid protein VP1

NASA Technical Reports Server (NTRS)

Chang, D.; Haynes, J. I. 2nd; Brady, J. N.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1992-01-01

A nuclear localization signal (NLS) has been identified in the N-terminal (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) amino acid sequence of the polyomavirus major capsid protein VP1. The importance of this amino acid sequence for nuclear transport of VP1 protein was demonstrated by a genetic "subtractive" study using the constructs pSG5VP1 (full-length VP1) and pSG5 delta 5'VP1 (truncated VP1, lacking amino acids Ala1-Cys11). These constructs were used to transfect COS-7 cells, and expression and intracellular localization of the VP1 protein was visualized by indirect immunofluorescence. These studies revealed that the full-length VP1 was expressed and localized in the nucleus, while the truncated VP1 protein was localized in the cytoplasm and not transported to the nucleus. These findings were substantiated by an "additive" approach using FITC-labeled conjugates of synthetic peptides homologous to the NLS of VP1 cross-linked to bovine serum albumin or immunoglobulin G. Both conjugates localized in the nucleus after microinjection into the cytoplasm of 3T6 cells. The importance of individual amino acids found in the basic sequence (Lys3-Arg-Lys5) of the NLS was also investigated. This was accomplished by synthesizing three additional peptides in which lysine-3 was substituted with threonine, arginine-4 was substituted with threonine, or lysine-5 was substituted with threonine. It was found that lysine-3 was crucial for nuclear transport, since substitution of this amino acid with threonine prevented nuclear localization of the microinjected, FITC-labeled conjugate.

Activation of the proteasomes of sand dollar eggs at fertilization depends on the intracellular pH rise.

PubMed

Chiba, K; Alderton, J M; Hoshi, M; Steinhardt, R A

1999-05-01

The mechanism of the activation of intracellular proteasomes at fertilization was measured in living sand dollar eggs using the membrane-impermeant fluorogenic substrate, succinyl-Phe-Leu-Arg-coumarylamido-4-methanesulfonic acid. When the substrate was microinjected into unfertilized eggs, the initial velocity of hydrolysis of the substrate (V0) was low. V0 measured 5 to 10 min after fertilization was five to nine times the prefertilization level and remained high throughout the first cell cycle. Hydrolysis of the substrate was inhibited by clasto-lactacystin beta-lactone, a specific inhibitor of the proteasome. There has been in vitro evidence that calcium may be involved in regulation of proteasome activity to either inhibit the increase in peptidase activity associated with PA 28 binding to the 20S proteasome or stimulate activity of the PA 700-proteasome complex. Since both intracellular free Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) increase after fertilization, hydrolysis of the proteasome substrate was measured under conditions in which [Ca2+]i and pHi were varied independently during activation. When the pHi of unfertilized eggs was elevated by exposure to 15 mM ammonium chloride in pH 9 seawater, V0 increased to a level comparable to that measured after fertilization. In contrast, [Ca2+]i elevation without pHi change, induced by calcium ionophore in sodium-free seawater, had no effect on V0 in the unfertilized egg. Moreover, when unfertilized eggs were microinjected with buffers modulating pHi, V0 increased in a pH-dependent manner. These results indicate that the pHi rise at fertilization is the necessary prerequisite for activation of the proteasome, an essential component in the regulation of the cell cycle. Copyright 1999 Academic Press.

Role of the parabrachial complex in the cardiorespiratory response evoked from hypothalamic defense area stimulation in the anesthetized rat.

PubMed

Díaz-Casares, Amelia; López-González, Manuel Víctor; Peinado-Aragonés, Carlos Antonio; Lara, José Pablo; González-Barón, Salvador; Dawid-Milner, Marc Stefan

2009-07-07

To analyze the role of parabrachial complex (PBc) in the modulation of cardiorespiratory response evoked from the hypothalamic defense area (HDA), cardiorespiratory changes were analyzed in spontaneously breathing anesthetised rats in response to electrical stimulation of the HDA (1 ms pulses, 30-50 microA, 100 Hz for 5 s) before and after the microinjection of muscimol (50 nl, 0.25 nmol, 5 s) within the PBc. HDA stimulation evoked an inspiratory facilitatory response, consisting of an increase in respiratory rate (p<0.001) due to a decrease in expiratory time (p<0.01). The respiratory response was accompanied by a pressor (p<0.001) and a tachycardic (p<0.001) response. Muscimol microinjection within the lateral parabrachial region (lPB) abolished the respiratory response to HDA stimulation (p<0.01) and decreased the pressor response (p<0.05). Muscimol within the medial parabrachial region and Kölliker-Fuse (mPB-KF) decreased the magnitude of the pressor (p<0.01) and tachycardic (p<0.05) responses to HDA stimulation. The respiratory response persisted unchanged. Finally, extracellular recording of putative neurons from these regions were obtained during HDA stimulation to confirm functional interaction between HDA and parabrachial regions. 105 pontine cells were recorded during HDA stimulation, 57 from the lPB and 48 from the mPB-KF. In mPB-KF 34/48 (71%) and in lPB 38/57 (67%) cells were influenced from HDA. The results indicate that neurons from different regions of the PBc have an important function in mediating the cardiorespiratory response evoked from the HDA. The possible mechanisms involved in these interactions are discussed.

Disinhibition of perifornical hypothalamic neurones activates noradrenergic neurones and blocks pontine carbachol-induced REM sleep-like episodes in rats

PubMed Central

Lu, Jackie W; Fenik, Victor B; Branconi, Jennifer L; Mann, Graziella L; Rukhadze, Irma; Kubin, Leszek

2007-01-01

Studies in behaving animals suggest that neurones located in the perifornical (PF) region of the posterior hypothalamus promote wakefulness and suppress sleep. Among such cells are those that synthesize the excitatory peptides, orexins (ORX). Lack of ORX, or their receptors, is associated with narcolepsy/cataplexy, a disorder characterized by an increased pressure for rapid eye movement (REM) sleep. We used anaesthetized rats in which pontine microinjections of a cholinergic agonist, carbachol, can repeatedly elicit REM sleep-like episodes to test whether activation of PF cells induced by antagonism of endogenous, GABAA receptor-mediated, inhibition suppresses the ability of the brainstem to generate REM sleep-like state. Microinjections of the GABAA receptor antagonist, bicuculline (20 nl, 1 mm), into the PF region elicited cortical and hippocampal activation, increased the respiratory rate and hypoglossal nerve activity, induced c-fos expression in ORX and other PF neurones, and increased c-fos expression in pontine A7 and other noradrenergic neurones. The ability of pontine carbachol to elicit any cortical, hippocampal or brainstem component of the REM sleep-like response was abolished during the period of bicuculline-induced activation. The activating and REM sleep-suppressing effect of PF bicuculline was not attenuated by systemic administration of the ORX type 1 receptor antagonist, SB334867. Thus, activation of PF neurones that are endogenously inhibited by GABAA receptors is sufficient to turn off the brainstem REM sleep-generating network; the effect is, at least in part, due to activation of pontine noradrenergic neurones, but is not mediated by ORX type 1 receptors. A malfunction of the pathway that originates in GABAA receptor-expressing PF neurones may cause narcolepsy/cataplexy. PMID:17495048

Glial interleukin-1β upregulates neuronal sodium channel 1.7 in trigeminal ganglion contributing to temporomandibular joint inflammatory hypernociception in rats.

PubMed

Zhang, Peng; Bi, Rui-Yun; Gan, Ye-Hua

2018-04-20

The proinflammatory cytokine interleukin-1β (IL-1β) drives pain by inducing the expression of inflammatory mediators; however, its ability to regulate sodium channel 1.7 (Nav1.7), a key driver of temporomandibular joint (TMJ) hypernociception, remains unknown. IL-1β induces cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2). We previously showed that PGE2 upregulated trigeminal ganglionic Nav1.7 expression. Satellite glial cells (SGCs) involve in inflammatory pain through glial cytokines. Therefore, we explored here in the trigeminal ganglion (TG) whether IL-1β upregulated Nav1.7 expression and whether the IL-1β located in the SGCs upregulated Nav1.7 expression in the neurons contributing to TMJ inflammatory hypernociception. We treated rat TG explants with IL-1β with or without inhibitors, including NS398 for COX-2, PF-04418948 for EP2, and H89 and PKI-(6-22)-amide for protein kinase A (PKA), or with adenylate cyclase agonist forskolin, and used real-time PCR, Western blot, and immunohistofluorescence to determine the expressions or locations of Nav1.7, COX-2, cAMP response element-binding protein (CREB) phosphorylation, and IL-1β. We used chromatin immunoprecipitation to examine CREB binding to the Nav1.7 promoter. Finally, we microinjected IL-1β into the TGs or injected complete Freund's adjuvant into TMJs with or without previous microinjection of fluorocitrate, an inhibitor of SGCs activation, into the TGs, and evaluated nociception and gene expressions. Differences between groups were examined by one-way analysis of variance (ANOVA) or independent samples t test. IL-1β upregulated Nav1.7 mRNA and protein expressions in the TG explants, whereas NS398, PF-04418948, H89, or PKI-(6-22)-amide could all block this upregulation, and forskolin could also upregulate Nav1.7 mRNA and protein expressions. IL-1β enhanced CREB binding to the Nav1.7 promoter. Microinjection of IL-1β into the TGs or TMJ inflammation both induced hypernociception of TMJ region and correspondingly upregulated COX-2, phospho-CREB, and Nav1.7 expressions in the TGs. Moreover, microinjection of fluorocitrate into the TGs completely blocked TMJ inflammation-induced activation of SGCs and the upregulation of IL-1β and COX-2 in the SGCs, and phospho-CREB and Nav1.7 in the neurons and alleviated inflammation-induced TMJ hypernociception. Glial IL-1β upregulated neuronal Nav1.7 expression via the crosstalk between signaling pathways of the glial IL-1β/COX-2/PGE2 and the neuronal EP2/PKA/CREB/Nav1.7 in TG contributing to TMJ inflammatory hypernociception.

Single-cell manipulation and DNA delivery technology using atomic force microscopy and nanoneedle.

PubMed

Han, Sung-Woong; Nakamura, Chikashi; Miyake, Jun; Chang, Sang-Mok; Adachi, Taiji

2014-01-01

The recent single-cell manipulation technology using atomic force microscopy (AFM) not only allows high-resolution visualization and probing of biomolecules and cells but also provides spatial and temporal access to the interior of living cells via the nanoneedle technology. Here we review the development and application of single-cell manipulations and the DNA delivery technology using a nanoneedle. We briefly describe various DNA delivery methods and discuss their advantages and disadvantages. Fabrication of the nanoneedle, visualization of nanoneedle insertion into living cells, DNA modification on the nanoneedle surface, and the invasiveness of nanoneedle insertion into living cells are described. Different methods of DNA delivery into a living cell, such as lipofection, microinjection, and nanoneedles, are then compared. Finally, single-cell diagnostics using the nanoneedle and the perspectives of the nanoneedle technology are outlined. The nanoneedle-based DNA delivery technology provides new opportunities for efficient and specific introduction of DNA and other biomolecules into precious living cells with a high spatial resolution within a desired time frame. This technology has the potential to be applied for many basic cellular studies and for clinical studies such as single-cell diagnostics.

Excitatory innervation of caudal hypoglossal nucleus from nucleus reticularis gigantocellularis in the rat.

PubMed

Yang, C C; Chan, J Y; Chan, S H

1995-03-01

We examined the possible innervation of the caudal hypoglossal nucleus by the nucleus reticularis gigantocellularis of the medulla oblongata, based on single-neuron recording and retrograde tracing experiments in Sprague-Dawley rats. Under pentobarbital sodium (50 mg/kg, i.p.) anesthesia, electrical stimulation of the caudal portion of the nucleus reticularis gigantocellularis with repetitive 0.5-ms rectangular pulses increased (46 of 51 neurons) the basal discharge frequency of spontaneously active cells, or evoked spike activity in silent, hypoglossal neurons located at the level of the obex. This excitatory effect was related to the intensity (25-100 microA) and/or frequency (0.5-20 Hz) of the stimulating pulses to the nucleus reticularis gigantocellularis. Perikaryal activation of neurons by microinjection of L-glutamate (0.5 nmol, 25 nl) into the caudal portion of the nucleus reticularis gigantocellularis similarly produced an excitatory action on eight of 14 hypoglossal neurons. Retrogradely labeled neurons were found bilaterally within the confines of the nucleus reticularis gigantocellularis following unilateral microinjection of wheatgerm agglutinin-conjugated horseradish peroxidase or Fast Blue into the corresponding hypoglossal recording sites. Furthermore, the distribution of labeled neurons in the nucleus reticularis gigantocellularis substantially overlapped with the loci of electrical or chemical stimulation. These complementary electrophysiological and neuroanatomical results support the conclusion that an excitatory link exists between the nucleus reticularis gigantocellularis and at least the caudal portion of the hypoglossal nucleus in the rat.

Overexpression of parkin in the rat nigrostriatal dopamine system protects against methamphetamine neurotoxicity.

PubMed

Liu, Bin; Traini, Roberta; Killinger, Bryan; Schneider, Bernard; Moszczynska, Anna

2013-09-01

Methamphetamine (METH) is a central nervous system psychostimulant with a high potential for abuse. At high doses, METH causes a selective degeneration of dopaminergic terminals in the striatum, sparing other striatal terminals and cell bodies. We previously detected a deficit in parkin after binge METH in rat striatal synaptosomes. Parkin is an ubiquitin-protein E3 ligase capable of protecting dopamine neurons from diverse cellular insults. Whether the deficit in parkin mediates the toxicity of METH and whether parkin can protect from toxicity of the drug is unknown. The present study investigated whether overexpression of parkin attenuates degeneration of striatal dopaminergic terminals exposed to binge METH. Parkin overexpression in rat nigrostriatal dopamine system was achieved by microinjection of adeno-associated viral transfer vector 2/6 encoding rat parkin (AAV2/6-parkin) into the substantia nigra pars compacta. The microinjections of AAV2/6-parkin dose-dependently increased parkin levels in both the substantia nigra pars compacta and striatum. The levels of dopamine synthesizing enzyme, tyrosine hydroxylase, remained at the control levels; therefore, tyrosine hydroxylase immunoreactivity was used as an index of dopaminergic terminal integrity. In METH-exposed rats, the increase in parkin levels attenuated METH-induced decreases in striatal tyrosine hydroxylase immunoreactivity in a dose-dependent manner, indicating that parkin can protect striatal dopaminergic terminals against METH neurotoxicity. Copyright © 2013 Elsevier Inc. All rights reserved.

Overexpression of parkin in rat nigrostriatal dopamine system protects against methamphetamine neurotoxicity

PubMed Central

Liu, Bin; Traini, Roberta; Killinger, Bryan; Schneider, Bernard; Moszczynska, Anna

2013-01-01

Methamphetamine (METH) is a central nervous system psychostimulant with a high potential for abuse. At high doses, METH causes a selective degeneration of dopaminergic terminals in the striatum, sparing other striatal terminals and cell bodies. We previously detected a deficit in parkin after binge METH in rat striatal synaptosomes. Parkin is an ubiquitin-protein E3 ligase capable of protecting dopamine neurons from diverse cellular insults. Whether the deficit in parkin mediates the toxicity of METH and whether parkin can protect from toxicity of the drug is unknown. The present study investigated whether overexpression of parkin attenuates degeneration of striatal dopaminergic terminals exposed to binge METH. Parkin overexpression in rat nigrostriatal dopamine system was achieved by microinjection of adeno-associated viral transfer vector 2/6 encoding rat parkin (AAV2/6-parkin) into the substantia nigra pars compacta. The microinjections of AAV2/6-parkin dose-dependently increased parkin levels in both the substantia nigra pars compacta and striatum. The levels of dopamine synthesizing enzyme, tyrosine hydroxylase, remained at the control levels; therefore, tyrosine hydroxylase immunoreactivity was used as an index of dopaminergic terminal integrity. In METH-exposed rats, the increase in parkin levels attenuated METH-induced decreases in striatal tyrosine hydroxylase immunoreactivity in a dose-dependent manner, indicating that parkin can protect striatal dopaminergic terminals against METH neurotoxicity. PMID:23313192

Cell response to long term mechanical interaction with nanopipettes

NASA Astrophysics Data System (ADS)

Orynbayeva, Zulfiya; Singhal, Riju; Vitol, Elina; Bouchard, Michael; Azizkhan-Clifford, Jane; Layton, Bradley; Friedman, Gary; Gogotsi, Yury

2009-03-01

Traditional microinjection into cells is performed over a relatively short term. Pipettes are typically withdrawn following any kind of injection. On the other hand, there is growing interest in using nanopipettes for cellular and subcellular probing. This interest is partly due to new developments in nanopipette technology which employ carbon nanotubes and provide robustness, flexibility, and biocompatibility. However, as far as we know, no systematic study of physiological, biochemical, and biophysical processes associated with cell response to lengthy mechanical stimulations by nanopipette probing have been performed so far. We present a detailed investigation of a wide range of effects of long term pipette insertion into a cell. Both traditional glass micropipettes and the novel carbon nanotube-tipped probes were involved in this study. The mechanism of Ca2+ response to the mechanical stimuli introduced by the nanopipette, and the role of different organelles in this mechanism were studied. We hypothesize that the calcium response is a function of cytoskeleton integrity and the mode of coupling between the cytoskeleton and the plasma membrane domains.

IL4/STAT6 Signaling Activates Neural Stem Cell Proliferation and Neurogenesis upon Amyloid-β42 Aggregation in Adult Zebrafish Brain.

PubMed

Bhattarai, Prabesh; Thomas, Alvin Kuriakose; Cosacak, Mehmet Ilyas; Papadimitriou, Christos; Mashkaryan, Violeta; Froc, Cynthia; Reinhardt, Susanne; Kurth, Thomas; Dahl, Andreas; Zhang, Yixin; Kizil, Caghan

2016-10-18

Human brains are prone to neurodegeneration, given that endogenous neural stem/progenitor cells (NSPCs) fail to support neurogenesis. To investigate the molecular programs potentially mediating neurodegeneration-induced NSPC plasticity in regenerating organisms, we generated an Amyloid-β42 (Aβ42)-dependent neurotoxic model in adult zebrafish brain through cerebroventricular microinjection of cell-penetrating Aβ42 derivatives. Aβ42 deposits in neurons and causes phenotypes reminiscent of amyloid pathophysiology: apoptosis, microglial activation, synaptic degeneration, and learning deficits. Aβ42 also induces NSPC proliferation and enhanced neurogenesis. Interleukin-4 (IL4) is activated primarily in neurons and microglia/macrophages in response to Aβ42 and is sufficient to increase NSPC proliferation and neurogenesis via STAT6 phosphorylation through the IL4 receptor in NSPCs. Our results reveal a crosstalk between neurons and immune cells mediated by IL4/STAT6 signaling, which induces NSPC plasticity in zebrafish brains. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Microspectrofluorometry for metabolic control analysis and the study of organelle morphogenesis in cell differentiation and transformation

NASA Astrophysics Data System (ADS)

Hirschberg, Joseph G.; Kohen, Elli; Kohen, Cahide; Pinon, Raul

1994-02-01

Microspectrofluorometry has been used in conjunction with fluorescence micrography for metabolic control analysis in normal and genetically deficient human fibroblasts, as well as human melanoma cells. These studies point to the role of mitochondria as the `cell's policeman' with regard to metabolic control. Cytotoxic agents active on mitochondrial structure and function (i.e. anthralin, azelaic acid) produce an unleashing of extramitochondrial pathways characterized by large and out-of-control NAD(P)H transients elicited by microinjected substrates. An interesting aspect has been the demonstration of an active nuclear energy metabolism, by NAD(P)H fluorescence excited at 365 nm, which may help to link cell bioenergetics to gene expression in the eukaryotes by the use of DNA probes. The metabolic control analysis of cell bioenergetics has been extended to the pathways involved in the cell's handling of cytotoxic agents. Non invasive fluorescence equipment offers possibilities for diagnostics and therapeutics in dermatology. Structure and function studies can be carried out at considerably enhanced resolution and with on-line interpretation by introducing scanning nearfield optics microscopy (SNOM) and real-time interactive parameter experimentation control (RIPEC).

In vivo oestrogenic modulation of Egr1 and Pitx1 gene expression in female rat pituitary gland.

PubMed

Gajewska, Alina; Herman, Andrzej P; Wolińska-Witort, Ewa; Kochman, Kazimierz; Zwierzchowski, Lech

2014-12-01

EGR1 and PITX1 are transcription factors required for gonadotroph cell Lhb promoter activation. To determine changes in Egr1 and Pitx1 mRNA levels in central and peripheral pituitary stimulations, an in vivo model based on i.c.v. pulsatile (1 pulse/0.5 h over 2 h) GnRH agonist (1.5 nM buserelin) or antagonist (2 nM antide) microinjections was used. The microinjections were given to ovariectomised and 17β-oestradiol (E2) (3×20 μg), ERA (ESR1) agonist propyl pyrazole triol (PPT) (3×0.5 mg), ERB (ESR2) agonist diarylpropionitrile (DPN) (3×0.5 mg) s.c. pre-treated rats 30 min after last pulse anterior pituitaries were excised. Relative mRNA expression was determined by quantitative RT-PCR (qRT-PCR). Results revealed a gene-specific response for GnRH and/or oestrogenic stimulations in vivo. Buserelin pulses enhanced Egr1 expression by 66% in ovariectomised rats, whereas the oestradiol-supplemented+i.c.v. NaCl-microinjected group showed a 50% increase in Egr1 mRNA expression. The oestrogenic signal was transmitted via ERA (ESR1) and ERB (ESR2) activation as administration of PPT and DPN resulted in 97 and 62%, respectively, elevation in Egr1 mRNA expression. A synergistic action of GnRH agonist and 17β-oestradiol (E2) stimulation of the Egr1 gene transcription in vivo were found. GnRHR activity did not affect Pitx1 mRNA expression; regardless of NaCl, buserelin or antide i.c.v. pulses, s.c. oestrogenic supplementation (with E2, PPT or DPN) consistently decreased (by -46, -48 and -41% respectively) the Pitx1 mRNA in the anterior pituitary gland. Orchestrated Egr1 and Pitx1 activities depending on specific central and peripheral regulatory inputs could be responsible for physiologically variable Lhb gene promoter activation in vivo. © 2014 Society for Endocrinology.

Properties of a U1 RNA enhancer-like sequence.

PubMed Central

Ciliberto, G; Palla, F; Tebb, G; Mattaj, I W; Philipson, L

1987-01-01

The properties of a X.laevis U1B snRNA gene enhancer have been studied by microinjection in Xenopus oocytes. The enhancer-like sequence, defined as a short DNA stretch that is able to activate transcription in an orientation independent manner, is interchangeable between different U snRNA genes. The enhancer sequence alone does not, however, efficiently activate transcription from an SV40 pol II promoter but regains its activity when combined with the U-gene specific proximal sequence element. DNase I protection experiments show that the X.laevis U1B enhancer can interact specifically with a nuclear factor present in mammalian cells. Images PMID:3031597

TRANSCUTANEOUS ELECTRICAL NERVE STIMULATION AT BOTH HIGH AND LOW FREQUENCIES ACTIVATES VENTROLATERAL PERIAQUEDUCTAL GREY TO DECREASE MECHANICAL HYPERALGESIA IN ARTHRITIC RATS

PubMed Central

Desantana, J. M.; Da Silva, L. F. S.; De Resende, M. A.; Sluka, K. A.

2014-01-01

Transcutaneous electric nerve stimulation (TENS) is widely used for the treatment of pain. TENS produces an opioid-mediated antinociception that utilizes the rostroventromedial medulla (RVM). Similarly, antinociception evoked from the periaqueductal grey (PAG) is opioid-mediated and includes a relay in the RVM. Therefore, we investigated whether the ventrolateral or dorsolateral PAG mediates antinociception produced by TENS in rats. Paw and knee joint mechanical withdrawal thresholds were assessed before and after knee joint inflammation (3% kaolin/carrageenan), and after TENS stimulation (active or sham). Cobalt chloride (CoCl2; 5 mM) or vehicle was microinjected into the ventrolateral periaqueductal grey (vlPAG) or dorsolateral periaqueductal grey (dlPAG) prior to treatment with TENS. Either high (100 Hz) or low (4 Hz) frequency TENS was then applied to the inflamed knee for 20 min. Active TENS significantly increased withdrawal thresholds of the paw and knee joint in the group microinjected with vehicle when compared to thresholds prior to TENS (P<0.001) or to sham TENS (P<0.001). The increases in withdrawal thresholds normally observed after TENS were prevented by microinjection of CoCl2 into the vlPAG, but not the dlPAG prior to TENS and were significantly lower than controls treated with TENS (P<0.001). In a separate group of animals, microinjection of CoCl2 into the vlPAG temporarily reversed the decreased mechanical withdrawal threshold suggesting a role for the vlPAG in the facilitation of joint pain. No significant difference was observed for dlPAG. We hypothesize that the effects of TENS are mediated through the vlPAG that sends projections through the RVM to the spinal cord to produce an opioid-mediated analgesia. PMID:19576962

Electroacupuncture modulation of reflex hypertension in rats: role of cholecystokinin octapeptide

PubMed Central

Tjen-A-Looi, Stephanie C.; Guo, Zhi-Ling; Longhurst, John C.

2013-01-01

Acupuncture or electroacupuncture (EA) potentially offers a nonpharmacological approach to reduce high blood pressure (BP). However, ∼70% of the patients and animal subjects respond to EA, while 30% do not. EA acts, in part, through an opioid mechanism in the rostral ventrolateral medulla (rVLM) to inhibit sympathoexcitatory reflexes induced by gastric distention. CCK-8 opposes the action of opioids during analgesia. Therefore, we hypothesized that CCK-8 in the rVLM antagonizes EA modulation of sympathoexcitatory cardiovascular reflex responses. Male rats anesthetized with ketamine and α-chloralose subjected to repeated gastric distension every 10 min were examined for their responsiveness to EA (2 Hz, 0.5 ms, 1–4 mA) at P5-P6 acupoints overlying median nerve. Repeated gastric distension every 10 min evoked consistent sympathoexcitatory responses. EA at P5-P6 modulated gastric distension-induced responses. Microinjection of CCK-8 in the rVLM reversed the EA effect in seven responders. The CCK1 receptor antagonist devazepide microinjected into the rVLM converted six nonresponders to responders by lowering the reflex response from 21 ± 2.2 to 10 ± 2.9 mmHg (first vs. second application of EA). The EA modulatory action in rats converted to responders with devazepide was reversed with rVLM microinjection of naloxone (n = 6). Microinjection of devazepide in the absence of a second application of EA did not influence the primary pressor reflexes of nonresponders. These data suggest that CCK-8 antagonizes EA modulation of sympathoexcitatory cardiovascular responses through an opioid mechanism and that inhibition of CCK-8 can convert animals that initially are unresponsive to EA to become responsive. PMID:23785073

Differential involvement of prelimbic and infralimbic medial prefrontal cortex in discrete cue-induced reinstatement of 3,4-methylenedioxymethamphetamine (MDMA; ecstasy) seeking in rats

PubMed Central

Ball, Kevin T.; Slane, Mylissa

2014-01-01

Rationale The amphetamine derivative 3,4-methylenedioxymethamphetamine(MDMA; ecstasy) is a widely abused drug, particularly in adolescent and young-adult populations. Although it was shown that MDMA-associated cues reinstate extinguished MDMA seeking in an animal relapse model, there is little information regarding the neural mechanisms underlying this behavior. Objectives Because the medial prefrontal cortex (mPFC) plays an important role in relapse to cocaine and methamphetamine seeking, we tested the effects of lidocaine inactivation of prelimbic(PL) and infralimbic (IL)subregions of mPFC on cue-induced relapse to MDMA seeking. Methods Rats were trained to respond for MDMA infusions (0.50 mg/kg/infusion, i.v.) paired with a discrete cue in daily 2-hr sessions. Responding was reinforced contingent on a modified fixed-ratio 5 schedule of reinforcement. Cue-induced reinstatement tests were conducted after responding was extinguished in the absence of MDMA and the conditioned cues. Prior to reinstatement tests, rats received bilateral microinjections of either lidocaine (100 µg/0.5 µl/side) or physiological saline (0.5 µl/side) delivered to either PL or IL mPFC. Results Microinjections of lidocaine into PL completely blocked reinstatement of MDMA-seeking behavior compared with saline microinjections into the same region. Lidocaine microinjections did not, however, have an effect on food-maintained responding, ruling out a nonspecific disruption of motor performance. Conversely, lidocaine inactivation of IL had no effect on reinstatement of MDMA seeking or food-maintained responding. Conclusions Our results provide direct support for PL activation in reinstatement of MDMA-seeking behavior. Moreover, akin to cocaine seeking, there appears to be differential involvement of PL and IL subregions in this behavior. PMID:22710489

Ponto-medullary nuclei involved in the generation of sequential pharyngeal swallowing and concomitant protective laryngeal adduction in situ

PubMed Central

Bautista, Tara G; Dutschmann, Mathias

2014-01-01

Both swallowing and respiration involve postinspiratory laryngeal adduction. Swallowing-related postinspiratory neurons are likely to be located in the nucleus of the solitary tract (NTS) and those involved in respiration are found in the Kölliker–Fuse nucleus (KF). The function of KF and NTS in the generation of swallowing and its coordination with respiration was investigated in perfused brainstem preparations of juvenile rats (n = 41). Orally injected water evoked sequential pharyngeal swallowing (s-PSW) seen as phasic, spindle-shaped bursting of vagal nerve activity (VNA) against tonic postinspiratory discharge. KF inhibition by microinjecting isoguvacine (GABAA receptor agonist) selectively attenuated tonic postinspiratory VNA (n = 10, P < 0.001) but had no effect on frequency or timing of s-PSW. KF disinhibition after bicuculline (GABAA receptor antagonist) microinjections caused an increase of the tonic VNA (n = 8, P < 0.01) resulting in obscured and delayed phasic s-PSW. Occurrence of spontaneous PSW significantly increased after KF inhibition (P < 0.0001) but not after KF disinhibition (P = 0.14). NTS isoguvacine microinjections attenuated the occurrence of all PSW (n = 5, P < 0.01). NTS bicuculline microinjections (n = 6) resulted in spontaneous activation of a disordered PSW pattern and long-lasting suppression of respiratory activity. Pharmacological manipulation of either KF or NTS also triggered profound changes in respiratory postinspiratory VNA. Our results indicate that the s-PSW comprises two functionally distinct components. While the primary s-PSW is generated within the NTS, a KF-mediated laryngeal adductor reflex safeguards the lower airways from aspiration. Synaptic interaction between KF and NTS is required for s-PSW coordination with respiration as well as for proper gating and timing of s-PSW. PMID:24639482

Involvement of prelimbic medial prefrontal cortex in panic-like elaborated defensive behaviour and innate fear-induced antinociception elicited by GABAA receptor blockade in the dorsomedial and ventromedial hypothalamic nuclei: role of the endocannabinoid CB1 receptor.

PubMed

Freitas, Renato Leonardo de; Salgado-Rohner, Carlos José; Hallak, Jaime Eduardo Cecílio; Crippa, José Alexandre de Souza; Coimbra, Norberto Cysne

2013-09-01

It has been shown that GABAA receptor blockade in the dorsomedial and ventromedial hypothalamic nuclei (DMH and VMH, respectively) induces elaborated defensive behavioural responses accompanied by antinociception, which has been utilized as an experimental model of panic attack. Furthermore, the prelimbic (PL) division of the medial prefrontal cortex (MPFC) has been related to emotional reactions and the processing of nociceptive information. The aim of the present study was to investigate the possible involvement of the PL cortex and the participation of local cannabinoid CB1 receptors in the elaboration of panic-like reactions and in innate fear-induced antinociception. Elaborated fear-induced responses were analysed during a 10-min period in an open-field test arena. Microinjection of the GABAA receptor antagonist bicuculline into the DMH/VMH evoked panic-like behaviour and fear-induced antinociception, which was decreased by microinjection of the non-selective synaptic contact blocker cobalt chloride in the PL cortex. Moreover, microinjection of AM251 (25, 100 or 400 pmol), an endocannabinoid CB1 receptor antagonist, into the PL cortex also attenuated the defensive behavioural responses and the antinociception that follows innate fear behaviour elaborated by DMH/VMH. These data suggest that the PL cortex plays an important role in the organization of elaborated forward escape behaviour and that this cortical area is also involved in the elaboration of innate fear-induced antinociception. Additionally, CB1 receptors in the PL cortex modulate both panic-like behaviours and fear-induced antinociception elicited by disinhibition of the DMH/VMH through microinjection of bicuculline.

Contribution of hippocampal area CA1 to acetone cyanohydrin-induced loss of motor coordination in rats.

PubMed

Rivadeneyra-Domínguez, E; Vázquez-Luna, A; Díaz-Sobac, R; Briones-Céspedes, E E; Rodríguez-Landa, J F

2017-05-01

Some vegetable foodstuffs contain toxic compounds that, when consumed, favour the development of certain diseases. Cassava (Manihot esculenta Crantz) is an important food source, but it contains cyanogenic glucosides (linamarin and lotaustralin) that have been associated with the development of tropical ataxic neuropathy and konzo. In rats, intraperitoneal administration of acetone cyanohydrin (a metabolite of linamarin) produces neurological disorders and neuronal damage in the hippocampus. However, it is unknown whether hippocampal area CA1 plays a role in neurological disorders associated with acetone cyanohydrin. A total of 32 male Wistar rats 3 months old were assigned to 4 groups (n=8 per group) as follows: vehicle (1μl physiological saline), and 3 groups with acetone cyanohydrin (1μl of 10, 15, and 20mM solution, respectively). The substances were microinjected intrahippocampally every 24hours for 7 consecutive days, and their effects on locomotor activity, rota-rod and swim tests were assessed daily. On the fifth day post-treatment, rats underwent further assessment with behavioural tests to identify or rule out permanent damage induced by acetone cyanohydrin. Microinjection of acetone cyanohydrin 20mM resulted in hyperactivity, motor impairment, and reduced exploration from the third day of treatment. All concentrations of acetone cyanohydrin produced rotational behaviour in the swim test from the first day of microinjection. The hippocampal area CA1 is involved in motor alterations induced by microinjection of acetone cyanohydrin, as has been reported for other cassava compounds. Copyright © 2015 Sociedad Española de Neurología. Publicado por Elsevier España, S.L.U. All rights reserved.

BDNF and AMPA receptors in the cNTS modulate the hyperglycemic reflex after local carotid body NaCN stimulation.

PubMed

Cuéllar, R; Montero, S; Luquín, S; García-Estrada, J; Melnikov, V; Virgen-Ortiz, A; Lemus, M; Pineda-Lemus, M; de Álvarez-Buylla, E

2017-07-01

The application of sodium cyanide (NaCN) to the carotid body receptors (CBR) (CBR stimulation) induces rapid blood hyperglycemia and an increase in brain glucose retention. The commissural nucleus tractus solitarius (cNTS) is an essential relay nucleus in this hyperglycemic reflex; it receives glutamatergic afferents (that also release brain derived neurotrophic factor, BDNF) from the nodose-petrosal ganglia that relays CBR information. Previous work showed that AMPA in NTS blocks hyperglycemia and brain glucose retention after CBR stimulation. In contrast, BDNF, which attenuates glutamatergic AMPA currents in NTS, enhances these glycemic responses. Here we investigated the combined effects of BDNF and AMPA (and their antagonists) in NTS on the glycemic responses to CBR stimulation. Microinjections of BDNF plus AMPA into the cNTS before CBR stimulation in anesthetized rats, induced blood hyperglycemia and an increase in brain arteriovenous (a-v) of blood glucose concentration difference, which we infer is due to increased brain glucose retention. By contrast, the microinjection of the TrkB antagonist K252a plus AMPA abolished the glycemic responses to CBR stimulation similar to what is observed after AMPA pretreatments. In BDNF plus AMPA microinjections preceding CBR stimulation, the number of c-fos immunoreactive cNTS neurons increased. In contrast, in the rats microinjected with K252a plus AMPA in NTS, before CBR stimulation, c-fos expression in cNTS decreased. The expression of AMPA receptors GluR2/3 did not change in any of the studied groups. These results indicate that BDNF in cNTS plays a key role in the modulation of the hyperglycemic reflex initiated by CBR stimulation. Copyright © 2017. Published by Elsevier B.V.

Ellagic acid ameliorates learning and memory deficits in a rat model of Alzheimer's disease: an exploration of underlying mechanisms.

PubMed

Kiasalari, Zahra; Heydarifard, Rana; Khalili, Mohsen; Afshin-Majd, Siamak; Baluchnejadmojarad, Tourandokht; Zahedi, Elham; Sanaierad, Ashkan; Roghani, Mehrdad

2017-06-01

Alzheimer's disease (AD) is a neurodegenerative disorder with irreversible loss of intellectual abilities. Current therapies for AD are still insufficient. In this study, the effect of ellagic acid on learning and memory deficits was evaluated in intrahippocampal amyloid beta (Aβ 25-35 )-microinjected rats and its modes of action were also explored. AD rat model was induced by bilateral intrahippocampal microinjection of Aβ 25-35 and ellagic acid was daily administered (10, 50, and 100 mg/kg), and learning, recognition memory, and spatial memory were evaluated in addition to histochemical assessment, oxidative stress, cholinesterases activity, and level of nuclear factor-kappaB (NF-κB), Toll-like receptor 4 (TLR4), and nuclear factor (erythroid-derived 2)-like 2 (Nrf2). The amyloid beta-microinjected rats showed a lower discrimination ratio in novel object and alternation score in Y maze tasks and exhibited an impairment of retention and recall capability in passive avoidance paradigm and higher working and reference memory errors in radial arm maze (RAM). In addition, amyloid beta group showed a lower number of Nissl-stained neurons in CA1 area in addition to enhanced oxidative stress, higher activity of cholinesterases, greater level of NF-κB and TLR4, and lower level of nuclear/cytoplasmic ratio for Nrf2 and ellagic acid at a dose of 100 mg/kg significantly prevented most of these abnormal alterations. Ellagic acid pretreatment of intrahippocampal amyloid beta-microinjected rats could dose-dependently improve learning and memory deficits via neuronal protection and at molecular level through mitigation of oxidative stress and acetylcholinesterase (AChE) activity and modulation of NF-κB/Nrf2/TLR4 signaling pathway.

Atypical antipsychotic clozapine reversed deficit on prepulse inhibition of the acoustic startle reflex produced by microinjection of DOI into the inferior colliculus in rats.

PubMed

de Oliveira, Rodolpho Pereira; Nagaishi, Karen Yuriko; Barbosa Silva, Regina Cláudia

2017-05-15

Dysfunctions of the serotonergic system have been suggested to be important in the neurobiology of schizophrenia. Patients with schizophrenia exhibit deficits in an operational measure of sensorimotor gating: prepulse inhibition (PPI) of startle. PPI is the normal reduction in the startle response caused by a low intensity non-startling stimulus (prepulse) which is presented shortly before the startle stimulus (pulse). The hallucinogen 2,5-dimethoxy-4-iodoamphetamine (DOI), a 5-hydroxytryptamine(HT) 2 receptor agonist disrupted PPI in rats. The inferior colliculus (IC) is a critical nucleus of the auditory pathway mediating acoustic PPI. The activation of the IC by the acoustic prepulse reduces startle magnitude. The present study investigated the role of serotonergic transmission in the IC on the expression of acoustic PPI. For that we investigated whether 5-HT2A receptor activation or blockade would affect this response. Unilateral microinjection of DOI (10μg/0.3μl) into the IC disrupted PPI, while microinjection of the 5-HT2A receptor antagonist ritanserin (4μg/0.3μl), into this structure did not alter PPI. We also examined the ability of the atypical antipsychotic clozapine (5.0mg/kg; I.P.) to reverse the disruption of PPI produced by unilateral microinjections of DOI into the IC of rats. Pretreatment with clozapine blocked DOI-induced disruption of PPI. Altogether, these results suggest that serotonin-mediated mechanisms of the IC are involved in the expression of PPI in rodents and that this response is sensitive to atypical antipsychotic clozapine. Copyright © 2017 Elsevier B.V. All rights reserved.

Food and water intake, body temperature and metabolic consequences of interleukin-1β microinjection into the cingulate cortex of the rat.

PubMed

Csetényi, B; Hormay, E; Szabó, I; Takács, G; Nagy, B; László, K; Karádi, Z

2017-07-28

In order to elucidate whether cytokine mechanisms of the cingulate cortex (cctx) are important in the central regulation of homeostasis, in the present study, feeding-metabolic effects of direct bilateral microinjection of interleukin-1β (IL-1β) into the cctx of the rat have been investigated. Short- (2h), medium (12h) and long-term (24h) food and water intakes and body temperature were measured after the intracerebral administration of this primary cytokine or vehicle solution, with or without paracetamol pretreatment. The effect of IL-1β on the blood glucose level of animals was examined in glucose tolerance test (GTT), and concentrations of relevant plasma metabolites (total cholesterol, HDL, LDH, triglycerides, uric acid) were additionally also determined following the above microinjections. In contrast to causing no major alteration in the food and water intakes, the cytokine treatment evoked significant increase in the body temperature of the rats. Prostaglandin-mediated mechanisms were shown to have important role in the mode of this action of IL-1β, since paracetamol pretreatment partially prevented the development of the above mentioned hyperthermia. In the GTT, no considerable difference was observed between the blood glucose levels of the cytokine treated and control animals. Following IL-1β microinjection, however, significant decrease of HDL and total cholesterol was found. Our present findings indicate that elucidating the IL-1β mediated homeostatic control mechanisms in the cingulate cortex may lead to the better understanding not only the regulatory entities of the healthy organism but also those found in obesity, diabetes mellitus and other worldwide rapidly spreading feeding-metabolic disorders. Copyright © 2017 Elsevier B.V. All rights reserved.

Atypical antipsychotic olanzapine reversed deficit on prepulse inhibition of the acoustic startle reflex produced by microinjection of dizocilpine (MK-801) into the inferior colliculus in rats.

PubMed

Zangrando, Julia; Carvalheira, Renata; Labbate, Giovanna; Medeiros, Priscila; Longo, Beatriz Monteiro; Melo-Thomas, Liana; Silva, Regina Claudia Barbosa

2013-11-15

Patients with schizophrenia exhibit deficits in an operational measure of sensorimotor gating: prepulse inhibition (PPI) of startle. PPI is the normal reduction in the startle response caused by a low intensity non-startling stimulus (prepulse) which is presented shortly before the startle stimulus (pulse). MK-801 is an NMDA receptor-antagonist known to produce hyperactivity, deficits in prepulse inhibition and social withdrawal, behaviors which correlate well with some of the positive, cognitive and negative symptoms of schizophrenia. The inferior colliculus (IC) is a critical part of the auditory pathway mediating acoustic PPI. The activation of the IC by the acoustic prepulse reduces startle magnitude. Thus, the purpose of the present study was to elucidate the role of glutamatergic transmission in the IC on the expression of acoustic PPI. For that we investigated whether NMDA receptor stimulation or blockade would affect this response. Unilateral microinjections of NMDA (30 nmol/0.5 μL) into the IC did not alter PPI while microinjections of MK-801 (30 nmol/0.5 μL) into this structure disrupted PPI. We also examined the ability of the atypical antipsychotic olanzapine (5.0mg/kg; i.p.) to reverse the disruption of pre-pulse inhibition produced by unilateral microinjections of MK-801 into the IC of rats. Pretreatment with olanzapine blocked MK-801-induced disruption of PPI. Altogether, these results suggest that glutamate-mediated mechanisms of the IC are involved in the expression of PPI in rodents and that this response is sensitive to atypical antipsychotic olanzapine. Copyright © 2013 Elsevier B.V. All rights reserved.

Which Cue to ‘Want?’ Central Amygdala Opioid Activation Enhances and Focuses Incentive Salience on a Prepotent Reward Cue

PubMed Central

Mahler, Stephen V.; Berridge, Kent C.

2009-01-01

The central nucleus of the amygdala (CeA) helps translate learning into motivation, and here we show that opioid stimulation of CeA magnifies and focuses learned incentive salience onto a specific reward cue (Pavlovian conditioned stimulus, or CS). This motivation enhancement makes that cue more attractive, noticeable, and liable to elicit appetitive and consummatory behaviors. To reveal the focusing of incentive salience, we exploited individual differences in an autoshaping paradigm in which a rat prefers to approach, nibble and sniff one of two reward-associated stimuli (its prepotent stimulus). The individually-prepotent cue is either a predictive CS+ that signals reward (8sec metal lever insertion), or instead the metal cup that delivers sucrose pellets (the reward source). Results indicated that CeA opioid activation by microinjection of the μ agonist DAMGO (0.1μg) selectively and reversibly enhanced the attractiveness of whichever reward CS was that rat's prepotent cue. CeA DAMGO microinjections made rats more vigorously approach their particular prepotent CS, and to energetically sniff and nibble it in a nearly frenzied consummatory fashion. Only the prepotent cue was enhanced as an incentive target, and alternative cues were not enhanced. Conversely, inactivation of CeA by muscimol microinjection (0.25μg) suppressed approach, nibbles and sniffs of the prepotent CS. Confirming modulation of incentive salience, unconditioned food intake was similarly increased by DAMGO microinjection and decreased by muscimol in CeA. We conclude that opioid neurotransmission in central amygdala helps determine which environmental stimuli become most ‘wanted,’ and how ‘wanted’ they become. This may powerfully guide reward-seeking behavior. PMID:19458221

Thermal nociception is decreased by hypocretin-1 and an adenosine A1 receptor agonist microinjected into the pontine reticular formation of Sprague Dawley rat.

PubMed

Watson, Sarah L; Watson, Christopher J; Baghdoyan, Helen A; Lydic, Ralph

2010-06-01

Clinical and preclinical data concur that sleep disruption causes hyperalgesia, but the brain mechanisms through which sleep and pain interact remain poorly understood. Evidence that pontine components of the ascending reticular activating system modulate sleep and nociception encouraged the present study testing the hypothesis that hypocretin-1 (orexin-A) and an adenosine receptor agonist administered into the pontine reticular nucleus, oral part (PnO) each alter thermal nociception. Adult male rats (n = 23) were implanted with microinjection guide tubes aimed for the PnO. The PnO was microinjected with saline (control), hypocretin-1, the adenosine A(1) receptor agonist N(6)-p-sulfophenyladenosine (SPA), the hypocretin receptor-1 antagonist N-(2-Methyl-6-benzoxazolyl)-N''-1,5-naphthyridin-4-yl-urea (SB-334867), and hypocretin-1 plus SB-334867. As an index of antinociceptive behavior, the latency (in seconds) to paw withdrawal away from a thermal stimulus was measured following each microinjection. Compared to control, antinociception was significantly increased by hypocretin-1 and by SPA. SB-334867 increased nociceptive responsiveness, and administration of hypocretin-1 plus SB-334867 blocked the antinociception caused by hypocretin-1. These results suggest for the first time that hypocretin receptors in rat PnO modulate nociception. Widely distributed and overlapping neural networks regulate states of sleep and pain. Specifying the brain regions and neurotransmitters through which pain and sleep interact is an essential step for developing adjunctive therapies that diminish pain without disrupting states of sleep and wakefulness. Copyright (c) 2010 American Pain Society. Published by Elsevier Inc. All rights reserved.

Stereotypic circling behavior in mice with vestibular dysfunction: asymmetrical effects of intrastriatal microinjection of a dopamine agonist.

PubMed

Ishiguro, Akio; Inagaki, Masumi; Kaga, Makiko

2007-07-01

Bronx Waltzer (bv) mouse, which has been used as a model of hearing and vestibular dysfunction, shows remarkable repetitive circling behavior. This study investigated whether the behavior is caused by the asymmetry of striatal function by observing the behavior of the bv mice following microinjection of dopamine D1 agonist, A68930 into the striatum ipsilaterally and contralaterally to the preferred direction of rotation separately. High dose of the drug induced opposite effects on ipsilateral rotations by the side of injections with statistical significance (p = .0026). These results suggested that the stereotypic circling behavior involves striatum and is based on striatal asymmetry.

A caprine chimera produced by injection of embryonic germ cells into a blastocyst.

PubMed

Jia, W; Yang, W; Lei, A; Gao, Z; Yang, C; Hua, J; Huang, W; Ma, X; Wang, H; Dou, Z

2008-02-01

This report details a chimeric goat derived by injecting caprine embryonic germ (EG) cells into a host blastocyst. The EG cells, isolated from the primordial genital ridge of white Guanzhong goat fetuses (28-42 days of pregnancy), had alkaline phosphatase activity and several stem cell markers, including SSEA-1, c-kit, and Nanog. Ten to 20EG cells were microinjected into the blastocoelic cavity of a host blastocyst collected from a black goat following natural service. Twenty-nine injected blastocysts were transferred into nine white surrogate goats. One of the recipients maintained pregnancy to term and gave birth to three kids: one male, one female, and a dead, malformed fetus of undetermined gender; all three fetuses were black, but the female and the malformed fetus each had a large white spot on their head. Based on PCR and microsatellite DNA assay, the female and the malformed fetus were monozygotic twins and chimeras. Microsatellite assay on various tissues from the dead fetus (including skin, blood, liver, placenta, lung, heart, spleen, muscle, and brain), revealed that these tissues and organs were chimeric and contained cells derived from EG cells. In conclusion, caprine EG cells differentiated into all three germ layers in vivo.

Analysis of gene expression in single live neurons.

PubMed Central

Eberwine, J; Yeh, H; Miyashiro, K; Cao, Y; Nair, S; Finnell, R; Zettel, M; Coleman, P

1992-01-01

We present here a method for broadly characterizing single cells at the molecular level beyond the more common morphological and transmitter/receptor classifications. The RNA from defined single cells is amplified by microinjecting primer, nucleotides, and enzyme into acutely dissociated cells from a defined region of rat brain. Further processing yields amplified antisense RNA. A second round of amplification results in greater than 10(6)-fold amplification of the original starting material, which is adequate for analysis--e.g., use as a probe, making of cDNA libraries, etc. We demonstrate this method by constructing expression profiles of single live cells from rat hippocampus. This profiling suggests that cells that appear to be morphologically similar may show marked differences in patterns of expression. In addition, we characterize several mRNAs from a single cell, some of which were previously undescribed, perhaps due to "rarity" when averaged over many cell types. Electrophysiological analysis coupled with molecular biology within the same cell will facilitate a better understanding of how changes at the molecular level are manifested in functional properties. This approach should be applicable to a wide variety of studies, including development, mutant models, aging, and neurodegenerative disease. Images PMID:1557406

[Effects of hypothalamic microinjections of 6-hydroxydopamine (6-OHDA) on estral cycle and morphology of the genital tract in the female rat (author's transl)].

PubMed

Sala, M A; Oteui, J T; Benedetti, W I

1975-01-01

To determine whether central catecholaminergic pathways are involved in the neural contral of gonadotrophin secretion, they were interrupted at the hypothalamic level by microinjections of 6-hydroxydopamine (6-OHDA). The effects on ovulation, estral cycle and ovarian and uterine histology were studied. Microinjections of 50 mug of 6-OHDA hydrobromyde were made bilaterally into the anterolateral hypothalamus in a group of rats. Another group was injected with 25 mug of 6-OHDA, while a control group recieved an equivalent volume (5 mul) of saline with ascorbic acid. Animals injected with 50 mug of 6-OHDA showed blockade of ovulation, vaginal cytology characteristics of persistent estrous, polyfollicular ovaries and enlarged uteri with hypertrophic endometrial glands. In the group injected with 25 mug, similiar effects were demonstrated, but the number of affected animals was smaller than that in the 50 mug group. Control animals dit not show modifications, either in estral cycle or in ovarian and uterine histology. These results suggest that 6-OHDA injected into the anterolateral hypothalmus interferes with catecholaminergic pathways that participate in the neural control of ovulation.

Microinfusion of nefazodone into the basolateral nucleus of the amygdala enhances defensive behavior induced by NMDA stimulation of the inferior colliculus.

PubMed

Maisonnette, S; Villela, C; Carotti, A P; Landeira-Fernandez, J

2000-01-01

The inferior colliculus is notably associated with defensive behavior. Electrical or pharmacological stimulation of the inferior colliculus induces aversive reactions such as running and jumping. Lesion of the basolateral nucleus of the amygdala decreases the threshold of aversive reactions induced by electrical stimulation of the inferior colliculus. The present work examined the influence of microinjections of nefazodone, a serotonin (5-HT(2)) antagonist, into the basolateral nucleus of amygdala on aversive reactions induced by N-methyl-D-aspartate (NMDA) microinjected into the inferior colliculus. Rats implanted with cannulae in the inferior colliculus and in the basolateral nucleus of the amygdala were submitted to the open-field test where defensive behaviors were observed. Results indicated that microinjection of nefazodone into the basolateral nucleus of the amygdala increases aversive responses induced by NMDA injections into the inferior colliculus. This result suggests that the inferior colliculus and the basolateral nucleus of the amygdala have a functional relationship on the neural circuitry of defensive behavior. Moreover, 5-HT(2) receptors located at the basolateral nucleus of the amygdala seem to play an inhibitory role on defensive behaviors induced by inferior colliculus stimulation.

Mirtazapine exerts an anxiolytic-like effect through activation of the median raphe nucleus-dorsal hippocampal 5-HT pathway in contextual fear conditioning in rats.

PubMed

An, Yan; Chen, Chong; Inoue, Takeshi; Nakagawa, Shin; Kitaichi, Yuji; Wang, Ce; Izumi, Takeshi; Kusumi, Ichiro

2016-10-03

The functional role of serotonergic projections from the median raphe nucleus (MRN) to the dorsal hippocampus (DH) in anxiety remains understood poorly. The purpose of the present research was to examine the functional role of this pathway, using the contextual fear conditioning (CFC) model of anxiety. We show that intra-MRN microinjection of mirtazapine, a noradrenergic and specific serotonergic antidepressant, reduced freezing in CFC without affecting general motor activity dose-dependently, suggesting an anxiolytic-like effect. In addition, intra-MRN microinjection of mirtazapine dose-dependently increased extracellular concentrations of serotonin (5-HT) but not dopamine in the DH. Importantly, intra-DH pre-microinjection of WAY-100635, a 5-HT1A antagonist, significantly attenuated the effect of mirtazapine on freezing. These results, for the first time, suggest that activation of the MRN-DH 5-HT1A pathway exerts an anxiolytic-like effect in CFC. This is consistent with the literature that the hippocampus is essential for retrieval of contextual memory and that 5-HT1A receptor activation in the hippocampus primarily exerts an inhibitory effect on the neuronal activity. Copyright © 2016 Elsevier Inc. All rights reserved.

Effect of dietary sodium intake on central angiotensinergic pathways.

PubMed

DiBona, Gerald F; Jones, Susan Y

2002-06-28

The role of central angiotensinergic pathways in the cardiovascular regulation has been examined using the microinjection of angiotensin peptides and angiotensin receptor antagonists. However, in such studies, neither the overall nor the local level of activity of the renin-angiotensin system is generally known. Herein, physiological changes in the endogenous level of activity of the renin-angiotensin system were produced by alterations in the dietary sodium intake. Microinjection of the angiotensin II AT1 receptor antagonists losartan or candesartan into the rostral ventrolateral medulla produced the bradycardic, depressor and renal sympathoinhibitory responses which were greater in low sodium diet rats with stimulated activity of the renin-angiotensin system than in high sodium diet rats with suppressed activity of the renin-angiotensin system activity. The renal sympathoexcitatory responses to activation of the paraventricular nucleus by microinjection of bicuculline, known to be dependent on the excitatory synaptic inputs to the rostral ventrolateral medulla mediated by AT1 receptors, were greater in low sodium diet rats than in high sodium rats. These observations support the view that physiologically regulated angiotensin peptides of the brain origin exert a local paracrine or autocrine action on sites that influence the renal sympathetic nerve activity.

Cross state-dependency of learning between arachidonylcyclopropylamide (ACPA) and muscimol in the mouse dorsal hippocampus.

PubMed

Jafari-Sabet, Majid; Karimi, Amir-Mohammad

2017-12-01

The aim of the present study was to examine cross state-dependent learning between ACPA (a selective cannabinoid CB1 receptor agonist) and muscimol (a selective GABAA receptor agonist) in the step-down inhibitory avoidance learning task. The dorsal hippocampal CA1 regions of adult male NMRI mice were bilaterally cannulated, and all drugs were microinjected into the intended sites of injection. Post-training and/or pre-test administration of ACPA (1 and 2ng/mouse) dose-dependently induced amnesia. Pre-test microinjection of the same doses of ACPA reversed the post-training ACPA-induced amnesia. This event has been named ACPA state-dependent learning (SDL). Post-training and/or pre-test microinjection of muscimol (0.05 and 0.1μg/mouse) dose-dependently induced amnesia. Pre-test administration of the same doses of muscimol reversed the post-training muscimol-induced amnesia, suggesting muscimol SDL. The amnesia induced by post-training administration of ACPA was reversed by pre-test administration of muscimol (0.05 and 0.1μg/mouse). Furthermore, the pre-test microinjection of muscimol (0.025 and 0.05μg/mouse) with an ineffective dose of ACPA (0.5ng/mouse) significantly restored memory retrieval and induced ACPA SDL. In another series of experiments, the amnesia induced by post-training administration of muscimol was reversed by pre-test administration of ACPA (1 and 2ng/mouse). Moreover, pre-test microinjection of ACPA (0.5 and 1ng/mouse) with an ineffective dose of muscimol (0.025μg/mouse) significantly restored memory retrieval and induced muscimol SDL. It is important to note that pre-test intra-CA1 injection of a selective GABAA receptor antagonist, bicuculline (0.125 and 0.25μg/mouse), 5min before the administration of muscimol (0.1μg/mouse) or ACPA (2ng/mouse) dose-dependently inhibited muscimol- and ACPA-induced SDL, respectively. Pre-test intra-CA1 administration of bicuculline (0.0625, 0.125 and 0.25μg/mouse) by itself did not affect memory retention. In conclusion, the data strongly revealed a cross SDL among ACPA and muscimol in the dorsal hippocampal CA1 regions. Copyright © 2017 Elsevier Inc. All rights reserved.

A novel pneumatic micropipette aspiration method using a balance pressure model.

PubMed

Zhao, Qili; Wu, Ming; Cui, Maosheng; Qin, Yanding; Yu, Jin; Sun, Mingzhu; Zhao, Xin; Feng, Xizeng

2013-12-01

This paper presents a novel micropipette aspiration (MA) method based on a common pneumatic micro-injection system. This method is the first to quantify the influence of capillary effect on aspiration pressure using a balance pressure model, and in return, uses the capillary effect to quantify the aspiration pressure. Subsequently, the seal between the cell and the micropipette is detected to judge and exclude the ineffective MA attempts. The rationality of the balance pressure model is validated by the designed micropipette-filling experiments. Through applied to elasticity-determination of the cells with different sizes, the feasibility and versatility of this MA method are proved. With abilities to quantify aspiration pressures and detect the seam between the cell and the micropipette, our method is expected to advance the application of the commercial pneumatic injector in the MA of cells. Moreover, with the quantified volume of the liquid entering into the micropipette during MA process, our method also has a potential applicability to the study of the permeability of the cell membrane in the future.

Monitoring mis-acylated tRNA suppression efficiency in mammalian cells via EGFP fluorescence recovery

PubMed Central

Ilegems, Erwin; Pick, Horst M.; Vogel, Horst

2002-01-01

A reporter assay was developed to detect and quantify nonsense codon suppression by chemically aminoacylated tRNAs in mammalian cells. It is based on the cellular expression of the enhanced green fluorescent protein (EGFP) as a reporter for the site-specific amino acid incorporation in its sequence using an orthogonal suppressor tRNA derived from Escherichia coli. Suppression of an engineered amber codon at position 64 in the EGFP run-off transcript could be achieved by the incorporation of a leucine via an in vitro aminoacylated suppressor tRNA. Microinjection of defined amounts of mutagenized EGFP mRNA and suppressor tRNA into individual cells allowed us to accurately determine suppression efficiencies by measuring the EGFP fluorescence intensity in individual cells using laser-scanning confocal microscopy. Control experiments showed the absence of natural suppression or aminoacylation of the synthetic tRNA by endogenous aminoacyl-tRNA synthetases. This reporter assay opens the way for the optimization of essential experimental parameters for expanding the scope of the suppressor tRNA technology to different cell types. PMID:12466560

Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by the TALEN and CRISPR/Cas systems.

PubMed

Yasue, Akihiro; Mitsui, Silvia Naomi; Watanabe, Takahito; Sakuma, Tetsushi; Oyadomari, Seiichi; Yamamoto, Takashi; Noji, Sumihare; Mito, Taro; Tanaka, Eiji

2014-07-16

Since the establishment of embryonic stem (ES) cell lines, the combined use of gene targeting with homologous recombination has aided in elucidating the functions of various genes. However, the ES cell technique is inefficient and time-consuming. Recently, two new gene-targeting technologies have been developed: the transcription activator-like effector nuclease (TALEN) system, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system. In addition to aiding researchers in solving conventional problems, these technologies can be used to induce site-specific mutations in various species for which ES cells have not been established. Here, by targeting the Fgf10 gene through RNA microinjection in one-cell mouse embryos with the TALEN and CRISPR/Cas systems, we produced the known limb-defect phenotypes of Fgf10-deficient embryos at the F0 generation. Compared to the TALEN system, the CRISPR/Cas system induced the limb-defect phenotypes with a strikingly higher efficiency. Our results demonstrate that although both gene-targeting technologies are useful, the CRISPR/Cas system more effectively elicits single-step biallelic mutations in mice.

Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

PubMed Central

Weyler, Linda; Engelbrecht, Mattias; Mata Forsberg, Manuel; Brehwens, Karl; Vare, Daniel; Vielfort, Katarina; Wojcik, Andrzej; Aro, Helena

2014-01-01

The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies. PMID:25460012

The use of specific antibodies to mediate fusion between Sendai virus envelopes and living cells.

PubMed

Loyter, A; Tomasi, M; Gitman, A G; Etinger, L; Nussbaum, O

1984-01-01

Incubation of Sendai virus particles with non-ionic detergents such as Triton X-100 completely solubilizes the viral envelopes. Removal of the detergent from the supernatant (which contains the two main viral glycoproteins) leads to the formation of fusogenic, reconstituted viral envelopes. Soluble macromolecules such as DNA or proteins can be enclosed within the reconstituted vesicles, while membrane components can be inserted into the viral envelopes. Fusion of such loaded or 'hybrid' reconstituted envelopes with living cells in culture results in either microinjection or transfer of the viral components to the recipient cells. Thus such reconstituted envelopes can serve as efficient carriers for the introduction of macromolecules of biological interest into living cells in culture. A more specific vehicle has been constructed by chemically coupling anti-cell membrane antibodies (anti-human erythrocyte antibody) to the viral envelope. Such antibody-bearing intact virus particles or reconstituted envelopes bound to and fused with virus receptor-depleted cells. In addition, anti-Sendai virus antibodies were coupled to neuraminidase-treated human erythrocytes. Such antibodies mediated the binding and fusion of intact Sendai virus particles and their reconstituted envelopes to virus receptor-depleted cells.

The chromokinesin Kid is necessary for chromosome arm orientation and oscillation, but not congression, on mitotic spindles

PubMed Central

Levesque, Aime A.; Compton, Duane A.

2001-01-01

Chromokinesins have been postulated to provide the polar ejection force needed for chromosome congression during mitosis. We have evaluated that possibility by monitoring chromosome movement in vertebrate-cultured cells using time-lapse differential interference contrast microscopy after microinjection with antibodies specific for the chromokinesin Kid. 17.5% of cells injected with Kid-specific antibodies have one or more chromosomes that remain closely opposed to a spindle pole and fail to enter anaphase. In contrast, 82.5% of injected cells align chromosomes in metaphase, progress to anaphase, and display chromosome velocities not significantly different from control cells. However, injected cells lack chromosome oscillations, and chromosome orientation is atypical because chromosome arms extend toward spindle poles during both congression and metaphase. Furthermore, chromosomes cluster into a mass and fail to oscillate when Kid is perturbed in cells containing monopolar spindles. These data indicate that Kid generates the polar ejection force that pushes chromosome arms away from spindle poles in vertebrate-cultured cells. This force increases the efficiency with which chromosomes make bipolar spindle attachments and regulates kinetochore activities necessary for chromosome oscillation, but is not essential for chromosome congression. PMID:11564754

The antiproliferative aspects of mortalin (review).

PubMed

Wadhwa, R; Mitsui, Y; Ide, T; Kaul, S

1995-07-01

Cellular mortal and immortal phenotypes as defined by the limited and the infinite capacity of cells to divide are the characteristics of normal and cancerous cells in culture. Numerous strategies that have been employed to understand the mechanism(s) of normal as well as tumor cell growth have revealed that these are genetically controlled, however, the genes and the synchronized regulations remain largely undefined so far. The present report reviews the identification of mortalin, a novel member of murine hsp70 family of proteins, as a gene involved in pathways that determine divisional phenotype of cells in vitro. In the present study, the anti-proliferative activity of mortalin is demonstrated also in human skin fibroblasts (TIG-73PD) by microinjection of anti-mortalin antibody. Furthermore, studies on the mortalin immunofluorescence patterns in SV40-immortalized pre-crisis and post-crisis human cells have revealed that the change in the intracellular distribution of mortalin is linked to the change in the divisional phenotype of cells. Thus, the studies to resolve the molecular basis of association of the cytosolically distributed form of mortalin with cellular mortal phenotype would be important in understanding of the mechanism(s) that determine replicative potential of cells in culture.

Simplified CRISPR tools for efficient genome editing and streamlined protocols for their delivery into mammalian cells and mouse zygotes.

PubMed

Jacobi, Ashley M; Rettig, Garrett R; Turk, Rolf; Collingwood, Michael A; Zeiner, Sarah A; Quadros, Rolen M; Harms, Donald W; Bonthuis, Paul J; Gregg, Christopher; Ohtsuka, Masato; Gurumurthy, Channabasavaiah B; Behlke, Mark A

2017-05-15

Genome editing using the CRISPR/Cas9 system requires the presence of guide RNAs bound to the Cas9 endonuclease as a ribonucleoprotein (RNP) complex in cells, which cleaves the host cell genome at sites specified by the guide RNAs. New genetic material may be introduced during repair of the double-stranded break via homology dependent repair (HDR) if suitable DNA templates are delivered with the CRISPR components. Early methods used plasmid or viral vectors to make these components in the host cell, however newer approaches using recombinant Cas9 protein with synthetic guide RNAs introduced directly as an RNP complex into cells shows faster onset of action with fewer off-target effects. This approach also enables use of chemically modified synthetic guide RNAs that have improved nuclease stability and reduces the risk of triggering an innate immune response in the host cell. This article provides detailed methods for genome editing using the RNP approach with synthetic guide RNAs using lipofection or electroporation in mammalian cells or using microinjection in murine zygotes, with or without addition of a single-stranded HDR template DNA. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Restriction endonucleases from invasive Neisseria gonorrhoeae cause double-strand breaks and distort mitosis in epithelial cells during infection.

PubMed

Weyler, Linda; Engelbrecht, Mattias; Mata Forsberg, Manuel; Brehwens, Karl; Vare, Daniel; Vielfort, Katarina; Wojcik, Andrzej; Aro, Helena

2014-01-01

The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies.

Two independent forms of endocytosis maintain embryonic cell surface homeostasis during early development

PubMed Central

Covian-Nares, J. Fernando; Smith, Robert M.; Vogel, Steven S.

2008-01-01

Eukaryotic cells have multiple forms of endocytosis which maintain cell surface homeostasis. One explanation for this apparent redundancy is to allow independent retrieval of surface membranes derived from different types of vesicles. Consistent with this hypothesis we find that sea urchin eggs have at least two types of compensatory endocytosis. One is associated with retrieving cortical vesicle membranes, and formed large endosomes by a mechanism that was inhibited by agatoxin, cadmium, staurosporine and FK506. The second type is thought to compensate for constitutive exocytosis, and formed small endosomes using a mechanism that was insensitive to the above mentioned reagents, but was inhibited by phenylarsine oxide (PAO), and by microinjection of mRNA encoding Src kinase. Both mechanisms could act concurrently, and account for all of the endocytosis occurring during early development. Inhibition of either form did not trigger compensation by the other form, and phorbol ester treatment rescued the endocytotic activity blocked by agatoxin, but not the retrieval blocked by PAO. PMID:18281031

Growth enhancement in transgenic tilapia by ectopic expression of tilapia growth hormone.

PubMed

Martínez, R; Estrada, M P; Berlanga, J; Guillén, I; Hernández, O; Cabrera, E; Pimentel, R; Morales, R; Herrera, F; Morales, A; Piña, J C; Abad, Z; Sánchez, V; Melamed, P; Lleonart, R; de la Fuente, J

1996-03-01

The generation of transgenic fish with the transfer of growth hormone (GH) genes has opened new possibilities for the manipulation of growth in economically important fish species. The tilapia growth hormone (tiGH) cDNA was linked to the human cytomegalovirus (CMV) enhancer-promoter and used to generate transgenic tilapia by microinjection into one-cell embryos. Five transgenic tilapia were obtained from 40 injected embryos. A transgenic animal containing one copy of the transgene per cell was selected to establish a transgenic line. The transgene was stably transmitted to F1 and F2 generations in a Mendelian fashion. Ectopic, low-level expression of tiGH was detected in gonad and muscle cells of F1 transgenic tilapia by immunohystochemical analysis of tissue sections. Nine-month-old transgenic F1 progeny were 82% larger than nontransgenic fish at p = .001. These results showed that low-level ectopic expression of tiGH resulted in a growth acceleration in transgenic tilapia. Tilapia GH gene transfer is an alternative for growth acceleration in tilapia.

ANG II receptor subtype 1a gene knockdown in the subfornical organ prevents increased drinking behavior in bile duct-ligated rats.

PubMed

Walch, Joseph D; Nedungadi, T Prashant; Cunningham, J Thomas

2014-09-15

Bile duct ligation (BDL) causes congestive liver failure that initiates hemodynamic changes, resulting in dilutional hyponatremia due to increased water intake and vasopressin release. This project tested the hypothesis that angiotensin signaling at the subfornical organ (SFO) augments drinking behavior in BDL rats. A genetically modified adeno-associated virus containing short hairpin RNA (shRNA) for ANG II receptor subtype 1a (AT1aR) gene was microinjected into the SFO of rats to knock down expression. Two weeks later, BDL or sham surgery was performed. Rats were housed in metabolic chambers for measurement of fluid and food intake and urine output. The rats were euthanized 28 days after BDL surgery for analysis. A group of rats was perfused for immunohistochemistry, and a second group was used for laser-capture microdissection for analysis of SFO AT1aR gene expression. BDL rats showed increased water intake that was attenuated in rats that received SFO microinjection of AT1aR shRNA. Among BDL rats treated with scrambled (control) and AT1aR shRNA, we observed an increased number of vasopressin-positive cells in the supraoptic nucleus that colocalized with ΔFosB staining, suggesting increased vasopressin release in both groups. These results indicate that angiotensin signaling through the SFO contributes to increased water intake, but not dilutional hyponatremia, during congestive liver failure. Copyright © 2014 the American Physiological Society.

Ca2+ release triggered by nicotinate adenine dinucleotide phosphate in intact sea urchin eggs.

PubMed Central

Perez-Terzic, C M; Chini, E N; Shen, S S; Dousa, T P; Clapham, D E

1995-01-01

Nicotinate adenine dinucleotide phosphate (NAADP) was recently identified [Lee and Aarhus (1995) J. Biol. Chem. 270, 2152-2157; Chini, Beers and Dousa (1995) J. Biol. Chem. 270, 3116-3223] as a potent Ca(2+)-releasing agent in sea urchin egg homogenates. NAADP triggered Ca2+ release by a mechanism that was distinct from inositol 1,4,5-trisphosphate (InsP3)- and cyclic ADP-ribose (cADPR)-induced Ca2+ release. When NAADP was microinjected into intact sea urchin eggs it induced a dose-dependent increase in cytoplasmic free Ca2+ which was independent of the extracellular [Ca2+]. The Ca2+ waves elicited by microinjections of NAADP originated at the site of injection and swept across the cytosol. As previously found in sea urchin egg homogenates, NAADP-induced Ca2+ release in intact eggs was not blocked by heparin or by prior desensitization to InsP3 or cADPR. Thio-NADP, a specific inhibitor of the NAADP-induced Ca2+ release in sea urchin homogenates [Chini, Beers and Dousa (1995) J. Biol. Chem. 270, 3116-3223] blocked NAADP (but not InsP3 or cADPR) injection-induced Ca2+ release in intact sea urchin eggs. Finally, fertilization of sea urchin eggs abrogated subsequent NAADP-induced Ca2+ release, suggesting that the NAADP-sensitive Ca2+ pool may participate in the fertilization response. This study demonstrates that NAADP acts as a selective Ca(2+)-releasing agonist in intact cells. Images Figure 2 PMID:8554544

Spatial Reorganization of the Endoplasmic Reticulum during Mitosis Relies on Mitotic Kinase Cyclin A in the Early Drosophila Embryo

PubMed Central

Bergman, Zane J.; Mclaurin, Justin D.; Eritano, Anthony S.; Johnson, Brittany M.; Sims, Amanda Q.; Riggs, Blake

2015-01-01

Mitotic cyclin-dependent kinase with their cyclin partners (cyclin:Cdks) are the master regulators of cell cycle progression responsible for regulating a host of activities during mitosis. Nuclear mitotic events, including chromosome condensation and segregation have been directly linked to Cdk activity. However, the regulation and timing of cytoplasmic mitotic events by cyclin:Cdks is poorly understood. In order to examine these mitotic cytoplasmic events, we looked at the dramatic changes in the endoplasmic reticulum (ER) during mitosis in the early Drosophila embryo. The dynamic changes of the ER can be arrested in an interphase state by inhibition of either DNA or protein synthesis. Here we show that this block can be alleviated by micro-injection of Cyclin A (CycA) in which defined mitotic ER clusters gathered at the spindle poles. Conversely, micro-injection of Cyclin B (CycB) did not affect spatial reorganization of the ER, suggesting CycA possesses the ability to initiate mitotic ER events in the cytoplasm. Additionally, RNAi-mediated simultaneous inhibition of all 3 mitotic cyclins (A, B and B3) blocked spatial reorganization of the ER. Our results suggest that mitotic ER reorganization events rely on CycA and that control and timing of nuclear and cytoplasmic events during mitosis may be defined by release of CycA from the nucleus as a consequence of breakdown of the nuclear envelope. PMID:25689737

[Microinjection Monitoring System Design Applied to MRI Scanning].

PubMed

Xu, Yongfeng

2017-09-30

A microinjection monitoring system applied to the MRI scanning was introduced. The micro camera probe was used to stretch into the main magnet for real-time video injection monitoring of injection tube terminal. The programming based on LabVIEW was created to analysis and process the real-time video information. The feedback signal was used for intelligent controlling of the modified injection pump. The real-time monitoring system can make the best use of injection under the condition that the injection device was away from the sample which inside the magnetic room and unvisible. 9.4 T MRI scanning experiment showed that the system in ultra-high field can work stability and doesn't affect the MRI scans.

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

PubMed Central

Molotkov, Dmitry A.; Yukin, Alexey Y.; Afzalov, Ramil A.; Khiroug, Leonard S.

2010-01-01

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells. The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices. PMID:20972387

CENP-A regulates chromosome segregation during the first meiosis of mouse oocytes.

PubMed

Li, Li; Qi, Shu-Tao; Sun, Qing-Yuan; Chen, Shi-Ling

2017-06-01

Proper chromosome separation in both mitosis and meiosis depends on the correct connection between kinetochores of chromosomes and spindle microtubules. Kinetochore dysfunction can lead to unequal distribution of chromosomes during cell division and result in aneuploidy, thus kinetochores are critical for faithful segregation of chromosomes. Centromere protein A (CENP-A) is an important component of the inner kinetochore plate. Multiple studies in mitosis have found that deficiencies in CENP-A could result in structural and functional changes of kinetochores, leading to abnormal chromosome segregation, aneuploidy and apoptosis in cells. Here we report the expression and function of CENP-A during mouse oocyte meiosis. Our study found that microinjection of CENP-A blocking antibody resulted in errors of homologous chromosome segregation and caused aneuploidy in eggs. Thus, our findings provide evidence that CENP-A is critical for the faithful chromosome segregation during mammalian oocyte meiosis.

Exciton-controlled fluorescence: application to hybridization-sensitive fluorescent DNA probe.

PubMed

Okamoto, Akimitsu; Ikeda, Shuji; Kubota, Takeshi; Yuki, Mizue; Yanagisawa, Hiroyuki

2009-01-01

A hybridization-sensitive fluorescent probe has been designed for nucleic acid detection, using the concept of fluorescence quenching caused by the intramolecular excitonic interaction of fluorescence dyes. We synthesized a doubly thiazole orange-labeled nucleotide showing high fluorescence intensity for a hybrid with the target nucleic acid and effective quenching for the single-stranded state. This exciton-controlled fluorescent probe was applied to living HeLa cells using microinjection to visualize intracellular mRNA localization. Immediately after injection of the probe into the cell, fluorescence was observed from the probe hybridizing with the target RNA. This fluorescence rapidly decreased upon addition of a competitor DNA. Multicoloring of this probe resulted in the simple simultaneous detection of plural target nucleic acid sequences. This probe realized a large, rapid, reversible change in fluorescence intensity in sensitive response to the amount of target nucleic acid, and facilitated spatiotemporal monitoring of the behavior of intracellular RNA.

Cofilin promotes stimulus-induced lamellipodium formation by generating an abundant supply of actin monomers

PubMed Central

Kiuchi, Tai; Ohashi, Kazumasa; Kurita, Souichi; Mizuno, Kensaku

2007-01-01

Cofilin stimulates actin filament disassembly and accelerates actin filament turnover. Cofilin is also involved in stimulus-induced actin filament assembly during lamellipodium formation. However, it is not clear whether this occurs by replenishing the actin monomer pool, through filament disassembly, or by creating free barbed ends, through its severing activity. Using photoactivatable Dronpa-actin, we show that cofilin is involved in producing more than half of all cytoplasmic actin monomers and that the rate of actin monomer incorporation into the tip of the lamellipodium is dependent on the size of this actin monomer pool. Finally, in cofilin-depleted cells, stimulus-induced actin monomer incorporation at the cell periphery is attenuated, but the incorporation of microinjected actin monomers is not. We propose that cofilin contributes to stimulus-induced actin filament assembly and lamellipodium extension by supplying an abundant pool of cytoplasmic actin monomers. PMID:17470633

Magnetic Carbon nanoparticles enabled efficient photothermal alteration of mammalian cells

NASA Astrophysics Data System (ADS)

Cardenas, Nelson; Thomas, Patrick; Yu, Lingfeng; Mohanty, Samarendra

2011-03-01

While cw near-infrared (NIR) laser beams have been finding widespread application in photothermal therapy of cancer and pulsed NIR laser microbeams are recently being used for optoporation of exogeneous impermeable materials into cells. Since, carbon nanomaterials are very good in photothermal conversion, we utilized carbon nanoparticles (CNP) doped with Fe, so that they can be localized in a defined area by two fold selectivity, (i) external magnetic field for retention of the CNP in targeted area and (ii) surface functionalization for binding the targeted cells. Here, we report efficient photothermal therapy as well as poration of cells using magnetic CNPs with very low power continuous wave laser beam. Localization of CNPs on cell membrane under application of magnetic field was confirmed by scanning electron microscopy. At different power levels, cells could be damaged or microinjected with fluorescence protein-encoding plasmids or impermeable dyes. Monte Carlo simulation showed that the dose of NIR laser beam is sufficient to elicit response for magnetic CNP based photothermal treatment at significant depth. The results of our study suggest that magnetic CNP based photothermal alteration is a viable approach to remotely guide treatments offering high efficiency with significantly reduced cytotoxicity.

Translation of globin messenger RNA by the mouse ovum

PubMed Central

Brinster, R. L.; Chen, H. Y.; Trumbauer, M. E.; Avarbock, M. R.

2016-01-01

It has been demonstrated that the Xenopus oocyte can translate rabbit haemoglobin messenger RNA (mRNA) following microinjection of the message into the cell1. The Xenopus oocyte has since been shown to be capable of translating a variety of messenger RNAs from different species2–4. This system has proved useful in understanding the mechanism of message translation and has also provided information about the translation capability of the Xenopus oocyte5,6. Several other cell types, including HeLa cells and fibroblasts, can also translate exogenous message injected into the cell7,8. However, there have been no reports of injection of mRNA into oocytes or fertilised one-cell ova of mammalian species. Nevertheless, the latter system could be of considerable use in studying the processing of exogenous messages in a mammalian system undergoing development, as well as providing insight into the way the early embryo processes injected messages and the protein products of such messages. We report here the results of injecting message into the fertilised one-cell mouse ovum and show that both mouse and rabbit globin mRNA are translated in this system. PMID:7352032

Pontine cholinergic reticular mechanisms cause state-dependent changes in the discharge of parabrachial neurons.

PubMed

Gilbert, K A; Lydic, R

1994-01-01

The present study examined the hypothesis that cholinoceptive reticular mechanisms in the gigantocellular tegmental field (FTG) of the medial pontine reticular formation cause state-dependent changes in the discharge of parabrachial neurons. In chronically implanted, unanesthetized cats, extracellular recordings were made from nonrespiratory and respiratory neurons in the parabrachial nuclear complex (PBNC) during the natural sleep-wake cycle and during the rapid eye movement (REM) sleeplike state caused by FTG microinjection of carbachol or neostigmine. PBNC cells that increased discharge during natural REM sleep (REM-on cells) revealed similar increased discharge during the carbachol-induced REM sleeplike state (DCarb). Cells that decreased discharge in natural REM sleep (REM-off cells) displayed decreased discharge during both DCarb and the neostigmine-induced REM sleeplike states. The limited sample of parabrachial respiratory neurons revealed significantly diminished discharge during the cholinergically induced REM sleeplike state. Thus cholinoceptive mechanisms localized to specific regions of the pontine reticular formation can cause state-dependent changes in the firing rates of respiratory and nonrespiratory neurons in the PBNC.

Intracellular targeting of isoproteins in muscle cytoarchitecture

PubMed Central

1988-01-01

Part of the muscle creatine kinase (MM-CK) in skeletal muscle of chicken is localized in the M-band of myofibrils, while chicken heart cells containing myofibrils and BB-CK, but not expressing MM-CK, do not show this association. The specificity of the MM-CK interaction was tested using cultured chicken heart cells as "living test tubes" by microinjection of in vitro generated MM-CK and hybrid M-CK/B-CK mRNA with SP6 RNA polymerase. The resulting translation products were detected in injected cells with isoprotein-specific antibodies. M-CK molecules and translation products of chimeric cDNA molecules containing the head half of the B-CK and the tail half of the M-CK coding regions were localized in the M-band of the myofibrils. The tail, but not the head portion of M-CK is essential for the association of M-CK with the M-band of myofibrils. We conclude that gross biochemical properties do not always coincide with a molecule's specific functions like the participation in cell cytoarchitecture which may depend on molecular targeting even within the same cellular compartment. PMID:3283147

Embryo Microinjection of Selenomethionine Reduces Hatchability and Modifies Oxidant Responsive Gene Expression in Zebrafish

NASA Astrophysics Data System (ADS)

Thomas, J. K.; Janz, D. M.

2016-05-01

In previous studies we demonstrated that exposure to selenomethionine (SeMet) causes developmental toxicities in zebrafish (Danio rerio). The objectives of this study were to establish a dose-response relationship for developmental toxicities in zebrafish after embryo microinjection of Se (8, 16 or 32 μg/g dry mass of eggs) in the form of SeMet, and to investigate potential underlying mechanism(s) of SeMet-induced developmental toxicities. A dose-dependent increase in frequencies of mortality and total deformities, and reduced hatchability were observed in zebrafish exposed to excess Se via embryo microinjection. The egg Se concentration causing 20% mortality was then used to investigate transcript abundance of proteins involved in antioxidant protection and methylation. Excess Se exposure modified gene expression of oxidant-responsive transcription factors (nuclear factor erythroid 2-related factor nrf2a and nrf2b), and enzymes involved in cellular methylation (methionine adenosyltransferase mat1a and mat2ab) in zebrafish larvae. Notably, excess Se exposure up-regulated transcript abundance of aryl hydrocarbon receptor 2 (ahr2), a signalling pathway involved in the toxicity of dioxin-related compounds. Our findings suggest that oxidative stress or modification of methylation, or a combination of these mechanisms, might be responsible for Se-induced developmental toxicities in fishes.

Cardiovascular effects of substance P receptor stimulation in the ventrolateral medullary pressor and depressor areas.

PubMed

Urbanski, R W; Murugaian, J; Krieger, A J; Sapru, H N

1989-07-10

The pressor (VLPA) and the depressor (VLDA) areas in the ventrolateral medulla were identified with the microinjection of L-glutamate (1.77 nmol/site) in artificially ventilated urethane-anesthetized male Wistar rats. Bilateral microinjection of a stable substance P (SP) agonist [pGlu5, MePhe8, Sar9]-SP(5-11)], abbreviated as DiMe, into the VLPA (6-600 pmol/site) produced a dose-dependent increase in blood pressure (BP). The effects on heart rate (HR) were variable. Intravenous pretreatment with a ganglionic blocker chlorisondamine (3.0 mg/kg, i.v.), but not with a vasopressin antagonist, blocked these responses. Similar microinjection of DiMe (6-600 pmol/site) into the VLDA produced a dose-dependent decrease in HR but had no effect on BP levels. The DiMe-induced bradycardic response elicited from the VLDA was blocked by i.v. pretreatment with atropine methylbromide (0.5 mg/kg, i.v.). These findings indicate that there are SP receptors localized on sympathoexcitatory neurons in the VLPA and that SP may be an excitatory neurotransmitter in this area. In the VLDA, the SP receptors appear to be localized on a subpopulation of neurons that affect vagal, but not sympathetic, outflow to the heart.

Angiotensin II in the paraventricular nucleus stimulates sympathetic outflow to the cardiovascular system and make vasopressin release in rat.

PubMed

Khanmoradi, Mehrangiz; Nasimi, Ali

2016-10-06

The hypothalamic paraventricular nucleus (PVN) plays essential roles in neuroendocrine and autonomic functions, including cardiovascular regulation. It was shown that microinjection of angiotensin II (AngII) into the PVN produced a pressor response. In this study, we explored the probable mechanisms of this pressor response. AngII was microinjected into the PVN and cardiovascular responses were recorded. Then, the responses were re-tested after systemic injection of a ganglionic blocker, Hexamethonium, or a vasopressin V1 receptor blocker. Hexamethonium pretreatment (i.v.) greatly and significantly attenuated the pressor response to AngII, with no significant effect on heart rate, indicating that the sympathetic system is involved in the cardiovascular effect of AngII in the PVN. Systemic pretreatment (i.v.) with V1 antagonist greatly and significantly attenuated the pressor response to AngII, with no significant effect on heart rate, indicating that vasopressin release is involved in the cardiovascular effect of AngII in the PVN. Overall, we found that AngII microinjected into the PVN produced a pressor response mediated by the sympathetic system and vasopressin release, indicating that other than circulating AngII, endogenous AngII of the PVN increases the vasopressin release from the PVN. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Analysis of molecular chaperones using a Xenopus oocyte protein refolding assay.

PubMed

Heikkila, John J; Kaldis, Angelo; Abdulle, Rashid

2006-01-01

Heat shock proteins (Hsps) are molecular chaperones that aid in the folding and translocation of protein under normal conditions and protect cellular proteins during stressful situations. A family of Hsps, the small Hsps, can maintain denatured target proteins in a folding-competent state such that they can be refolded and regain biological activity in the presence of other molecular chaperones. Previous assays have employed cellular lysates as a source of molecular chaperones involved in folding. In this chapter, we describe the production and purification of a Xenopus laevis recombinant small Hsp, Hsp30C, and an in vivo luciferase (LUC) refolding assay employing microinjected Xenopus oocytes. This assay tests whether LUC can be maintained in a folding-competent state when heat denatured in the presence of a small Hsp or other molecular chaperone. For example, micro-injection of heat-denatured LUC alone into oocytes resulted in minimal reactivation of enzyme activity. However, LUC heat denatured in the presence of Hsp30C resulted in 100% recovery of enzyme activity after microinjection. The in vivo oocyte refolding system is more sensitive and requires less molecular chaperone than in vitro refolding assays. Also, this protocol is not limited to testing Xenopus molecular chaperones because small Hsps from other organisms have been used successfully.

Testing protozoacidal activity of ligand-lytic peptides against termite gut protozoa in vitro (protozoa culture) and in vivo (microinjection into termite hindgut).

PubMed

Husseneder, Claudia; Sethi, Amit; Foil, Lane; Delatte, Jennifer

2010-12-29

We are developing a novel approach to subterranean termite control that would lead to reduced reliance on the use of chemical pesticides. Subterranean termites are dependent on protozoa in the hindguts of workers to efficiently digest wood. Lytic peptides have been shown to kill a variety of protozoan parasites (Mutwiri et al. 2000) and also protozoa in the gut of the Formosan subterranean termite, Coptotermes formosanus (Husseneder and Collier 2009). Lytic peptides are part of the nonspecific immune system of eukaryotes, and destroy the membranes of microorganisms (Leuschner and Hansel 2004). Most lytic peptides are not likely to harm higher eukaryotes, because they do not affect the electrically neutral cholesterol-containing cell membranes of higher eukaryotes (Javadpour et al. 1996). Lytic peptide action can be targeted to specific cell types by the addition of a ligand. For example, Hansel et al. (2007) reported that lytic peptides conjugated with cancer cell membrane receptor ligands could be used to destroy breast cancer cells, while lytic peptides alone or conjugated with non-specific peptides were not effective. Lytic peptides also have been conjugated to human hormones that bind to receptors on tumor cells for targeted destruction of prostate and testicular cancer cells (Leuschner and Hansel 2004). In this article we present techniques used to demonstrate the protozoacidal activity of a lytic peptide (Hecate) coupled to a heptapeptide ligand that binds to the surface membrane of protozoa from the gut of the Formosan subterranean termite. These techniques include extirpation of the gut from termite workers, anaerobic culture of gut protozoa (Pseudotrichonympha grassii, Holomastigotoides hartmanni,Spirotrichonympha leidyi), microscopic confirmation that the ligand marked with a fluorescent dye binds to the termite gut protozoa and other free-living protozoa but not to bacteria or gut tissue. We also demonstrate that the same ligand coupled to a lytic peptide efficiently kills termite gut protozoa in vitro (protozoa culture) and in vivo (microinjection into hindgut of workers), but is less bacteriacidal than the lytic peptide alone. The loss of protozoa leads to the death of the termites in less than two weeks. In the future, we will genetically engineer microorganisms that can survive in the termite hindgut and spread through a termite colony as "Trojan Horses" to express ligand-lytic peptides that would kill the protozoa in the termite gut and subsequently kill the termites in the colony. Ligand-lytic peptides also could be useful for drug development against protozoan parasites.

Neuromodulatory effects of the dorsal hippocampal endocannabinoid system in dextromethorphan/morphine-induced amnesia.

PubMed

Ghasemzadeh, Zahra; Rezayof, Ameneh

2017-01-05

Dextromethorphan which is an active ingredient in many cough medicines has been previously shown to potentiate amnesic effect of morphine in rats. However, the effect of dextromethorphan, that is also a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, in combination with morphine on hippocampus-based long term memory has not been well characterized. The aim of the present study was to assess the possible role of endocannabinoid system of the dorsal hippocampus in dextromethorphan /morphine-induced amnesia. Our results showed that intraperitoneal (i.p.) injection of morphine (5mg/kg) or dextromethorphan (5-15mg/kg) before testing the passive avoidance learning induced amnesia. Combination of ineffective doses of dextromethorphan (7.5mg/kg, i.p.) and morphine (2mg/kg, i.p.) also produced amnesia, suggesting the enhancing effects of the drugs. To assess the effect of the activation or inhibition of the dorsal hippocampal cannabinoid CB 1 receptors on this amnesia, ACPA or AM251 as selective receptor agonists or antagonists were respectively injected into the CA1 regions before systemic injection of dextromethorphan and morphine. Interestingly, intra-CA1 microinjection of ACPA (0.5-1ng/rat) improved the amnesic effect of dextromethorphan /morphine combination. The microinjection of AM251 into the CA1 region enhanced the response of the combination of dextromethorphan /morphine in inducing amnesia. Moreover, Intra-CA1 microinjection of AM251 inhibited the improving effect of ACPA on dextromethorphan /morphine-induced amnesia. It is important to note that intra-CA1 microinjection of the same doses of the agonist or antagonist by itself had no effects on memory formation. Thus, it can be concluded that the dorsal hippocampal endocannabinoid system, via CB 1 receptor-dependent mechanism, may be involved in morphine/dextromethorphan -induced amnesia. Copyright © 2016 Elsevier B.V. All rights reserved.

A critical period in the supraspinal control of pain: opioid-dependent changes in brainstem rostroventral medulla function in preadolescence

PubMed Central

Hathway, Gareth J.; Vega-Avelaira, David; Fitzgerald, Maria

2012-01-01

We have previously shown that the balance of electrically evoked descending brainstem control of spinal nociceptive reflexes undergoes a switch from excitation to inhibition in preadolescent rats. Here we show that the same developmental switch occurs when μ-opioid receptor agonists are microinjected into the rostroventral medulla (RVM). Microinjections of the μ-opioid receptor agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) into the RVM of lightly anaesthetised adult rats produced a dose-dependent decrease in mechanical nociceptive hindlimb reflex electromyographic activity. However, in preadolescent (postnatal day 21 [P21]) rats, the same doses of DAMGO produced reflex facilitation. RVM microinjection of δ-opioid receptor or GABAA receptor agonists, on the other hand, caused reflex depression at both ages. The μ-opioid receptor-mediated descending facilitation is tonically active in naive preadolescent rats, as microinjection of the μ-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) into the RVM at this age decreases spinal nociceptive reflexes while having no effect in adults. To test whether tonic opioid central activity is required for the preadolescent switch in RVM descending control, naloxone hydrochloride was delivered continuously from subcutaneous osmotic mini-pumps for 7-day periods, at various postnatal stages. Blockade of tonic opioidergic activity from P21 to P28, but not at earlier or later ages, prevented the normal development of descending RVM inhibitory control of spinal nociceptive reflexes. Enhancing opioidergic activity with chronic morphine over P7 to P14 accelerated this development. These results show that descending facilitation of spinal nociception in young animals is mediated by μ-opioid receptor pathways in the RVM. Furthermore, the developmental transition from RVM descending facilitation to inhibition of pain is determined by activity in central opioid networks at a critical period of periadolescence. PMID:22325744

Spatio-temporal imaging of EGF-induced activation of protein kinase A by FRET in living cells

NASA Astrophysics Data System (ADS)

Wang, Jin Jun; Chen, Xiao-Chuan; Xing, Da

2004-07-01

Intracellular molecular interaction is important for the study of cell physiology, yet current relevant methods require fixation or microinjection and lack temporal or spatial resolution. We introduced a new method -- fluorescence resonance energy transfer (FRET) to detect molecular interaction in living cells. On the basis of FRET principle, A-kinase activity reporter (AKAR) protein was designed to consist of the fusions of cyan fluorescent protein (CFP), a phosphoamino acid binding domain, a consensus substrate for protein kinase-A (PKA), and yellow fluorescent protein (YFP). In this study, the designed pAKAR plasmid was used to transfect a human lung cancer cell line (ASTC-a-1). When the AKAR-transfected cells were treated by forskolin (Fsk), we were able to observe the efficient transfer of energy from excited CFP to YFP within the AKAR molecule by fluorescence microcopy, whereas no FRET was detected in the transfected cells without the treatment of Fsk. When the cells were treated by Epidermal growth factor (EGF), the change of FRET was observed at different subcellular locations, reflecting PKA activation inside the cells upon EGF stimulation. The successful design of a fluorescence reporter of PKA activation and its application demonstrated the superiority of this technology in the research of intracellular protein-protein interaction.

ABA Renewal Involves Enhancements in Both GluA2-Lacking AMPA Receptor Activity and GluA1 Phosphorylation in the Lateral Amygdala

PubMed Central

Park, Sungmo; Kim, Jihye; An, Bobae; Lee, Hyun Woo; Lee, Seungbok; Kim, Hyun; Lee, Justin C.; Lee, Sukwon; Choi, Sukwoo

2014-01-01

Fear renewal, the context-specific relapse of fear following fear extinction, is a leading animal model of post-traumatic stress disorders (PTSD) and fear-related disorders. Although fear extinction can diminish fear responses, this effect is restricted to the context where the extinction is carried out, and the extinguished fear strongly relapses when assessed in the original acquisition context (ABA renewal) or in a context distinct from the conditioning and extinction contexts (ABC renewal). We have previously identified Ser831 phosphorylation of GluA1 subunit in the lateral amygdala (LA) as a key molecular mechanism for ABC renewal. However, molecular mechanisms underlying ABA renewal remain to be elucidated. Here, we found that both the excitatory synaptic efficacy and GluA2-lacking AMPAR activity at thalamic input synapses onto the LA (T-LA synapses) were enhanced upon ABA renewal. GluA2-lacking AMPAR activity was also increased during low-threshold potentiation, a potential cellular substrate of renewal, at T-LA synapses. The microinjection of 1-naphtylacetyl-spermine (NASPM), a selective blocker of GluA2-lacking AMPARs, into the LA attenuated ABA renewal, suggesting a critical role of GluA2-lacking AMPARs in ABA renewal. We also found that Ser831 phosphorylation of GluA1 in the LA was increased upon ABA renewal. We developed a short peptide mimicking the Ser831-containing C-tail region of GluA1, which can be phosphorylated upon renewal (GluA1S); thus, the phosphorylated GluA1S may compete with Ser831-phosphorylated GluA1. This GluA1S peptide blocked the low-threshold potentiation when dialyzed into a recorded neuron. The microinjection of a cell-permeable form of GluA1S peptide into the LA attenuated ABA renewal. In support of the GluA1S experiments, a GluA1D peptide (in which the serine at 831 is replaced with a phosphomimetic amino acid, aspartate) attenuated ABA renewal when microinjected into the LA. These findings suggest that enhancements in both the GluA2-lacking AMPAR activity and GluA1 phosphorylation at Ser831 are required for ABA renewal. PMID:24925360

ABA renewal involves enhancements in both GluA2-lacking AMPA receptor activity and GluA1 phosphorylation in the lateral amygdala.

PubMed

Park, Kyungjoon; Song, Beomjong; Kim, Jeongyeon; Hong, Ingie; Song, Sangho; Lee, Junuk; Park, Sungmo; Kim, Jihye; An, Bobae; Lee, Hyun Woo; Lee, Seungbok; Kim, Hyun; Lee, Justin C; Lee, Sukwon; Choi, Sukwoo

2014-01-01

Fear renewal, the context-specific relapse of fear following fear extinction, is a leading animal model of post-traumatic stress disorders (PTSD) and fear-related disorders. Although fear extinction can diminish fear responses, this effect is restricted to the context where the extinction is carried out, and the extinguished fear strongly relapses when assessed in the original acquisition context (ABA renewal) or in a context distinct from the conditioning and extinction contexts (ABC renewal). We have previously identified Ser831 phosphorylation of GluA1 subunit in the lateral amygdala (LA) as a key molecular mechanism for ABC renewal. However, molecular mechanisms underlying ABA renewal remain to be elucidated. Here, we found that both the excitatory synaptic efficacy and GluA2-lacking AMPAR activity at thalamic input synapses onto the LA (T-LA synapses) were enhanced upon ABA renewal. GluA2-lacking AMPAR activity was also increased during low-threshold potentiation, a potential cellular substrate of renewal, at T-LA synapses. The microinjection of 1-naphtylacetyl-spermine (NASPM), a selective blocker of GluA2-lacking AMPARs, into the LA attenuated ABA renewal, suggesting a critical role of GluA2-lacking AMPARs in ABA renewal. We also found that Ser831 phosphorylation of GluA1 in the LA was increased upon ABA renewal. We developed a short peptide mimicking the Ser831-containing C-tail region of GluA1, which can be phosphorylated upon renewal (GluA1S); thus, the phosphorylated GluA1S may compete with Ser831-phosphorylated GluA1. This GluA1S peptide blocked the low-threshold potentiation when dialyzed into a recorded neuron. The microinjection of a cell-permeable form of GluA1S peptide into the LA attenuated ABA renewal. In support of the GluA1S experiments, a GluA1D peptide (in which the serine at 831 is replaced with a phosphomimetic amino acid, aspartate) attenuated ABA renewal when microinjected into the LA. These findings suggest that enhancements in both the GluA2-lacking AMPAR activity and GluA1 phosphorylation at Ser831 are required for ABA renewal.

The site of action of corticosteroid antipyresis in the rabbit.

PubMed Central

Willies, G H; Woolf, C J

1980-01-01

1. The antipyretic effects of corticosteroids on the fevers produced by bacterial and endogenous pyrogens in the rabbit were investigated. 2. Intravenous infusions of hydrocortisone and methyl prednisolone, when administered simultaneously with bacterial or endogenous pyrogens, failed to produce an antipyresis. 3. Pretreatment of rabbits with methyl prednisolone for 3 days diminished the febrile effect of both bacterial and endogenous pyrogens. 4. The fever produced by intrahypothalamic micro-injections of endogenous pyrogen was significantly attenuated by the simultaneous micro-injection of methyl prednisolone. 5. These results indicate that the antipyretic effect of steroids in the rabbit is the result not of a peripheral inhibition of endogenous pyrogen production, but rather of an action on the central nervous system. PMID:7381781

Microfluidic valve with cored glass microneedle for microinjection.

PubMed

Lee, Sanghoon; Jeong, Wonje; Beebe, David J

2003-08-01

In this paper, a new microinjection device was constructed by fusing a glass microneedle and a PDMS-based microvalve. The microneedle was fabricated via traditional micropipette pulling. The PDMS-based microvalve regulates the fluid flow in the microchannel and microneedle. The 'ON/OFF' operation of the valve was controlled by manually supplied pneumatic pressure. The valve membrane utilized a two level geometry to improve control at low flow rates. The relation between pressure and flow was measured and the results showed that very small volumes of fluid (>1 nl) could be controlled. The valve operation was investigated by monitoring the tip of the needle and pneumatic pressure simultaneously and it demonstrated very stable 'ON/OFF' operation to the pressure change.

Prolonged enhancement of REM sleep produced by carbachol microinjection into the amygdala.

PubMed

Calvo, J M; Simón-Arceo, K; Fernández-Mas, R

1996-01-31

The effect on sleep organization of carbachol microinjected into different amygdaloid nuclei was analysed in 12 cats. Single carbachol doses of 8 micrograms in 0.50 microliter saline were delivered unilaterally or bilaterally into the central, basal, lateral or basolateral amygdaloid nucleus. Carbachol administration into the central nucleus induced a prolonged (5 days) enhancement of both REM sleep and its preceeding slow wave sleep episodes with PGO waves (sommeil phasique a ondes lentes, SPHOL), which was more pronounced following bilateral than unilateral carbachol administration. However, neither SPHOL nor REM sleep changes were produced by administration of carbachol into the other amygdaloid nuclei. We conclude that cholinergic activation of the central amygdaloid nucleus produces a long-term facilitation of REM sleep occurrence.

Electrochemical attosyringe.

PubMed

Laforge, François O; Carpino, James; Rotenberg, Susan A; Mirkin, Michael V

2007-07-17

The ability to manipulate ultrasmall volumes of liquids is essential in such diverse fields as cell biology, microfluidics, capillary chromatography, and nanolithography. In cell biology, it is often necessary to inject material of high molecular weight (e.g., DNA, proteins) into living cells because their membranes are impermeable to such molecules. All techniques currently used for microinjection are plagued by two common problems: the relatively large injector size and volume of injected fluid, and poor control of the amount of injected material. Here we demonstrate the possibility of electrochemical control of the fluid motion that allows one to sample and dispense attoliter-to-picoliter (10(-18) to 10(-12) liter) volumes of either aqueous or nonaqueous solutions. By changing the voltage applied across the liquid/liquid interface, one can produce a sufficient force to draw solution inside a nanopipette and then inject it into an immobilized biological cell. A high success rate was achieved in injections of fluorescent dyes into cultured human breast cells. The injection of femtoliter-range volumes can be monitored by video microscopy, and current/resistance-based approaches can be used to control injections from very small pipettes. Other potential applications of the electrochemical syringe include fluid dispensing in nanolithography and pumping in microfluidic systems.

Electrochemical attosyringe

PubMed Central

Laforge, François O.; Carpino, James; Rotenberg, Susan A.; Mirkin, Michael V.

2007-01-01

The ability to manipulate ultrasmall volumes of liquids is essential in such diverse fields as cell biology, microfluidics, capillary chromatography, and nanolithography. In cell biology, it is often necessary to inject material of high molecular weight (e.g., DNA, proteins) into living cells because their membranes are impermeable to such molecules. All techniques currently used for microinjection are plagued by two common problems: the relatively large injector size and volume of injected fluid, and poor control of the amount of injected material. Here we demonstrate the possibility of electrochemical control of the fluid motion that allows one to sample and dispense attoliter-to-picoliter (10−18 to 10−12 liter) volumes of either aqueous or nonaqueous solutions. By changing the voltage applied across the liquid/liquid interface, one can produce a sufficient force to draw solution inside a nanopipette and then inject it into an immobilized biological cell. A high success rate was achieved in injections of fluorescent dyes into cultured human breast cells. The injection of femtoliter-range volumes can be monitored by video microscopy, and current/resistance-based approaches can be used to control injections from very small pipettes. Other potential applications of the electrochemical syringe include fluid dispensing in nanolithography and pumping in microfluidic systems. PMID:17620612

Acetylcholine from the mesopontine tegmental nuclei differentially affects methamphetamine induced locomotor activity and neurotransmitter levels in the mesolimbic pathway

PubMed Central

Dobbs, Lauren K.; Mark, Gregory P.

2012-01-01

Methamphetamine (MA) increases dopamine (DA) levels within the mesolimbic pathway and acetylcholine (ACh), a neurotransmitter known to increase DA cell firing and release and mediate reinforcement, within the ventral tegmental area (VTA). The laterodorsal tegmental (LDT) and pedunculopontine tegmental (PPT) nuclei provide cholinergic input to the VTA; however, the contribution of LDT- and PPT-derived ACh to MA-induced DA and ACh levels and locomotor activation remains unknown. The first experiment examined the role of LDT-derived ACh in MA locomotor activation by reversibly inhibiting these neurons with bilateral intra-LDT microinjections of the M2 receptor agonist oxotremorine (OXO). Male C57BL/6 J mice were given a bilateral 0.1 µl OXO (0, 1, or 10 nM/side) microinjection immediately prior to IP saline or MA (2 mg/kg). The highest OXO concentration significantly inhibited both saline-and MA-primed locomotor activity. In a second set of experiments we characterized the individual contributions of ACh originating in the LDT or pedunculopontine tegmental nucleus (PPT) to MA-induced levels of ACh and DA by administering intra-LDT or PPT OXO and performing in vivo microdialysis in the VTA and NAc. Intra-LDT OXO dose-dependently attenuated the MA-induced increase in ACh within the VTA but had no effect on DA in NAc. Intra-PPT OXO had no effect on ACh or DA levels within the VTA or NAc, respectively. We conclude that LDT, but not PPT, ACh is important in locomotor behavior and the cholinergic, but not dopaminergic, response to systemic MA. PMID:21945297

Cholinergic stimulation of the pons depresses respiration in decerebrate cats.

PubMed

Kimura, H; Kubin, L; Davies, R O; Pack, A I

1990-12-01

The injection of carbachol into the pontine tegmentum of decerebrate cats evokes a postural motor atonia that has many of the characteristics of the atonia of natural rapid-eye-movement (REM) sleep (Morales et al. J. Neurophysiol. 57: 1118-1129, 1987). We have used the carbachol-injected decerebrate cat to study the changes in respiratory neuronal activity that accompany the atonia. The activities of representative respiratory motor nerves--phrenic, intercostal, and hypoglossal--and that of a motor branch of C4 were recorded in decerebrate, vagotomized, paralyzed, and artificially ventilated cats. After the microinjection of carbachol, there was a profound suppression of activity in all the nerves and a decrease in respiratory rate. This was a consistent stereotyped response in which the magnitude of the suppression of respiratory-related activity was phrenic (to approximately 65% of control) less than inspiratory intercostal (approximately 50%) less than hypoglossal (approximately 10%) less than expiratory intercostal (approximately 5%). The decrease in respiratory rate (to approximately 70% of control) was caused by a prolongation of both inspiratory and expiratory durations. Complete reversal of the carbachol effect was elicited by the microinjection of atropine into the same site as the carbachol injection. This allowed us to produce a second episode of atonia by the injection of carbachol into the contralateral pons. Thus we have demonstrated the existence of neural pathways originating in the cholinoceptive cells of the pons that have the potential to powerfully and differentially depress various respiratory motoneuronal pools and to reduce the respiratory rate. These pathways are likely to be activated along with the atonia of REM sleep.

Paraventricular Stimulation with Glutamate Elicits Bradycardia and Pituitary Responses

NASA Technical Reports Server (NTRS)

Darlington, Daniel N.; Miyamoto, Michael; Keil, Lanny C.; Dallman, Mary F.

1989-01-01

The excitatory neurotransmitter, L-glutamate (0.5 M, pH 7.4), or the organic acid, acetate (0.5 M, pH 7.4), was microinjected (50 nl over 2 min) directly into the paraventricular nuclei (PVN) of pentobarbital sodium-anesthetized rats while arterial blood pressure and heart rate and plasma adrenocorticotropic hormone (ACTH), vasopressin, and oxytocin were measured. Activation of PVN neurons with L-glutamate led to increases in plasma ACTH, vasopressin, and oxytocin and a profound bradycardia (-80 beats/min) with little change in arterial blood pressure. Microinjection of acetate had no effect on the above variables. The decrease in heart rate was shown to be dependent on the concentration of glutamate injected and the volume of injectate. The bradycardia was mediated through the autonomic nervous system because ganglionic blockade (pentolinium tartrate) eliminated the response; atropine and propranolol severely attenuated the bradycardia. The bradycardia was greatest when L-glutamate was microinjected into the caudal PVN. Injections into the rostral PVN or into nuclei surrounding the PVN led to small or nonsignificant decreases in heart rate. Focal electric stimulation (2-50 pA) of the PVN also led to decreases in heart rate and arterial blood pressure. These data suggest that activation of PVN neurons leads to the release of ACTH, vasopressin, and oxytocin from the pituitary and a bradycardia that is mediated by the autonomic nervous system.

Microinjection of urocortin into the rat nucleus tractus solitarii decreases arterial blood pressure.

PubMed

Yamazaki, Toshiya; Waki, Hidefumi; Kohsaka, Akira; Nakamura, Takeshi; Cui, He; Yukawa, Kazunori; Maeda, Masanobu

2008-11-03

Systemic administration of urocortin I (Ucn I), a member of the corticotrophin-releasing factor (CRF) peptide family, modulates cardiovascular system. In the central nervous system, Ucn I is found in the nucleus tractus solitarii (NTS), which plays an important role in regulating arterial blood pressure (ABP) and heart rate (HR) in response to activation of the baroreceptor afferents. In this study, we examined the effects of Ucn I, which has a high affinity for both type 1 and type 2 CRF receptors (i.e. CRF-R1 and -R2), on cardiovascular functions at the level of the NTS. A specific agonist of CRF-R1 (i.e. CRF) and a specific agonist of CRF-R2 (i.e. Urocortin II) were also tested to identify the specific cardiovascular effects induced by individual activation of either CRF-R1 or -R2. We found that Ucn I microinjected into the rat NTS produced a significant reduction in both ABP and HR. Both agonists for CRF-R1 and -R2 microinjected into the NTS also reduced ABP and HR. Our results suggest that Ucn I in the NTS may play an important role in cardiovascular regulation and the cardiovascular effects of Ucn I may be mediated by activation of both CRF-R1 and -R2, which are known to be present in the NTS.

[Effect of 5-HT1A receptors in the hippocampal DG on active avoidance learning in rats].

PubMed

Jiang, Feng-ze; Lv, Jing; Wang, Dan; Jiang, Hai-ying; Li, Ying-shun; Jin, Qing-hua

2015-01-01

To investigate the effects of serotonin (5-HTIA) receptors in the hippocampal dentate gyrus (DG) on active avoidance learning in rats. Totally 36 SD rats were randomly divided into control group, antagonist group and agonist group(n = 12). Active avoidance learning ability of rats was assessed by the shuttle box. The extracellular concentrations of 5-HT in the DG during active avoidance conditioned reflex were measured by microdialysis and high performance liquid chromatography (HPLC) techniques. Then the antagonist (WAY-100635) or agonist (8-OH-DPAT) of the 5-HT1A receptors were microinjected into the DG region, and the active avoidance learning was measured. (1) During the active avoidance learning, the concentration of 5-HT in the hippocampal DG was significantly increased in the extinction but not establishment in the conditioned reflex, which reached 164.90% ± 26.07% (P <0.05) of basal level. (2) The microinjection of WAY-100635 (an antagonist of 5-HT1A receptor) into the DG did not significantly affect the active avoidance learning. (3) The microinjection of 8-OH-DPAT(an agonist of 5-HT1A receptor) into the DG significantly facilitated the establishment process and inhibited the extinction process during active avoidance conditioned reflex. The data suggest that activation of 5-HT1A receptors in hipocampal DG may facilitate active avoidance learning and memory in rats.

Glutamate microinjection in the medial septum of rats decreases paradoxical sleep and increases slow wave sleep.

PubMed

Mukherjee, Didhiti; Kaushik, Mahesh K; Jaryal, Ashok Kumar; Kumar, Velayudhan Mohan; Mallick, Hruda Nanda

2012-05-09

The role of the medial septum in suppressing paradoxical sleep and promoting slow wave sleep was suggested on the basis of neurotoxic lesion studies. However, these conclusions need to be substantiated with further experiments, including chemical stimulation studies. In this report, the medial septum was stimulated in adult male rats by microinjection of L-glutamate. Sleep-wakefulness was electrophysiologically recorded, through chronically implanted electrodes, for 2 h before the injection and 4 h after the injection. There was a decrease in paradoxical sleep during the first hour and an increase in slow wave sleep during the second hour after the injection. The present findings not only supported the lesion studies but also showed that the major role of the medial septum is to suppress paradoxical sleep.

Rho kinase inhibitor Y-27632 and Accutase dramatically increase mouse embryonic stem cell derivation.

PubMed

Zhang, Peng; Wu, Xinglong; Hu, Chunchao; Wang, Pengbo; Li, Xiangyun

2012-01-01

Although it has been 30 yr since the development of derivation methods for mouse embryonic stem (ES) cells, the biology of derivation of ES cells is poorly understood and the efficiency varies dramatically between cell lines. Recently, the Rho kinase inhibitor Y-27632 and the cell dissociation reagent Accutase were reported to significantly inhibit apoptosis of human ES cells during passaging. Therefore, in the current study, C57BL/6×129/Sv mouse blastocysts were used to evaluate the effect of the combination of the two reagents instead of using the conventional 129 line in mouse ES cell derivation. The data presented in this study suggests that the combination of Y-27632 and Accutase significantly increases the efficiency of mouse ES cell derivation; furthermore, no negative side effects were observed with Y-27632 and Accutase treatment. The newly established ES cell lines retain stable karyotype, surface markers expression, formed teratomas, and contributed to viable chimeras and germline transmission by tetraploid complementation assay. In addition, Y-27632 improved embryoid body formation of ES cells. During ES cell microinjection, Y-27632 prevented the formation of dissociation-induced cell blebs and facilitates the selection and the capture of intact cells. The methods presented in this study clearly demonstrate that inhibition of Rho kinase with Y-27632 and Accutase dissociation improve the derivation efficiently and reproducibility of mouse ES cell generation which is essential for reducing variability in the results obtained from different cell lines.

Bim, a Proapoptotic Protein, Up-regulated via Transcription Factor E2F1-dependent Mechanism, Functions as a Prosurvival Molecule in Cancer*

PubMed Central

Gogada, Raghu; Yadav, Neelu; Liu, Junwei; Tang, Shaohua; Zhang, Dianmu; Schneider, Andrea; Seshadri, Athul; Sun, Leimin; Aldaz, C. Marcelo; Tang, Dean G.; Chandra, Dhyan

2013-01-01

Proapoptotic Bcl-2 homology 3-only protein Bim plays an important role in Bax/Bak-mediated cytochrome c release and apoptosis. Here, we provide evidence for a novel prosurvival function of Bim in cancer cells. Bim was constitutively overexpressed in multiple prostate and breast cancer cells as well as in primary tumor cells. Quantitative real time PCR analysis showed that Bim was transcriptionally up-regulated. We have identified eight endogenous E2F1-binding sites on the Bim promoter using in silico analysis. Luciferase assay demonstrated that Bim expression was E2F1-dependent as mutation of the E2F1-binding sites on the Bim promoter inhibited luciferase activities. In support, E2F1 silencing led to the loss of Bim expression in cancer cells. Bim primarily localized to mitochondrial and cytoskeleton-associated fractions. Bim silencing or microinjection of anti-Bim antibodies into the cell cytoplasm resulted in cell rounding, detachment, and subsequent apoptosis. We observed up-regulation of prosurvival proteins Bcl-xL and Mcl-1, which sequester Bim in cancer cells. In addition, a phosphorylated form of Bim was also elevated in cancer cells. These findings suggest that the constitutively overexpressed Bim may function as a prosurvival molecule in epithelial cancer cells, and phosphorylation and association with Bcl-xL/Mcl-1 block its proapoptotic functions. PMID:23152504

Genome editing reveals a role for OCT4 in human embryogenesis.

PubMed

Fogarty, Norah M E; McCarthy, Afshan; Snijders, Kirsten E; Powell, Benjamin E; Kubikova, Nada; Blakeley, Paul; Lea, Rebecca; Elder, Kay; Wamaitha, Sissy E; Kim, Daesik; Maciulyte, Valdone; Kleinjung, Jens; Kim, Jin-Soo; Wells, Dagan; Vallier, Ludovic; Bertero, Alessandro; Turner, James M A; Niakan, Kathy K

2017-10-05

Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.

High diadenosine tetraphosphate (Ap4A) level in germ cells and embryos of sea urchin and Xenopus and its effect on DNA synthesis.

PubMed

Weinmann-Dorsch, C; Grummt, F

1985-09-01

Ap4A levels in sperms, eggs and different developmental stages of sea urchin (Psammechinus miliaris) and (Xenopus laevis) were determined by a method based on ATP measurement with luciferin/luciferase after splitting diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) into ATP and AMP. Appreciable storage pools of Ap4A were found in unfertilized eggs of Psammechinus and Xenopus as well as in sea urchin sperms. The actual Ap4A concentration of 28 microM in sperm represents the highest Ap4A level so far observed in eukaryotic cells. Upon fertilization an instant onset of de novo synthesis of Ap4A was demonstrated. Ap4A levels during early embryogenesis of P. miliaris and X. laevis (2.5-4 microM) are higher than those in exponentially growing mammalian culture cells and mammalian fetuses. Microinjection of Ap4A into unfertilized eggs of Psammechinus miliaris caused a 3-7 fold increase of DNA synthesis in comparison with mock-injected eggs.

MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems.

PubMed

Sakuma, Tetsushi; Nakade, Shota; Sakane, Yuto; Suzuki, Ken-Ichi T; Yamamoto, Takashi

2016-01-01

Programmable nucleases enable engineering of the genome by utilizing endogenous DNA double-strand break (DSB) repair pathways. Although homologous recombination (HR)-mediated gene knock-in is well established, it cannot necessarily be applied in every cell type and organism because of variable HR frequencies. We recently reported an alternative method of gene knock-in, named the PITCh (Precise Integration into Target Chromosome) system, assisted by microhomology-mediated end-joining (MMEJ). MMEJ harnesses independent machinery from HR, and it requires an extremely short homologous sequence (5-25 bp) for DSB repair, resulting in precise gene knock-in with a more easily constructed donor vector. Here we describe a streamlined protocol for PITCh knock-in, including the design and construction of the PITCh vectors, and their delivery to either human cell lines by transfection or to frog embryos by microinjection. The construction of the PITCh vectors requires only a few days, and the entire process takes ∼ 1.5 months to establish knocked-in cells or ∼ 1 week from injection to early genotyping in frog embryos.

Targeted intracellular delivery of proteins with spatial and temporal control.

PubMed

Morales, Demosthenes P; Braun, Gary B; Pallaoro, Alessia; Chen, Renwei; Huang, Xiao; Zasadzinski, Joseph A; Reich, Norbert O

2015-02-02

While a host of methods exist to deliver genetic materials or small molecules to cells, very few are available for protein delivery to the cytosol. We describe a modular, light-activated nanocarrier that transports proteins into cells by receptor-mediated endocytosis and delivers the cargo to the cytosol by light triggered endosomal escape. The platform is based on hollow gold nanoshells (HGN) with polyhistidine tagged proteins attached through an avidity-enhanced, nickel chelation linking layer; here, we used green fluorescent protein (GFP) as a model deliverable cargo. Endosomal uptake of the GFP loaded nanocarrier was mediated by a C-end Rule (CendR) internalizing peptide fused to the GFP. Focused femtosecond pulsed-laser excitation triggered protein release from the nanocarrier and endosome disruption, and the released protein was capable of targeting the nucleoli, a model intracellular organelle. We further demonstrate the generality of the approach by loading and releasing Sox2 and p53. This method for targeting of individual cells, with resolution similar to microinjection, provides spatial and temporal control over protein delivery.

Fever produced by endotoxin injected into the hypothalamus of the monkey and its antagonism by salicylate

PubMed Central

Myers, R. D.; Rudy, T. A.; Yaksh, T. L.

1974-01-01

1. A suspension of the killed cell bodies of either E. coli, S. dysenteriae or S. typhosa was micro-injected through cannulae implanted chronically at specific sites within the diencephalon and mid-brain of the unanaesthetized monkey. A biphasic, monophasic or an undifferentiated fever could be induced by each type of micro-organism, but the type of response depended solely upon the locus of injection. 2. Although little difference in the potency of the three pyrogens was found, the rise in body temperature was in each instance dependent upon the concentration of the endotoxin. A more intense fever was accompanied by shivering, vasoconstriction of the ear vessels, piloerection and huddling behaviour. Tolerance to the pyrexic effect of repeated injections of endotoxin did not develop. 3. The febrile response having the shortest latency, greatest maximum rise in temperature and largest 10-hr fever index was evoked by micro-injections into the anterior hypothalamic, preoptic area. The incidence of biphasic fevers was also greater after endotoxin was injected into this same region. Endotoxin given similarly in the posterior hypothalamus or in the mesencephalon had either no effect or produced a smaller elevation in temperature after a longer latency. The distance of an injection site from the coronal plane formed by the optic chiasm and anterior commissure correlated significantly with the latency and magnitude of the temperature change as well as the fever index. 4. When given intravenously, endotoxin in a quantity at least 100 times greater was required to evoke a fever similar to that produced when the pyrogen was micro-injected into the anterior hypothalamic, preoptic region. However, a biphasic fever was evoked with a latency of from 3 to 15 min when a larger amount of endotoxin was injected intravenously. Tolerance developed rapidly to the febrile effect of endotoxin administered by this route although toxic reactions were not observed. 5. After the fever evoked by the hypothalamic injection of endotoxin had reached a plateau, 300-1200 mg sodium salicylate administered intragastrically produced a dose-dependent fall in temperature, but had no effect on the body temperature of an afebrile monkey. 6. It is concluded that in the rhesus monkey, a bacterial pyrogen can evoke a fever which is mediated entirely by an action on the central nervous system, the principal site being the anterior hypothalamic, preoptic area. The first phase of a biphasic fever caused by bacteria acting either by the central or peripheral route seems to be due either to a direct action of the pyrogen on the cells of the anterior hypothalamus, or to the secondary release within this region of an intermediary thermogenic substance such as 5-hydroxytryptamine or prostaglandin. The finding that sodium salicylate counteracts a centrally evoked fever is not compatible with the hypothesis that an antipyretic exerts its action by preventing a pyrogen that is circulating in the blood stream from entering the central nervous system. PMID:4615138

Opto-injection into single living cells by femtosecond near-infrared laser

NASA Astrophysics Data System (ADS)

Peng, Cheng

This dissertation presents a novel technique to deliver membrane impermeable molecules into single living cells with the assistance of femtosecond (fs) near-infrared (NIR) laser pulses. This approach merges ultrafast laser technology with key biological, biomedical, and medical applications, such as gene transfection, gene therapy and drug delivery. This technique promises several major advantages, namely, very high transfection efficiency, high cell survival rate (≈100%) and fully preserved cell viabilities. It is also a promising method to deliver molecules into cells that are difficult or even completely resistant to established physical methods, such as microinjection by glass pipettes, electroporation, and biolistics. In this work, the system for fs NIR opto-injection was designed and built. Successful fs NIR opto-injection has been performed on several cell systems including single mammalian cells (bovine aortic endothelial cells), marine animal eggs (Spisula solidissima oocytes), and human cancer cells (fibrosarcoma HT1080) cultured in a tissue-like environment. The connections between laser parameters and cell responses were explored through further experiments and in-depth analyses, especially the relationship between dye uptake rate and incident laser intensity, and the relationship between pore size created on cell membranes and incident laser intensity. Dye uptake rate of the target cells was observed to depend on incident laser intensity. Pore size was found dependent on incident laser intensity. The conclusion was made that laser-induced breakdown and plasma-induced ablation in cell membrane are the physical principles that govern the process of fs NIR opto-injection.

Reciprocal phosphorylation and glycosylation recognition motifs control NCAPP1 interaction with pumpkin phloem proteins and their cell-to-cell movement.

PubMed

Taoka, Ken-Ichiro; Ham, Byung-Kook; Xoconostle-Cázares, Beatriz; Rojas, Maria R; Lucas, William J

2007-06-01

In plants, cell-to-cell trafficking of non-cell-autonomous proteins (NCAPs) involves protein-protein interactions, and a role for posttranslational modification has been implicated. In this study, proteins contained in pumpkin (Cucurbita maxima cv Big Max) phloem sap were used as a source of NCAPs to further explore the molecular basis for selective NCAP trafficking. Protein overlay assays and coimmunoprecipitation experiments established that phosphorylation and glycosylation, on both Nicotiana tabacum NON-CELL-AUTONOMOUS PATHWAY PROTEIN1 (Nt-NCAPP1) and the phloem NCAPs, are essential for their interaction. Detailed molecular analysis of a representative phloem NCAP, Cm-PP16-1, identified the specific residues on which glycosylation and phosphorylation must occur for effective binding to NCAPP1. Microinjection studies confirmed that posttranslational modification on these residues is essential for cell-to-cell movement of Cm-PP16-1. Lastly, a glutathione S-transferase (GST)-Cm-PP16-1 fusion protein system was employed to test whether the peptide region spanning these residues was required for cell-to-cell movement. These studies established that a 36-amino acid peptide was sufficient to impart cell-to-cell movement capacity to GST, a normally cell-autonomous protein. These findings are consistent with the hypothesis that a phosphorylation-glycosylation recognition motif functions to control the binding of a specific subset of phloem NCAPs to NCAPP1 and their subsequent transport through plasmodesmata.

Reciprocal Phosphorylation and Glycosylation Recognition Motifs Control NCAPP1 Interaction with Pumpkin Phloem Proteins and Their Cell-to-Cell Movement[W

PubMed Central

Taoka, Ken-ichiro; Ham, Byung-Kook; Xoconostle-Cázares, Beatriz; Rojas, Maria R.; Lucas, William J.

2007-01-01

In plants, cell-to-cell trafficking of non-cell-autonomous proteins (NCAPs) involves protein–protein interactions, and a role for posttranslational modification has been implicated. In this study, proteins contained in pumpkin (Cucurbita maxima cv Big Max) phloem sap were used as a source of NCAPs to further explore the molecular basis for selective NCAP trafficking. Protein overlay assays and coimmunoprecipitation experiments established that phosphorylation and glycosylation, on both Nicotiana tabacum NON-CELL-AUTONOMOUS PATHWAY PROTEIN1 (Nt-NCAPP1) and the phloem NCAPs, are essential for their interaction. Detailed molecular analysis of a representative phloem NCAP, Cm-PP16-1, identified the specific residues on which glycosylation and phosphorylation must occur for effective binding to NCAPP1. Microinjection studies confirmed that posttranslational modification on these residues is essential for cell-to-cell movement of Cm-PP16-1. Lastly, a glutathione S-transferase (GST)–Cm-PP16-1 fusion protein system was employed to test whether the peptide region spanning these residues was required for cell-to-cell movement. These studies established that a 36–amino acid peptide was sufficient to impart cell-to-cell movement capacity to GST, a normally cell-autonomous protein. These findings are consistent with the hypothesis that a phosphorylation-glycosylation recognition motif functions to control the binding of a specific subset of phloem NCAPs to NCAPP1 and their subsequent transport through plasmodesmata. PMID:17601822

Activation of the cholinergic system of the striatum improves attention to conditioned reflex stimuli.

PubMed

Shapovalova, K B

1999-01-01

Chronic experiments were performed on 16 dogs using a model of an operant defensive reflex associated with maintenance of a flexion pose to study the effects of uni- and bilateral microinjections of the acetylcholine agonist carbacholine (0.05-0.4 microg) and the choline receptor blocker scopolamine (0.5 microg) into the dorsolateral part of the head of the caudate nucleus and CM-Pf intralaminar thalamic nuclei. These experiments produced data showing that the cholinergic system of the striatum has an important role in realizing the sensory and motor components of the learned movement. Activation of the cholinergic system of the dorsal striatum led to general calming of behavior and inhibition of intersignal limb elevation and the phasic components of the movement, along with ordering and stabilizing of the pose and an increase in the tonic component of the operant response. This suggests that the cholinergic system of the striatum receives an indirect efferent output via motor structures and takes part in preparing the motor apparatus needed for transferring attention to significant stimuli. Microinjections of scopolamine had the opposite effects. Use of differential signals in the same behavioral model, along with special tests for attention, showed that the cholinergic system of the striatum plays an important role in the sensory control of attention. Activation of the striatal cholinergic system led to a significant improvement in responses to differential signals and defensive signals of intensity 2-3 times slower than normal signals, and these changes were accompanied by clearer responses in special tests for attention. Scopolamine microinjections had the opposite effects. Carbacholine microinjections into the intralaminar thalamic nuclei potentiated the effects of cholinergic activation of the striatum. These data indicate that the dorsal striatum can be regarded not only as a parallel level of information processing, but also as a control system for passing this information to various levels of both sensory and motor structures. One important result of this type of control may be that of improving attention to significant stimuli.

Enhanced noradrenergic activity in the amygdala contributes to hyperarousal in an animal model of PTSD.

PubMed

Ronzoni, Giacomo; Del Arco, Alberto; Mora, Francisco; Segovia, Gregorio

2016-08-01

Increased activity of the noradrenergic system in the amygdala has been suggested to contribute to the hyperarousal symptoms associated with post-traumatic stress disorder (PTSD). However, only two studies have examined the content of noradrenaline or its metabolites in the amygdala of rats previously exposed to traumatic stress showing inconsistent results. The aim of this study was to investigate the effects of an inescapable foot shock (IFS) procedure (1) on reactivity to novelty in an open-field (as an index of hyperarousal), and (2) on noradrenaline release in the amygdala during an acute stress. To test the role of noradrenaline in amygdala, we also investigated the effects of microinjections of propranolol, a β-adrenoreceptor antagonist, and clenbuterol, a β-adrenoreceptor agonist, into the amygdala of IFS and control animals. Finally, we evaluated the expression of mRNA levels of β-adrenoreceptors (β1 and β2) in the amygdala, the hippocampus and the prefrontal cortex. Male Wistar rats (3 months) were stereotaxically implanted with bilateral guide cannulae. After recovering from surgery, animals were exposed to IFS (10 shocks, 0.86mA, and 6s per shock) and seven days later either microdialysis or microinjections were performed in amygdala. Animals exposed to IFS showed a reduced locomotion compared to non-shocked animals during the first 5min in the open-field. In the amygdala, IFS animals showed an enhanced increase of noradrenaline induced by stress compared to control animals. Bilateral microinjections of propranolol (0.5μg) into the amygdala one hour before testing in the open-field normalized the decreased locomotion observed in IFS animals. On the other hand, bilateral microinjections of clenbuterol (30ng) into the amygdala of control animals did not change the exploratory activity induced by novelty in the open field. IFS modified the mRNA expression of β1 and β2 adrenoreceptors in the prefrontal cortex and the hippocampus. These results suggest that an increased noradrenergic activity in the amygdala contributes to the expression of hyperarousal in an animal model of PTSD. Copyright © 2016 Elsevier Ltd. All rights reserved.

Glutamate receptors of the A5 region modulate cardiovascular responses evoked from the dorsomedial hypothalamic nucleus and perifornical area.

PubMed

López-González, M V; Díaz-Casares, A; González-García, M; Peinado-Aragonés, C A; Barbancho, M A; Carrillo de Albornoz, M; Dawid-Milner, M S

2018-05-01

To assess the possible function of glutamate in the interaction between the dorsomedial hypothalamic nucleus-perifornical area (DMH-PeF) and the A5 pontine region (A5), cardiovascular and respiratory changes were studied in response to electrical stimulation of the DMH-PeF (1 ms pulses, 30-50 μA given at 100 Hz for 5 s) before and after the microinjection of kynurenic acid (non-specific glutamate receptor antagonist; 50 nl, 5 nmol), MK-801 (NMDA receptor antagonist; 50 nl, 50 nmol), CNQX (non-NMDA receptor antagonist; 50 nl, 50 nmol) or MCPG (metabotropic glutamate receptor antagonist; 50 nl, 5 nmol) within the A5 region. DMH-PeF electrical stimulation elicited a pressor (p < 0.001) and tachycardic response (p < 0.001) which was accompanied by an inspiratory facilitation characterised by an increase in respiratory rate (p < 0.001) due to a decrease in expiratory time (p < 0.01). Kynurenic acid within the A5 region decreased the tachycardia (p < 0.001) and the intensity of the blood pressure response (p < 0.001) to DMH-PeF stimulation. After the microinjection of MK-801 and CNQX into the A5 region, the magnitude of the tachycardia and the pressor response were decreased (p < 0.05 and p < 0.01; p < 0.001 and p < 0.05, respectively). After MCPG microinjection into the A5 region, a decrease in the tachycardia (p < 0.001) with no changes in the pressor response was observed during DMH-PeF stimulation. The respiratory response elicited by DMH-PeF stimulation was not changed after the microinjection of kynurenic acid, MK-801, CNQX or MCPG within the A5 region. These results suggest that A5 region glutamate receptors play a role in the cardiovascular response elicited from the DMH-PeF. The possible mechanisms involved in these interactions are discussed.

Behavioral and EEG effects of GABAergic manipulation of the nigro-tectal pathway in the Wistar audiogenic rat (WAR) strain II: an EEG wavelet analysis and retrograde neuronal tracer approach.

PubMed

Rossetti, Franco; Rodrigues, Marcelo Cairrão Araújo; Marroni, Simone S; Fernandes, Artur; Foresti, Maira Licia; Romcy-Pereira, Rodrigo N; de Araújo, Dráulio Barros; Garcia-Cairasco, Norberto

2012-08-01

The role of the substantia nigra pars reticulata (SNPr) and superior colliculus (SC) network in rat strains susceptible to audiogenic seizures still remain underexplored in epileptology. In a previous study from our laboratory, the GABAergic drugs bicuculline (BIC) and muscimol (MUS) were microinjected into the deep layers of either the anterior SC (aSC) or the posterior SC (pSC) in animals of the Wistar audiogenic rat (WAR) strain submitted to acoustic stimulation, in which simultaneous electroencephalographic (EEG) recording of the aSC, pSC, SNPr and striatum was performed. Only MUS microinjected into the pSC blocked audiogenic seizures. In the present study, we expanded upon these previous results using the retrograde tracer Fluorogold (FG) microinjected into the aSC and pSC in conjunction with quantitative EEG analysis (wavelet transform), in the search for mechanisms associated with the susceptibility of this inbred strain to acoustic stimulation. Our hypothesis was that the WAR strain would have different connectivity between specific subareas of the superior colliculus and the SNPr when compared with resistant Wistar animals and that these connections would lead to altered behavior of this network during audiogenic seizures. Wavelet analysis showed that the only treatment with an anticonvulsant effect was MUS microinjected into the pSC region, and this treatment induced a sustained oscillation in the theta band only in the SNPr and in the pSC. These data suggest that in WAR animals, there are at least two subcortical loops and that the one involved in audiogenic seizure susceptibility appears to be the pSC-SNPr circuit. We also found that WARs presented an increase in the number of FG+ projections from the posterior SNPr to both the aSC and pSC (primarily to the pSC), with both acting as proconvulsant nuclei when compared with Wistar rats. We concluded that these two different subcortical loops within the basal ganglia are probably a consequence of the WAR genetic background. Copyright © 2012 Elsevier Inc. All rights reserved.

Progress and biotechnological prospects in fish transgenesis.

PubMed

Tonelli, Fernanda M P; Lacerda, Samyra M S N; Tonelli, Flávia C P; Costa, Guilherme M J; de França, Luiz Renato; Resende, Rodrigo R

2017-11-01

The history of transgenesis is marked by milestones such as the development of cellular transdifferentiation, recombinant DNA, genetic modification of target cells, and finally, the generation of simpler genetically modified organisms (e.g. bacteria and mice). The first transgenic fish was developed in 1984, and since then, continuing technological advancements to improve gene transfer have led to more rapid, accurate, and efficient generation of transgenic animals. Among the established methods are microinjection, electroporation, lipofection, viral vectors, and gene targeting. Here, we review the history of animal transgenesis, with an emphasis on fish, in conjunction with major developments in genetic engineering over the past few decades. Importantly, spermatogonial stem cell modification and transplantation are two common techniques capable of revolutionizing the generation of transgenic fish. Furthermore, we discuss recent progress and future biotechnological prospects of fish transgenesis, which has strong applications for the aquaculture industry. Indeed, some transgenic fish are already available in the current market, validating continued efforts to improve economically important species with biotechnological advancements. Copyright © 2017. Published by Elsevier Inc.

The zinc spark is an inorganic signature of human egg activation.

PubMed

Duncan, Francesca E; Que, Emily L; Zhang, Nan; Feinberg, Eve C; O'Halloran, Thomas V; Woodruff, Teresa K

2016-04-26

Egg activation refers to events required for transition of a gamete into an embryo, including establishment of the polyspermy block, completion of meiosis, entry into mitosis, selective recruitment and degradation of maternal mRNA, and pronuclear development. Here we show that zinc fluxes accompany human egg activation. We monitored calcium and zinc dynamics in individual human eggs using selective fluorophores following activation with calcium-ionomycin, ionomycin, or hPLCζ cRNA microinjection. These egg activation methods, as expected, induced rises in intracellular calcium levels and also triggered the coordinated release of zinc into the extracellular space in a prominent "zinc spark." The ability of the gamete to mount a zinc spark response was meiotic-stage dependent. Moreover, chelation of intracellular zinc alone was sufficient to induce cell cycle resumption and transition of a meiotic cell into a mitotic one. Together, these results demonstrate critical functions for zinc dynamics and establish the zinc spark as an extracellular marker of early human development.

The zinc spark is an inorganic signature of human egg activation

PubMed Central

Duncan, Francesca E.; Que, Emily L.; Zhang, Nan; Feinberg, Eve C.; O’Halloran, Thomas V.; Woodruff, Teresa K.

2016-01-01

Egg activation refers to events required for transition of a gamete into an embryo, including establishment of the polyspermy block, completion of meiosis, entry into mitosis, selective recruitment and degradation of maternal mRNA, and pronuclear development. Here we show that zinc fluxes accompany human egg activation. We monitored calcium and zinc dynamics in individual human eggs using selective fluorophores following activation with calcium-ionomycin, ionomycin, or hPLCζ cRNA microinjection. These egg activation methods, as expected, induced rises in intracellular calcium levels and also triggered the coordinated release of zinc into the extracellular space in a prominent “zinc spark.” The ability of the gamete to mount a zinc spark response was meiotic-stage dependent. Moreover, chelation of intracellular zinc alone was sufficient to induce cell cycle resumption and transition of a meiotic cell into a mitotic one. Together, these results demonstrate critical functions for zinc dynamics and establish the zinc spark as an extracellular marker of early human development. PMID:27113677

Antagonizing the Spemann organizer: role of the homeobox gene Xvent-1.

PubMed Central

Gawantka, V; Delius, H; Hirschfeld, K; Blumenstock, C; Niehrs, C

1995-01-01

We have identified a novel homeobox gene, Xvent-1, that is differentially expressed in the ventral marginal zone of the early Xenopus gastrula. Evidence is presented from mRNA microinjection experiments for a role for this gene in dorsoventral patterning of mesoderm. First, Xvent-1 is induced by BMP-4, a gene known to be a key regulator of ventral mesoderm development. Second, Xvent-1 and the organizer-specific gene goosecoid are able to interact, directly or indirectly, in a cross-regulatory loop suppressing each other's expression, consistent with their mutually exclusive expression in the marginal zone. Third, microinjection of Xvent-1 mRNA ventralizes dorsal mesoderm. The results suggest that Xvent-1 functions in a ventral signaling pathway that maintains the ventral mesodermal state and antagonizes the Spemann organizer. Images PMID:8557046

AAV-Mediated Gene Transfer to Dorsal Root Ganglion.

PubMed

Yu, Hongwei; Fischer, Gregory; Hogan, Quinn H

2016-01-01

Transferring genetic molecules into the peripheral sensory nervous system to manipulate nociceptive pathophysiology is a powerful approach for experimental modulation of sensory signaling and potentially for translation into therapy for chronic pain. This can be efficiently achieved by the use of recombinant adeno-associated virus (rAAV) in conjunction with nociceptor-specific regulatory transgene cassettes. Among different routes of delivery, direct injection into the dorsal root ganglia (DRGs) offers the most efficient AAV-mediated gene transfer selectively into the peripheral sensory nervous system. Here, we briefly discuss the advantages and applications of intraganglionic microinjection, and then provide a detailed approach for DRG injection, including a list of the necessary materials and description of a method for performing DRG microinjection experiments. We also discuss our experience with several adeno-associated virus (AAV) options for in vivo transgene expression in DRG neurons.

Brain nicotinic acetylcholine receptors are involved in stress-induced potentiation of nicotine reward in rats.

PubMed

Javadi, Parastoo; Rezayof, Ameneh; Sardari, Maryam; Ghasemzadeh, Zahra

2017-07-01

The aim of the present study was to examine the possible role of nicotinic acetylcholine receptors of the dorsal hippocampus (CA1 regions), the medial prefrontal cortex or the basolateral amygdala in the effect of acute or sub-chronic stress on nicotine-induced conditioned place preference. Our results indicated that subcutaneous administration of nicotine (0.2 mg/kg) induced significant conditioned place preference. Exposure to acute or sub-chronic elevated platform stress potentiated the response of an ineffective dose of nicotine. Pre-conditioning intra-CA1 (0.5-4 µg/rat) or intra-medial prefrontal cortex (0.2-0.3 µg/rat) microinjection of mecamylamine (a non-selective nicotinic acetylcholine receptor antagonist) reversed acute stress-induced potentiation of nicotine reward as measured in the conditioned place preference paradigm. By contrast, pre-conditioning intra-basolateral amygdala microinjection of mecamylamine (4 µg/rat) potentiated the effects of acute stress on nicotine reward. Our findings also showed that intra-CA1 or intra-medial prefrontal cortex, but not intra-basolateral amygdala, microinjection of mecamylamine (4 µg/rat) prevented the effect of sub-chronic stress on nicotine reward. These findings suggest that exposure to elevated platform stress potentiates the rewarding effect of nicotine which may be associated with the involvement of nicotinic acetylcholine receptors. It seems that there is a different contribution of the basolateral amygdala, the medial prefrontal cortex or the CA1 nicotinic acetylcholine receptors in stress-induced potentiation of nicotine-induced conditioned place preference.

Methods for rapid transfer and localization of lyme disease pathogens within the tick gut.

PubMed

Kariu, Toru; Coleman, Adam S; Anderson, John F; Pal, Utpal

2011-02-14

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle. A tick-borne murine model has been developed to study Lyme disease in the laboratory. While naíve ticks can be infected with B. burgdorferi by feeding them on infected mice, the molting process takes several weeks to months to complete. Therefore, development of more rapid and efficient tick infection techniques, such as a microinjection-based procedure, is an important tool for the study of Lyme disease. The procedure requires only hours to generate infected ticks and allows control over the delivery of equal quantities of spirochetes in a cohort of ticks. This is particularly important as the generation of B. burgdorferi infected ticks by the natural feeding process using mice fails to ensure 100% infection rate and potentially results in variation of pathogen burden amongst fed ticks. Furthermore, microinjection can be used to infect ticks with B. burgdorferi isolates in cases where an attenuated strain is unable to establish infection in mice and thus can not be naturally acquired by ticks. This technique can also be used to deliver a variety of other biological materials into ticks, for example, specific antibodies or double stranded RNA. In this article, we will demonstrate the microinjection of nymphal ticks with in vitro-grown B. burgdorferi. We will also describe a method for localization of Lyme disease pathogens in the tick gut using confocal immunofluorescence microscopy.

Antagonistic effects of somatostatin and substance P on respiratory regulation in the rat ventrolateral medulla oblongata.

PubMed

Chen, Z B; Engberg, G; Hedner, T; Hedner, J

1991-08-09

Substance P (SP) in the dose range 0.75-1.5 nmol exerts a potent stimulatory effect on ventilation after microinjection into the rat ventrolateral medulla oblongata (VLM; n. reticularis lateralis, n. paragigantocellularis lateralis). A significant but less pronounced effect is also seen in the dorsal medulla (DM; n. tractus solitarius). Somatostatin (0.6-1.8 nmol) inhibited ventilation and induced apnoea after microinjection into the VLM but not the DM. Serial microinjections of the two peptides showed a reciprocal antagonistic action in the VLM but not in the DM. The apnoea-inducing effect of SOM was blunted by SP while SOM reduced the ventilatory stimulation induced by SP. Extracellular single unit recordings were performed following the microiontophoretic application of SP and/or SOM to respiratory-related and non-respiratory-related neurons in the VLM and DM. Although a heterogeneous population of neurons were recorded from, the majority of respiratory-related units in the VLM responded with excitation to SP and inhibitory to SOM. A direct interaction between the peptides was seen in some respiratory-related units. The neurons not responding to either of the peptides were usually non-respiratory. Dorsal to the VLM, the type of response to the two peptides was less likely to be antagonistic and a wider distribution of response types were recorded. The results indicate a direct physiological antagonism between SP and SOM regarding their effects on respiratory regulation elicited in the VLM.

From the Cover: Microfabricated needles for transdermal delivery of macromolecules and nanoparticles: Fabrication methods and transport studies

NASA Astrophysics Data System (ADS)

McAllister, Devin V.; Wang, Ping M.; Davis, Shawn P.; Park, Jung-Hwan; Canatella, Paul J.; Allen, Mark G.; Prausnitz, Mark R.

2003-11-01

Arrays of micrometer-scale needles could be used to deliver drugs, proteins, and particles across skin in a minimally invasive manner. We therefore developed microfabrication techniques for silicon, metal, and biodegradable polymer microneedle arrays having solid and hollow bores with tapered and beveled tips and feature sizes from 1 to 1,000 μm. When solid microneedles were used, skin permeability was increased in vitro by orders of magnitude for macromolecules and particles up to 50 nm in radius. Intracellular delivery of molecules into viable cells was also achieved with high efficiency. Hollow microneedles permitted flow of microliter quantities into skin in vivo, including microinjection of insulin to reduce blood glucose levels in diabetic rats. transdermal drug delivery | skin | microelectromechanical systems | solid microneedle | hollow needle injection

Effects of the phosphatase inhibitors, okadaic acid, ATPgammaS, and calyculin A on the dividing sand dollar egg.

PubMed

Hamaguchi, Yukihisa; Kuriyama, Ryoko

2002-06-01

The effects of the phosphatase inhibitors, okadaic acid (OA), adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), and calyculin A (CL-A) on anaphase chromosome movement, cytokinesis, and cytoskeletal structures at cell division were examined by being microinjected into mitotic sand dollar eggs. When OA was injected, chromosome movement was inhibited and, moreover, chromosomes were ejected from the polar regions of the mitotic apparatus. By immunofluorescence, microtubules were observed to be severed in the OA-injected eggs, causing the smooth cell surface to be changed to an irregular surface. When ATPgammaS and CL-A were injected, the effect on cell shape was remarkable: In dividing eggs, furrowing stopped within several seconds after injection, small blebs appeared on the cell surface and became large, spherical or dumbbell cell shapes then changed to irregular forms, and subsequently cytoplasmic flow occurred. Microfilament detection revealed that actin accumulation in the cortex, which was not limited to the furrow cortex, occurred shortly after injection. Cortical accumulation of actin is thought to induce force generation and random cortical contraction, and accordingly to result in bleb extrusion from the cortex. Consequently, the phosphatase inhibitors inhibited the transition from mitosis to interphase by mediating cortical accumulation of actin filaments and/or fragmentation of microtubules.

The measurement of ultrasound scattering from individual micron-sized objects and its application in single cell scattering.

PubMed

Falou, Omar; Rui, Min; El Kaffas, Ahmed; Kumaradas, J Carl; Kolios, Michael C

2010-08-01

The measurement of the ultrasound backscatter from individual micron-sized objects such as cells is required for various applications such as tissue characterization. However, performing such a measurement remains a challenge. For example, the presence of air bubbles in a suspension of cells during the measurements may lead to the incorrect interpretation of the acoustic signals. This work introduces a technique for measuring the ultrasound backscatter from individual micron-sized objects by combining a microinjection system with a co-registered optical microscope and an ultrasound imaging device. This allowed the measurement of the ultrasound backscatter response from a single object under optical microscope guidance. The optical and ultrasonic data were used to determine the size of the object and to deduce its backscatter responses, respectively. In order to calibrate the system, the backscatter frequency responses from polystyrene microspheres were measured and compared to theoretical predictions. A very good agreement was found between the measured backscatter responses of individual microspheres and theoretical predictions of an elastic sphere. The backscatter responses from single OCI-AML-5 cells were also investigated. It was found that the backscatter responses from AML cells are best modeled using the fluid sphere model. The advantages, limitations, and future applications of the developed technique are discussed.

A technology platform to assess multiple cancer agents simultaneously within a patient's tumor

PubMed Central

Klinghoffer, Richard A.; Frazier, Jason P.; Moreno-Gonzalez, Alicia; Strand, Andrew D.; Kerwin, William S.; Casalini, Joseph R.; Thirstrup, Derek J.; You, Sheng; Morris, Shelli M.; Watts, Korashon L.; Veiseh, Mandana; Grenley, Marc O.; Tretyak, Ilona; Dey, Joyoti; Carleton, Michael; Beirne, Emily; Pedro, Kyle D.; Ditzler, Sally H.; Girard, Emily J.; Deckwerth, Thomas L.; Bertout, Jessica A.; Meleo, Karri A.; Filvaroff, Ellen H.; Chopra, Rajesh; Press, Oliver W.; Olson, James M.

2016-01-01

A fundamental problem in cancer drug development is that antitumor efficacy in preclinical cancer models does not translate faithfully to patient outcomes. Much of early cancer drug discovery is performed under in vitro conditions in cell-based models that poorly represent actual malignancies. To address this inconsistency, we have developed a technology platform called CIVO, which enables simultaneous assessment of up to eight drugs or drug combinations within a single solid tumor in vivo. The platform is currently designed for use in animal models of cancer and patients with superficial tumors but can be modified for investigation of deeper-seated malignancies. In xenograft lymphoma models, CIVO microinjection of well-characterized anticancer agents (vincristine, doxorubicin, mafosfamide, and prednisolone) induced spatially defined cellular changes around sites of drug exposure, specific to the known mechanisms of action of each drug. The observed localized responses predicted responses to systemically delivered drugs in animals. In pair-matched lymphoma models, CIVO correctly demonstrated tumor resistance to doxorubicin and vincristine and an unexpected enhanced sensitivity to mafosfamide in multidrug-resistant lymphomas compared with chemotherapy-naïve lymphomas. A CIVO-enabled in vivo screen of 97 approved oncology agents revealed a novel mTOR (mammalian target of rapamycin) pathway inhibitor that exhibits significantly increased tumor-killing activity in the drug-resistant setting compared with chemotherapy-naïve tumors. Finally, feasibility studies to assess the use of CIVO in human and canine patients demonstrated that microinjection of drugs is toxicity-sparing while inducing robust, easily tracked, drug-specific responses in autochthonous tumors, setting the stage for further application of this technology in clinical trials. PMID:25904742

Regulatory mechanism of body temperature in the central nervous system during the maintenance phase of hibernation in Syrian hamsters: involvement of β-endorphin.

PubMed

Tamura, Yutaka; Shintani, Mitsuteru; Inoue, Hirofumi; Monden, Mayuko; Shiomi, Hirohito

2012-04-11

We have shown previously that intracerebroventricular (icv) injection of naloxone (a non-selective opioid receptor antagonist) or naloxonazine (a selective μ1-opioid receptor antagonist) at the maintenance phase of hibernation arouses Syrian hamsters from hibernation. This study was designed to clarify the role of β-endorphin (an endogenous μ-opioid receptor ligand) on regulation of body temperature (T(b)) during the maintenance phase of hibernation. The number of c-Fos-positive cells and β-endorphin-like immunoreactivity increased in the arcuate nucleus (ARC) after hibernation onset. In contrast, endomorphin-1 (an endogenous μ-opioid receptor ligand)-like immunoreactivity observed on the anterior hypothalamus decreased after hibernation onset. In addition, hibernation was interrupted by icv injection of anti-β-endorphin antiserum at the maintenance phase of hibernation. The mRNA expression level of proopiomelanocortin (a precursor of β-endorphin) on ARC did not change throughout the hibernation phase. However, the mRNA expression level of prohormone convertase-1 increased after hibernation onset. [D-Ala2,N-MePhe4,Gly-ol5] enkephalin (DAMGO, a selective μ-opioid receptor agonist) microinjection into the dorsomedial hypothalamus (DMH) elicited the most marked T(b) decrease than other sites such as the preoptic area (PO), anterior hypothalamus (AH), lateral hypothalamus (LH), ventromedial hypothalamus and posterior hypothalamus (PH). However, microinjected DAMGO into the medial septum indicated negligible changes in T(b). These results suggest that β-endorphin which synthesizes in ARC neurons regulates T(b) during the maintenance phase of hibernation by activating μ-opioid receptors in PO, AH, VMH, DMH and PH. Copyright © 2012 Elsevier B.V. All rights reserved.

The gelatinous extracellular matrix facilitates transport studies in kelp: visualization of pressure-induced flow reversal across sieve plates

PubMed Central

Knoblauch, Jan; Peters, Winfried S.; Knoblauch, Michael

2016-01-01

Background and Aims In vascular plants, important questions regarding phloem function remain unanswered due to problems with invasive experimental procedures in this highly sensitive tissue. Certain brown algae (kelps; Laminariales) also possess sieve tubes for photoassimilate transport, but these are embedded in large volumes of a gelatinous extracellular matrix which isolates them from neighbouring cells. Therefore, we hypothesized that kelp sieve tubes might tolerate invasive experimentation better than their analogues in higher plants, and sought to establish Nereocystis luetkeana as an experimental system. Methods The predominant localization of cellulose and the gelatinous extracellular matrix in N. luetkeana was verified using specific fluorescent markers and confocal laser scanning microscopy. Sieve tubes in intact specimens were loaded with fluorescent dyes, either passively (carboxyfluorescein diacetate; CFDA) or by microinjection (rhodamine B), and the movement of the dyes was monitored by fluorescence microscopy. Key Results Application of CFDA demonstrated source to sink bulk flow in N. luetkeana sieve tubes, and revealed the complexity of sieve tube structure, with branches, junctions and lateral connections. Microinjection into sieve elements proved comparatively easy. Pulsed rhodamine B injection enabled the determination of flow velocity in individual sieve elements, and the direct visualization of pressure-induced reversals of flow direction across sieve plates. Conclusions The reversal of flow direction across sieve plates by pressurizing the downstream sieve element conclusively demonstrates that a critical requirement of the Münch theory is satisfied in kelp; no such evidence exists for tracheophytes. Because of the high tolerance of its sieve elements to experimental manipulation, N. luetkeana is a promising alternative to vascular plants for studying the fluid mechanics of sieve tube networks. PMID:26929203

The gelatinous extracellular matrix facilitates transport studies in kelp: visualization of pressure-induced flow reversal across sieve plates.

PubMed

Knoblauch, Jan; Peters, Winfried S; Knoblauch, Michael

2016-04-01

In vascular plants, important questions regarding phloem function remain unanswered due to problems with invasive experimental procedures in this highly sensitive tissue. Certain brown algae (kelps; Laminariales) also possess sieve tubes for photoassimilate transport, but these are embedded in large volumes of a gelatinous extracellular matrix which isolates them from neighbouring cells. Therefore, we hypothesized that kelp sieve tubes might tolerate invasive experimentation better than their analogues in higher plants, and sought to establish Nereocystis luetkeana as an experimental system. The predominant localization of cellulose and the gelatinous extracellular matrix in N. luetkeana was verified using specific fluorescent markers and confocal laser scanning microscopy. Sieve tubes in intact specimens were loaded with fluorescent dyes, either passively (carboxyfluorescein diacetate; CFDA) or by microinjection (rhodamine B), and the movement of the dyes was monitored by fluorescence microscopy. Application of CFDA demonstrated source to sink bulk flow in N. luetkeana sieve tubes, and revealed the complexity of sieve tube structure, with branches, junctions and lateral connections. Microinjection into sieve elements proved comparatively easy. Pulsed rhodamine B injection enabled the determination of flow velocity in individual sieve elements, and the direct visualization of pressure-induced reversals of flow direction across sieve plates. The reversal of flow direction across sieve plates by pressurizing the downstream sieve element conclusively demonstrates that a critical requirement of the Münch theory is satisfied in kelp; no such evidence exists for tracheophytes. Because of the high tolerance of its sieve elements to experimental manipulation, N. luetkeana is a promising alternative to vascular plants for studying the fluid mechanics of sieve tube networks. © The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Systemic inflammation induces anxiety disorder through CXCL12/CXCR4 pathway.

PubMed

Yang, L; Wang, M; Guo, Y Y; Sun, T; Li, Y J; Yang, Q; Zhang, K; Liu, S B; Zhao, M G; Wu, Y M

2016-08-01

It is evidenced that inflammation is involved in the pathogenesis of anxiety disorder, as well as the dysfunction of glutamate neurotransmission in the central nervous system (CNS). Chemokine CXCL12 has been reported taking part in the regulation of neurotransmitter release, however, the roles of CXCL12 in the development of anxiety are still unclear. In this study, we found that intraperitoneal (i.p) injection of lipopolysaccharide (LPS) induced anxiety-like behaviors in adult mice as measured by elevated plus-maze test (EPM) and open field test (OFT). Astrocytes were responsible for CXCL12 induction upon LPS challenge in hippocampus and amygdala, and microinjection of CXCL12 into amygdala induced mice anxiety-like behaviors. AMD3100, which is an antagonist for CXCL12 receptor CXCR4, prevented the anxiety behaviors induced by microinjection of CXCL12 into amygdala as well as injection i.p of LPS. Knockdown of CXCR4 expression in neurons using short hairpin RNAs (shRNAs) significantly blocked anxiety behaviors mediated by CXCL12 i.c injection. Furthermore, AMD3100 or shCXCR4 prevented the impairment of nesting ability induced by CXCL12 in mice. Whole-cell patch-clamp recordings in the neurons of basolateral amygdala (BLA) revealed that CXCL12 enhanced glutamatergic transmission by increasing sEPSC frequency in the amygdala. AMD3100 inhibited the excitatory glutamatergic neural transmission and involved in the development of anxiety through CXCR4. These findings provide direct evidence that alterations of CXCL12 in BLA play critical roles in the development of anxiety induced by systemic inflammation and that CXCR4 may be a potential therapeutic target for inflammation-induced anxiety. Copyright © 2016 Elsevier Inc. All rights reserved.

VEGF Triggers the Activation of Cofilin and the Arp2/3 Complex within the Growth Cone

PubMed Central

Schlau, Matthias; Terheyden-Keighley, Daniel; Theis, Verena; Mannherz, Hans Georg; Theiss, Carsten

2018-01-01

A crucial neuronal structure for the development and regeneration of neuronal networks is the axonal growth cone. Affected by different guidance cues, it grows in a predetermined direction to reach its final destination. One of those cues is the vascular endothelial growth factor (VEGF), which was identified as a positive effector for growth cone movement. These positive effects are mainly mediated by a reorganization of the actin network. This study shows that VEGF triggers a tight colocalization of cofilin and the Arp2/3 complex to the actin cytoskeleton within chicken dorsal root ganglia (DRG). Live cell imaging after microinjection of GFP (green fluorescent protein)-cofilin and RFP (red fluorescent protein)-LifeAct revealed that both labeled proteins rapidly redistributed within growth cones, and showed a congruent distribution pattern after VEGF supplementation. Disruption of signaling upstream of cofilin via blocking LIM-kinase (LIMK) activity resulted in growth cones displaying regressive growth behavior. Microinjection of GFP-p16b (a subunit of the Arp2/3 complex) and RFP-LifeAct revealed that both proteins redistributed into lamellipodia of the growth cone within minutes after VEGF stimulation. Disruption of the signaling to the Arp2/3 complex in the presence of VEGF by inhibition of N-WASP (neuronal Wiskott–Aldrich–Scott protein) caused retraction of growth cones. Hence, cofilin and the Arp2/3 complex appear to be downstream effector proteins of VEGF signaling to the actin cytoskeleton of DRG growth cones. Our data suggest that VEGF simultaneously affects different pathways for signaling to the actin cytoskeleton, since activation of cofilin occurs via inhibition of LIMK, whereas activation of Arp2/3 is achieved by stimulation of N-WASP. PMID:29382077

GABAA- and glycine-mediated inhibitory modulation of the cough reflex in the caudal nucleus tractus solitarii of the rabbit.

PubMed

Cinelli, Elenia; Iovino, Ludovica; Bongianni, Fulvia; Pantaleo, Tito; Mutolo, Donatella

2016-09-01

Cough-related sensory inputs from rapidly adapting receptors (RARs) and C fibers are processed by second-order neurons mainly located in the caudal nucleus tractus solitarii (NTS). Both GABAA and glycine receptors have been proven to be involved in the inhibitory control of second-order cells receiving RAR projections. We investigated the role of these receptors within the caudal NTS in the modulation of the cough reflex induced by either mechanical or chemical stimulation of the tracheobronchial tree in pentobarbital sodium-anesthetized, spontaneously breathing rabbits. Bilateral microinjections (30-50 nl) of the receptor antagonists bicuculline and strychnine as well as of the receptor agonists muscimol and glycine were performed. Bicuculline (0.1 mM) and strychnine (1 mM) caused decreases in peak abdominal activity and marked increases in respiratory frequency due to decreases in both inspiratory time (Ti) and expiratory time (Te), without concomitant changes in arterial blood pressure. Noticeably, these microinjections induced potentiation of the cough reflex consisting of increases in the cough number associated with decreases either in cough-related Ti after bicuculline or in both cough-related Ti and Te after strychnine. The effects caused by muscimol (0.1 mM) and glycine (10 mM) were in the opposite direction to those produced by the corresponding antagonists. The results show that both GABAA and glycine receptors within the caudal NTS mediate a potent inhibitory modulation of the pattern of breathing and cough reflex responses. They strongly suggest that disinhibition is one important mechanism underlying cough regulation and possibly provide new hints for novel effective antitussive strategies. Copyright © 2016 the American Physiological Society.

Neurons of the A5 region are required for the tachycardia evoked by electrical stimulation of the hypothalamic defence area in anaesthetized rats.

PubMed

López-González, M V; Díaz-Casares, A; Peinado-Aragonés, C A; Lara, J P; Barbancho, M A; Dawid-Milner, M S

2013-08-01

In order to assess the possible interactions between the pontine A5 region and the hypothalamic defence area (HDA), we have examined the pattern of double staining for c-Fos protein immunoreactivity (c-Fos-ir) and tyrosine hydroxylase, throughout the rostrocaudal extent of the A5 region in spontaneously breathing anaesthetized male Sprague-Dawley rats during electrical stimulation of the HDA. Activation of the HDA elicited a selective increase in c-Fos-ir with an ipsilateral predominance in catecholaminergic and non-catecholaminergic A5 somata (P < 0.001 in both cases). A second group of experiments was done to examine the importance of the A5 region in modulating the cardiorespiratory response evoked from the HDA. Cardiorespiratory changes were analysed in response to electrical stimulation of the HDA before and after ipsilateral microinjection of muscimol within the A5 region. Stimulation of the HDA evoked an inspiratory facilitatory response, consisting of an increase in respiratory rate (P < 0.001) due to a decrease in expiratory time (P < 0.01). The respiratory response was accompanied by a pressor response (P < 0.001) and tachycardia (P < 0.001). After muscimol microinjection within the A5 region, pressor and heart rate responses to HDA stimulation were reduced (P < 0.01 and P < 0.001, respectively). The respiratory response persisted unchanged. Finally, to confirm functional interactions between the HDA and the A5 region, extracellular recordings of putative A5 neurones were obtained during HDA stimulation. Seventy-five A5 cells were recorded, 35 of which were affected by the HDA (47%). These results indicate that neurones of the A5 region participate in the cardiovascular response evoked from the HDA. The possible mechanisms involved in these interactions are discussed.

From synapse to gene product: Prolonged expression of c-fos induced by a single microinjection of carbachol in the pontomesencephalic tegmentum

PubMed Central

Quattrochi, James J.; Bazalakova, Mihaela; Hobson, J. Allan

2006-01-01

It is not known how the brain modifies its regulatory systems in response to the application of a drug, especially over the long term of weeks and months. We have developed a model system approach to this question by manipulating cholinergic cell groups of the laterodorsal and pedunculopontine tegmental (LDT/PPT) nuclei in the pontomesencephalic tegmentum (PMT), which are known to be actively involved in the timing and quantity of rapid eye movement (REM) sleep. In a freely moving feline model, a single microinjection of the cholinergic agonist carbachol conjugated to a latex nanosphere delivery system into the caudolateral PMT elicits a long-term enhancement of one distinguishing phasic event of REM sleep, ponto-geniculo-occipital (PGO) waves, lasting 5 days but without any significant change in REM sleep or other behavioral state. Here, we test the hypothesis that cholinergic activation within the caudolateral PMT alters the postsynaptic excitability of the PGO network, stimulating the prolonged expression of c-fos that underlies this long-term PGO enhancement (LTPE) effect. Using quantitative Fos immunohistochemistry, we found that the number of Fos-immunoreactive (Fos-IR) neurons surrounding the caudolateral PMT injection site decreased sharply by postcarbachol day 03, while the number of Fos-IR neurons in the more rostral LDT/PPT increased >30-fold and remained at a high level following the course of LTPE. These results demonstrate a sustained c-fos expression in response to pharmacological stimulation of the brain and suggest that carbachol's acute effects induce LTPE via cholinergic receptors, with subsequent transsynaptic activation of the LDT/PPT maintaining the LTPE effect. PMID:15893601

Kai Xin San aqueous extract improves Aβ1-40-induced cognitive deficits on adaptive behavior learning by enhancing memory-related molecules expression in the hippocampus.

PubMed

Lu, Cong; Shi, Zhe; Sun, Xiuping; Pan, Ruile; Chen, Shanguang; Li, Yinghui; Qu, Lina; Sun, Lihua; Dang, Haixia; Bu, Lanlan; Chen, Lingling; Liu, Xinmin

2017-04-06

Kai Xin San (KXS), a traditional formula of Chinese medicine, has been used to treat dementia. The present study aimed to investigate its ameliorating effects on Aβ 1-40 -induced cognitive impairment in rats using a series of novel reward-directed instrumental learning tasks, and to determine its possible mechanism of action. Rats were pretreated with KXS aqueous extract (0.72 and 1.44g/kg, p.o.) for 10 days, and were trained to gain reward reinforcement by lever pressing at the meantime. Thereafter, rats received a bilateral microinjection of Aβ 1-40 in CA1 regions of the hippocampus. Cognitive performance was evaluated with the goal directed (higher response ratio) and habit (visual signal discrimination and extinction) learning tasks, as well as on the levels of memory-related biochemical parameters and molecules. Our findings first demonstrated that KXS can improve Aβ 1-40 -induced amnesia in RDIL via enhancing the comprehension of action-outcome association and the utilization of cue information to guide behavior. Then, its ameliorating effects should be attributed to the modulation of memory-related molecules in the hippocampus. In conclusion, KXS has the potential to prevent and/or delay the deterioration of cognitive impairment in AD. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Diazepam reduces excitability of amygdala and further influences auditory cortex following sodium salicylate treatment in rats.

PubMed

Song, Yu; Liu, Junxiu; Ma, Furong; Mao, Lanqun

2016-12-01

Diazepam can reduce the excitability of lateral amygdala and eventually suppress the excitability of the auditory cortex in rats following salicylate treatment, indicating the regulating effect of lateral amygdala to the auditory cortex in the tinnitus procedure. To study the spontaneous firing rates (SFR) of the auditory cortex and lateral amygdala regulated by diazepam in the tinnitus rat model induced by sodium salicylate. This study first created a tinnitus rat modal induced by sodium salicylate, and recorded SFR of both auditory cortex and lateral amygdala. Then diazepam was intraperitoneally injected and the SFR changes of lateral amygdala recorded. Finally, diazepam was microinjected on lateral amygdala and the SFR changes of the auditory cortex recorded. Both SFRs of the auditory cortex and lateral amygdala increased after salicylate treatment. SFR of lateral amygdala decreased after intraperitoneal injection of diazepam. Microinjecting diazepam to lateral amygdala decreased SFR of the auditory cortex ipsilaterally and contralaterally.

Leptin replacement restores supraspinal cholinergic antinociception in leptin-deficient obese mice.

PubMed

Wang, Wenfei; Baghdoyan, Helen A; Lydic, Ralph

2009-08-01

A single gene deletion causes lack of leptin and obesity in B6.V-Lep(ob) (obese; ob) mice compared with wild-type C57BL/6J (B6) mice. This study compared the phenotype of nociception and supraspinal antinociception in obese and B6 mice by testing 2 hypotheses: (1) microinjection of cholinomimetics or an adenosine receptor agonist, but not morphine, into the pontine reticular formation (PRF) is antinociceptive in B6 but not obese mice, and (2) leptin replacement in obese mice attenuates differences in nociceptive responses between obese and B6 mice. Adult male mice (n = 22) were implanted with microinjection guide tubes aimed for the PRF. The PRF was injected with neostigmine, carbachol, nicotine, N(6)-p-sulfophenyladenosine (SPA), morphine, or saline (control), and latency to paw withdrawal (PWL) from a thermal stimulus was recorded. B6 and ob mice did not differ in PWL after saline microinjection into the PRF. Neostigmine, carbachol, and SPA caused PWL to increase significantly in B6 but not obese mice. An additional 15 obese mice were implanted with osmotic pumps that delivered leptin for 7 days. Leptin replacement in obese mice restored the analgesic effect of PRF neostigmine to the level displayed by B6 mice. The results show for the first time that leptin significantly alters supraspinal cholinergic antinociception. This study specifies a brain region (the pontine reticular formation), cholinergic neurotransmission, and a protein (leptin) modulating thermal nociception. The results are relevant for efforts to understand the association between obesity, disordered sleep, and hyperalgesia.

Dorsal Vagal Complex Modulates Neurogenic Airway Inflammation in a Guinea Pig Model With Esophageal Perfusion of HCl.

PubMed

Chen, Zhe; Sun, Lejia; Chen, Hui; Gu, Dachuan; Zhang, Weitao; Yang, Zifeng; Peng, Tao; Dong, Rong; Lai, Kefang

2018-01-01

Neurogenic airway inflammation in chronic cough and bronchial asthma related to gastroesophageal reflux (GER) is involved in the esophageal-bronchial reflex, but it is unclear whether this reflex is mediated by central neurons. This study aimed to investigate the regulatory effects of the dorsal vagal complex (DVC) on airway inflammation induced by the esophageal perfusion of hydrochloric acid (HCl) following the microinjection of nuclei in the DVC in guinea pigs. Airway inflammation was evaluated by measuring the extravasation of Evans blue dye (EBD) and substance P (SP) expression in the airway. Neuronal activity was indicated by Fos expression in the DVC. The neural pathways from the lower esophagus to the DVC and the DVC to the airway were identified using DiI tracing and pseudorabies virus Bartha (PRV-Bartha) retrograde tracing, respectively. HCl perfusion significantly increased plasma extravasation, SP expression in the trachea, and the expression of SP and Fos in the medulla oblongata nuclei, including the nucleus of the solitary tract (NTS) and the dorsal motor nucleus of the vagus (DMV). The microinjection of glutamic acid (Glu) or exogenous SP to enhance neuronal activity in the DVC significantly potentiated plasma extravasation and SP release induced by intra-esophageal perfusion. The microinjection of γ-aminobutyric acid (GABA), lidocaine to inhibit neuronal activity or anti-SP serum in the DVC alleviated plasma extravasation and SP release. In conclusion, airway inflammation induced by the esophageal perfusion of HCl is regulated by DVC. This study provides new insight for the mechanism of airway neurogenic inflammation related to GER.

Dorsal Vagal Complex Modulates Neurogenic Airway Inflammation in a Guinea Pig Model With Esophageal Perfusion of HCl

PubMed Central

Chen, Zhe; Sun, Lejia; Chen, Hui; Gu, Dachuan; Zhang, Weitao; Yang, Zifeng; Peng, Tao; Dong, Rong; Lai, Kefang

2018-01-01

Neurogenic airway inflammation in chronic cough and bronchial asthma related to gastroesophageal reflux (GER) is involved in the esophageal–bronchial reflex, but it is unclear whether this reflex is mediated by central neurons. This study aimed to investigate the regulatory effects of the dorsal vagal complex (DVC) on airway inflammation induced by the esophageal perfusion of hydrochloric acid (HCl) following the microinjection of nuclei in the DVC in guinea pigs. Airway inflammation was evaluated by measuring the extravasation of Evans blue dye (EBD) and substance P (SP) expression in the airway. Neuronal activity was indicated by Fos expression in the DVC. The neural pathways from the lower esophagus to the DVC and the DVC to the airway were identified using DiI tracing and pseudorabies virus Bartha (PRV-Bartha) retrograde tracing, respectively. HCl perfusion significantly increased plasma extravasation, SP expression in the trachea, and the expression of SP and Fos in the medulla oblongata nuclei, including the nucleus of the solitary tract (NTS) and the dorsal motor nucleus of the vagus (DMV). The microinjection of glutamic acid (Glu) or exogenous SP to enhance neuronal activity in the DVC significantly potentiated plasma extravasation and SP release induced by intra-esophageal perfusion. The microinjection of γ-aminobutyric acid (GABA), lidocaine to inhibit neuronal activity or anti-SP serum in the DVC alleviated plasma extravasation and SP release. In conclusion, airway inflammation induced by the esophageal perfusion of HCl is regulated by DVC. This study provides new insight for the mechanism of airway neurogenic inflammation related to GER. PMID:29867575

Role of hydrogen sulfide within the dorsal motor nucleus of the vagus in the control of gastric function in rats.

PubMed

Sun, H-Z; Yu, K-H; Ai, H-B

2015-05-01

Hydrogen sulfide (H2 S) is a gaseous messenger and serves as an important neuromodulator in the central nervous system. This study aimed to clarify the role of H2 S within the dorsal motor nucleus of the vagus (DMV) in the control of gastric function in rats. Cystathionine β-synthetase (CBS) is an important generator of endogenous H2 S in the brain. We investigated the distribution of CBS in the DMV using immunohistochemical method, and the effects of H2 S on gastric motility and on gastric acid secretion. CBS-immunoreactive (IR) neurons were detected in the rostral, intermediate and caudal DMV, with the highest number of CBS-IR neurons in the caudal DMV, and the lowest in the intermediate DMV. We also found that microinjection of the exogenous H2 S donor NaHS (0.04 and 0.08 mol/L; 0.1 μL; n = 6; p < 0.05) into the DMV significantly inhibited gastric motility with a dose-dependent trend, and promoted gastric acid secretion in Wistar rats. Microinjection of the same volume of physiological saline (PS; 0.1 μL, n = 6, p > 0.05) at the same location did not noticeably change gastric motility and acid secretion. The data from these experiments suggest that the CBS that produces H2 S is present in the DMV, and microinjection of NaHS into the DMV inhibited gastric motility and enhanced gastric acid secretion in rats. © 2015 John Wiley & Sons Ltd.

Functional cardiovascular action of L-cysteine microinjected into pressor sites of the rostral ventrolateral medulla of the rat.

PubMed

Takemoto, Yumi

2014-04-01

The endogenous sulfur-containing amino acid L-cysteine injected into the cerebrospinal fluid space of the cisterna magna increases arterial blood pressure (ABP) and heart rate (HR) in the freely moving rat. The present study examined (1) cardiovascular responses to L-cysteine microinjected into the rostral ventrolateral medulla (RVLM), where a group of neurons regulate activities of cardiovascular sympathetic neurons and (2) involvement of ionotropic excitatory amino acid (iEAA) receptors in response. In the RVLM of urethane-anesthetized rats accessed ventrally and identified with pressor responses to L-glutamate (10 mM, 34 nl), microinjections of L-cysteine increased ABP and HR dose dependently (3-100 mM, 34 nl). The cardiovascular responses to L-cysteine (30 mM) were not attenuated by a prior injection of either antagonist alone, MK801 (20 mM, 68 nl) for the NMDA type of iEAA receptors, or CNQX (2 mM) for the non-NMDA type. However, inhibition of both NMDA and non-NMDA receptors with additional prior injection of either antagonist completely blocked those responses to L-cysteine. The results indicate that L-cysteine has functional cardiovascular action in the RVLM of the anesthetized rat, and the responses to L-cysteine involve both NMDA and non-NMDA receptors albeit in a mutually exclusive parallel fashion. The findings may suggest endogenous roles of L-cysteine indirectly via iEAA receptors in the neuronal network of the RVLM for cardiovascular regulation in physiological and pathological situations.

Oxytocin in the amygdala and not the prefrontal cortex enhances fear and impairs extinction in the juvenile rat.

PubMed

Kritman, Milly; Lahoud, Nisrine; Maroun, Mouna

2017-05-01

A growing body of evidence suggests that the hypothalamic neuropeptide oxytocin (OT), aside from its central role in the regulation of social behavior, reduces fear and anxiety. The functional and opposing interactions of the medial prefrontal cortex (mPFC) and the amygdala in regulation of fear provide a unique experimental setting to examine the effects of OT on fear and extinction. Recent evidence suggests that in the adult animal OT can play a dual role in the regulation of fear leading to contrasting effects on fear depending on the manipulated brain region and the time of manipulations. The OT system is one of the systems that undergoes major changes throughout development, however, its role in regulating fear in young animals has not been widely explored. We recently showed that the mechanisms of extinction, and specifically engagement of the mPFC in extinction, are not identical in adult and juvenile animals. Thus, the purpose of this study was to elucidate the effects of OT on fear and extinction in juvenile animals. To that end, we determine extinction, by measuring freezing at different time points, following microinjection of the OT agonist, TGOT, into the mPFC, the basolateral and the central nuclei of the amygdala (BLA and CeA, respectively). The results show that whereas TGOT microinjections into the IL-mPFC did not affect extinction, microinjections into the amygdala were mainly associated with enhanced fear and impaired extinction. These results further emphasize the differences between adult and juvenile brains. Copyright © 2017. Published by Elsevier Inc.

Melanin-Concentrating Hormone: A New Sleep Factor?

PubMed Central

Torterolo, Pablo; Lagos, Patricia; Monti, Jaime M.

2011-01-01

Neurons containing the neuropeptide melanin-concentrating hormone (MCH) are mainly located in the lateral hypothalamus and the incerto-hypothalamic area, and have widespread projections throughout the brain. While the biological functions of this neuropeptide are exerted in humans through two metabotropic receptors, the MCHR1 and MCHR2, only the MCHR1 is present in rodents. Recently, it has been shown that the MCHergic system is involved in the control of sleep. We can summarize the experimental findings as follows: (1) The areas related to the control of sleep and wakefulness have a high density of MCHergic fibers and receptors. (2) MCHergic neurons are active during sleep, especially during rapid eye movement (REM) sleep. (3) MCH knockout mice have less REM sleep, notably under conditions of negative energy balance. Animals with genetically inactivated MCHR1 also exhibit altered vigilance state architecture and sleep homeostasis. (4) Systemically administered MCHR1 antagonists reduce sleep. (5) Intraventricular microinjection of MCH increases both slow wave sleep (SWS) and REM sleep; however, the increment in REM sleep is more pronounced. (6) Microinjection of MCH into the dorsal raphe nucleus increases REM sleep time. REM seep is inhibited by immunoneutralization of MCH within this nucleus. (7) Microinjection of MCH in the nucleus pontis oralis of the cat enhances REM sleep time and reduces REM sleep latency. All these data strongly suggest that MCH has a potent role in the promotion of sleep. Although both SWS and REM sleep are facilitated by MCH, REM sleep seems to be more sensitive to MCH modulation. PMID:21516258

Region-specific role of Rac in nucleus accumbens core and basolateral amygdala in consolidation and reconsolidation of cocaine-associated cue memory in rats.

PubMed

Ding, Zeng-Bo; Wu, Ping; Luo, Yi-Xiao; Shi, Hai-Shui; Shen, Hao-Wei; Wang, Shen-Jun; Lu, Lin

2013-08-01

Drug reinforcement and the reinstatement of drug seeking are associated with the pathological processing of drug-associated cue memories that can be disrupted by manipulating memory consolidation and reconsolidation. Ras-related C3 botulinum toxin substrate (Rac) is involved in memory processing by regulating actin dynamics and neural structure plasticity. The nucleus accumbens (NAc) and amygdala have been implicated in the consolidation and reconsolidation of emotional memories. Therefore, we hypothesized that Rac in the NAc and amygdala plays a role in the consolidation and reconsolidation of cocaine-associated cue memory. Conditioned place preference (CPP) and microinjection of Rac inhibitor NSC23766 were used to determine the role of Rac in the NAc and amygdala in the consolidation and reconsolidation of cocaine-associated cue memory in rats. Microinjections of NSC23766 into the NAc core but not shell, basolateral (BLA), or central amygdala (CeA) after each cocaine-conditioning session inhibited the consolidation of cocaine-induced CPP. A microinjection of NSC23766 into the BLA but not CeA, NAc core, or NAc shell immediately after memory reactivation induced by exposure to a previously cocaine-paired context disrupted the reconsolidation of cocaine-induced CPP. The effect of memory disruption on cocaine reconsolidation was specific to reactivated memory, persisted at least 2 weeks, and was not reinstated by a cocaine-priming injection. Our findings indicate that Rac in the NAc core and BLA are required for the consolidation and reconsolidation of cocaine-associated cue memory, respectively.

Serotonin in the solitary tract nucleus shortens the laryngeal chemoreflex in anesthetized neonatal rats

PubMed Central

Donnelly, William T.; Bartlett, Donald; Leiter, J.C.

2017-01-01

The laryngeal chemoreflex (LCR), an airway protective reflex that causes apnea and bradycardia, has long been suspected as an initiating event in the sudden infant death syndrome (SIDS). Serotonin (5-HT) and 5-HT receptors may be deficient in the brainstems of babies who die of SIDS, and 5-HT seems to be important in terminating apneas directly or in causing arousals or as part of the process of autoresuscitation. We hypothesized that 5-HT in the brainstem would limit the duration of the LCR. We studied anesthetized rat pups between 7 and 21 days of age and made microinjections into the cisterna magna or into the nucleus of the solitary tract (NTS). Focal, bilateral microinjections of 5-HT into the caudal NTS significantly shortened the LCR. The 5-HT 1a receptor antagonist, WAY 100635, did not affect the LCR consistently, nor did a 5-HT2 receptor antagonist, ketanserin, alter the duration of the LCR. The 5-HT3 specific agonist, 1-(3-chlorophenyl)-biguanide, microinjected bilaterally into the caudal NTS significantly shortened the LCR. Thus, endogenous 5-HT released within the NTS may curtail the respiratory depression that is part of the LCR, and serotonergic shortening of the LCR may be attributed to activation of 5-HT3 receptors within the NTS. 5-HT3 receptors are expressed presynaptically on C-fiber afferents of the superior laryngeal nerve, and serotonergic shortening of the LCR may be mediated presynaptically by enhanced activation of inhibitory interneurons within the NTS that terminate during the LCR. PMID:27121960

REM Sleep–like Atonia of Hypoglossal (XII) Motoneurons Is Caused by Loss of Noradrenergic and Serotonergic Inputs

PubMed Central

Fenik, Victor B.; Davies, Richard O.; Kubin, Leszek

2017-01-01

Rationale Studies of hypoglossal (XII) motoneurons that innervate the genioglossus muscle, an upper airway dilator, suggested that the suppression of upper airway motor tone during REM sleep is caused by withdrawal of excitation mediated by norepinephrine and serotonin. Objectives Our objectives were to determine whether antagonism of aminergic receptors located in the XII nucleus region can abolish the REM sleep–like atonia of XII motoneurons, and whether both serotonergic and noradrenergic antagonists are required to achieve this effect. Methods REM sleep–like episodes were elicited in anesthetized rats by pontine carbachol injections before and at various times after microinjection of prazosin and methysergide combined, or of only one of the drugs, into the XII nucleus. Measurements and Main Results Spontaneous XII nerve activity was significantly reduced, by 35 to 81%, by each antagonist alone and in combination, indicating that XII motoneurons were under both noradrenergic and serotonergic endogenous excitatory drives. During the 32 to 81 min after microinjections of both antagonists, pontine carbachol caused no depression of XII nerve activity, whereas other characteristic effects (activation of the hippocampal and cortical EEG, and slowing of the respiratory rate) remained intact. A partial recovery of the depressant effect of carbachol then occurred parallel to the recovery of spontaneous XII nerve activity from the depressant effect of the antagonists. Microinjections of either antagonist alone did not eliminate the depressant effect of carbachol. Conclusions The REM sleep–like depression of XII motoneuronal activity induced by pontine carbachol can be fully accounted for by the combined withdrawal of noradrenergic and serotonergic effects on XII motoneurons. PMID:16100007

Dorsolateral neostriatum contribution to incentive salience: Opioid or dopamine stimulation makes one reward cue more motivationally attractive than another

PubMed Central

DiFeliceantonio, Alexandra G.; Berridge, Kent C.

2016-01-01

Pavlovian cues for rewards can become attractive incentives: approached and ‘wanted’ as the rewards themselves. The motivational attractiveness of a previously learned cue is not fixed, but can be dynamically amplified during re-encounter by simultaneous activation of brain limbic circuitry. Here we report that opioid or dopamine microinjections in the dorsolateral quadrant of the neostriatum (DLS) of rats selectively amplify attraction toward a previously learned Pavlovian cue in an individualized fashion, at the expense of a competing cue. In an autoshaping (sign-tracking vs goal-tracking) paradigm, microinjection of the mu opioid receptor agonist (DAMGO) or dopamine indirect agonist (amphetamine) in DLS of sign-tracker individuals selectively enhanced their sign-tracking attraction toward the reward-predictive lever cue. By contrast, DAMGO or amphetamine in DLS of goal-trackers selectively enhanced prepotent attraction toward the reward-proximal cue of sucrose dish. Amphetamine also enhanced goal-tracking in some sign-tracker individuals (if they ever defected to the dish even once). That DLS enhancement of cue attraction was due to stronger motivation, not stronger habits was suggested by: 1) sign-trackers flexibly followed their cue to a new location when the lever was suddenly moved after DLS DAMGO microinjection, and 2) DAMGO in DLS also made sign-trackers work harder on a new instrumental nose-poke response required to earn presentations of their Pavlovian lever cue (instrumental conditioned reinforcement). Altogether, our results suggest that DLS circuitry can enhance the incentive salience of a Pavlovian reward cue, selectively making that cue a stronger motivational magnet. PMID:26924040

MHC class I D(k) expression in hematopoietic and nonhematopoietic cells confers natural killer cell resistance to murine cytomegalovirus.

PubMed

Xie, Xuefang; Stadnisky, Michael D; Coats, Ebony R; Ahmed Rahim, Mir Munir; Lundgren, Alyssa; Xu, Wenhao; Makrigiannis, Andrew P; Brown, Michael G

2010-05-11

NK cell-mediated murine cytomegalovirus (MCMV) resistance (Cmv(r)) is under H-2(k) control in MA/My mice, but the underlying gene(s) is unclear. Prior genetic analysis mapped Cmv(r) to the MHC class I (MHC-I) D(k) gene interval. Because NK cell receptors are licensed by and responsive to MHC class I molecules, D(k) itself is a candidate gene. A 10-kb genomic D(k) fragment was subcloned and microinjected into MCMV-susceptible (Cmv(s)) (MA/My.L-H2(b) x C57L)F(1) or (B6 x DBA/2)F(2) embryos. Transgenic founders, which are competent for D(k) expression and germline transgene transmission, were identified and further backcrossed to MA/My.L-H2(b) or C57L mice. Remarkably, D(k) expression delivered NK-mediated resistance in either genetic background. Further, NK cells with cognate inhibitory Ly49G receptors for self-MHC-I D(k) were licensed and critical in protection against MCMV infection. In radiation bone marrow chimeras, NK resistance was significantly diminished when MHC-I D(k) expression was restricted to only hematopoietic or nonhematopoietic cells. Thus, MHC-I D(k) is the H-2(k)-linked Cmv(r) locus; these findings suggest a role for NK cell interaction with D(k)-bearing hematopoietic and nonhematopoietic cells to shape NK-mediated virus immunity.

Impact of CdSe/ZnS quantum dots on the development of zebrafish embryos

NASA Astrophysics Data System (ADS)

Lei, Yong; Xiao, Qi; Huang, Shan; Xu, Wansu; Zhang, Zhe; He, Zhike; Liu, Yi; Deng, Fengjiao

2011-12-01

Due to their unique fluorescent characteristics, quantum dots (QDs) have been successfully applied in the fields of biotechnology and medicine, but there is very limited information regarding their biodistribution and chronic toxicity in vivo. In this article, the biological behavior and toxic effects of mercaptoacetic acid-CdSe/ZnS QDs (MAA-QDs) in developing zebrafish embryos were investigated by in vivo tests. The MAA-QDs were introduced into zebrafish through microinjection at early stage. The results showed that the MAA-QDs at certain concentrations influenced the survival of zebrafish embryos, but treated embryos without developmental defects were also observed. MAA-QDs injected into the cytoplasm at the one-cell stage were allocated to progeny blastoderm cells during proliferation and almost never entered the yolk. The formation of notochord and primordial germ cells with normal morphologies was detected in the treated embryos by whole-mount in situ hybridization. Furthermore, traces of the element cadmium were mainly discovered in the tissue of liver and kidney of 3-month-old-treated zebrafish by quantitative assessment with inductively coupled plasma mass spectrometry. Thus, we hypothesized that low concentration MAA-QDs have chronic toxicities when they were delivered into zebrafish organs.

Disruption of the nucleolus mediates stabilization of p53 in response to DNA damage and other stresses

PubMed Central

Rubbi, Carlos P.; Milner, Jo

2003-01-01

p53 protects against cancer through its capacity to induce cell cycle arrest or apoptosis under a large variety of cellular stresses. It is not known how such diversity of signals can be integrated by a single molecule. However, the literature reveals that a common denominator in all p53-inducing stresses is nucleolar disruption. We thus postulated that the impairment of nucleolar function might stabilize p53 by preventing its degradation. Using micropore irradiation, we demonstrate that large amounts of nuclear DNA damage fail to stabilize p53 unless the nucleolus is also disrupted. Forcing nucleolar disruption by anti-upstream binding factor (UBF) microinjection (in the absence of DNA damage) also causes p53 stabilization. We propose that the nucleolus is a stress sensor responsible for maintenance of low levels of p53, which are automatically elevated as soon as nucleolar function is impaired in response to stress. Our model integrates all known p53-inducing agents and also explains cell cycle-related variations in p53 levels which correlate with established phases of nucleolar assembly/disassembly through the cell cycle. PMID:14609953

Generation of transgenic Hydra by embryo microinjection.

PubMed

Juliano, Celina E; Lin, Haifan; Steele, Robert E

2014-09-11

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology(1). Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Serotonin in the solitary tract nucleus shortens the laryngeal chemoreflex in anaesthetized neonatal rats.

PubMed

Donnelly, William T; Bartlett, Donald; Leiter, J C

2016-07-01

What is the central question of this study? Failure to terminate apnoea and arouse is likely to contribute to sudden infant death syndrome (SIDS). Serotonin is deficient in the brainstems of babies who died of SIDS. Therefore, we tested the hypothesis that serotonin in the nucleus of the solitary tract (NTS) would shorten reflex apnoea. What is the main finding and its importance? Serotonin microinjected into the NTS shortened the apnoea and respiratory inhibition associated with the laryngeal chemoreflex. Moreover, this effect was achieved through a 5-HT3 receptor. This is a new insight that is likely to be relevant to the pathogenesis of SIDS. The laryngeal chemoreflex (LCR), an airway-protective reflex that causes apnoea and bradycardia, has long been suspected as an initiating event in the sudden infant death syndrome. Serotonin (5-HT) and 5-HT receptors may be deficient in the brainstems of babies who die of sudden infant death syndrome, and 5-HT seems to be important in terminating apnoeas directly or in causing arousals or as part of the process of autoresuscitation. We hypothesized that 5-HT in the brainstem would limit the duration of the LCR. We studied anaesthetized rat pups between 7 and 21 days of age and made microinjections into the cisterna magna or into the nucleus of the solitary tract (NTS). Focal, bilateral microinjections of 5-HT into the caudal NTS significantly shortened the LCR. The 5-HT1a receptor antagonist, WAY 100635, did not affect the LCR consistently, nor did a 5-HT2 receptor antagonist, ketanserin, alter the duration of the LCR. The 5-HT3 specific agonist, 1-(3-chlorophenyl)-biguanide, microinjected bilaterally into the caudal NTS significantly shortened the LCR. Thus, endogenous 5-HT released within the NTS may curtail the respiratory depression that is part of the LCR, and serotonergic shortening of the LCR may be attributed to activation of 5-HT3 receptors within the NTS. 5-HT3 receptors are expressed presynaptically on C fibre afferents of the superior laryngeal nerve, and serotonergic shortening of the LCR may be mediated presynaptically by enhanced activation of inhibitory interneurons within the NTS. © 2016 The Authors. Experimental Physiology © 2016 The Physiological Society.

Nucleus accumbens corticotropin-releasing factor increases cue-triggered motivation for sucrose reward: paradoxical positive incentive effects in stress?

PubMed

Peciña, Susana; Schulkin, Jay; Berridge, Kent C

2006-04-13

Corticotropin-releasing factor (CRF) is typically considered to mediate aversive aspects of stress, fear and anxiety. However, CRF release in the brain is also elicited by natural rewards and incentive cues, raising the possibility that some CRF systems in the brain mediate an independent function of positive incentive motivation, such as amplifying incentive salience. Here we asked whether activation of a limbic CRF subsystem magnifies the increase in positive motivation for reward elicited by incentive cues previously associated with that reward, in a way that might exacerbate cue-triggered binge pursuit of food or other incentives? We assessed the impact of CRF microinjections into the medial shell of nucleus accumbens using a pure incentive version of Pavlovian-Instrumental transfer, a measure specifically sensitive to the incentive salience of reward cues (which it separates from influences of aversive stress, stress reduction, frustration and other traditional explanations for stress-increased behavior). Rats were first trained to press one of two levers to obtain sucrose pellets, and then separately conditioned to associate a Pavlovian cue with free sucrose pellets. On test days, rats received microinjections of vehicle, CRF (250 or 500 ng/0.2 microl) or amphetamine (20 microg/0.2 microl). Lever pressing was assessed in the presence or absence of the Pavlovian cues during a half-hour test. Microinjections of the highest dose of CRF (500 ng) or amphetamine (20 microg) selectively enhanced the ability of Pavlovian reward cues to trigger phasic peaks of increased instrumental performance for a sucrose reward, each peak lasting a minute or so before decaying after the cue. Lever pressing was not enhanced by CRF microinjections in the baseline absence of the Pavlovian cue or during the presentation without a cue, showing that the CRF enhancement could not be explained as a result of generalized motor arousal, frustration or stress, or by persistent attempts to ameliorate aversive states. We conclude that CRF in nucleus accumbens shell amplifies positive motivation for cued rewards, in particular by magnifying incentive salience that is attributed to Pavlovian cues previously associated with those rewards. CRF-induced magnification of incentive salience provides a novel explanation as to why stress may produce cue-triggered bursts of binge eating, drug addiction relapse, or other excessive pursuits of rewards.

Nucleus accumbens corticotropin-releasing factor increases cue-triggered motivation for sucrose reward: paradoxical positive incentive effects in stress?

PubMed Central

Peciña, Susana; Schulkin, Jay; Berridge, Kent C

2006-01-01

Background Corticotropin-releasing factor (CRF) is typically considered to mediate aversive aspects of stress, fear and anxiety. However, CRF release in the brain is also elicited by natural rewards and incentive cues, raising the possibility that some CRF systems in the brain mediate an independent function of positive incentive motivation, such as amplifying incentive salience. Here we asked whether activation of a limbic CRF subsystem magnifies the increase in positive motivation for reward elicited by incentive cues previously associated with that reward, in a way that might exacerbate cue-triggered binge pursuit of food or other incentives? We assessed the impact of CRF microinjections into the medial shell of nucleus accumbens using a pure incentive version of Pavlovian-Instrumental transfer, a measure specifically sensitive to the incentive salience of reward cues (which it separates from influences of aversive stress, stress reduction, frustration and other traditional explanations for stress-increased behavior). Rats were first trained to press one of two levers to obtain sucrose pellets, and then separately conditioned to associate a Pavlovian cue with free sucrose pellets. On test days, rats received microinjections of vehicle, CRF (250 or 500 ng/0.2 μl) or amphetamine (20 μg/0.2 μl). Lever pressing was assessed in the presence or absence of the Pavlovian cues during a half-hour test. Results Microinjections of the highest dose of CRF (500 ng) or amphetamine (20 μg) selectively enhanced the ability of Pavlovian reward cues to trigger phasic peaks of increased instrumental performance for a sucrose reward, each peak lasting a minute or so before decaying after the cue. Lever pressing was not enhanced by CRF microinjections in the baseline absence of the Pavlovian cue or during the presentation without a cue, showing that the CRF enhancement could not be explained as a result of generalized motor arousal, frustration or stress, or by persistent attempts to ameliorate aversive states. Conclusion We conclude that CRF in nucleus accumbens shell amplifies positive motivation for cued rewards, in particular by magnifying incentive salience that is attributed to Pavlovian cues previously associated with those rewards. CRF-induced magnification of incentive salience provides a novel explanation as to why stress may produce cue-triggered bursts of binge eating, drug addiction relapse, or other excessive pursuits of rewards. PMID:16613600

Electrical Detection of Cellular Penetration during Microinjection with Carbon Nanopipettes

PubMed Central

Anderson, Sean E.; Bau, Haim H.

2014-01-01

The carbon nanopipette (CNP) is comprised of a pulled-glass pipette terminating with a nanoscale (tens to hundreds of nm) diameter carbon pipe. The entire inner glass surface of the CNP is coated with a carbon film, providing an electrically conductive path from the carbon tip to the distal, macroscopic end of the pipette. The CNP can double as a nanoelectrode, enabling electrical measurements through its carbon lining, and as a nanoinjector, facilitating reagent injection through its hollow bore. With the aid of a lock-in amplifier, we measured, in real time and with millisecond resolution, variations in impedance as the CNP penetrated into the cytoplasm and nucleus of adherent human osteosarcoma (U20S) cells. The capacitance change associated with nucleus penetration was, on average, 1.5 times greater than the one associated with cell membrane penetration. The experimental data was compared and favorably agreed with theoretical predictions based on a simple electrical network model. As a proof of concept, the cytoplasm and nucleus were transfected with fluorescent tRNA, enabling real-time monitoring of tRNA trafficking across the nuclear membrane. The CNP provides a robust and reliable means to detect cell and nucleus penetration, and trigger injection, thereby enabling the automation of cell injection. PMID:24859799

Perturbation of Incenp function impedes anaphase chromatid movements and chromosomal passenger protein flux at centromeres

PubMed Central

Ahonen, Leena J.; Kukkonen, Anu M.; Pouwels, Jeroen; Bolton, Margaret A.; Jingle, Christopher D.; Stukenberg, P. Todd; Kallio, Marko J.

2012-01-01

Incenp is an essential mitotic protein that, together with Aurora B, Survivin, and Borealin, forms the core of the chromosomal passenger protein complex (CPC). The CPC regulates various mitotic processes and functions to maintain genomic stability. The proper subcellular localization of the CPC and its full catalytic activity require the presence of each core subunit in the complex. We have investigated the mitotic tasks of the CPC using a function blocking antibody against Incenp microinjected into cells at different mitotic phases. This method allowed temporal analysis of CPC functions without perturbation of complex assembly or activity prior to injection. We have also studied the dynamic properties of Incenp and Aurora B using fusion protein photobleaching. We found that in early mitotic cells, Incenp and Aurora B exhibit dynamic turnover at centromeres, which is prevented by the anti-Incenp antibody. In these cells, the loss of centromeric CPC turnover is accompanied by forced mitotic exit without the execution of cytokinesis. Introduction of anti-Incenp antibody into early anaphase cells causes abnormalities in sister chromatid separation through defects in anaphase spindle functions. In summary, our data uncovers new mitotic roles for the CPC in anaphase and proposes that CPC turnover at centromeres modulates spindle assembly checkpoint signaling. PMID:18784935

Perturbation of Incenp function impedes anaphase chromatid movements and chromosomal passenger protein flux at centromeres.

PubMed

Ahonen, Leena J; Kukkonen, Anu M; Pouwels, Jeroen; Bolton, Margaret A; Jingle, Christopher D; Stukenberg, P Todd; Kallio, Marko J

2009-02-01

Incenp is an essential mitotic protein that, together with Aurora B, Survivin, and Borealin, forms the core of the chromosomal passenger protein complex (CPC). The CPC regulates various mitotic processes and functions to maintain genomic stability. The proper subcellular localization of the CPC and its full catalytic activity require the presence of each core subunit in the complex. We have investigated the mitotic tasks of the CPC using a function blocking antibody against Incenp microinjected into cells at different mitotic phases. This method allowed temporal analysis of CPC functions without perturbation of complex assembly or activity prior to injection. We have also studied the dynamic properties of Incenp and Aurora B using fusion protein photobleaching. We found that in early mitotic cells, Incenp and Aurora B exhibit dynamic turnover at centromeres, which is prevented by the anti-Incenp antibody. In these cells, the loss of centromeric CPC turnover is accompanied by forced mitotic exit without the execution of cytokinesis. Introduction of anti-Incenp antibody into early anaphase cells causes abnormalities in sister chromatid separation through defects in anaphase spindle functions. In summary, our data uncovers new mitotic roles for the CPC in anaphase and proposes that CPC turnover at centromeres modulates spindle assembly checkpoint signaling.

Parvovirus Induced Alterations in Nuclear Architecture and Dynamics

PubMed Central

Ihalainen, Teemu O.; Niskanen, Einari A.; Jylhävä, Juulia; Paloheimo, Outi; Dross, Nicolas; Smolander, Hanna; Langowski, Jörg; Timonen, Jussi; Vihinen-Ranta, Maija

2009-01-01

The nucleus of interphase eukaryotic cell is a highly compartmentalized structure containing the three-dimensional network of chromatin and numerous proteinaceous subcompartments. DNA viruses induce profound changes in the intranuclear structures of their host cells. We are applying a combination of confocal imaging including photobleaching microscopy and computational methods to analyze the modifications of nuclear architecture and dynamics in parvovirus infected cells. Upon canine parvovirus infection, expansion of the viral replication compartment is accompanied by chromatin marginalization to the vicinity of the nuclear membrane. Dextran microinjection and fluorescence recovery after photobleaching (FRAP) studies revealed the homogeneity of this compartment. Markedly, in spite of increase in viral DNA content of the nucleus, a significant increase in the protein mobility was observed in infected compared to non-infected cells. Moreover, analyzis of the dynamics of photoactivable capsid protein demonstrated rapid intranuclear dynamics of viral capsids. Finally, quantitative FRAP and cellular modelling were used to determine the duration of viral genome replication. Altogether, our findings indicate that parvoviruses modify the nuclear structure and dynamics extensively. Intranuclear crowding of viral components leads to enlargement of the interchromosomal domain and to chromatin marginalization via depletion attraction. In conclusion, parvoviruses provide a useful model system for understanding the mechanisms of virus-induced intranuclear modifications. PMID:19536327

Efficient Cargo Delivery into Adult Brain Tissue Using Short Cell-Penetrating Peptides.

PubMed

Kizil, Caghan; Iltzsche, Anne; Thomas, Alvin Kuriakose; Bhattarai, Prabesh; Zhang, Yixin; Brand, Michael

2015-01-01

Zebrafish brains can regenerate lost neurons upon neurogenic activity of the radial glial progenitor cells (RGCs) that reside at the ventricular region. Understanding the molecular events underlying this ability is of great interest for translational studies of regenerative medicine. Therefore, functional analyses of gene function in RGCs and neurons are essential. Using cerebroventricular microinjection (CVMI), RGCs can be targeted efficiently but the penetration capacity of the injected molecules reduces dramatically in deeper parts of the brain tissue, such as the parenchymal regions that contain the neurons. In this report, we tested the penetration efficiency of five known cell-penetrating peptides (CPPs) and identified two- polyR and Trans - that efficiently penetrate the brain tissue without overt toxicity in a dose-dependent manner as determined by TUNEL staining and L-Plastin immunohistochemistry. We also found that polyR peptide can help carry plasmid DNA several cell diameters into the brain tissue after a series of coupling reactions using DBCO-PEG4-maleimide-based Michael's addition and azide-mediated copper-free click reaction. Combined with the advantages of CVMI, such as rapidness, reproducibility, and ability to be used in adult animals, CPPs improve the applicability of the CVMI technique to deeper parts of the central nervous system tissues.

The kinesin KIF9 and reggie/flotillin proteins regulate matrix degradation by macrophage podosomes

PubMed Central

Cornfine, Susanne; Himmel, Mirko; Kopp, Petra; el Azzouzi, Karim; Wiesner, Christiane; Krüger, Marcus; Rudel, Thomas; Linder, Stefan

2011-01-01

Podosomes are actin-based matrix contacts in a variety of cell types, most notably monocytic cells, and are characterized by their ability to lyse extracellular matrix material. Besides their dependence on actin regulation, podosomes are also influenced by microtubules and microtubule-dependent transport processes. Here we describe a novel role for KIF9, a previously little-characterized member of the kinesin motor family, in the regulation of podosomes in primary human macrophages. We find that small interfering RNA (siRNA)/short-hairpin RNA–induced knockdown of KIF9 significantly affects both numbers and matrix degradation of podosomes. Overexpression and microinjection experiments reveal that the unique C-terminal region of KIF9 is crucial for these effects, presumably through binding of specific interactors. Indeed, we further identify reggie-1/flotillin-2, a signaling mediator between intracellular vesicles and the cell periphery, as an interactor of the KIF9 C-terminus. Reggie-1 dynamically colocalizes with KIF9 in living cells, and, consistent with KIF9-mediated effects, siRNA-induced knockdown of reggies/flotillins significantly impairs matrix degradation by podosomes. In sum, we identify the kinesin KIF9 and reggie/flotillin proteins as novel regulators of macrophage podosomes and show that their interaction is critical for the matrix-degrading ability of these structures. PMID:21119006

Tissue Motion and Assembly During Early Cardiovascular Morphogenesis

NASA Astrophysics Data System (ADS)

Rongish, Brenda

2010-03-01

Conventional dogma in the field of cardiovascular developmental biology suggests that cardiac precursor cells migrate to the embryonic midline to form a tubular heart. These progenitors are believed to move relative to their extracellular matrix (ECM); responding to stimulatory and inhibitory cues in their environment. The tubular heart that is formed by 30 hours post fertilization is comprised of two concentric layers: the muscular myocardium and the endothelial-like endocardium, which are separated by a thick layer of ECM believed to be secreted predominantly by the myocardial cells. Here we describe the origin and motility of fluorescently tagged endocardial precursors in transgenic (Tie1-YFP) quail embryos (R. Lansford, Caltech) using epifluorescence time-lapse imaging. To visualize the environment of migrating endocardial progenitors, we labeled two ECM components, fibronectin and fibrillin-2, via in vivo microinjection of fluorochrome-conjugated monoclonal antibodies. Dynamic imaging was performed at stages encompassing tubular heart assembly and early looping. We established the motion of endocardial precursor cells and presumptive cardiac ECM fibrils using both object tracking and particle image velocimetry (image cross correlation). We determined the relative importance of directed cell autonomous motility versus passive tissue movements in endocardial morphogenesis. The data show presumptive endocardial cells and cardiac ECM fibrils are swept passively into the anterior and posterior poles of the elongating tubular heart. These quantitative data indicate the contribution of cell autonomous motility displayed by endocardial precursors is limited. Thus, tissue motion drives most of the cell displacements during endocardial morphogenesis.

CC chemokine receptor 4 is required for experimental autoimmune encephalomyelitis by regulating GM-CSF and IL-23 production in dendritic cells

PubMed Central

Poppensieker, Karola; Otte, David-Marian; Schürmann, Britta; Limmer, Andreas; Dresing, Philipp; Drews, Eva; Schumak, Beatrix; Klotz, Luisa; Raasch, Jennifer; Mildner, Alexander; Waisman, Ari; Scheu, Stefanie; Knolle, Percy; Förster, Irmgard; Prinz, Marco; Maier, Wolfgang; Zimmer, Andreas; Alferink, Judith

2012-01-01

Dendritic cells (DCs) are pivotal for the development of experimental autoimmune encephalomyelitis (EAE). However, the mechanisms by which they control disease remain to be determined. This study demonstrates that expression of CC chemokine receptor 4 (CCR4) by DCs is required for EAE induction. CCR4−/− mice presented enhanced resistance to EAE associated with a reduction in IL-23 and GM-CSF expression in the CNS. Restoring CCR4 on myeloid cells in bone marrow chimeras or intracerebral microinjection of CCR4-competent DCs, but not macrophages, restored EAE in CCR4−/− mice, indicating that CCR4+ DCs are cellular mediators of EAE development. Mechanistically, CCR4−/− DCs were less efficient in GM-CSF and IL-23 production and also TH-17 maintenance. Intraspinal IL-23 reconstitution restored EAE in CCR4−/− mice, whereas intracerebral inoculation using IL-23−/− DCs or GM-CSF−/− DCs failed to induce disease. Thus, CCR4-dependent GM-CSF production in DCs required for IL-23 release in these cells is a major component in the development of EAE. Our study identified a unique role for CCR4 in regulating DC function in EAE, harboring therapeutic potential for the treatment of CNS autoimmunity by targeting CCR4 on this specific cell type. PMID:22355103

Differential expression of conserved germ line markers and delayed segregation of male and female primordial germ cells in a hermaphrodite, the leech helobdella.

PubMed

Cho, Sung-Jin; Vallès, Yvonne; Weisblat, David A

2014-02-01

In sexually reproducing animals, primordial germ cells (PGCs) are often set aside early in embryogenesis, a strategy that minimizes the risk of genomic damage associated with replication and mitosis during the cell cycle. Here, we have used germ line markers (piwi, vasa, and nanos) and microinjected cell lineage tracers to show that PGC specification in the leech genus Helobdella follows a different scenario: in this hermaphrodite, the male and female PGCs segregate from somatic lineages only after more than 20 rounds of zygotic mitosis; the male and female PGCs share the same (mesodermal) cell lineage for 19 rounds of zygotic mitosis. Moreover, while all three markers are expressed in both male and female reproductive tissues of the adult, they are expressed differentially between the male and female PGCs of the developing embryo: piwi and vasa are expressed preferentially in female PGCs at a time when nanos is expressed preferentially in male PGCs. A priori, the delayed segregation of male and female PGCs from somatic tissues and from one another increases the probability of mutations affecting both male and female PGCs of a given individual. We speculate that this suite of features, combined with a capacity for self-fertilization, may contribute to the dramatically rearranged genome of Helobdella robusta relative to other animals.

Differential Expression of Conserved Germ Line Markers and Delayed Segregation of Male and Female Primordial Germ Cells in a Hermaphrodite, the Leech Helobdella

PubMed Central

Cho, Sung-Jin; Vallès, Yvonne; Weisblat, David A.

2014-01-01

In sexually reproducing animals, primordial germ cells (PGCs) are often set aside early in embryogenesis, a strategy that minimizes the risk of genomic damage associated with replication and mitosis during the cell cycle. Here, we have used germ line markers (piwi, vasa, and nanos) and microinjected cell lineage tracers to show that PGC specification in the leech genus Helobdella follows a different scenario: in this hermaphrodite, the male and female PGCs segregate from somatic lineages only after more than 20 rounds of zygotic mitosis; the male and female PGCs share the same (mesodermal) cell lineage for 19 rounds of zygotic mitosis. Moreover, while all three markers are expressed in both male and female reproductive tissues of the adult, they are expressed differentially between the male and female PGCs of the developing embryo: piwi and vasa are expressed preferentially in female PGCs at a time when nanos is expressed preferentially in male PGCs. A priori, the delayed segregation of male and female PGCs from somatic tissues and from one another increases the probability of mutations affecting both male and female PGCs of a given individual. We speculate that this suite of features, combined with a capacity for self-fertilization, may contribute to the dramatically rearranged genome of Helobdella robusta relative to other animals. PMID:24217283

Acute phase lipocalin Ex-FABP is involved in heart development and cell survival.

PubMed

Gentili, C; Tutolo, G; Zerega, B; Di Marco, E; Cancedda, R; Cancedda, F Descalzi

2005-03-01

Ex-FABP is an extracellular fatty acid binding protein, expressed during chicken embryo development in cartilage, muscle fibers, and blood granulocytes. Transfection of chondrocytes and myoblasts with anti-sense Ex-FABP cDNA results in inhibition of cell proliferation and apoptosis induction. Ex-FABP expression is dramatically enhanced by inflammatory stimuli and in pathological conditions. In this paper, by in situ whole mount and immunohistochemistry analysis we show that, at early developmental stage, Ex-FABP is diffuse in all tissues of chick embryos. Particularly high level of transcript and protein are expressed in the heart. During acute phase response (APR) induced by endotoxin LPS injection, a marked increase of Ex-FABP mRNA was observed in embryos, highest Ex-FABP expression being in heart and liver. To investigate in vivo the biological role of Ex-FABP, we have directly microinjected chicken embryos with antibody against Ex-FABP. Almost 70% of chicken embryos died and the target tissue was the heart. We detected in heart of the treated embryos a significant increase of apoptotic cells and high level of fatty acids. We propose that the accumulation of fatty acid, specific ligand of Ex-FABP, in the cell microenvironment is responsible of heart cell death, and we suggest that Ex-FABP may act as a survival protein by playing a role as scavenger for fatty acids. 2004 Wiley-Liss, Inc.

Comparative Therapeutic Effects of Minocycline Treatment and Bone Marrow Mononuclear Cell Transplantation following Striatal Stroke

PubMed Central

Souza, Celice C.; da Silva, Michelle Castro; Lopes, Rosana Telma; Cardoso, Marcelo M.; Santos, Adriano Guimarães; dos Santos, Ijair Rogério

2017-01-01

We explored the comparative effects of minocycline treatment and intrastriatal BMMC transplantation after experimental striatal stroke in adult rats. Male Wistar adult rats were divided as follows: saline-treated (N = 5), minocycline-treated (N = 5), and BMMC-transplanted (N = 5) animals. Animals received intrastriatal microinjections of 80 pmol of endothelin-1 (ET-1). Behavioral tests were performed at 1, 3, and 7 days postischemia. Animals were treated with minocycline (50 mg/kg, i.p.) or intrastriatal transplants of 106 BMMCs at 24 h postischemia. Animals were perfused at 7 days after ischemic induction. Coronal sections were stained with cresyl violet for gross histopathological analysis and immunolabeled for the identification of neuronal bodies (NeuN), activated microglia/macrophages (ED1), and apoptotic cells (active caspase-3). BMMC transplantation and minocycline reduced the number of ED1+ cells (p < 0.05, ANOVA-Tukey), but BMMC afforded better results. Both treatments afforded comparable levels of neuronal preservation compared to control (p > 0.05). BMMC transplantation induced a higher decrease in the number of apoptotic cells compared to control and minocycline treatment. Both therapeutic approaches improved functional recovery in ischemic animals. The results suggest that BMMC transplantation is more effective in modulating microglial activation and reducing apoptotic cell death than minocycline, although both treatments are equally efficacious on improving neuronal preservation. PMID:28713482

Comparative Therapeutic Effects of Minocycline Treatment and Bone Marrow Mononuclear Cell Transplantation following Striatal Stroke.

PubMed

Souza, Celice C; da Silva, Michelle Castro; Lopes, Rosana Telma; Cardoso, Marcelo M; de Souza, Lucas Lacerda; Santos, Adriano Guimarães; Dos Santos, Ijair Rogério; Franco, Edna C S; Gomes-Leal, Walace

2017-01-01

We explored the comparative effects of minocycline treatment and intrastriatal BMMC transplantation after experimental striatal stroke in adult rats. Male Wistar adult rats were divided as follows: saline-treated ( N = 5), minocycline-treated ( N = 5), and BMMC-transplanted ( N = 5) animals. Animals received intrastriatal microinjections of 80 pmol of endothelin-1 (ET-1). Behavioral tests were performed at 1, 3, and 7 days postischemia. Animals were treated with minocycline (50 mg/kg, i.p.) or intrastriatal transplants of 106 BMMCs at 24 h postischemia. Animals were perfused at 7 days after ischemic induction. Coronal sections were stained with cresyl violet for gross histopathological analysis and immunolabeled for the identification of neuronal bodies (NeuN), activated microglia/macrophages (ED1), and apoptotic cells (active caspase-3). BMMC transplantation and minocycline reduced the number of ED1+ cells ( p < 0.05, ANOVA-Tukey), but BMMC afforded better results. Both treatments afforded comparable levels of neuronal preservation compared to control ( p > 0.05). BMMC transplantation induced a higher decrease in the number of apoptotic cells compared to control and minocycline treatment. Both therapeutic approaches improved functional recovery in ischemic animals. The results suggest that BMMC transplantation is more effective in modulating microglial activation and reducing apoptotic cell death than minocycline, although both treatments are equally efficacious on improving neuronal preservation.

Daam1 regulates fascin for actin assembly in mouse oocyte meiosis.

PubMed

Lu, Yujie; Zhang, Yu; Pan, Meng-Hao; Kim, Nam-Hyung; Sun, Shao-Chen; Cui, Xiang-Shun

2017-07-18

As a formin protein, Daam1 (Dishevelled-associated activator of morphogenesis 1) is reported to regulate series of cell processes like endocytosis, cell morphology and migration via its effects on actin assembly in mitosis. However, whether Daam1 plays roles in female meiosis remains uncertain. In this study, we investigated the expression and functions of Daam1 during mouse oocyte meiosis. Our results indicated that Daam1 localized at the cortex of oocytes, which was similar with actin filaments. After Daam1 morpholino (MO) microinjection, the expression of Daam1 significantly decreased, which resulted in the failure of oocyte polar body extrusion. These results might be due to the defects of actin assembly, since the decreased fluorescence intensity of actin filaments in oocyte cortex and cytoplasm were observed. However, Daam1 knockdown seemed not to affect the meiotic spindle movement. In addition, we found that fascin might be the down effector of Daam1, since the protein expression of fascin decreased after Daam1 knockdown. Thus, our data suggested that Daam1 affected actin assembly during oocyte meiotic division via the regulation of fascin expression.

Evaluation of the damage of cell wall and cell membrane for various extracellular polymeric substance extractions of activated sludge.

PubMed

Guo, Xuesong; Liu, Junxin; Xiao, Benyi

2014-10-20

Extracellular polymeric substances (EPS) are susceptible to contamination by intracellular substances released during the extraction of EPS owing to the damage caused to microbial cell structures. The damage to cell walls and cell membranes in nine EPS extraction processes of activated sludge was evaluated in this study. The extraction of EPS (including proteins, carbohydrates and DNA) was the highest using the NaOH extraction method and the lowest using formaldehyde extraction. All nine EPS extraction methods in this study resulted in cell wall and membrane damage. The damage to cell walls, evaluated by 2-keto-3-deoxyoctonate (KDO) and N-acetylglucosamine content changes in extracted EPS, was the most significant in the NaOH extraction process. Formaldehyde extraction showed a similar extent of damage to cell walls to those detected in the control method (centrifugation), while those in the formaldehyde-NaOH and cation exchange resin extractions were slightly higher than those detected in the control. N-acetylglucosamine was more suitable than KDO for the evaluation of cell wall damage in the EPS extraction of activated sludge. The damage to cell membranes was characterized by two fluorochromes (propidium iodide and FITC Annexin V) with flow cytometry (FCM) measurement. The highest proportion of membrane-damaged cells was detected in NaOH extraction (26.54% of total cells) while membrane-damaged cells comprised 8.19% of total cells in the control. Copyright © 2014 Elsevier B.V. All rights reserved.

Vagally mediated effects of brain stem dopamine on gastric tone and phasic contractions of the rat.

PubMed

Anselmi, L; Toti, L; Bove, C; Travagli, R A

2017-11-01

Dopamine (DA)-containing fibers and neurons are embedded within the brain stem dorsal vagal complex (DVC); we have shown previously that DA modulates the membrane properties of neurons of the dorsal motor nucleus of the vagus (DMV) via DA1 and DA2 receptors. The vagally dependent modulation of gastric tone and phasic contractions, i.e., motility, by DA, however, has not been characterized. With the use of microinjections of DA in the DVC while recording gastric tone and motility, the aims of the present study were 1 ) assess the gastric effects of brain stem DA application, 2 ) identify the DA receptor subtype, and, 3 ) identify the postganglionic pathway(s) activated. Dopamine microinjection in the DVC decreased gastric tone and motility in both corpus and antrum in 29 of 34 rats, and the effects were abolished by ipsilateral vagotomy and fourth ventricular treatment with the selective DA2 receptor antagonist L741,626 but not by application of the selective DA1 receptor antagonist SCH 23390. Systemic administration of the cholinergic antagonist atropine attenuated the inhibition of corpus and antrum tone in response to DA microinjection in the DVC. Conversely, systemic administration of the nitric oxide synthase inhibitor nitro-l-arginine methyl ester did not alter the DA-induced decrease in gastric tone and motility. Our data provide evidence of a dopaminergic modulation of a brain stem vagal neurocircuit that controls gastric tone and motility. NEW & NOTEWORTHY Dopamine administration in the brain stem decreases gastric tone and phasic contractions. The gastric effects of dopamine are mediated via dopamine 2 receptors on neurons of the dorsal motor nucleus of the vagus. The inhibitory effects of dopamine are mediated via inhibition of the postganglionic cholinergic pathway. Copyright © 2017 the American Physiological Society.

Involvement of ERK phosphorylation in brainstem neurons in modulation of swallowing reflex in rats

PubMed Central

Tsujimura, Takanori; Kondo, Masahiro; Kitagawa, Junichi; Tsuboi, Yoshiyuki; Saito, Kimiko; Tohara, Haruka; Ueda, Koichiro; Sessle, Barry J; Iwata, Koichi

2009-01-01

In order to evaluate the neuronal mechanisms underlying functional abnormalities of swallowing in orofacial pain patients, this study investigated the effects of noxious orofacial stimulation on the swallowing reflex, phosphorylated extracellular signal-regulated kinase (pERK) and γ-aminobutyric acid (GABA) immunohistochemical features in brainstem neurons, and also analysed the effects of brainstem lesioning and of microinjection of GABA receptor agonist or antagonist into the nucleus tractus solitarii (NTS) on the swallowing reflex in anaesthetized rats. The swallowing reflex elicited by topical administration of distilled water to the pharyngolaryngeal region was inhibited after capsaicin injection into the facial (whisker pad) skin or lingual muscle. The capsaicin-induced inhibitory effect on the swallowing reflex was itself depressed after the intrathecal administration of MAPK kinase (MEK) inhibitor. No change in the capsaicin-induced inhibitory effect was observed after trigeminal spinal subnucleus caudalis lesioning, but the inhibitory effect was diminished by paratrigeminal nucleus (Pa5) lesioning. Many pERK-like immunoreactive neurons in the NTS showed GABA immunoreactivity. The local microinjection of the GABAA receptor agonist muscimol into the NTS produced a significant reduction in swallowing reflex, and the capsaicin-induced depression of the swallowing reflex was abolished by microinjection of the GABAA receptor antagonist bicuculline into the NTS. The present findings suggest that facial skin–NTS, lingual muscle–NTS and lingual muscle–Pa5–NTS pathways are involved in the modulation of swallowing reflex by facial and lingual pain, respectively, and that the activation of GABAergic NTS neurons is involved in the inhibition of the swallowing reflex following noxious stimulation of facial and intraoral structures. PMID:19124539

L-Cysteine and L-AP4 microinjections in the rat caudal ventrolateral medulla decrease arterial blood pressure.

PubMed

Takemoto, Yumi

2014-12-01

The thiol amino acid L-cysteine increases arterial blood pressure (ABP) when injected into the cerebrospinal fluid space in conscious rats, indicating a pressor response to centrally acting L-cysteine. A prior synaptic membrane binding assay suggests that L-cysteine has a strong affinity for the L-2-amino-4-phosphonobutyric acid (L-AP4) binding site. The central action of L-cysteine may be vial-AP4 sensitive receptors. The present study investigated cardiovascular responses to L-cysteine and L-ap4 microinjected into the autonomic area of the caudal ventrolateral medulla (CVLM) where inhibitory neurons regulate ABP via pre-sympathetic vasomotor neurons. Both the injection of L-cysteine and L-AP4 in the CVLM sites identified with L-glutamate produced the same depressor and bradycardic responses in urethane-anesthetized rats. Neither a prior antagonist microinjection of MK801 for the N-methyl-D-aspartate (NMDA) receptor nor CNQX for the non-NMDA receptor attenuated the responses to L-cysteine, but the combination of the two receptor blocking with an additional prior injection abolished the response. In contrast, either receptor blockade alone abolished the response to L-AP4, indicating distinct mechanisms between responses to L-cysteine and L-AP4 in the CVLM. The results indicate that the CVLM is a central active site for L-cysteine's cardiovascular response. Central L-cysteine's action could be independent of the L-AP4 sensitive receptors. Cardiovascular regulation may involve endogenous L-cysteine in the CVLM. Further multidisciplinary examinations are required to elaborate on L-cysteine's functional roles in the CVLM. Copyright © 2014 Elsevier B.V. All rights reserved.

Motor tics evoked by striatal disinhibition in the rat

PubMed Central

Bronfeld, Maya; Yael, Dorin; Belelovsky, Katya; Bar-Gad, Izhar

2013-01-01

Motor tics are sudden, brief, repetitive movements that constitute the main symptom of Tourette syndrome (TS). Multiple lines of evidence suggest the involvement of the cortico-basal ganglia system, and in particular the basal ganglia input structure—the striatum in tic formation. The striatum receives somatotopically organized cortical projections and contains an internal GABAergic network of interneurons and projection neurons' collaterals. Disruption of local striatal GABAergic connectivity has been associated with TS and was found to induce abnormal movements in model animals. We have previously described the behavioral and neurophysiological characteristics of motor tics induced in monkeys by local striatal microinjections of the GABAA antagonist bicuculline. In the current study we explored the abnormal movements induced by a similar manipulation in freely moving rats. We targeted microinjections to different parts of the dorsal striatum, and examined the effects of this manipulation on the induced tic properties, such as latency, duration, and somatic localization. Tics induced by striatal disinhibition in monkeys and rats shared multiple properties: tics began within several minutes after microinjection, were expressed solely in the contralateral side, and waxed and waned around a mean inter-tic interval of 1–4 s. A clear somatotopic organization was observed only in rats, where injections to the anterior or posterior striatum led to tics in the forelimb or hindlimb areas, respectively. These results suggest that striatal disinhibition in the rat may be used to model motor tics such as observed in TS. Establishing this reliable and accessible animal model could facilitate the study of the neural mechanisms underlying motor tics, and the testing of potential therapies for tic disorders. PMID:24065893

Vasoactive intestinal polypeptide microinjections into the oral pontine tegmentum enhance rapid eye movement sleep in the rat.

PubMed

Bourgin, P; Lebrand, C; Escourrou, P; Gaultier, C; Franc, B; Hamon, M; Adrien, J

1997-03-01

Rapid eye movement sleep can be elicited in the rat by microinjection of the cholinergic agonist carbachol into the oral pontine reticular nucleus. Intracerebroventricular administration, during the light period, of vasoactive intestinal peptide enhances rapid eye movement sleep in several species. Since this peptide is co-localized with acetylcholine in many neurons in the central nervous system, it was assumed that the oral pontine tegmentum could also be one target for vasoactive intestinal peptide to induce rapid eye movement sleep. This hypothesis was tested by recording the sleep-wakefulness cycle in freely-moving rats injected with vasoactive intestinal peptide or its fragments (1-12 and 10-28) directly into the oral pontine reticular nucleus. when administered into the posterior part of this nucleus, vasoactive intestinal peptide at 1 and 10 ng (in 0.1 microliter of saline), but not its fragments, induced a 2-fold enhancement of rapid eye movement sleep during 4 h, at the expense of wakefulness. At the dose of 10 ng, a significant increase in rapid eye movement sleep persisted for up to 8 h. Moreover, when the peptide was injected into the centre of the positive zone, rapid eye movement sleep was enhanced during three to eight consecutive days. These data provide the first evidence that rapid eye movement sleep can be elicited at both short- and long-term by a single intracerebral microinjection of vasoactive intestinal peptide. Peptidergic mechanisms, possibly in association with cholinergic mechanisms, within the caudal part of the oral pontine reticular nucleus may play a critical role in the long-term regulation of rapid eye movement sleep in rats.

Activation of NMDA receptors in the brainstem, rostral ventromedial medulla, and nucleus reticularis gigantocellularis mediates mechanical hyperalgesia produced by repeated intramuscular injections of acidic saline in rats.

PubMed

Da Silva, Luis F; Desantana, Josimari M; Sluka, Kathleen A

2010-04-01

Repeated injections of acidic saline into the gastrocnemius muscle induce both muscle and cutaneous hypersensitivity. We have previously shown that microinjection of local anesthetic into either the rostral ventromedial medulla (RVM) or the nucleus reticularis gigantocellularis (NGC) reverses this muscle and cutaneous hypersensitivity. Although prior studies show that NMDA receptors in the RVM play a clear role in mediating visceral and inflammatory hypersensitivity, the role of NMDA receptors in the NGC or in noninflammatory muscle pain is unclear. Therefore, the present study evaluated involvement of the NMDA receptors in the RVM and NGC in muscle and cutaneous hypersensitivity induced by repeated intramuscular injections of acidic saline. Repeated intramuscular injections of acidic saline, 5 days apart, resulted in a bilateral decrease in the withdrawal thresholds of the paw and muscle in all groups 24 hours after the second injection. Microinjection of NMDA receptor antagonists into the RVM reversed both the muscle and cutaneous hypersensitivity. However, microinjection of NMDA receptor antagonists into the NGC only reversed cutaneous but not muscle hypersensitivity. These results suggest that NMDA receptors in the RVM mediate both muscle and cutaneous hypersensitivity, but those in the NGC mediate only cutaneous hypersensitivity after muscle insult. The current study shows that NMDA receptors in supraspinal facilitatory sites maintain noninflammatory muscle pain. Clinical studies in people with chronic widespread, noninflammatory pain, similarly, show alterations in central excitability. Thus, understanding mechanisms in an animal model could lead to improved treatment for patients with chronic muscle pain. Copyright 2010 American Pain Society. Published by Elsevier Inc. All rights reserved.

Involvement of AMPA/Kainate Glutamate Receptor in the Extinction and Reinstatement of Morphine-Induced Conditioned Place Preference: A Behavioral and Molecular Study.

PubMed

Siahposht-Khachaki, Ali; Fatahi, Zahra; Yans, Asal; Khodagholi, Fariba; Haghparast, Abbas

2017-03-01

Glutamate receptors in mesolimbic areas such as the nucleus accumbens, ventral tegmental area, prefrontal cortex (PFC), and hippocampus (HIP) are a component of the mechanisms of drug-induced reward and can modulate the firing pattern of dopaminergic neurons in the reward system. In addition, several lines of study have indicated that cAMP response element-binding protein (CREB) and c-fos have important role in morphine-induced conditioned place preference (CPP) induced by drugs of abuse, such as morphine, cocaine, nicotine, and alcohol. Therefore, in the present study, we investigated the changes in phosphorylated CREB (p-CREB) and c-fos induction within the nucleus accumbens (NAc), HIP, and PFC after intracerebroventricular (ICV) administration of different doses of CNQX or vehicle during extinction period or reinstatement of morphine-induced CPP. In all groups, the CPP procedure was done; afterward, the conditioning scores were recorded by Ethovision software. After behavioral test recording, we dissected out the NAc, HIP, and PFC regions and measured the p-CREB/CREB ratio and c-fos level by Western blot analysis. Our results showed that administration of CNQX significantly shortened the extinction of morphine CPP. Besides, ICV microinjection of CNQX following extinction period decreased the reinstatement of morphine CPP in extinguished rats. In molecular section, in treatment group, all mentioned factors were dose-dependently decreased in comparison with vehicle group (DMSO) after ICV microinjection of different doses of CNQX but not in pre-extinction microinjection. These findings suggested that antagonism of AMPA receptor decreased p-CREB/CREB ratio and c-fos level in the PFC, NAc, and HIP. Modulation of the drug memory reconsolidation may be useful for faster extinction of drug-induced reward and attenuation of drug-seeking behavior.

elPBN neurons regulate rVLM activity through elPBN-rVLM projections during activation of cardiac sympathetic afferent nerves

PubMed Central

Longhurst, John C.; Tjen-A-Looi, Stephanie C.; Fu, Liang-Wu

2016-01-01

The external lateral parabrachial nucleus (elPBN) within the pons and rostral ventrolateral medulla (rVLM) contributes to central processing of excitatory cardiovascular reflexes during stimulation of cardiac sympathetic afferent nerves (CSAN). However, the importance of elPBN cardiovascular neurons in regulation of rVLM activity during CSAN activation remains unclear. We hypothesized that CSAN stimulation excites the elPBN cardiovascular neurons and, in turn, increases rVLM activity through elPBN-rVLM projections. Compared with controls, in rats subjected to microinjection of retrograde tracer into the rVLM, the numbers of elPBN neurons double-labeled with c-Fos (an immediate early gene) and the tracer were increased after CSAN stimulation (P < 0.05). The majority of these elPBN neurons contain vesicular glutamate transporter 3. In cats, epicardial bradykinin and electrical stimulation of CSAN increased the activity of elPBN cardiovascular neurons, which was attenuated (n = 6, P < 0.05) after blockade of glutamate receptors with iontophoresis of kynurenic acid (Kyn, 25 mM). In separate cats, microinjection of Kyn (1.25 nmol/50 nl) into the elPBN reduced rVLM activity evoked by both bradykinin and electrical stimulation (n = 5, P < 0.05). Excitation of the elPBN with microinjection of dl-homocysteic acid (2 nmol/50 nl) significantly increased basal and CSAN-evoked rVLM activity. However, the enhanced rVLM activity induced by dl-homocysteic acid injected into the elPBN was reversed following iontophoresis of Kyn into the rVLM (n = 7, P < 0.05). These data suggest that cardiac sympathetic afferent stimulation activates cardiovascular neurons in the elPBN and rVLM sequentially through a monosynaptic (glutamatergic) excitatory elPBN-rVLM pathway. PMID:27225950

Dopamine or opioid stimulation of nucleus accumbens similarly amplify cue-triggered 'wanting' for reward: entire core and medial shell mapped as substrates for PIT enhancement.

PubMed

Peciña, Susana; Berridge, Kent C

2013-05-01

Pavlovian cues [conditioned stimulus (CS+)] often trigger intense motivation to pursue and consume related reward [unconditioned stimulus (UCS)]. But cues do not always trigger the same intensity of motivation. Encountering a reward cue can be more tempting on some occasions than on others. What makes the same cue trigger more intense motivation to pursue reward on a particular encounter? The answer may be the level of incentive salience ('wanting') that is dynamically generated by mesocorticolimbic brain systems, influenced especially by dopamine and opioid neurotransmission in the nucleus accumbens (NAc) at that moment. We tested the ability of dopamine stimulation (by amphetamine microinjection) vs. mu opioid stimulation [by d-Ala, nMe-Phe, Glyol-enkephalin (DAMGO) microinjection] of either the core or shell of the NAc to amplify cue-triggered levels of motivation to pursue sucrose reward, measured with a Pavlovian-Instrumental Transfer (PIT) procedure, a relatively pure assay of incentive salience. Cue-triggered 'wanting' in PIT was enhanced by amphetamine or DAMGO microinjections equally, and also equally at nearly all sites throughout the entire core and medial shell (except for a small far-rostral strip of shell). NAc dopamine/opioid stimulations specifically enhanced CS+ ability to trigger phasic peaks of 'wanting' to obtain UCS, without altering baseline efforts when CS+ was absent. We conclude that dopamine/opioid stimulation throughout nearly the entire NAc can causally amplify the reactivity of mesocorticolimbic circuits, and so magnify incentive salience or phasic UCS 'wanting' peaks triggered by a CS+. Mesolimbic amplification of incentive salience may explain why a particular cue encounter can become irresistibly tempting, even when previous encounters were successfully resisted before. © 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

GLP-1 and estrogen conjugate acts in the supramammillary nucleus to reduce food-reward and body weight.

PubMed

Vogel, Heike; Wolf, Stefanie; Rabasa, Cristina; Rodriguez-Pacheco, Francisca; Babaei, Carina S; Stöber, Franziska; Goldschmidt, Jürgen; DiMarchi, Richard D; Finan, Brian; Tschöp, Matthias H; Dickson, Suzanne L; Schürmann, Annette; Skibicka, Karolina P

2016-11-01

The obesity epidemic continues unabated and currently available pharmacological treatments are not sufficiently effective. Combining gut/brain peptide, GLP-1, with estrogen into a conjugate may represent a novel, safe and potent, strategy to treat diabesity. Here we demonstrate that the central administration of GLP-1-estrogen conjugate reduced food reward, food intake, and body weight in rats. In order to determine the brain location of the interaction of GLP-1 with estrogen, we avail of single-photon emission computed tomography imaging of regional cerebral blood flow and pinpoint a brain site unexplored for its role in feeding and reward, the supramammillary nucleus (SUM) as a potential target of the conjugated GLP-1-estrogen. We confirm that conjugated GLP-1 and estrogen directly target the SUM with site-specific microinjections. Additional microinjections of GLP-1-estrogen into classic energy balance controlling nuclei, the lateral hypothalamus (LH) and the nucleus of the solitary tract (NTS) revealed that the metabolic benefits resulting from GLP-1-estrogen injections are mediated through the LH and to some extent by the NTS. In contrast, no additional benefit of the conjugate was noted on food reward when the compound was microinjected into the LH or the NTS, identifying the SUM as the only neural substrate identified here to underlie the reward reducing benefits of GLP-1 and estrogen conjugate. Collectively we discover a surprising neural substrate underlying food intake and reward effects of GLP-1 and estrogen and uncover a new brain area capable of regulating energy balance and reward. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Role of ionotropic GABA, glutamate and glycine receptors in the tonic and reflex control of cardiac vagal outflow in the rat

PubMed Central

2010-01-01

Background Cardiac vagal preganglionic neurons (CVPN) are responsible for the tonic, reflex and respiratory modulation of heart rate (HR). Although CVPN receive GABAergic and glutamatergic inputs, likely involved in respiratory and reflex modulation of HR respectively, little else is known regarding the functions controlled by ionotropic inputs. Activation of g-protein coupled receptors (GPCR) alters these inputs, but the functional consequence is largely unknown. The present study aimed to delineate how ionotropic GABAergic, glycinergic and glutamatergic inputs contribute to the tonic and reflex control of HR and in particular determine which receptor subtypes were involved. Furthermore, we wished to establish how activation of the 5-HT1A GPCR affects tonic and reflex control of HR and what ionotropic interactions this might involve. Results Microinjection of the GABAA antagonist picrotoxin into CVPN decreased HR but did not affect baroreflex bradycardia. The glycine antagonist strychnine did not alter HR or baroreflex bradycardia. Combined microinjection of the NMDA antagonist, MK801, and AMPA antagonist, CNQX, into CVPN evoked a small bradycardia and abolished baroreflex bradycardia. MK801 attenuated whereas CNQX abolished baroreceptor bradycardia. Control intravenous injections of the 5-HT1A agonist 8-OH-DPAT evoked a small bradycardia and potentiated baroreflex bradycardia. These effects were still observed following microinjection of picrotoxin but not strychnine into CVPN. Conclusions We conclude that activation of GABAA receptors set the level of HR whereas AMPA to a greater extent than NMDA receptors elicit baroreflex changes in HR. Furthermore, activation of 5-HT1A receptors evokes bradycardia and enhances baroreflex changes in HR due to interactions with glycinergic neurons involving strychnine receptors. This study provides reference for future studies investigating how diseases alter neurochemical inputs to CVPN. PMID:20939929

Dorsolateral neostriatum contribution to incentive salience: opioid or dopamine stimulation makes one reward cue more motivationally attractive than another.

PubMed

DiFeliceantonio, Alexandra G; Berridge, Kent C

2016-05-01

Pavlovian cues for rewards can become attractive incentives: approached and 'wanted' as the rewards themselves. The motivational attractiveness of a previously learned cue is not fixed, but can be dynamically amplified during re-encounter by simultaneous activation of brain limbic circuitry. Here it was reported that opioid or dopamine microinjections in the dorsolateral quadrant of the neostriatum (DLS) of rats selectively amplify attraction toward a previously learned Pavlovian cue in an individualized fashion, at the expense of a competing cue. In an autoshaping (sign-tracking vs. goal-tracking) paradigm, microinjection of the mu opioid receptor agonist (DAMGO) or dopamine indirect agonist (amphetamine) in the DLS of sign-tracker individuals selectively enhanced their sign-tracking attraction toward the reward-predictive lever cue. By contrast, DAMGO or amphetamine in the DLS of goal-trackers selectively enhanced prepotent attraction toward the reward-proximal cue of sucrose dish. Amphetamine also enhanced goal-tracking in some sign-tracker individuals (if they ever defected to the dish even once). That DLS enhancement of cue attraction was due to stronger motivation, not stronger habits, was suggested by: (i) sign-trackers flexibly followed their cue to a new location when the lever was suddenly moved after DLS DAMGO microinjection; and (ii) DAMGO in the DLS also made sign-trackers work harder on a new instrumental nose-poke response required to earn presentations of their Pavlovian lever cue (instrumental conditioned reinforcement). Altogether, the current results suggest that DLS circuitry can enhance the incentive salience of a Pavlovian reward cue, selectively making that cue a stronger motivational magnet. © 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Neurokinin receptor modulation of respiratory activity in the rabbit.

PubMed

Bongianni, Fulvia; Mutolo, Donatella; Cinelli, Elenia; Pantaleo, Tito

2008-06-01

The respiratory role of neurokinin (NK) receptors was investigated in alpha-chloralose-urethane-anaesthetized, vagotomized, paralysed and artificially ventilated rabbits by using bilateral microinjections (30-50 nL) of NK receptor agonists and antagonists. Microinjections were performed in a region located just caudal to the rostral expiratory neurons. This region displayed features similar to those of the pre-Bötzinger complex (pre-BötC) of adult cats and rats, and proved to produce excitatory respiratory effects in response to microinjections of D,L-homocysteic acid. We used as agonists (0.1, 0.5 and 5 mM) substance P (SP), the NK1 receptor agonists [Sar(9), Met(O2)(11)]-SP and GR 73632, the NK2 receptor agonist NKA, the NK3 receptor agonist senktide, and as antagonists (5 mM) the NK1 receptor antagonist CP-99,994 and the NK2 receptor antagonist MEN 10376. SP always increased respiratory frequency, but NK1 receptor agonists did not change respiratory variables. NKA and senktide at 5 mm increased respiratory frequency. CP-99,994 caused increases in respiratory frequency and did not antagonize the effects of SP. MEN 10376 prevented the respiratory responses induced by NKA and reduced those provoked by SP. SP or the NK1 receptor agonists (5 mM) injected (1 microL) into the IV ventricle caused marked excitatory effects on respiration. The results suggest that NK2 and NK3, but not NK1, receptors are involved in the excitatory modulation of inspiratory activity within the investigated region and are consistent with the notion that the pre-BötC neurons are important components of the inspiratory rhythm-generating mechanisms.

Scopolamine into the anterior cingulate cortex diminishes nociception in a neuropathic pain model in the rat: an interruption of 'nociception-related memory acquisition'?

PubMed

Ortega-Legaspi, J Manuel; López-Avila, Alberto; Coffeen, Ulises; del Angel, Rosendo; Pellicer, Francisco

2003-01-01

The cingulate cortex plays a key role in the affective component related to pain perception. This structure receives cholinergic projections and also plays a role in memory processing. Therefore, we propose that the cholinergic system in the anterior cingulate cortex is involved in the nociceptive memory process. We used scopolamine (10 microg in 0.25 mircrol/saline) microinjected into the anterior cingulate cortex, either before thermonociception followed by a sciatic denervation, between thermonociception and denervation or after both procedures (n=10 each). The vehicle group (saline solution 0.9%, n=14) was microinjected before thermonociception. Chronic nociception was measured by the autotomy score, which onset and incidence were also determined. Group scopolamine-thermonociception-denervation (STD) presented the lowest autotomy score as compared to vehicle and group thermonociception-denervation-scopolamine (TDS) (vehicle vs. STD, p=0.002, STD vs. TDS, p=0.001). Group thermonociception-scopolamine-denervation (TSD) showed a diminished autotomy score when compared to TDS (p=0.053). STD group showed a delay in the onset of AB as compared to the rest of the groups. Group TSD presented a significative delay (p=0.048) in AB onset when compared to group TDS. There were no differences in the incidence between groups. The results show that nociception-related memory processed in the anterior cingulate cortex is susceptible of being modified by the cholinergic transmission blockade. When scopolamine is microinjected prior to the nociceptive stimuli, nociception-related memory acquisition is prevented. The evidence obtained in this study shows the role of the anterior cingulate cortex in the acquisition of nociception-related memory.

Activation of cannabinoid CB1 receptors in the dorsolateral periaqueductal gray induces anxiolytic effects in rats submitted to the Vogel conflict test.

PubMed

Lisboa, Sabrina F; Resstel, Leonardo B M; Aguiar, Daniele C; Guimarães, Francisco S

2008-09-28

There are contradictory results concerning the effects of systemic injections of cannabinoid agonists in anxiety-induced behavioral changes. Direct drug administration into brain structures related to defensive responses could help to clarify the role of cannabinoids in these changes. Activation of cannabinoid CB(1) receptors in the dorsolateral periaqueductal gray induces anxiolytic-like effects in the elevated plus maze. The aim of this work was to verify if facilitation of endocannabinoid-mediated neurotransmission in this region would also produce anxiolytic-like effects in another model of anxiety, the Vogel conflict test. Male Wistar rats (n=5-9/group) with cannulae aimed at the dorsolateral periaqueductal gray were water deprived for 24 h and pre-exposed to the apparatus where they were allowed to drink for 3 min. After another 24 h-period of water deprivation, they received the microinjections and, 10 min later, were placed into the experimental box. In this box an electrical shock (0.5 mA, 2 s) was delivered in the spout of a drinking bottle at every twenty licks. The animals received a first microinjection of vehicle (0.2 microl) or AM251 (a cannabinoid CB(1) receptor antagonist; 100 pmol) followed, 5 min later, by a second microinjection of vehicle, anandamide (an endocannabinoid, 5 pmol), AM404 (an inhibitor of anandamide uptake, 50 pmol) or URB597 (an inhibitor of Fatty Acid Amide Hydrolase, 0.01 or 0.1 nmol). Anandamide, AM404 and URB597 (0.01 nmol) increased the total number of punished licks. These effects were prevented by AM251. The results give further support to the proposal that facilitation of CB(1) receptor-mediated endocannabinoid neurotransmission in the dorsolateral periaqueductal gray modulates defensive responses.

Calpain expression and activity during lens fiber cell differentiation.

PubMed

De Maria, Alicia; Shi, Yanrong; Kumar, Nalin M; Bassnett, Steven

2009-05-15

In animal models, the dysregulated activity of calcium-activated proteases, calpains, contributes directly to cataract formation. However, the physiological role of calpains in the healthy lens is not well defined. In this study, we examined the expression pattern of calpains in the mouse lens. Real time PCR and Western blotting data indicated that calpain 1, 2, 3, and 7 were expressed in lens fiber cells. Using controlled lysis, depth-dependent expression profiles for each calpain were obtained. These indicated that, unlike calpain 1, 2, and 7, which were most abundant in cells near the lens surface, calpain 3 expression was strongest in the deep cortical region of the lens. We detected calpain activities in vitro and showed that calpains were active in vivo by microinjecting fluorogenic calpain substrates into cortical fiber cells. To identify endogenous calpain substrates, membrane/cytoskeleton preparations were treated with recombinant calpain, and cleaved products were identified by two-dimensional difference electrophoresis/mass spectrometry. Among the calpain substrates identified by this approach was alphaII-spectrin. An antibody that specifically recognized calpain-cleaved spectrin was used to demonstrate that spectrin is cleaved in vivo, late in fiber cell differentiation, at or about the time that lens organelles are degraded. The generation of the calpain-specific spectrin cleavage product was not observed in lens tissue from calpain 3-null mice, indicating that calpain 3 is uniquely activated during lens fiber differentiation. Our data suggest a role for calpains in the remodeling of the membrane cytoskeleton that occurs with fiber cell maturation.

Analgesia induced by morphine microinjected into the nucleus raphe magnus: effects on tonic pain.

PubMed

Dualé, Christian; Sierralta, Fernando; Dallel, Radhouane

2007-07-01

One of the possible sites of action of the analgesic effect of morphine is the Nucleus Raphe Magnus, as morphine injected into this structure induces analgesia in transient pain models. In order to test if morphine in the Nucleus Raphe Magnus is also analgesic in a tonic pain model, 5 microg of morphine or saline (control) were microinjected into the Nucleus Raphe Magnus of the rat. Analgesic effects were assessed following nociceptive stimulation using transient heating of the tail (phasic pain) and subcutaneous orofacial injection of 1.5 % formalin (tonic pain). While morphine was strongly analgesic for the tail-flick response (p <0.0001 compared to control), analgesia on the response to formalin was also observed for both early (p = 0.007) and late responses (p = 0.02). However, the response to formalin was not completely blunted. These results suggest that the Nucleus Raphe Magnus is not the exclusive site of action of morphine-induced analgesia in clinical conditions.

Ceramic micro-injection molded nozzles for serial femtosecond crystallography sample delivery

NASA Astrophysics Data System (ADS)

Beyerlein, K. R.; Adriano, L.; Heymann, M.; Kirian, R.; Knoška, J.; Wilde, F.; Chapman, H. N.; Bajt, S.

2015-12-01

Serial femtosecond crystallography (SFX) using X-ray Free-Electron Lasers (XFELs) allows for room temperature protein structure determination without evidence of conventional radiation damage. In this method, a liquid suspension of protein microcrystals can be delivered to the X-ray beam in vacuum as a micro-jet, which replenishes the crystals at a rate that exceeds the current XFEL pulse repetition rate. Gas dynamic virtual nozzles produce the required micrometer-sized streams by the focusing action of a coaxial sheath gas and have been shown to be effective for SFX experiments. Here, we describe the design and characterization of such nozzles assembled from ceramic micro-injection molded outer gas-focusing capillaries. Trends of the emitted jet diameter and jet length as a function of supplied liquid and gas flow rates are measured by a fast imaging system. The observed trends are explained by derived relationships considering choked gas flow and liquid flow conservation. Finally, the performance of these nozzles in a SFX experiment is presented, including an analysis of the observed background.

Effector peptides of the renin-angiotensin system in the central mechanisms of acquired and innate behavior in thirst in rats.

PubMed

Vlasenko, R Ya; Kotov, A V

2007-03-01

We report here a comparative analysis of the involvement of a number of components of the renin-angiotensin system in the performance of simple and complex forms of drinking behavior and thirst-associated non-drinking types of behavior. On central (intracerebroventricular) microinjection, [des-Asp1]-angiotensin I at doses equieffective to those of angiotensins II and III was found to be involved only in the performance of simple (taking water from the bowl) and linked forms of activity (comfort behavior, stress grooming, orientational-investigative, and feeding behavior). Angiotensin II was involved in the central mechanisms of complex acquired drinking behavior, selectively modulating its key stages (initial, final), while angiotensin III was involved only in the mechanisms of reproduction of the complex skill. All three substances induced "innate patterns of behavior" specific for each compound, these occurring at fixed periods of time after intracerebral microinjection. The effects of these substances were selectively suppressed by the AT1 receptor blocker losartan potassium.

Microinjection of l-glutamate into the nucleus ambiguus partially inhibits gastric motility through the NMDA receptor - nitric oxide pathway.

PubMed

Sun, Hong-Zhao; Zhao, Shu-Zhen; Ai, Hong-Bin

2014-06-01

We have previously reported that both l-glutamate (l-Glu) and nitric oxide (NO) modulate gastric motility in the nucleus ambiguus (NA). The aim of this study is to explore the potential correlation between the l-Glu and NO. A latex balloon connected to a pressure transducer was inserted into the pylorus through the fundus of anesthetized male Wistar rats to continuously record changes in gastric smooth muscle contractile curves. Pretreatment with the NO-synthase inhibitor N-nitro-l-arginine methylester (l-NAME) did not completely abolish the inhibitory effect of l-Glu on gastric motility, but intravenous injection of the ganglionic blocker hexamethonium bromide (Hb) did. By using a specific N-methyl-d-aspartic acid (NMDA) receptor antagonist, we blocked the inhibitory effect of the NO-donor sodium nitroprusside (SNP) on gastric motility. These results suggest that microinjections of l-Glu into the NA inhibits gastric motility by activating the cholinergic preganglionic neurons, partially through the NMDA receptor - NO pathway.

Muscimol microinjected in the arcuate nucleus affects metabolism, body temperature & ventilation.

PubMed

Schlenker, Evelyn H

2016-06-15

Effects of microinjection of 2 doses of γ-aminobutyric acid (GABA)A receptor agonist, muscimol (M), into the hypothalamic arcuate nucleus on oxygen consumption and control of ventilation over time and body temperature (BT) at the end of the experiment were compared in adult male and female rats. Relative to cerebrospinal fluid (CSF, 0 nmol), BT was decreased only in male rats with both doses of M, while in female rats, the 5 nmol dose depressed oxygen consumption. Ventilation was depressed by 5 nmol M in male and 10 nmol M in female rats by decreasing tidal volume. M did not affect the ventilatory response of male or female rats to hypoxia, whereas in females 5 and 10 nmol M and in males 10 nmol M depressed the ventilatory response to hypercapnia. Thus, in rats GABAA receptors in the arcuate nucleus modulate BT, oxygen consumption, and ventilation in air and in response to hypercapnia in a sexually dimorphic manner. Copyright © 2016 Elsevier B.V. All rights reserved.

Assessment of the synergic effect of immunomodulation on nerve repair using multiparametric magnetic resonance imaging.

PubMed

Zheng, Chu-Shan; Zhang, Xiang; Chen, Yue-Yao; Zhang, Fang; Duan, Xiao-Hui; Chen, Mei-Wei; Lu, Lie-Jing; Shen, Jun

2018-01-01

The immune system plays a pivotal role in nerve injury. The aim of this study was to determine the role of multiparametric magnetic resonance imaging (MRI) in evaluation of the synergic effect of immunomodulation on nerve regeneration in neurotmesis. Rats with sciatic nerve neurotmesis and surgical repair underwent serial multiparametric MR examinations over an 8-week period after subepineurial microinjection of lipopolysaccharide (LPS) and subsequent subcutaneous injection of FK506 or subepineurial microinjection of LPS or phosphate-buffered saline (PBS) alone. Nerves treated with immunomodulation showed more prominent regeneration than those treated with LPS or PBS alone and more rapid restoration toward normal T2, fractional anisotropy (FA), and radial diffusivity (RD) values than nerves injected with LPS or PBS. Nerves treated with immunomodulation exert synergic beneficial effects on nerve regeneration that can be predicted by T2 measurements and FA and RD values. Muscle Nerve 57: E38-E45, 2018. © 2017 Wiley Periodicals, Inc.

The Role of Mesopontine NGF in Sleep and Wakefulness

PubMed Central

Ramos, Oscar V.; Torterolo, Pablo; Lim, Vincent; Chase, Michael H.; Sampogna, Sharon; Yamuy, Jack

2011-01-01

The microinjection of nerve growth factor (NGF) into the cat pontine tegmentum rapidly induces rapid eye movement (REM) sleep. To determine if NGF is involved in naturally-occurring REM sleep, we examined whether it is present in mesopontine cholinergic structures that promote the initiation of REM sleep, and whether the blockade of NGF production in these structures suppresses REM sleep. We found that cholinergic neurons in the cat dorsolateral mesopontine tegmentum exhibited NGF-like immunoreactivity. In addition, the microinjection of an oligodeoxyribonucleotide (OD) directed against cat NGF mRNA into this region resulted in a reduction in the time spent in REM sleep in conjunction with an increase in the time spent in wakefulness. Sleep and wakefulness returned to baseline conditions 2 to 5 days after antisense OD administration. The preceding antisense OD-induced effects occurred in conjunction with the suppression of NGF-like immunoreactivity within the site of antisense OD injection. These data support the hypothesis that NGF is involved in the modulation of naturally-occurring sleep and wakefulness. PMID:21840513

Computer-Assisted Transgenesis of Caenorhabditis elegans for Deep Phenotyping

PubMed Central

Gilleland, Cody L.; Falls, Adam T.; Noraky, James; Heiman, Maxwell G.; Yanik, Mehmet F.

2015-01-01

A major goal in the study of human diseases is to assign functions to genes or genetic variants. The model organism Caenorhabditis elegans provides a powerful tool because homologs of many human genes are identifiable, and large collections of genetic vectors and mutant strains are available. However, the delivery of such vector libraries into mutant strains remains a long-standing experimental bottleneck for phenotypic analysis. Here, we present a computer-assisted microinjection platform to streamline the production of transgenic C. elegans with multiple vectors for deep phenotyping. Briefly, animals are immobilized in a temperature-sensitive hydrogel using a standard multiwell platform. Microinjections are then performed under control of an automated microscope using precision robotics driven by customized computer vision algorithms. We demonstrate utility by phenotyping the morphology of 12 neuronal classes in six mutant backgrounds using combinations of neuron-type-specific fluorescent reporters. This technology can industrialize the assignment of in vivo gene function by enabling large-scale transgenic engineering. PMID:26163188

Altered respiratory response to substance P and reduced NK1 receptor binding in the nucleus of the solitary tract of aged rats.

PubMed

Mazzone, S B; Geraghty, D P

1999-04-24

The respiratory response to microinjection of substance P (SP) into the commissural nucleus of the solitary tract (cNTS) and binding of [125I]-Bolton-Hunter SP ([125I]-BHSP) to brain stem NK1 receptors were compared in young and aged rats. Injection of SP (750 pmol) into the cNTS of young rats (2 months) increased tidal volume (VT) but had no effect on respiratory rate (f). In aged rats (19-21 months), injection of SP had no significant effect on f or VT. The NTS of aged rats displayed significantly lower specific [125I]-BHSP binding than young rats, indicating a reduction in the number in NK1 receptors. These findings show that the respiratory response to microinjection of SP into the cNTS of aged rats is severely blunted and that this phenomenon may be due to a decrease in the number of NK1 receptors in the NTS. Copyright 1999 Elsevier Science B.V.

Physical non-viral gene delivery methods for tissue engineering.

PubMed

Mellott, Adam J; Forrest, M Laird; Detamore, Michael S

2013-03-01

The integration of gene therapy into tissue engineering to control differentiation and direct tissue formation is not a new concept; however, successful delivery of nucleic acids into primary cells, progenitor cells, and stem cells has proven exceptionally challenging. Viral vectors are generally highly effective at delivering nucleic acids to a variety of cell populations, both dividing and non-dividing, yet these viral vectors are marred by significant safety concerns. Non-viral vectors are preferred for gene therapy, despite lower transfection efficiencies, and possess many customizable attributes that are desirable for tissue engineering applications. However, there is no single non-viral gene delivery strategy that "fits-all" cell types and tissues. Thus, there is a compelling opportunity to examine different non-viral vectors, especially physical vectors, and compare their relative degrees of success. This review examines the advantages and disadvantages of physical non-viral methods (i.e., microinjection, ballistic gene delivery, electroporation, sonoporation, laser irradiation, magnetofection, and electric field-induced molecular vibration), with particular attention given to electroporation because of its versatility, with further special emphasis on Nucleofection™. In addition, attributes of cellular character that can be used to improve differentiation strategies are examined for tissue engineering applications. Ultimately, electroporation exhibits a high transfection efficiency in many cell types, which is highly desirable for tissue engineering applications, but electroporation and other physical non-viral gene delivery methods are still limited by poor cell viability. Overcoming the challenge of poor cell viability in highly efficient physical non-viral techniques is the key to using gene delivery to enhance tissue engineering applications.

Physical non-viral gene delivery methods for tissue engineering

PubMed Central

Mellott, Adam J.; Forrest, M. Laird; Detamore, Michael S.

2016-01-01

The integration of gene therapy into tissue engineering to control differentiation and direct tissue formation is not a new concept; however, successful delivery of nucleic acids into primary cells, progenitor cells, and stem cells has proven exceptionally challenging. Viral vectors are generally highly effective at delivering nucleic acids to a variety of cell populations, both dividing and non-dividing, yet these viral vectors are marred by significant safety concerns. Non-viral vectors are preferred for gene therapy, despite lower transfection efficiencies, and possess many customizable attributes that are desirable for tissue engineering applications. However, there is no single non-viral gene delivery strategy that “fits-all” cell types and tissues. Thus, there is a compelling opportunity to examine different non-viral vectors, especially physical vectors, and compare their relative degrees of success. This review examines the advantages and disadvantages of physical non-viral methods (i.e., microinjection, ballistic gene delivery, electroporation, sonoporation, laser irradiation, magnetofection, and electric field-induced molecular vibration), with particular attention given to electroporation because of its versatility, with further special emphasis on Nucleofection™. In addition, attributes of cellular character that can be used to improve differentiation strategies are examined for tissue engineering applications. Ultimately, electroporation exhibits a high transfection efficiency in many cell types, which is highly desirable for tissue engineering applications, but electroporation and other physical non-viral gene delivery methods are still limited by poor cell viability. Overcoming the challenge of poor cell viability in highly efficient physical non-viral techniques is the key to using gene delivery to enhance tissue engineering applications. PMID:23099792

Cancer-suppressive potential of extracts of endemic plant Helichrysum zivojinii: effects on cell migration, invasion and angiogenesis.

PubMed

Matić, Ivana Z; Aljancić, Ivana; Vajs, Vlatka; Jadranin, Milka; Gligorijević, Nevenka; Milosavljević, Slobodan; Juranić, Zorica D

2013-09-01

Helichrysum zivojinii Cernjavski & Soska is an endemic plant species that grows in the National Park Galicica in Macedonia. Five extracts were isolated as fractions from the aerial parts of the plant: a n-hexane extract (1), a dichloromethane extract (2), an ethyl-acetate extract (3), a n-butanol extract (4) and a methanol extract (5). A dose-dependent cytotoxic activity of the extracts on MDA-MB-231 and EA.hy926 cells was observed. Extracts exhibited more pronounced cytotoxic actions on MDA-MB-231 cells than on EA.hy926 cells. The n-hexane extract (1), at a non-toxic concentration, exhibited an inhibitory effect on the migration as well the invasiveness of MDA-MB-231 cells. The dichloromethane extract (2), at a non-toxic concentration, demonstrated inhibition of MDA-MB-231 cells invasion. Each of the five extracts applied at non-toxic concentrations inhibited migration of EA.hy926 cells. The prominent inhibitory effect of the n-hexane extract on EA.hy926 cells migration was associated with a notable anti-angiogenic action of this extract. The other four tested extracts demonstrated mild anti-angiogenic activity. Our data highlight the prominent anticancer potential of n-hexane (1) and dichloromethane (2) extracts, which could be attributed to their very pronounced and selective cytotoxic activities as well as their anti-invasive and anti-angiogenic properties.

Frontrunners of T cell activation: Initial, localized Ca2+ signals mediated by NAADP and the type 1 ryanodine receptor.

PubMed

Wolf, Insa M A; Diercks, Björn-Philipp; Gattkowski, Ellen; Czarniak, Frederik; Kempski, Jan; Werner, René; Schetelig, Daniel; Mittrücker, Hans-Willi; Schumacher, Valéa; von Osten, Manuel; Lodygin, Dimitri; Flügel, Alexander; Fliegert, Ralf; Guse, Andreas H

2015-10-13

The activation of T cells is the fundamental on switch for the adaptive immune system. Ca(2+) signaling is essential for T cell activation and starts as initial, short-lived, localized Ca(2+) signals. The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) forms rapidly upon T cell activation and stimulates early Ca(2+) signaling. We developed a high-resolution imaging technique using multiple fluorescent Ca(2+) indicator dyes to characterize these early signaling events and investigate the channels involved in NAADP-dependent Ca(2+) signals. In the first seconds of activation of either primary murine T cells or human Jurkat cells with beads coated with an antibody against CD3, we detected Ca(2+) signals with diameters close to the limit of detection and that were close to the activation site at the plasma membrane. In Jurkat cells in which the ryanodine receptor (RyR) was knocked down or in primary T cells from RyR1(-/-) mice, either these early Ca(2+) signals were not detected or the number of signals was markedly reduced. Local Ca(2+) signals observed within 20 ms upon microinjection of Jurkat cells with NAADP were also sensitive to RyR knockdown. In contrast, TRPM2 (transient receptor potential channel, subtype melastatin 2), a potential NAADP target channel, was not required for the formation of initial Ca(2+) signals in primary T cells. Thus, through our high-resolution imaging method, we characterized early Ca(2+) release events in T cells and obtained evidence for the involvement of RyR and NAADP in such signals. Copyright © 2015, American Association for the Advancement of Science.

Characterization of an in vitro system for the synthesis of mRNA from human parainfluenza virus type 3.

PubMed

De, B P; Galinski, M S; Banerjee, A K

1990-03-01

A cell extract derived from human parainfluenza virus type 3-infected human lung carcinoma (HLC) cells synthesized mRNA in vitro. Under optimal conditions, the extract was able to support transcription of all virus-encoded genes as determined by hybridization analyses. The RNA products contained full-length poly(A)-containing mRNA species similar to those observed in acutely infected cells. Further purification of the viral nucleocapsids from the infected HLC cell extract resulted in total loss of the capacity of the extract to synthesize mRNA in vitro. However, the addition of cytoplasmic extracts from uninfected HLC cells to the nucleocapsid preparations restored transcription to levels observed in the infected cell lysates, indicating requirement of a host factor(s) in the human parainfluenza virus type 3 transcription process. In distinction to the abundant transcription observed in the cell extract from HLC cells, cell extract prepared from CV-1 cells failed to support transcription in vitro. High levels of RNase activity in the cell extract from CV-1 cells appears to be the principal reason for this difference.

Conserved elements in the nanos3 3'UTR of olive flounder are responsible for the selective retention of RNA in germ cells.

PubMed

Li, Meijie; Tan, Xungang; Sui, Yulei; Jiao, Shuang; Wu, Zhihao; You, Feng

2016-08-01

In teleost fish, primordial germ cells (PGCs) are specified very early during embryogenesis and migrate to the site that gonads are formed. A previous study indicated that nanos3 is specifically expressed in PGCs, and the 3' untranslated region (UTR) of nanos3 is responsible for the localization of mRNA in these cells. In this study, we aimed to investigate the functional regions of nanos3 3'UTR in olive flounder using truncated and mutated nanos3 3'UTRs fused to chimeric RNAs and microinjected into fertilized zebrafish eggs. The results indicated that a 68-bp functional element in the nanos3 3'UTR of olive flounder played important roles in the protection and degradation of RNA. Within this element, a U-rich region was identified to be responsible for the protection of RNA in PGCs and two GCAC sites for the degradation of RNA in somatic cells. The first GCAC was located adjacently to the U-rich region and the second GCAC within the U-rich region. Overall, we concluded that the two GCACs were the binding sites of miR-430, a microRNA that suppresses translation, whereas the U-rich region was the binding site of Dnd, a protein that antagonizes the miR-430-mediated silencing of mRNA. Copyright © 2016 Elsevier Inc. All rights reserved.

Maternal program of apoptosis activated shortly after midblastula transition by overexpression of S-adenosylmethionine decarboxylase in Xenopus early embryos.

PubMed

Shiokawa, K; Kai, M; Higo, T; Kaito, C; Yokoska, J; Yasuhiko, Y; Kajita, E; Nagano, M; Yamada, Y; Shibata, M; Muto, T; Shinga, J; Hara, H; Takayama, E; Fukamachi, H; Yaoita, Y; Igarashi, K

2000-06-01

When we studied polyamine metabolism in Xenopus embryos, we cloned the cDNA for Xenopus S-adenosylmethionine decarboxylase (SAMDC), which converts SAM (S-adenosylmethionine), the methyl donor, into decarboxylated SAM (dcSAM), the aminopropyl donor, and microinjected its in vitro transcribed mRNA into Xenopus fertilized eggs. We found here that the mRNA injection induces a SAM deficient state in early embryos due to over-function of the overexpressed SAMDC, which in turn induces inhibition of protein synthesis. Such embryos developed quite normally until blastula stage, but stopped development at the early gastrula stage, due to induction of massive cell dissociation and cell autolysis, irrespective of the dosage and stage of the mRNA injection. We found that the dissociated cells were TUNEL-positive, contained fragmented nuclei with ladder-forming DNA, and furthermore, rescued completely by coinjection of Bcl-2 mRNA. Thus, overexpression of SAMDC in Xenopus embryos appeared to switch on apoptotic program, probably via inhibition of protein synthesis. Here, we briefly review our results together with those reported from other laboratories. After discussing the general importance of this newly discovered apoptotic program, we propose that the maternal program of apoptosis serves as a surveillance mechanism to eliminate metabolically severely-damaged cells and functions as a 'fail-safe' mechanism for normal development in Xenopus embryos.

Cytotoxicity and apoptotic inducibility of Vitex agnus-castus fruit extract in cultured human normal and cancer cells and effect on growth.

PubMed

Ohyama, Kunio; Akaike, Takenori; Hirobe, Chieko; Yamakawa, Toshio

2003-01-01

A crude extract was prepared with ethanol from dried ripened Vitex agnus-castus fruits growing in Israel (Vitex extract). Cytotoxicity of the extract against human uterine cervical canal fibroblast (HCF), human embryo fibroblast (HE-21), ovarian cancer (MCF-7), cervical carcinoma (SKG-3a), breast carcinoma (SKOV-3), gastric signet ring carcinoma (KATO-III), colon carcinoma (COLO 201), and small cell lung carcinoma (Lu-134-A-H) cells was examined. After culture for 24 h (logarithmic growth phase) or 72 h (stationary growth phase), the cells were treated with various concentrations of Vitex extract. In both growth phases, higher growth activity of cells and more cytotoxic activity of Vitex extract were seen. The cytotoxic activity against stationary growth-phase cells was less than that against logarithmic growth-phase cells. DNA fragmentation of Vitex extract-treated cells was seen in SKOV-3, KATO-III, COLO 201, and Lu-134-A-H cells. The DNA fragmentation in Vitex extract-treated KATO-III cells was inhibited by the presence of the antioxidative reagent pyrrolidine dithiocarbamate or N-acetyl-L-cysteine (NAC). Western blotting analysis showed that in Vitex extract-treated KATO-III cells, the presence of NAC also inhibited the expression of heme oxygenase-1 and the active forms of caspases-3, -8 and -9. It is concluded that the cytotoxic activity of Vitex extract may be attributed to the effect on cell growth, that cell death occurs through apoptosis, and that this apoptotic cell death may be attributed to increased intracellular oxidation by Vitex extract treatment.

Somatic cell nuclear transfer cloning: practical applications and current legislation.

PubMed

Niemann, H; Lucas-Hahn, A

2012-08-01

Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding. Significant improvements have been made in SCNT protocols in the past years which now allow to embarking on practical applications. The main areas of application of SCNT are: Reproductive cloning, therapeutic cloning and basic research. A great application potential of SCNT based cloning is the production of genetically modified (transgenic) animals. Somatic cell nuclear transfer based transgenic animal production has significant advances over the previously employed microinjection of foreign DNA into pronuclei of zygotes. This cell based transgenesis is compatible with gene targeting and allows both, the addition of a specific gene and the deletion of an endogenous gene. Efficient transgenic animal production provides numerous opportunities for agriculture and biomedicine. Regulatory agencies around the world have agreed that food derived from cloned animals and their offspring is safe and there is no scientific basis for questioning this. Commercial application of somatic cloning within the EU is via the Novel Food regulation EC No. 258/97. Somatic cloning raises novel questions regarding the ethical and moral status of animals and their welfare which has prompted a controversial discussion in Europe which has not yet been resolved. © 2012 Blackwell Verlag GmbH.

The Kinesin-Related Protein, Hset, Opposes the Activity of Eg5 and Cross-Links Microtubules in the Mammalian Mitotic Spindle

PubMed Central

Mountain, Vicki; Simerly, Calvin; Howard, Louisa; Ando, Asako; Schatten, Gerald; Compton, Duane A.

1999-01-01

We have prepared antibodies specific for HSET, the human homologue of the KAR3 family of minus end-directed motors. Immuno-EM with these antibodies indicates that HSET frequently localizes between microtubules within the mammalian metaphase spindle consistent with a microtubule cross-linking function. Microinjection experiments show that HSET activity is essential for meiotic spindle organization in murine oocytes and taxol-induced aster assembly in cultured cells. However, inhibition of HSET did not affect mitotic spindle architecture or function in cultured cells, indicating that centrosomes mask the role of HSET during mitosis. We also show that (acentrosomal) microtubule asters fail to assemble in vitro without HSET activity, but simultaneous inhibition of HSET and Eg5, a plus end-directed motor, redresses the balance of forces acting on microtubules and restores aster organization. In vivo, centrosomes fail to separate and monopolar spindles assemble without Eg5 activity. Simultaneous inhibition of HSET and Eg5 restores centrosome separation and, in some cases, bipolar spindle formation. Thus, through microtubule cross-linking and oppositely oriented motor activity, HSET and Eg5 participate in spindle assembly and promote spindle bipolarity, although the activity of HSET is not essential for spindle assembly and function in cultured cells because of centrosomes. PMID:10525540

Physical Methods for Intracellular Delivery: Practical Aspects from Laboratory Use to Industrial-Scale Processing

PubMed Central

Meacham, J. Mark; Durvasula, Kiranmai; Degertekin, F. Levent; Fedorov, Andrei G.

2015-01-01

Effective intracellular delivery is a significant impediment to research and therapeutic applications at all processing scales. Physical delivery methods have long demonstrated the ability to deliver cargo molecules directly to the cytoplasm or nucleus, and the mechanisms underlying the most common approaches (microinjection, electroporation, and sonoporation) have been extensively investigated. In this review, we discuss established approaches, as well as emerging techniques (magnetofection, optoinjection, and combined modalities). In addition to operating principles and implementation strategies, we address applicability and limitations of various in vitro, ex vivo, and in vivo platforms. Importantly, we perform critical assessments regarding (1) treatment efficacy with diverse cell types and delivered cargo molecules, (2) suitability to different processing scales (from single cell to large populations), (3) suitability for automation/integration with existing workflows, and (4) multiplexing potential and flexibility/adaptability to enable rapid changeover between treatments of varied cell types. Existing techniques typically fall short in one or more of these criteria; however, introduction of micro-/nanotechnology concepts, as well as synergistic coupling of complementary method(s), can improve performance and applicability of a particular approach, overcoming barriers to practical implementation. For this reason, we emphasize these strategies in examining recent advances in development of delivery systems. PMID:23813915

Multiple functional attributes of glucose-monitoring neurons in the medial orbitofrontal (ventrolateral prefrontal) cortex.

PubMed

Szabó, István; Hormay, Edina; Csetényi, Bettina; Nagy, Bernadett; Lénárd, László; Karádi, Zoltán

2018-02-01

Multiple functional attributes of glucose-monitoring neurons in the medial orbitofrontal (ventrolateral prefrontal) cortex. NEUROSCI BIOBEHAV REV 73(1) XXX-XXX, 2017.- Special chemosensory cells, the glucose-monitoring (GM) neurons, reportedly involved in the central feeding control, exist in the medial orbitofrontal (ventrolateral prefrontal) cortex (mVLPFC). Electrophysiological, metabolic and behavioral studies reveal complex functional attributes of these cells and raise their homeostatic significance. Single neuron recordings, by means of the multibarreled microelectrophoretic technique, elucidate differential sensitivities of limbic forebrain neurons in the rat and the rhesus monkey to glucose and other chemicals, whereas gustatory stimulations demonstrate their distinct taste responsiveness. Metabolic examinations provide evidence for alteration of blood glucose level in glucose tolerance test and elevation of plasma triglyceride concentration after destruction of the local GM cells by streptozotocin (STZ). In behavioral studies, STZ microinjection into the mVLPFC fails to interfere with the acquisition of saccharin conditioned taste avoidance, does cause, however, taste perception deficit in taste reactivity tests. Multiple functional attributes of GM neurons in the mVLPFC, within the frame of the hierarchically organized central GM neuronal network, appear to play important role in the maintenance of the homeostatic balance. Copyright © 2017 Elsevier Ltd. All rights reserved.

Noradrenaline Triggers GABAA Inhibition of Bed Nucleus of the Stria Terminalis Neurons Projecting to the Ventral Tegmental Area

PubMed Central

Dumont, Éric C.; Williams, John T.

2014-01-01

The lateral part of the ventral bed nucleus of the stria terminalis (vlBNST) is a critical site for the antiaversive effects of noradrenergic drugs during opioid withdrawal. The objective of the present study is to identify the cellular action(s) of noradrenaline in the vlBNST after withdrawal from a 5 d treatment with morphine. The vlBNST is a heterogeneous cell group with multiple efferent projections. Therefore, neurons projecting to the midbrain were identified by retrograde transport of fluorescent microspheres injected in the ventral tegmental area (VTA). Whole-cell voltage clamp recordings of these neurons and of those sharing physiological properties were done in brain slices. Noradrenaline activated α1-adrenergic receptors to increase GABAA-IPSC frequency. Noradrenaline produced a similar increase in GABAA-IPSCs during acute opioid withdrawal, but this increase resulted from activation of β-adrenergic receptors, adenylyl cyclase, and protein kinase A, as well as α1-adrenergic receptors. Given that neurons in the vlBNST send an excitatory projection to the VTA, noradrenaline may reduce excitatory drive to mesolimbic dopamine cells. This mechanism might contribute to the withdrawal-induced inhibition of dopamine neurons and explain how noradrenergic drugs microinjected into the vlBNST reduce aversive aspects of opioid withdrawal. PMID:15385602

Establishment and characterization of CAG/EGFP transgenic rabbit line.

PubMed

Takahashi, Ri-ichi; Kuramochi, Takashi; Aoyagi, Kazuki; Hashimoto, Shu; Miyoshi, Ichiro; Kasai, Noriyuki; Hakamata, Yoji; Kobayashi, Eiji; Ueda, Masatsugu

2007-02-01

Cell marking is a very important procedure for identifying donor cells after cell and/or organ transplantation in vivo. Transgenic animals expressing marker proteins such as enhanced green fluorescent protein (EGFP) in their tissues are a powerful tool for research in fields of tissue engineering and regenerative medicine. The purpose of this study was to establish transgenic rabbit lines that ubiquitously express EGFP under the control of the cytomegalovirus immediate early enhancer/beta-actin promoter (CAG) to provide a fluorescent transgenic animal as a bioresource. We microinjected the EGFP expression vector into 945 rabbit eggs and 4 independent transgenic candidate pups were obtained. Two of them died before sexual maturation and one was infertile. One transgenic male candidate founder rabbit was obtained and could be bred by artificial insemination. The rabbit transmitted the transgene in a Mendelian manner. Using fluorescence in situ hybridization analysis, we detected the transgene at 7q11 on chromosome 7 as a large centromeric region in two F1 offspring (one female and one male). Eventually, one transgenic line was established. Ubiquitous EGFP fluorescence was confirmed in all examined organs. There were no gender-related differences in fluorescence. The established CAG/EGFP transgenic rabbit will be an important bioresource and a useful tool for various studies in tissue engineering and regenerative medicine.

Modulation of Ca2+ Activity in Cardiomyocytes through Caveolae-Gαq Interactions

PubMed Central

Guo, Yuanjian; Golebiewska, Urszula; Scarlata, Suzanne

2011-01-01

Cardiomyocytes have a complex Ca2+ behavior and changes in this behavior may underlie certain disease states. Intracellular Ca2+ activity can be regulated by the phospholipase Cβ–Gαq pathway localized on the plasma membrane. The plasma membranes of cardiomycoytes are rich in caveolae domains organized by caveolin proteins. Caveolae may indirectly affect cell signals by entrapping and localizing specific proteins. Recently, we found that caveolin may specifically interact with activated Gαq, which could affect Ca2+ signals. Here, using fluorescence imaging and correlation techniques we show that Gαq-Gβγ subunits localize to caveolae in adult ventricular canine cardiomyoctyes. Carbachol stimulation releases Gβγ subunits from caveolae with a concurrent stabilization of activated Gαq by caveolin-3 (Cav3). These cells show oscillating Ca2+ waves that are not seen in neonatal cells that do not contain Cav3. Microinjection of a peptide that disrupts Cav3-Gαq association, but not a control peptide, extinguishes the waves. Furthermore, these waves are unchanged with rynaodine treatment, but not seen with treatment of a phospholipase C inhibitor, implying that Cav3-Gαq is responsible for this Ca2+ activity. Taken together, these studies show that caveolae play a direct and active role in regulating basal Ca2+ activity in cardiomyocytes. PMID:21463572

Developmental potential of elongating and elongated spermatids obtained after in-vitro maturation of isolated round spermatids.

PubMed

Cremades, N; Sousa, M; Bernabeu, R; Barros, A

2001-09-01

Round spermatid injections are associated with disappointing clinical outcomes, and although these cells have been shown to mature into late spermatids in vitro, the developmental potential of such gametes remains to be demonstrated. Round spermatids were isolated from 12 testicle samples of patients with obstructive azoospermia, hypoplasia, complete maturation arrest, and incomplete Sertoli cell-only syndrome. They were cultured for 7 days at 32 degrees C, 5% CO(2)in air, in microdrops of Vero cell-conditioned medium containing 10% synthetic serum substitute. From the 238 round spermatids cultured, 25.2% attained the elongating and 5.5% the elongated spermatid stage (3-4 days per step). Relatively higher maturation rates were found in cases with obstructive azoospermia, but differences were significant only for elongated spermatids (9.3%). No differences were found in maturation rates between cases with non-obstructive azoospermia (4.3% of elongated spermatids). Experimental microinjections with elongating and elongated spermatids revealed a low fertilization rate (40.9%) but a normal blastocyst formation rate (60%). Late spermatids resulting from in-vitro culture of round spermatids in conditioned medium, either in controls in cases with a spermiogenetic block, appeared able to successfully fertilize the human oocyte and elicit normal embryo development.

Antitumor activity of ethanol extract from Hippophae rhamnoides L. leaves towards human acute myeloid leukemia cells in vitro.

PubMed

Zhamanbaeva, G T; Murzakhmetova, M K; Tuleukhanov, S T; Danilenko, M P

2014-12-01

We studied the effects of ethanol extract from Hippophae rhamnoides L. leaves on the growth and differentiation of human acute myeloid leukemia cells (KG-1a, HL60, and U937). The extract of Hippophae rhamnoides L. leaves inhibited cell growth depending on the cell strain and extract dose. In a high concentration (100 μg/ml), the extract also exhibited a cytotoxic effect on HL60 cells. Hippophae rhamnoides L. leaves extract did not affect cell differentiation and did not modify the differentiating effect of calcitriol, active vitamin D metabolite. Inhibition of cell proliferation was paralleled by paradoxical accumulation of phase S cells (synthetic phase) with a reciprocal decrease in the count of G1 cells (presynthetic phase). The extract in a concentration of 100 μg/ml induced the appearance of cells with a subdiploid DNA content (sub-G1 phase cells), which indicated induction of apoptosis. The antiproliferative effect of Hippophae rhamnoides L. extract on acute myeloid leukemia cells was at least partially determined by activation of the S phase checkpoint, which probably led to deceleration of the cell cycle and apoptosis induction.

Eggshell ultrastructure and delivery of pharmacological inhibitors to the early embryo of R. prolixus by ethanol permeabilization of the extraembryonic layers.

PubMed

Bomfim, Larissa; Vieira, Priscila; Fonseca, Ariene; Ramos, Isabela

2017-01-01

Most vectors of arthropod-borne diseases produce large eggs with hard and opaque eggshells. In several species, it is still not possible to induce molecular perturbations to the embryo by delivery of molecules using microinjections or eggshell permeabilization without losing embryo viability, which impairs basic studies regarding development and population control. Here we tested the properties and permeability of the eggshell of R. prolixus, a Chagas disease vector, with the aim to deliver pharmacological inhibitors to the egg cytoplasm and allow controlled molecular changes to the embryo. Using field emission scanning and transmission electron microscopy we found that R. prolixus egg is coated by three main layers: exochorion, vitelline layer and the plasma membrane, and that the pores that allow gas exchange (aeropiles) have an average diameter of 10 μm and are found in the rim of the operculum at the anterior pole of the egg. We tested if different solvents could permeate through the aeropiles and reach the egg cytoplasm/embryo and found that immersions of the eggs in ethanol lead to its prompt penetration through the aeropiles. A single five minute-immersion of the eggs/embryos in pharmacological inhibitors, such as azide, cyanide and cycloheximide, solubilized in ethanol resulted in impairment of embryogenesis in a dose dependent manner and DAPI-ethanol solutions were also able to label the embryo cells, showing that ethanol penetration was able to deliver those molecules to the embryo cells. Multiple immersions of the embryo in the same solutions increased the effect and tests using bafilomycin A1 and Pepstatin A, known inhibitors of the yolk proteolysis, were also able to impair embryogenesis and the yolk protein degradation. Additionally, we found that ethanol pre-treatments of the egg make the aeropiles more permeable to aqueous solutions, so drugs diluted in water can be carried after the eggs are pre-treated with ethanol. Thus, we found that delivery of pharmacological inhibitors to the embryo of R. prolixus can be performed simply by submersing the fertilized eggs in ethanol with no need for additional methods such as microinjections or electroporation. We discuss the potential importance of this methodology to the study of this vector developmental biology and population control.

Functional characterization of three MicroRNAs of the Asian Tiger Mosquito, Aedes albopictus

PubMed Central

2013-01-01

Background Temporal and stage specific expression of microRNAs (miRNAs) in embryos, larvae, pupae and adults of Aedes albopictus showed differential expression levels across the four developmental stages, indicating their potential regulatory roles in mosquito development. The functional characterization of these miRNAs was not known. Accordingly our study evaluated the functional characterization of three miRNAs, which are temporally up-regulated in the various developmental stages of Ae. albopictus mosquitoes. Methods miRNA mimics, inhibitors and negative controls were designed and their knock-in and knock-down efficiency were analyzed by qRT-PCR after transfecting the mosquito cell lines C6/36, and also by injecting in their specific developmental stages. The functional role of each individual miRNA was analyzed with various parameters of development such as, hatching rate and hatching time in embryos, eclosion rate in larvae, longevity and fecundity in the adult mosquitoes. Results The knock-in with the specifically designed miRNA mimics showed increased levels of expression of miRNA compared with their normal controls. We confirmed these findings using qRT-PCR, both by in vitro expression in C6/36 mosquito cell lines after transfection as well as in in vivo expression in developmental stages of mosquitoes by microinjection. The knock-down of expression with the corresponding inhibitors showed a considerable decrease in the expression levels of these miRNAs and obvious functional effects in Ae. albopictus development, detected by a decrease in the hatching rate of embryos and eclosion rate in larvae and a marked reduction in longevity and fecundity in adults. Conclusion This study carried out by knock-in and knock-down of specifically and temporally expressed miRNAs in Ae. albopictus by microinjection is a novel study to delineate the importance of the miRNA expression in regulating mosquito development. The knock-down and loss of function of endogenously expressed miRNAs by the miRNA inhibitors in specific developmental stages had considerable effects on development, but enhancement of their gain of function was not observed on knock-in of these specific miRNAs. Hence, our study indicates that an optimal level of endogenous expression of miRNA is indispensable for the normal development and maintenance of the vectorial population density and pathogen transmissibility of this mosquito vector. PMID:23924583

Development of RNAi methods for Peregrinus maidis, the corn planthopper.

PubMed

Yao, Jianxiu; Rotenberg, Dorith; Afsharifar, Alireza; Barandoc-Alviar, Karen; Whitfield, Anna E

2013-01-01

The corn planthopper, Peregrinus maidis, is a major pest of agronomically-important crops. Peregrinus maidis has a large geographical distribution and transmits Maize mosaic rhabdovirus (MMV) and Maize stripe tenuivirus (MSpV). The objective of this study was to develop effective RNAi methods for P. maidis. Vacuolar-ATPase (V-ATPase) is an essential enzyme for hydrolysis of ATP and for transport of protons out of cells thereby maintaining membrane ion balance, and it has been demonstrated to be an efficacious target for RNAi in other insects. In this study, two genes encoding subunits of P. maidis V-ATPase (V-ATPase B and V-ATPase D) were chosen as RNAi target genes. The open reading frames of V-ATPase B and D were generated and used for constructing dsRNA fragments. Experiments were conducted using oral delivery and microinjection of V-ATPase B and V-ATPase D dsRNA to investigate the effectiveness of RNAi in P. maidis. Real-time quantitative reverse transcriptase-PCR (qRT-PCR) analysis indicated that microinjection of V-ATPase dsRNA led to a minimum reduction of 27-fold in the normalized abundance of V-ATPase transcripts two days post injection, while ingestion of dsRNA resulted in a two-fold reduction after six days of feeding. While both methods of dsRNA delivery resulted in knockdown of target transcripts, the injection method was more rapid and effective. The reduction in V-ATPase transcript abundance resulted in observable phenotypes. Specifically, the development of nymphs injected with 200 ng of either V-ATPase B or D dsRNA was impaired, resulting in higher mortality and lower fecundity than control insects injected with GFP dsRNA. Microscopic examination of these insects revealed that female reproductive organs did not develop normally. The successful development of RNAi in P. maidis to target specific genes will enable the development of new insect control strategies and functional analysis of vital genes and genes associated with interactions between P. maidis and MMV.

Using a Time-Driven Activity-Based Costing Model To Determine the Actual Cost of Services Provided by a Transgenic Core.

PubMed

Gerwin, Philip M; Norinsky, Rada M; Tolwani, Ravi J

2018-03-01

Laboratory animal programs and core laboratories often set service rates based on cost estimates. However, actual costs may be unknown, and service rates may not reflect the actual cost of services. Accurately evaluating the actual costs of services can be challenging and time-consuming. We used a time-driven activity-based costing (ABC) model to determine the cost of services provided by a resource laboratory at our institution. The time-driven approach is a more efficient approach to calculating costs than using a traditional ABC model. We calculated only 2 parameters: the time required to perform an activity and the unit cost of the activity based on employee cost. This method allowed us to rapidly and accurately calculate the actual cost of services provided, including microinjection of a DNA construct, microinjection of embryonic stem cells, embryo transfer, and in vitro fertilization. We successfully implemented a time-driven ABC model to evaluate the cost of these services and the capacity of labor used to deliver them. We determined how actual costs compared with current service rates. In addition, we determined that the labor supplied to conduct all services (10,645 min/wk) exceeded the practical labor capacity (8400 min/wk), indicating that the laboratory team was highly efficient and that additional labor capacity was needed to prevent overloading of the current team. Importantly, this time-driven ABC approach allowed us to establish a baseline model that can easily be updated to reflect operational changes or changes in labor costs. We demonstrated that a time-driven ABC model is a powerful management tool that can be applied to other core facilities as well as to entire animal programs, providing valuable information that can be used to set rates based on the actual cost of services and to improve operating efficiency.

Solid state lift for micrometering in a fuel injector

DOEpatents

Milam, David M.; Carroll, Thomas S.; Lee, Chien-Chang; Miller, Charles R.

2002-01-01

A fuel injector performs main fuel injection by raising fuel pressure in a nozzle chamber to lift a check valve member to a fully open position, and performs preinjection or microinjection by operating a solid state motor to lift the check valve member a much smaller distance.

Methylphenidate Enhances Extinction of Contextual Fear

ERIC Educational Resources Information Center

Abraham, Antony D.; Cunningham, Christopher L.; Lattal, K. Matthew

2012-01-01

Methylphenidate (MPH, Ritalin) is a norepinephrine and dopamine transporter blocker that is widely used in humans for treatment of attention deficit disorder and narcolepsy. Although there is some evidence that targeted microinjections of MPH may enhance fear acquisition, little is known about the effect of MPH on fear extinction. Here, we show…

MICROINJECTION OF DYNORPHIN INTO THE HIPPOCAMPUS IMPAIRS SPATIAL LEARNING IN RATS

EPA Science Inventory

The effect of hippocampal dynorphin administration on learning and memory was examined in spatial and nonspatial tasks. ilateral infusion of dynorphin A(1-8)(DYN; 10 or 20 ug in one ul) into the dorsal hippocampus resulted in dose-related impairment of spatial working memory in a...

Transfected connexin45 alters gap junction permeability in cells expressing endogenous connexin43

PubMed Central

1995-01-01

Many cells express multiple connexins, the gap junction proteins that interconnect the cytosol of adjacent cells. Connexin43 (Cx43) channels allow intercellular transfer of Lucifer Yellow (LY, MW = 443 D), while connexin45 (Cx45) channels do not. We transfected full-length or truncated chicken Cx45 into a rat osteosarcoma cell line ROS-17/2.8, which expresses endogenous Cx43. Both forms of Cx45 were expressed at high levels and colocalized with Cx43 at plasma membrane junctions. Cells transfected with full-length Cx45 (ROS/Cx45) and cells transfected with Cx45 missing the 37 carboxyl-terminal amino acids (ROS/Cx45tr) showed 30-60% of the gap junctional conductance exhibited by ROS cells. Intercellular transfer of three negatively charged fluorescent reporter molecules was examined. In ROS cells, microinjected LY was transferred to an average of 11.2 cells/injected cell, while dye transfer between ROS/Cx45 cells was reduced to 3.9 transfer between ROS/Cx45 cells was reduced to 3.9 cells. In contrast, ROS/Cx45tr cells transferred LY to > 20 cells. Transfer of calcein (MW = 623 D) was also reduced by approximately 50% in ROS/Cx45 cells, but passage of hydroxycoumarin carboxylic acid (HCCA; MW = 206 D) was only reduced by 35% as compared to ROS cells. Thus, introduction of Cx45 altered intercellular coupling between cells expressing Cx43, most likely the result of direct interaction between Cx43 and Cx45. Transfection of Cx45tr and Cx45 had different effects in ROS cells, consistent with a role of the carboxyl-terminal domain of Cx45 in determining gap junction permeability or interactions between connexins. These data suggest that coexpression of multiple connexins may enable cells to achieve forms of intercellular communication that cannot be attained by expression of a single connexin. PMID:7642714

Identification of Adeno-Associated Viral Vectors That Target Neonatal and Adult Mammalian Inner Ear Cell Subtypes

PubMed Central

Shu, Yilai; Tao, Yong; Wang, Zhengmin; Tang, Yong; Li, Huawei; Dai, Pu; Gao, Guangping; Chen, Zheng-Yi

2016-01-01

The mammalian inner ear consists of diverse cell types with important functions. Gene mutations in these diverse cell types have been found to underlie different forms of genetic hearing loss. Targeting these mutations for gene therapy development represents a future therapeutic strategy to treat hearing loss. Adeno-associated viral (AAV) vectors have become the vector of choice for gene delivery in animal models in vivo. To identify AAV vectors that target inner ear cell subtypes, we systemically screened 12 AAV vectors with different serotypes (AAV1, 2, 5, 6, 6.2, 7, 8, 9, rh.8, rh.10, rh.39, and rh.43) that carry a reporter gene GFP in neonatal and adult mice by microinjection in vivo. We found that most AAVs infect both neonatal and adult inner ear, with different specificities and expression levels. The inner ear cochlear sensory epithelial region, which includes auditory hair cells and supporting cells, is most frequently targeted for gene delivery. Expression of the transgene is sustained, and neonatal inner ear delivery does not adversely affect hearing. Adult inner ear injection of AAV has a similar infection pattern as the younger inner ear, with the exception that outer hair cell death caused by the injection procedure can lead to hearing loss. In the adult, more so than in the neonatal mice, cell types infected and efficiency of infection are correlated with the site of injection. Most infected cells survive in neonatal and adult inner ears. The study adds to the list of AAV vectors that transduce the mammalian inner ear efficiently, providing the tools that are important to study inner ear gene function and for the development of gene therapy to treat hearing loss. PMID:27342665

Danshen extract circumvents drug resistance and represses cell growth in human oral cancer cells.

PubMed

Yang, Cheng-Yu; Hsieh, Cheng-Chih; Lin, Chih-Kung; Lin, Chun-Shu; Peng, Bo; Lin, Gu-Jiun; Sytwu, Huey-Kang; Chang, Wen-Liang; Chen, Yuan-Wu

2017-12-29

Danshen is a common traditional Chinese medicine used to treat neoplastic and chronic inflammatory diseases in China. However, the effects of Danshen on human oral cancer cells remain relatively unknown. This study investigated the antiproliferative effects of a Danshen extract on human oral cancer SAS, SCC25, OEC-M1, and KB drug-resistant cell lines and elucidated the possible underlying mechanism. We investigated the anticancer potential of the Danshen extract in human oral cancer cell lines and an in vivo oral cancer xenograft mouse model. The expression of apoptosis-related molecules was evaluated through Western blotting, and the concentration of in vivo apoptotic markers was measured using immunohistochemical staining. The antitumor effects of 5-fluorouracil and the Danshen extract were compared. Cell proliferation assays revealed that the Danshen extract strongly inhibited oral cancer cell proliferation. Cell morphology studies revealed that the Danshen extract inhibited the growth of SAS, SCC25, and OEC-M1 cells by inducing apoptosis. The Flow cytometric analysis indicated that the Danshen extract induced cell cycle G0/G1 arrest. Immunoblotting analysis for the expression of active caspase-3 and X-linked inhibitor of apoptosis protein indicated that Danshen extract-induced apoptosis in human oral cancer SAS cells was mediated through the caspase pathway. Moreover, the Danshen extract significantly inhibited growth in the SAS xenograft mouse model. Furthermore, the Danshen extract circumvented drug resistance in KB drug-resistant oral cancer cells. The study results suggest that the Danshen extract could be a potential anticancer agent in oral cancer treatment.

Role of Hydroalcoholic Extract of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), in Facilitating Cellular Acclimatization in a Low-Oxygen Microenvironment.

PubMed

Koganti, Praveen; Tulsawani, Rajkumar; Sharma, Purva; Sharma, Manish; Arora, Shivani; Misra, Kshipra

2018-01-01

Ganoderma lucidum is known to exert many health benefits including effects to improve oxygen utilization. Therefore, this study was designed to evaluate the role of a hydroalcoholic G. lucidum extract in providing tolerance to HT22 cells grown under hypoxic conditions. HT22 cells were exposed to 0.5% O2 in the presence or absence of the extract for 24 hours. At the end of the exposure period, we performed cell viability assays, cell cycle analysis, and biochemical and protein expression studies. The extract-treated cells revealed less cell death, minimized caspase 3 and reactive oxygen species levels, and relieved G0/G1 cell cycle arrest compared with hypoxic cells cultured without the extract. Further, extract-treated cells showed improved expression of Nrf2, heme oxygenase 1, and metallothionein and stabilized levels of hypoxia-inducible factor 1α. Moreover, lower levels of nuclear factor-κB and tumor necrosis factor a were evident in extract-treated cells. Overall, the G. lucidum extract reduced hypoxia-induced cell death and augmented transcription factors (HIF-1α and Nrf2), conferring tolerance to hypoxia.

An efficient method for extraction of astaxanthin from green alga Haematococcus pluvialis.

PubMed

Sarada, R; Vidhyavathi, R; Usha, D; Ravishankar, G A

2006-10-04

Haematococcus pluvialis is one of the potent organisms for production of astaxanthin, a high value ketocarotenoid. Astaxanthin is accumulated in thick-walled cyst cells of Haematococcus. The thick cell wall is made up of sporopollenin-like material, algaenan, which hinders solvent extraction of astaxanthin. In the present study, an improved method for extraction of astaxanthin without homogenization of cells is reported. Extractability of astaxanthin from cyst cells was evaluated by treating cells with various solvents and pretreating the cells with organic and mineral acids at 70 degrees C followed by acetone extraction. Hydrochloric acid treatment facilitated 86-94% extractability of astaxanthin. Treatment time, temperature, and concentration of the acid were found to be critical factors for maximum extractability. The treatment did not affect the astaxanthin ester profile and the treated cells can be preserved until further use.

The interactions of intracellular Protista and their host cells, with special reference to heterotrophic organisms.

PubMed

Bannister, L H

1979-04-11

Intracellular genera are found in all the major groups of Protista, but are particularly common among the dinoflagellates, trypanosomatid zooflagellates and suctorian ciliates; the Sporozoa are nearly all intracellular at some stage of their life, and the Microspora entirely so. Intracellular forms can dwell in the nucleus, within phagosomal or other vacuoles or may lie free in the hyaloplasm of their host cells. Organisms tend to select their hosts from a restricted taxonomic range although there are some notable exceptions. There is also great variation in the types of host cell inhabited. There are various reasons for both host and cell selectivity including recognition phenomena at the cell surfaces. Invasion of host cells is usually preceded by surface interactions with the invader. Some organisms depend upon phagocytosis for entry, but others induce host cells to engulf them by non-phagocytic means or invade by microinjection through the host plasma membrane. Protista avoid lysosomal destruction by their resistance to enzyme attack, by surrounding themselves with lysosome-inhibiting vacuoles, by escaping from the phagosomal system into the hyaloplasm and by choosing host cells which lack lysosomes. Nutrition of intracellular heterotrophic organisms involves some degree of competition with the host cell's metabolism as well as erosion of host cell cytoplasm. In Plasmodium infections, red cells are made more permeable to required nutrients by the action of the parasite on the host cell membrane. The parasite is often dependent upon the host cell for complex nutrients which it cannot synthesize for itself. Intracellular forms often profoundly modify the structure and metabolism of the host cell or interfere with its growth and multiplication. This may result in the final lysis of the host cell at the end of the intracellular phase or before the infection of other cells. Certain types of intracellular organisms may have arisen initially as forms attached to the cell surface of digestive or other organs, but the intracellular habit appears to have arisen independently in several groups of Protista.

Moringa Oleifera aqueous leaf extract down-regulates nuclear factor-kappaB and increases cytotoxic effect of chemotherapy in pancreatic cancer cells.

PubMed

Berkovich, Liron; Earon, Gideon; Ron, Ilan; Rimmon, Adam; Vexler, Akiva; Lev-Ari, Shahar

2013-08-19

Fewer than 6% patients with adenocarcinoma of the pancreas live up to five years after diagnosis. Chemotherapy is currently the standard treatment, however, these tumors often develop drug resistance over time. Agents for increasing the cytotoxic effects of chemotherapy or reducing the cancer cells' chemo-resistance to the drugs are required to improve treatment outcome. Nuclear factor kappa B (NF-kB), a pro-inflammatory transcription factor, reportedly plays a significant role in the resistance of pancreatic cancer cells to apoptosis-based chemotherapy. This study investigated the effect of aqueous Moringa Oleifera leaf extract on cultured human pancreatic cancer cells - Panc-1, p34, and COLO 357, and whether it can potentiates the effect of cisplatin chemotherapy on these cells. The effect of Moringa Oleifera leaf extract alone and in combination with cisplatin on the survival of cultured human pancreatic cancer cells was evaluated by XTT-based colorimetric assay. The distribution of Panc-1 cells in the cell cycle following treatment with Moringa leaf extract was evaluated by flow cytometry, and evaluations of protein levels were via immunoblotting. Data of cell survival following combined treatments were analyzed with Calcusyn software. Moringa Oleifera leaf extract inhibited the growth of all pancreatic cell lines tested. This effect was significant in all cells following exposure to ≥0.75 mg/ml of the extract. Exposure of Panc-1 cells to Moringa leaf extract induced an elevation in the sub-G1 cell population of the cell-cycle, and reduced the expression of p65, p-IkBα and IkBα proteins in crude cell extracts. Lastly, Moringa Oleifera leaf extract synergistically enhanced the cytotoxic effect of cisplatin on Panc-1 cells. Moringa Oleifera leaf extract inhibits the growth of pancreatic cancer cells, the cells NF-κB signaling pathway, and increases the efficacy of chemotherapy in human pancreatic cancer cells.

Cytotoxic effects of Cochlospermum regium (Mart & Schrank) Pilger aqueous root extract on mammalian cells.

PubMed

Ceschini, Livônios; Campos, Elida Geralda

2006-01-16

We investigated the effect of Cochlospermum regium (Mart & Schrank) Pilger aqueous root extract on Chinese hamster ovarian (CHO)-K1 cells. The extract significantly decreased proliferation of CHO-K1 cells (EC(50)=1.5mg/mL). Apoptosis induction was analysed by fluorescent microscopy. Cell cultures treated with Cochlospermum regium extract for 4h contained 13.6% apoptotic cells after 24h (investigated by fluorescent DNA-microscopy with acridine orange/ethidium bromide staining). Characteristic chromatin condensation and fragmentation, verified by 4',6-diamidino-2-phenylindole (DAPI) staining, was observed in the cells after treatment with Cochlospermum regium extract. The results confirm the toxicity of Cochlospermum regium root extract to immortal, non-tumorigenic mammalian cells in vitro.

[Inhibition effects of black rice pericarp extracts on cell proliferation of PC-3 cells].

PubMed

Jiang, Weiwei; Yu, Xudong; Ren, Guofeng

2013-05-01

To observe the inhibitive effects of black rice pericarp extracts on cell proliferation of human prostate cancer cell PC-3 and to explore its effecting mechanism. The black rice pericarp extract was used to treat the PC-3 cells. The inhibitory effect of black rice pericarp extract on cells proliferation of PC-3 was tested by MTT method. Cell apoptosis rates and cell cycle were measured by flow cytometric assay (FCM). Western blot was used to study the protein expression levels of p38, p-p38, JNK, p-JNK. A dose-dependent and time-dependent proliferation inhibition of black rice pericarp extract was demonstrated in PC-3. The most prominent experiment condition was inhibitory concentration with 300microg/ml and treated for 72 h. The experiment result of flow cytometry analysis demonstrates that the apoptosis rate of PC-3 cells increased along with the increasing of black rice pericarp extract concentration, and a G1-S cell cycle arrest was induced in a dose-dependent manner. After PC-3 cell was treated with black rice pericarp extract for 72 h, the expressions of p-p38, p-JNK protein increased. Black rice pericarp extract could inhibit proliferation, change the cell cycle distributions and induce apoptosis in human prostatic cancer cell PC-3. Its inhibitory effect may be through promoting activation of the JNK, p38 signaling pathway. These results suggest that black rice pericarp extract maybe has an inhibitory effect on prostatic cancer.

Moringa Oleifera aqueous leaf extract down-regulates nuclear factor-kappaB and increases cytotoxic effect of chemotherapy in pancreatic cancer cells

PubMed Central

2013-01-01

Background Fewer than 6% patients with adenocarcinoma of the pancreas live up to five years after diagnosis. Chemotherapy is currently the standard treatment, however, these tumors often develop drug resistance over time. Agents for increasing the cytotoxic effects of chemotherapy or reducing the cancer cells’ chemo-resistance to the drugs are required to improve treatment outcome. Nuclear factor kappa B (NF-kB), a pro-inflammatory transcription factor, reportedly plays a significant role in the resistance of pancreatic cancer cells to apoptosis-based chemotherapy. This study investigated the effect of aqueous Moringa Oleifera leaf extract on cultured human pancreatic cancer cells - Panc-1, p34, and COLO 357, and whether it can potentiates the effect of cisplatin chemotherapy on these cells. Methods The effect of Moringa Oleifera leaf extract alone and in combination with cisplatin on the survival of cultured human pancreatic cancer cells was evaluated by XTT-based colorimetric assay. The distribution of Panc-1 cells in the cell cycle following treatment with Moringa leaf extract was evaluated by flow cytometry, and evaluations of protein levels were via immunoblotting. Data of cell survival following combined treatments were analyzed with Calcusyn software. Results Moringa Oleifera leaf extract inhibited the growth of all pancreatic cell lines tested. This effect was significant in all cells following exposure to ≥0.75 mg/ml of the extract. Exposure of Panc-1 cells to Moringa leaf extract induced an elevation in the sub-G1 cell population of the cell-cycle, and reduced the expression of p65, p-IkBα and IkBα proteins in crude cell extracts. Lastly, Moringa Oleifera leaf extract synergistically enhanced the cytotoxic effect of cisplatin on Panc-1 cells. Conclusion Moringa Oleifera leaf extract inhibits the growth of pancreatic cancer cells, the cells NF-κB signaling pathway, and increases the efficacy of chemotherapy in human pancreatic cancer cells. PMID:23957955

Growth-inhibitory effects of the red alga Gelidium amansii on cultured cells.

PubMed

Chen, Yue-Hwa; Tu, Ching-Jung; Wu, Hsiao-Ting

2004-02-01

The objective of this study was to investigate the effects of Gelidium amansii, an edible red agar cultivated off the northeast coast of Taiwan, on the growth of two lines of cancer cells, murine hepatoma (Hepa-1) and human leukemia (HL-60) cells, as well as a normal cell line, murine embryo fibroblast cells (NIH-3T3). The potential role of G. amansii on the induction of apoptosis was also examined. The results indicated that all extracts from G. amansii, including phosphate-buffered saline (PBS) and methanol extracts from dried algae as well as the dimethyl sulfoxide (DMSO) extract from freeze-dried G. amansii agar, inhibited the growth of Hepa-1 and NIH-3T3 cells, but not the growth of HL-60 cells. Annexin V-positive cells were observed in methanol and DMSO extract-treated, but not PBS extract-treated Hepa-1 and NIH-3T3 cells, suggesting that the lipid-soluble extracts of G. amansii induced apoptosis. In summary, extracts of G. amansii from various preparations exhibited antiproliferative effects on Hepa-1 and NIH-3T3 cells, and apoptosis may play a role in the methanol and DMSO extract-induced inhibitory effects. However, the antiproliferative effects of PBS extracts was not through apoptosis. Moreover, the growth-inhibitory effects of G. amansii were not specific to cancer cells.

Live cell imaging reveals different modes of cytotoxic action of extracts derived from commonly used luting cements.

PubMed

Trumpaitė-Vanagienė, Rita; Čebatariūnienė, Alina; Tunaitis, Virginijus; Pūrienė, Alina; Pivoriūnas, Augustas

2018-02-01

To compare cytotoxicity of extracts derived from commonly used luting cements: Hoffmann's Zinc Phosphate (ZPC), GC Fuji Plus Resin Modified Glass Ionomer (RMGIC) and 3M ESPE RelyX Unicem Resin Cement (RC) on primary human gingival fibroblasts (HGFs). HGFs were exposed to different concentrations of the ZPC, RMGIC and RC extracts. The cytotoxicity was assessed with the PrestoBlue Cell Viability Reagent and viable cells were counted by a haemocytometer using the trypan blue exclusion test. In order to determine the primary mechanism of the cell death induced by extracts from different luting cements, the real-time monitoring of caspase-3/-7 activity and membrane integrity of cells was employed. The extracts from the RMGIC and ZPC decreased the metabolic activity and numbers of viable cells. Unexpectedly, the extracts from the RC evoked only small effects on the metabolic activity of HGFs with a decreasing number of viable cells in a dose-and time-dependent manner. The live cell imaging revealed that the apoptosis was the primary mechanism of a cell death induced by the extracts derived from the RMGIC, whereas the extracts from the RC and ZPC induced a cell death through a necrotic and caspase-independent pathway. The apoptosis was the primary mechanism of the cell death induced by the extracts derived from the RMGIC, whereas the extracts from the RC and ZPC induced a cell death via a necrotic pathway. We suggest that metabolic assays commonly used to assess the cytotoxicity of luting cements should be validated by alternative methods. Copyright © 2017 Elsevier Ltd. All rights reserved.

Phosphorylation of zona occludens-2 by protein kinase C epsilon regulates its nuclear exportation.

PubMed

Chamorro, David; Alarcón, Lourdes; Ponce, Arturo; Tapia, Rocio; González-Aguilar, Héctor; Robles-Flores, Martha; Mejía-Castillo, Teresa; Segovia, José; Bandala, Yamir; Juaristi, Eusebio; González-Mariscal, Lorenza

2009-09-01

Here, we have analyzed the subcellular destiny of newly synthesized tight junction protein zona occludens (ZO)-2. After transfection in sparse cells, 74% of cells exhibit ZO-2 at the nucleus, and after 18 h the value decreases to 17%. The mutation S369A located within the nuclear exportation signal 1 of ZO-2 impairs the nuclear export of the protein. Because Ser369 represents a putative protein kinase C (PKC) phosphorylation site, we tested the effect of PKC inhibition and stimulation on the nuclear export of ZO-2. Our results strongly suggest that the departure of ZO-2 from the nucleus is regulated by phosphorylation at Ser369 by novel PKCepsilon. To test the route taken by ZO-2 from synthesis to the plasma membrane, we devised a novel nuclear microinjection assay in which the nucleus served as a reservoir for anti-ZO-2 antibody. Through this assay, we demonstrate that a significant amount of newly synthesized ZO-2 goes into the nucleus and is later relocated to the plasma membrane. These results constitute novel information for understanding the mechanisms that regulate the intracellular fate of ZO-2.

Kif2a regulates spindle organization and cell cycle progression in meiotic oocytes.

PubMed

Yi, Zi-Yun; Ma, Xue-Shan; Liang, Qiu-Xia; Zhang, Teng; Xu, Zhao-Yang; Meng, Tie-Gang; Ouyang, Ying-Chun; Hou, Yi; Schatten, Heide; Sun, Qing-Yuan; Quan, Song

2016-12-19

Kif2a is a member of the Kinesin-13 microtubule depolymerases. Here, we report the expression, subcellular localization and functions of Kif2a during mouse oocyte meiotic maturation. Immunoblotting analysis showed that Kif2a was gradually increased form GV to the M I stages, and then decreased slightly at the M II stage. Confocal microscopy identified that Kif2a localized to the meiotic spindle, especially concentrated at the spindle poles and inner centromeres in metaphase and translocated to the midbody at telophase. Kif2a depletion by siRNA microinjection generated severely defective spindles and misaligned chromosomes, reduced microtubule depolymerization, which led to significant pro-M I/M Iarrest and failure of first polar body (PB1) extrusion. Kif2a-depleted oocytes were also defective in spindle pole localization of γ-tubulin and showed spindle assembly checkpoint (SAC) protein Bub3 at the kinetochores even after 10 hr extended culture. These results demonstrate that Kif2a may act as a microtubule depolymerase, regulating microtubule dynamics, spindle assembly and chromosome congression, and thus cell cycle progression during mouse oocyte meiotic maturation.

The mushroom ribosome-inactivating protein lyophyllin exerts deleterious effects on mouse embryonic development in vitro.

PubMed

Chan, W Y; Ng, T B; Lam, Joyce S Y; Wong, Jack H; Chu, K T; Ngai, P H K; Lam, S K; Wang, H X

2010-01-01

Earlier investigations disclose that some plant ribosome-inactivating proteins (RIPs) adversely affect mouse embryonic development. In the present study, a mushroom RIP, namely lyophyllin from Lyophyllum shimeji, was isolated, partially sequenced, and its translation inhibitory activity determined. Its teratogenicity was studied by using a technique entailing microinjection and postimplantation whole-embryo culture. It was found that embryonic abnormalities during the period of organogenesis from E8.5 to E9.5 were induced by lyophyllin at a concentration as low as 50 microg/ml, and when the lyophyllin concentration was raised, the number of abnormal embryos increased, the final somite number decreased, and the abnormalities increased in severity. The affected embryonic structures included the cranial neural tube, forelimb buds, branchial arches, and body axis, while optic and otic placodes were more resistant. Lyophyllin at a concentration higher than 500 microg/ml also induced forebrain blisters within the cranial mesenchyme. When the abnormal embryos were examined histologically, an increase of cell death was found to be associated with abnormal structures, indicating that cell death may be one of the underlying causes of teratogenicity of the mushroom RIP. This constitutes the first report on the teratogenicity of a mushroom RIP.

Nucleation promoting factors regulate the expression and localization of Arp2/3 complex during meiosis of mouse oocytes.

PubMed

Liu, Jun; Wang, Qiao-Chu; Wang, Fei; Duan, Xing; Dai, Xiao-Xin; Wang, Teng; Liu, Hong-Lin; Cui, Xiang-Shun; Kim, Nam-Hyung; Sun, Shao-Chen

2012-01-01

The actin nucleation factor Arp2/3 complex is a main regulator of actin assembly and is involved in multiple processes like cell migration and adhesion, endocytosis, and the establishment of cell polarity in mitosis. Our previous work showed that the Arp2/3 complex was involved in the actin-mediated mammalian oocyte asymmetric division. However, the regulatory mechanisms and signaling pathway of Arp2/3 complex in meiosis is still unclear. In the present work, we identified that the nucleation promoting factors (NPFs) JMY and WAVE2 were necessary for the expression and localization of Arp2/3 complex in mouse oocytes. RNAi of both caused the degradation of actin cap intensity, indicating the roles of NPFs in the formation of actin cap. Moreover, JMY and WAVE2 RNAi decreased the expression of ARP2, a key component of Arp2/3 complex. However, knock down of Arp2/3 complex by Arpc2 and Arpc3 siRNA microinjection did not affect the expression and localization of JMY and WAVE2. Our results indicate that the NPFs, JMY and WAVE2, are upstream regulators of Arp2/3 complex in mammalian oocyte asymmetric division.

Cytotoxicity of extracts of spices to cultured cells.

PubMed

Unnikrishnan, M C; Kuttan, R

1988-01-01

The cytotoxicity of the extracts from eight different spices used in the Indian diet was determined using Dalton's lymphoma ascites tumor cells and human lymphocytes in vitro and Chinese Hamster Ovary cells and Vero cells in tissue culture. Alcoholic extracts of the spices were found to be more cytotoxic to these cells than their aqueous extracts. Alcoholic extracts of several spices inhibited cell growth at concentrations of 0.2-1 mg/ml in vitro and 0.12-0.3 mg/ml in tissue culture. Ginger, pippali (native to India; also called dried catkins), pepper, and garlic showed the highest activity followed by asafetida, mustard, and horse-gram (native to India). These extracts also inhibited the thymidine uptake into DNA.

The Effect of Sericin from Various Extraction Methods on Cell Viability and Collagen Production

PubMed Central

Aramwit, Pornanong; Kanokpanont, Sorada; Nakpheng, Titpawan; Srichana, Teerapol

2010-01-01

Silk sericin (SS) can accelerate cell proliferation and attachment; however, SS can be extracted by various methods, which result in SS exhibiting different physical and biological properties. We found that SS produced from various extraction methods has different molecular weights, zeta potential, particle size and amino acid content. The MTT assay indicated that SS from all extraction methods had no toxicity to mouse fibroblast cells at concentrations up to 40 μg/mL after 24 h incubation, but SS obtained from some extraction methods can be toxic at higher concentrations. Heat-degraded SS was the least toxic to cells and activated the highest collagen production, while urea-extracted SS showed the lowest cell viability and collagen production. SS from urea extraction was severely harmful to cells at concentrations higher than 100 μg/mL. SS from all extraction methods could still promote collagen production in a concentration-dependent manner, even at high concentrations that are toxic to cells. PMID:20559510

Effects of garlic extract on spreading depression: In vitro and in vivo investigations.

PubMed

Marschollek, Claudia; Karimzadeh, Fariba; Jafarian, Maryam; Ahmadi, Milad; Mohajeri, Seyed Mohammad Reza; Rahimi, Sadegh; Speckmann, Erwin-Josef; Gorji, Ali

2017-02-01

The potential use of garlic for prevention and treatment of different types of headaches has been suggested by several medieval literatures. Cortical spreading depression (CSD), a propagating wave of neuroglial depolarization, was established as a target for anti-migraine drugs. This study was designed to investigate the effect of garlic extract on CSD in adult rats. CSD was induced by KCl microinjection in the somatosensory cortex. The effects of five different concentrations of garlic oil (1-500 μl/l) were tested on different characteristic features of CSD in necocortical slices. In in vivo experiments, the effects of garlic oil on electrophysiological and morphological changes induced by CSD were investigated. Garlic oil in a dose-dependent manner decreased the amplitude of CSD but not its duration and velocity in neocortical brain slices. Garlic oil at concentration of 500 μl/l reversibly reduced the amplitude of the field excitatory post-synaptic potentials and inhibited induction of long-term potentiation in the third layer of neocortical slices. In in vivo studies, systemic application of garlic oil (1 ml/l) for three consecutive days reduced the amplitude and repetition rate of CSD. Garlic oil also prevented of CSD-induced reactive astrocytosis in the neocortex. Garlic oil suppresses CSD, likely via inhibition of synaptic plasticity, and prevents its harmful effects on astrocyte. Further studies are required to identify the exact active ingredient(s) of garlic oil that inhibit CSD and may have the potential to use in treatment of CSD-related disorders.

Urea enhances cell lysis of Schizosaccharomyces pombe ura4 mutants.

PubMed

Nishino, Kohei; Kushima, Misaki; Kaino, Tomohiro; Matsuo, Yasuhiro; Kawamukai, Makoto

2017-07-01

Cell lysis is induced in Schizosaccharomyces pombe ∆ura4 cells grown in YPD medium, which contains yeast extract, polypeptone, and glucose. To identify the medium components that induce cell lysis, we first tested various kinds of yeast extracts from different suppliers. Cell lysis of ∆ura4 cells on YE medium was observed when yeast extracts from OXOID, BD, Oriental, and Difco were used, but not when using yeast extract from Kyokuto. To determine which compounds induced cell lysis, we subjected yeast extract and polypeptone to GC-MS analysis. Ten kinds of compounds were detected in OXOID and BD yeast extracts, but not in Kyokuto yeast extract. Among them was urea, which was also present in polypeptone, and it clearly induced cell lysis. Deletion of the ure2 gene, which is responsible for utilizing urea, abolished the lytic effect of urea. The effect of urea was suppressed by deletion of pub1, and a similar phenotype was observed in the presence of polypeptone. Thus, urea is an inducer of cell lysis in S. pombe ∆ura4 cells.

Improving mixing efficiency of a polymer micromixer by use of a plastic shim divider

NASA Astrophysics Data System (ADS)

Li, Lei; Lee, L. James; Castro, Jose M.; Yi, Allen Y.

2010-03-01

In this paper, a critical modification to a polymer based affordable split-and-recombination static micromixer is described. To evaluate the improvement, both the original and the modified design were carefully investigated using an experimental setup and numerical modeling approach. The structure of the micromixer was designed to take advantage of the process capabilities of both ultraprecision micromachining and microinjection molding process. Specifically, the original and the modified design were numerically simulated using commercial finite element method software ANSYS CFX to assist the re-designing of the micromixers. The simulation results have shown that both designs are capable of performing mixing while the modified design has a much improved performance. Mixing experiments with two different fluids were carried out using the original and the modified mixers again showed a significantly improved mixing uniformity by the latter. The measured mixing coefficient for the original design was 0.11, and for the improved design it was 0.065. The developed manufacturing process based on ultraprecision machining and microinjection molding processes for device fabrication has the advantage of high-dimensional precision, low cost and manufacturing flexibility.

Cognitive Activation by Central Thalamic Stimulation: The Yerkes-Dodson Law Revisited.

PubMed Central

Mair, Robert G.; Onos, Kristen D.; Hembrook, Jacqueline R.

2011-01-01

Central thalamus regulates forebrain arousal, influencing activity in distributed neural networks that give rise to organized actions during alert, wakeful states. Central thalamus has been implicated in working memory by the effects of lesions and microinjected drugs in this part of the brain. Lesions and drugs that inhibit neural activity have been found to impair working memory. Drugs that increase activity have been found to enhance and impair memory depending on the dose tested. Electrical deep brain stimulation (DBS) similarly enhances working memory at low stimulating currents and impairs it at higher currents. These effects are time dependent. They were observed when DBS was applied during the memory delay (retention) or choice response (retrieval) but not earlier in trials during the sample (acquisition) phase. The effects of microinjected drugs and DBS are consistent with the Yerkes-Dodson law, which describes an inverted-U relationship between arousal and behavioral performance. Alternatively these results may reflect desensitization associated with higher levels of stimulation, spread of drugs or current to adjacent structures, or activation of less sensitive neurons or receptors at higher DBS currents or drug doses. PMID:22013395

Fluzone® Intradermal Quadrivalent Influenza Vaccine.

PubMed

Robertson, Corwin A; Tsang, Peter; Landolfi, Victoria A; Greenberg, David P

2016-10-01

An intradermal version of Fluzone® split-virion inactivated trivalent influenza vaccine, containing 9 µg hemagglutinin per strain of A/H1N1, A/H3N2, and one B lineage virus (Fluzone Intradermal, Sanofi Pasteur), became available in the US during the 2011-2012 influenza season for adults 18-64 years of age. In advance of the 2015-2016 season, Fluzone Intradermal was replaced with Fluzone Intradermal Quadrivalent vaccine, which contains 9 µg hemagglutinin per strain of the two A-strain viruses and both B-strain lineage viruses (Victoria and Yamagata). This literature review summarizes the history and mechanism of intradermal vaccination, discusses the clinical trial results supporting the immunogenicity and safety of Fluzone Intradermal Quadrivalent vaccine, and describes the unique microinjection system used to deliver Fluzone Intradermal Quadrivalent. Expert commentary: Fluzone Intradermal Quadrivalent may boost confidence in influenza vaccination with the addition of a second B-lineage strain. By using an innovative microinjection system, the vaccine is also designed to address some of the logistic challenges faced by healthcare providers administering immunizations.

Comparative Evaluation of Different Cell Lysis and Extraction Methods for Studying Benzo(a)pyrene Metabolism in HT-29 Colon Cancer Cell Cultures

PubMed Central

Myers, Jeremy N.; Rekhadevi, Perumalla V.; Ramesh, Aramandla

2011-01-01

Lysis and extraction of cells are essential sample processing steps for investigations pertaining to metabolism of xenobiotics in cell culture studies. Of particular importance to these procedures are maintaining high lysis efficiency and analyte integrity as they influence the qualitative and quantitative distribution of drug and toxicant metabolites in the intra- and extracellular milieus. In this study we have compared the efficiency of different procedures viz. homogenization, sonication, bead beating, and molecular grinding resin treatment for disruption of HT-29 colon cells exposed to benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH) compound and a suspected colon carcinogen. Also, we have evaluated the efficiency of various procedures for extracting BaP parent compound/metabolites from colon cells and culture media prior to High Performance Liquid Chromatography (HPLC) analyses. The extraction procedures include solid phase extraction, solid-supported liquid- liquid extraction, liquid-liquid extraction, and homogeneous liquid- liquid extraction. Our findings showed that bead-beating in combination with detergent treatment of cell pellet coupled with liquid-liquid extraction yielded greater concentrations of BaP metabolites compared to the other methods employed. Our method optimization strategy revealed that disruption of HT-29 colon cells by a combination of mechanical and chemical lysis followed by liquid-liquid extraction is efficient and robust enough for analyzing BaP metabolites from cell culture studies. PMID:21865728

Nanoparticles of Selaginella doederleinii leaf extract inhibit human lung cancer cells A549

NASA Astrophysics Data System (ADS)

Syaefudin; Juniarti, A.; Rosiyana, L.; Setyani, A.; Khodijah, S.

2016-01-01

The aim of the present study is to evaluate cytotoxicity effect of nanoparticles of Selaginella doederleinii (S. doederleinii) leaves extract. S. doederleinii was extracted by maceration method using 70%(v/v) ethanol as solvent. Phytochemical content was analyzed qualitatively by using Harborne and Thin Layer Chromatography (TLC) methods. Nanoparticle extract was prepared by ionic gelation using chitosan as encapsulant agent. Anticancer activity was performed by using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The results showed that S. doederleinii contains of flavonoids. Nanoparticle of S. doederleinii leaves extract greatly inhibited A549 cells growth (cancer cells), with IC50 of 3% or 1020 μg/ml. These nanoparticles extract also inhibited the growth of Chang cells (normal cells), with IC50 of 4% or 1442 μg/ml. The effective concentration of nanoparticles extract which inhibits cancer cells without harming the normal cells is 0.5% or 167 μg/ml. Further studies are needed to obtain the concentration of nanoparticles extract which can selectively suppress cancer cells.

In vitro antitumor actions of extracts from endemic plant Helichrysum zivojinii

PubMed Central

2013-01-01

Background The aim of this research was to determine the intensity and mechanisms of the cytotoxic actions of five extracts isolated from the endemic plant species Helichrysum zivojinii Černjavski & Soška (family Asteraceae) against specific cancer cell lines. In order to evaluate the sensitivity of normal immunocompetent cells implicated in the antitumor immune response, the cytotoxicity of extracts was also tested against healthy peripheral blood mononuclear cells (PBMC). Methods The aerial parts of the plants were air-dried, powdered, and successively extracted with solvents of increasing polarity to obtain hexane, dichloromethane, ethyl-acetate, n-butanol and methanol extracts. The cytotoxic activities of the extracts against human cervix adenocarcinoma HeLa, human melanoma Fem-x, human myelogenous leukemia K562, human breast adenocarcinoma MDA-MB-361 cells and PBMC were evaluated by the MTT test. The mode of HeLa cell death was investigated by morphological analysis. Changes in the cell cycle of HeLa cells treated with the extracts were analyzed by flow cytometry. The apoptotic mechanisms induced by the tested extracts were determined using specific caspase inhibitors. Results The investigated Helichrysum zivojinii extracts exerted selective dose-dependent cytotoxic actions against selected cancer cell lines and healthy immunocompetent PBMC stimulated to proliferate, while the cytotoxic actions exerted on unstimulated PBMC were less pronounced. The tested extracts exhibited considerably stronger cytotoxic activities towards HeLa, Fem-x and K562 cells in comparison to resting and stimulated PBMC. It is worth noting that the cytotoxicity of the extracts was weaker against unstimulated PBMC in comparison to stimulated PBMC. Furthermore, each of the five extracts induced apoptosis in HeLa cells, through the activation of both intrinsic and extrinsic signaling pathways. Conclusion Extracts obtained from the endemic plant Helichrysum zivojinii may represent an important source of novel potential antitumor agents due to their pronounced and selective cytotoxic actions towards malignant cells. PMID:23414290

In vitro antitumor actions of extracts from endemic plant Helichrysum zivojinii.

PubMed

Matić, Ivana Z; Aljančić, Ivana; Žižak, Željko; Vajs, Vlatka; Jadranin, Milka; Milosavljević, Slobodan; Juranić, Zorica D

2013-02-18

The aim of this research was to determine the intensity and mechanisms of the cytotoxic actions of five extracts isolated from the endemic plant species Helichrysum zivojinii Černjavski & Soška (family Asteraceae) against specific cancer cell lines. In order to evaluate the sensitivity of normal immunocompetent cells implicated in the antitumor immune response, the cytotoxicity of extracts was also tested against healthy peripheral blood mononuclear cells (PBMC). The aerial parts of the plants were air-dried, powdered, and successively extracted with solvents of increasing polarity to obtain hexane, dichloromethane, ethyl-acetate, n-butanol and methanol extracts. The cytotoxic activities of the extracts against human cervix adenocarcinoma HeLa, human melanoma Fem-x, human myelogenous leukemia K562, human breast adenocarcinoma MDA-MB-361 cells and PBMC were evaluated by the MTT test. The mode of HeLa cell death was investigated by morphological analysis. Changes in the cell cycle of HeLa cells treated with the extracts were analyzed by flow cytometry. The apoptotic mechanisms induced by the tested extracts were determined using specific caspase inhibitors. The investigated Helichrysum zivojinii extracts exerted selective dose-dependent cytotoxic actions against selected cancer cell lines and healthy immunocompetent PBMC stimulated to proliferate, while the cytotoxic actions exerted on unstimulated PBMC were less pronounced. The tested extracts exhibited considerably stronger cytotoxic activities towards HeLa, Fem-x and K562 cells in comparison to resting and stimulated PBMC. It is worth noting that the cytotoxicity of the extracts was weaker against unstimulated PBMC in comparison to stimulated PBMC. Furthermore, each of the five extracts induced apoptosis in HeLa cells, through the activation of both intrinsic and extrinsic signaling pathways. Extracts obtained from the endemic plant Helichrysum zivojinii may represent an important source of novel potential antitumor agents due to their pronounced and selective cytotoxic actions towards malignant cells.

Pao Pereira Extract Suppresses Castration-Resistant Prostate Cancer Cell Growth, Survival, and Invasion Through Inhibition of NFκB Signaling.

PubMed

Chang, Cunjie; Zhao, Wei; Xie, Bingxian; Deng, Yongming; Han, Tao; Cui, Yangyan; Dai, Yundong; Zhang, Zhen; Gao, Jimin; Guo, Hongqian; Yan, Jun

2014-05-01

Pao extract, derived from bark of Amazonian tree Pao Pereira, is commonly used in South American medicine. A recent study showed that Pao extract repressed androgen-dependent LNCaP prostate cancer cell growth. We hypothesize that Pao extract asserts its anticancer effects on metastatic castration-resistant prostate cancer (CRPC) cells. Pao extract suppressed CRPC PC3 cell growth in a dose- and time-dependent manner, through induction of apoptosis and cell cycle arrest. Pao extract treatment induced cell cycle inhibitors, p21 and p27, and repressed PCNA, Cyclin A and Cyclin D1. Furthermore, Pao extract also induced the upregulation of pro-apoptotic Bax, reduction of anti-apoptotic Bcl-2, Bcl-xL, and XIAP expression, which were associated with the cleavage of PARP protein. Moreover, Pao extract treatment blocked PC3 cell migration and invasion. Mechanistically, Pao extract suppressed phosphorylation levels of AKT and NFκB/p65, NFκB DNA binding activity, and luciferase reporter activity. Pao inhibited TNFα-induced relocation of NFκB/p65 to the nucleus, NFκB/p65 transcription activity, and MMP9 activity as shown by zymography. Consistently, NFκB/p65 downstream targets involved in proliferation (Cyclin D1), survival (Bcl-2, Bcl-xL, and XIAP), and metastasis (VEGFa, MMP9, and GROα/CXCL1) were also downregulated by Pao extract. Finally, forced expression of NFκB/p65 reversed the growth inhibitory effect of Pao extract. Overall, Pao extract induced cell growth arrest, apoptosis, partially through inhibiting NFκB activation in prostate cancer cells. These data suggest that Pao extract may be beneficial for protection against CRPC. © The Author(s) 2013.

Cytotoxic effects of Mangifera indica L. kernel extract on human breast cancer (MCF-7 and MDA-MB-231 cell lines) and bioactive constituents in the crude extract.

PubMed

Abdullah, Al-Shwyeh Hussah; Mohammed, Abdulkarim Sabo; Abdullah, Rasedee; Mirghani, Mohamed Elwathig Saeed; Al-Qubaisi, Mothanna

2014-06-25

Waterlily Mango (Mangifera indica L.) is thought to be antioxidant-rich, conferred by its functional phytochemicals. The potential anticancer effects of the ethanolic kernel extract on breast cancer cells (MDA-MB-231 and MCF-7) using MTT, anti-proliferation, neutral red (NR) uptake and lactate dehydrogenase (LDH) release assays were evaluated. Cytological studies on the breast cancer cells were also conducted, and phytochemical analyses of the extract were carried out to determine the likely bioactive compounds responsible for such effects. Results showed the extract induced cytotoxicity in MDA-MB-231 cells and MCF-7 cells with IC50 values of 30 and 15 μg/mL, respectively. The extract showed significant toxicity towards both cell lines, with low toxicity to normal breast cells (MCF-10A). The cytotoxic effects on the cells were further confirmed by the NR uptake, antiproliferative and LDH release assays. Bioactive analyses revealed that many bioactives were present in the extract although butylated hydroxytoluene, a potent antioxidant, was the most abundant with 44.65%. M. indica extract appears to be more cytoxic to both estrogen positive and negative breast cancer cell lines than to normal breast cells. Synergistic effects of its antioxidant bioactives could have contributed to the cytotoxic effects of the extract. The extract of M. indica, therefore, has potential anticancer activity against breast cancer cells. This potential is worth studying further, and could have implications on future studies and eventually management of human breast cancers.

Cytotoxic effects of Mangifera indica L. kernel extract on human breast cancer (MCF-7 and MDA-MB-231 cell lines) and bioactive constituents in the crude extract

PubMed Central

2014-01-01

Background Waterlily Mango (Mangifera indica L.) is thought to be antioxidant-rich, conferred by its functional phytochemicals. Methods The potential anticancer effects of the ethanolic kernel extract on breast cancer cells (MDA-MB-231 and MCF-7) using MTT, anti-proliferation, neutral red (NR) uptake and lactate dehydrogenase (LDH) release assays were evaluated. Cytological studies on the breast cancer cells were also conducted, and phytochemical analyses of the extract were carried out to determine the likely bioactive compounds responsible for such effects. Results Results showed the extract induced cytotoxicity in MDA-MB-231 cells and MCF-7 cells with IC50 values of 30 and 15 μg/mL, respectively. The extract showed significant toxicity towards both cell lines, with low toxicity to normal breast cells (MCF-10A). The cytotoxic effects on the cells were further confirmed by the NR uptake, antiproliferative and LDH release assays. Bioactive analyses revealed that many bioactives were present in the extract although butylated hydroxytoluene, a potent antioxidant, was the most abundant with 44.65%. Conclusions M. indica extract appears to be more cytoxic to both estrogen positive and negative breast cancer cell lines than to normal breast cells. Synergistic effects of its antioxidant bioactives could have contributed to the cytotoxic effects of the extract. The extract of M. indica, therefore, has potential anticancer activity against breast cancer cells. This potential is worth studying further, and could have implications on future studies and eventually management of human breast cancers. PMID:24962691

Focal inhibitory interneuron loss and principal cell hyperexcitability in the rat hippocampus after microinjection of a neurotoxic conjugate of saporin and a peptidase-resistant analog of Substance P.

PubMed

Martin, J L; Sloviter, R S

2001-07-23

Episodes of prolonged seizures or head trauma produce chronic hippocampal network hyperexcitability hypothesized to result primarily from inhibitory interneuron loss or dysfunction. The possibly causal role of inhibitory neuron failure in the development of epileptiform pathophysiology remains unclear because global neurologic injuries produce such a multitude of effects. The recent finding that Substance P receptors (SPRs) are expressed exclusively in the rat hippocampus by inhibitory interneurons provided the rationale for attempting to ablate interneurons selectively by using neurotoxic conjugates of SPR ligands and the ribosome inactivating protein saporin that specifically target Substance P receptor-expressing cells. Whereas intrahippocampal microinjection of a conjugate of native SP and saporin produced significant nonspecific damage at concentrations needed to produce even limited selective loss of SPR-positive cells, a conjugate of saporin and the more potent and peptidase-resistant SP analog [Sar(9), Met(O(2))(11)] Substance P (SSP-saporin) caused negligible nonspecific damage at the injection site, and a virtually complete loss of SPR-like immunoreactivity (LI) up to 1 mm from the injection site. Within the SPR depletion zone, immunoreactivities for most GABA-, parvalbumin-, somatostatin-, and cholecystokinin-immunoreactive cells and fibers were eliminated. The few interneurons detectable within the affected zone were devoid of SPR-LI. The apparent loss of interneurons was selective in that calbindin- and glutamate receptor subunit 2 (GluR2) -positive principal cells survived within the affected zone, as did myelinated fibers and the extrinsic calretinin- and tyrosine hydroxylase--immunoreactive terminals of subcortical afferents. An apparent lack of reactive synaptic reorganization in response to interneuron loss was indicated by zinc transporter-3 (ZnT3)-- and beta-synuclein--LI, as well as by Timm staining, all of which revealed relatively normal patterns of excitatory terminal distribution. Control injections produced minor damage at the injection site, but no apparent specific loss of SPR-LI. One to 12 weeks after injection of SSP-saporin, extracellular electrophysiological field responses recorded in the CA1 pyramidal and dentate granule cell layers in response to afferent stimulation were blindly evaluated simultaneously in two sites 1-2 mm apart along the longitudinal hippocampal axis. SSP-saporin-treated rats exhibited relatively normal responses in some sites, whereas disinhibition and hyperexcitability indistinguishable from the pathophysiology produced by experimental status epilepticus were simultaneously recorded at adjacent sites. Anatomic analysis of the recording sites in each animal revealed that epileptiform pathophysiology was consistently observed only within areas of SPR ablation, whereas relatively normal evoked responses were recorded from immediately adjacent and relatively unaffected regions. These data establish the efficacy of [Sar(9), Met(O(2))(11)] Substance P-saporin for producing a selective and spatially extensive ablation of hippocampal inhibitory interneurons in vivo and a highly focal disinhibition that was restricted to the site of interneuron loss. These results also demonstrate that the "epileptic" pathophysiology produced by experimental status epilepticus or head trauma can be replicated by focal interneuron loss per se, without involving principal cell loss and other interpretive confounds inherent in the use of global neurologic injury models. Copyright 2001 Wiley-Liss, Inc.

Pro-oxidative and pro-apoptotic action of defatted seeds of Oenothera paradoxa on human skin melanoma cells.

PubMed

Jaszewska, Edyta; Kośmider, Anita; Kiss, Anna K; Naruszewicz, Marek

2009-09-23

Three extracts of defatted seeds of Oenothera paradoxa Hudziok, aqueous extract, 60% ethanolic extract, and 30% isopropanolic extract, differing by their total content of phenolic compounds and by their contents of individual polyphenols, were investigated in this study. The extracts exerted cytotoxic action on HTB-140 human skin melanoma cells. After 24 h of incubation, IC(50) values of 169.7 +/- 5.9 micog/mL, 72.4 +/- 3.8 microg/mL, and 155.3 +/- 6.3 microg/mL were obtained for HTB-140 cells with the aqueous extract, 60% ethanolic extract, and 30% isopropanolic extract at the tested concentrations (5-200 microg/mL), respectively, while IC(50) for normal fibroblast cells NHDFs was not attained. Moreover, for HTB-140 cells, LD(50) (concentration at which 50% of cells were dead) of 89.2 +/- 4.3 microg/mL and 181.4 +/- 6.5 microg/mL were obtained with 60% ethanolic extract and 30% isopropanolic extract, respectively. In melanoma cells, all three extracts caused a concentration-dependent increase of ROS production, GSH, and ATP lowering, and appearance of phosphatidylserine on the external surface of cellular membranes where it was bound to annexin V-FITC; furthermore, apoptosis without activation of caspase-3 took place. The most effective was 60% ethanolic extract, which had the greatest total content of phenolic compounds and the greatest content of pentagalloyloglucose (PGG).

Lipid extraction from isolated single nerve cells

NASA Technical Reports Server (NTRS)

Krasnov, I. V.

1977-01-01

A method of extracting lipids from single neurons isolated from lyophilized tissue is described. The method permits the simultaneous extraction of lipids from 30-40 nerve cells and for each cell provides equal conditions of solvent removal at the conclusion of extraction.

Neuropeptide glutamic acid-isoleucine (NEI)-induced paradoxical sleep in rats.

PubMed

Fujimoto, Moe; Fukuda, Satoru; Sakamoto, Hidetoshi; Takata, Junko; Sawamura, Shigehito

2017-01-01

Neuropeptideglutamic acid-isoleucine (NEI) as well as melanin concentrating hormone (MCH) is cleaved from the 165 amino acid protein, prepro-melanin concentrating hormone (prepro-MCH). Among many physiological roles of MCH, we demonstrated that intracerebroventricular (icv) injection of MCH induced increases in REM sleep episodes as well as in non REM sleep episodes. However, there are no studies on the effect of NEI on the sleep-wake cycle. As for the sites of action of MCH for induction of REM sleep, the ventrolateral periaqueductal gray (vlPAG) has been reported to be one of its site of action. Although MCH neurons contain NEI, GABA, MCH, and other neuropeptides, we do not know which transmitter(s) might induce REM sleep by acting on the vlPAG. Thus, we first examined the effect of icv injection of NEI on the sleep-wake cycle, and investigated how microinjection of either NEI, MCH, or GABA into the vlPAG affected REM sleep in rats. Icv injection of NEI (0.61μg/5μl: n=7) significantly increased the time spent in REM episodes compared to control (saline: 5μl; n=6). Microinjection of either NEI (61ng/0.2μl: n=7), MCH (100ng/0.2μl: n=6) or GABA (250mM/0.2μl: n=7) into the vlPAG significantly increased the time spent in REM episodes and the AUC. Precise hourly analysis of REM sleep also revealed that after those microinjections, NEI and MCH increased REM episodes at the latter phase, compared to GABA which increased REM episodes at the earlier phase. This result suggests that NEI and MCH may induce sustained REM sleep, while GABA may initiate REM sleep. In conclusion, our findings demonstrate that NEI, a cleaved peptide from the same precursor, prepro-MCH, as MCH, induce REM sleep at least in part through acting on the vlPAG. Copyright © 2016 Elsevier Inc. All rights reserved.

Rat Nucleus Accumbens Core Astrocytes Modulate Reward and the Motivation to Self-Administer Ethanol after Abstinence

PubMed Central

Bull, Cecilia; Freitas, Kelen CC; Zou, Shiping; Poland, Ryan S; Syed, Wahab A; Urban, Daniel J; Minter, Sabrina C; Shelton, Keith L; Hauser, Kurt F; Negus, S Stevens; Knapp, Pamela E; Bowers, M Scott

2014-01-01

Our understanding of the active role that astrocytes play in modulating neuronal function and behavior is rapidly expanding, but little is known about the role that astrocytes may play in drug-seeking behavior for commonly abused substances. Given that the nucleus accumbens is critically involved in substance abuse and motivation, we sought to determine whether nucleus accumbens astrocytes influence the motivation to self-administer ethanol following abstinence. We found that the packing density of astrocytes that were expressing glial fibrillary acidic protein increased in the nucleus accumbens core (NAcore) during abstinence from EtOH self-administration. No change was observed in the nucleus accumbens shell. This increased NAcore astrocyte density positively correlated with the motivation for ethanol. Astrocytes can communicate with one another and influence neuronal activity through gap-junction hemichannels. Because of this, the effect of blocking gap-junction hemichannels on the motivation for ethanol was examined. The motivation to self-administer ethanol after 3 weeks abstinence was increased following microinjection of gap-junction hemichannel blockers into the NAcore at doses that block both neuronal and astrocytic channels. In contrast, no effect was observed following microinjection of doses that are not thought to block astrocytic channels or following microinjection of either dose into the nucleus accumbens shell. Additionally, the motivation for sucrose after 3 weeks abstinence was unaffected by NAcore gap-junction hemichannel blockers. Next, Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) were selectively expressed in NAcore astrocytes to test the effect of astrocyte stimulation. DREADD activation increased cytosolic calcium in primary astrocytes, facilitated responding for rewarding brain stimulation, and reduced the motivation for ethanol after 3 weeks abstinence. This is the first work to modulate drug-seeking behavior with astrocyte-specific DREADDs. Taken together, our findings demonstrate that NAcore astrocytes can shape the motivation to self-administer ethanol; suggesting that the development of ligands which selectively stimulate astrocytes may be a successful strategy to abate ethanol-seeking behavior. PMID:24903651

Rat nucleus accumbens core astrocytes modulate reward and the motivation to self-administer ethanol after abstinence.

PubMed

Bull, Cecilia; Freitas, Kelen C C; Zou, Shiping; Poland, Ryan S; Syed, Wahab A; Urban, Daniel J; Minter, Sabrina C; Shelton, Keith L; Hauser, Kurt F; Negus, S Stevens; Knapp, Pamela E; Bowers, M Scott

2014-11-01

Our understanding of the active role that astrocytes play in modulating neuronal function and behavior is rapidly expanding, but little is known about the role that astrocytes may play in drug-seeking behavior for commonly abused substances. Given that the nucleus accumbens is critically involved in substance abuse and motivation, we sought to determine whether nucleus accumbens astrocytes influence the motivation to self-administer ethanol following abstinence. We found that the packing density of astrocytes that were expressing glial fibrillary acidic protein increased in the nucleus accumbens core (NAcore) during abstinence from EtOH self-administration. No change was observed in the nucleus accumbens shell. This increased NAcore astrocyte density positively correlated with the motivation for ethanol. Astrocytes can communicate with one another and influence neuronal activity through gap-junction hemichannels. Because of this, the effect of blocking gap-junction hemichannels on the motivation for ethanol was examined. The motivation to self-administer ethanol after 3 weeks abstinence was increased following microinjection of gap-junction hemichannel blockers into the NAcore at doses that block both neuronal and astrocytic channels. In contrast, no effect was observed following microinjection of doses that are not thought to block astrocytic channels or following microinjection of either dose into the nucleus accumbens shell. Additionally, the motivation for sucrose after 3 weeks abstinence was unaffected by NAcore gap-junction hemichannel blockers. Next, Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) were selectively expressed in NAcore astrocytes to test the effect of astrocyte stimulation. DREADD activation increased cytosolic calcium in primary astrocytes, facilitated responding for rewarding brain stimulation, and reduced the motivation for ethanol after 3 weeks abstinence. This is the first work to modulate drug-seeking behavior with astrocyte-specific DREADDs. Taken together, our findings demonstrate that NAcore astrocytes can shape the motivation to self-administer ethanol; suggesting that the development of ligands which selectively stimulate astrocytes may be a successful strategy to abate ethanol-seeking behavior.

Topographic and functional neuroanatomical study of GABAergic disinhibitory striatum-nigral inputs and inhibitory nigrocollicular pathways: neural hodology recruiting the substantia nigra, pars reticulata, for the modulation of the neural activity in the inferior colliculus involved with panic-like emotions.

PubMed

Castellan-Baldan, Lissandra; da Costa Kawasaki, Mateus; Ribeiro, Sandro José; Calvo, Fabrício; Corrêa, Vani Maria Alves; Coimbra, Norberto Cysne

2006-08-01

Considering the influence of the substantia nigra on mesencephalic neurons involved with fear-induced reactions organized in rostral aspects of the dorsal midbrain, the present work investigated the topographical and functional neuroanatomy of similar influence on caudal division of the corpora quadrigemina, addressing: (a) the neural hodology connecting the neostriatum, the substantia nigra, periaqueductal gray matter and inferior colliculus (IC) neural networks; (b) the influence of the inhibitory neostriatonigral-nigrocollicular GABAergic links on the control of the defensive behavior organized in the IC. The effects of the increase or decrease of activity of nigrocollicular inputs on defensive responses elicited by either electrical or chemical stimulation of the IC were also determined. Electrolytic or chemical lesions of the substantia nigra, pars reticulata (SNpr), decreased the freezing and escape behaviors thresholds elicited by electrical stimulation of the IC, and increased the behavioral responses evoked by the GABAA blockade in the same sites of the mesencephalic tectum (MT) electrically stimulated. These findings were corroborated by similar effects caused by microinjections of the GABAA-receptor agonist muscimol in the SNpr, followed by electrical and chemical stimulations of the IC. The GABAA blockade in the SNpr caused a significant increase in the defensive behavior thresholds elicited by electrical stimulation of the IC and a decrease in the mean incidence of panic-like responses induced by microinjections of bicuculline in the mesencephalic tectum (inferior colliculus). These findings suggest that the substantia nigra receives GABAergic inputs that modulate local and also inhibitory GABAergic outputs toward the IC. In fact, neurotracing experiments with fast blue and iontophoretic microinjections of biotinylated dextran amine either into the inferior colliculus or in the reticular division of the substantia nigra demonstrated a neural link between these structures, as well as between the neostriatum and SNpr.

Epigenetic regulation of dorsal raphe GABA(B1a) associated with isolation-induced abnormal responses to social stimulation in mice.

PubMed

Araki, Ryota; Hiraki, Yosuke; Nishida, Shoji; Kuramoto, Nobuyuki; Matsumoto, Kinzo; Yabe, Takeshi

2016-02-01

In isolation-reared mice, social encounter stimulation induces locomotor hyperactivity and activation of the dorsal raphe nucleus (DRN), suggesting that dysregulation of dorsal raphe function may be involved in abnormal behaviors. In this study, we examined the involvement of dorsal raphe GABAergic dysregulation in the abnormal behaviors of isolation-reared mice. We also studied an epigenetic mechanism underlying abnormalities of the dorsal raphe GABAergic system. Both mRNA and protein levels of GABA(B1a), a GABA(B) receptor subunit, were increased in the DRN of isolation-reared mice, compared with these levels in group-reared mice. In contrast, mRNA levels for other GABAergic system-related genes (GABA(A) receptor α1, β2 and γ2 subunits, GABA(B) receptor 1b and 2 subunits, and glutamate decarboxylase 67 and 65) were unchanged. Intra-DRN microinjection of 0.06 nmol baclofen (a GABA(B) receptor agonist) exacerbated encounter-induced hyperactivity and aggressive behavior, while microinjection of 0.3 nmol phaclofen (a GABA(B) receptor antagonist) attenuated encounter-induced hyperactivity and aggressive behavior in isolation-reared mice. Furthermore, microinjection of 0.06 nmol baclofen elicited encounter-induced hyperactivity in group-reared mice. Neither baclofen nor phaclofen affected immobility time in the forced swim test and hyperactivity in a novel environment of isolation reared mice. Bisulfite sequence analyses revealed that the DNA methylation level of the CpG island around the transcription start site (TSS) of GABA(B1a) was decreased in the DRN of isolation-reared mice. Chromatin immunoprecipitation analysis showed that histone H3 was hyperacetylated around the TSS of GABA(B1a) in the DRN of isolation-reared mice. These findings indicate that an increase in dorsal raphe GABA(B1a) expression via epigenetic regulation is associated with abnormal responses to social stimulation such as encounter-induced hyperactivity and aggressive behavior in isolation-reared mice. Copyright © 2015 Elsevier Ltd. All rights reserved.