Cerebellar GABAergic progenitors adopt an external granule cell-like phenotype in the absence of Ptf1a transcription factor expression.

PubMed

Pascual, Marta; Abasolo, Ibane; Mingorance-Le Meur, Ana; Martínez, Albert; Del Rio, José A; Wright, Christopher V E; Real, Francisco X; Soriano, Eduardo

2007-03-20

We report in this study that, in the cerebellum, the pancreatic transcription factor Ptf1a is required for the specific generation of Purkinje cells (PCs) and interneurons. Moreover, granule cell progenitors in the external GCL (EGL) appear to be unaffected by deletion of Ptf1a. Cell lineage analysis in Ptf1a(Cre/Cre) mice was used to establish that, in the absence of Ptf1a expression, ventricular zone progenitors, normally fated to produce PCs and interneurons, aberrantly migrate to the EGL and express typical markers of these cells, such as Math1, Reelin, and Zic1/2. Furthermore, these cells have a fine structure typical of EGL progenitors, indicating that they adopt an EGL-like cell phenotype. These findings indicate that Ptf1a is necessary for the specification and normal production of PCs and cerebellar interneurons. Moreover, our results suggest that Ptf1a is also required for the suppression of the granule cell specification program in cerebellar ventricular zone precursors.

Proneurotrophin-3 promotes cell cycle withdrawal of developing cerebellar granule cell progenitors via the p75 neurotrophin receptor.

PubMed

Zanin, Juan Pablo; Abercrombie, Elizabeth; Friedman, Wilma J

2016-07-19

Cerebellar granule cell progenitors (GCP) proliferate extensively in the external granule layer (EGL) of the developing cerebellum prior to differentiating and migrating. Mechanisms that regulate the appropriate timing of cell cycle withdrawal of these neuronal progenitors during brain development are not well defined. The p75 neurotrophin receptor (p75(NTR)) is highly expressed in the proliferating GCPs, but is downregulated once the cells leave the cell cycle. This receptor has primarily been characterized as a death receptor for its ability to induce neuronal apoptosis following injury. Here we demonstrate a novel function for p75(NTR) in regulating proper cell cycle exit of neuronal progenitors in the developing rat and mouse EGL, which is stimulated by proNT3. In the absence of p75(NTR), GCPs continue to proliferate beyond their normal period, resulting in a larger cerebellum that persists into adulthood, with consequent motor deficits.

A low-density culture method of cerebellar granule neurons with paracrine support applicable for the study of neuronal morphogenesis.

PubMed

Kubota, Kenta; Seno, Takeshi; Konishi, Yoshiyuki

2013-11-20

Cerebellar granule neuronal cultures have been used to study the molecular mechanisms underlying neuronal functions, including neuronal morphogenesis. However, a limitation of this system is the difficulty to analyze isolated neurons because these are required to be maintained at a high density. Therefore, in the present study, we aimed to develop a simple and cost-effective method for culturing low-density cerebellar granule neurons. Cerebellar granule cells at two different densities (low- and high-density) were co-cultivated in order for the low-density culture to be supported by the paracrine signals from the high-density culture. This method enabled morphology analysis of isolated cerebellar granule neurons without astrocytic feeder cultures or supplements such as B27. Using this method, we investigated the function of a polarity factor. Studies using hippocampal neurons suggested that glycogen synthase kinase-3 (GSK-3) is an essential regulator of neuronal polarity, and inhibition of GSK-3 results in the formation of multiple axons. Pharmacological inhibitors for GSK-3 (6-bromoindirubin-3'-oxime and lithium chloride) did not cause the formation of multiple axons of cerebellar granule neurons but significantly reduced their length. Consistent results were obtained by introducing kinase-dead form of GSK-3 beta (K85A). These results indicated that GSK-3 is not directly involved in the control of neuronal polarity in cerebellar granule neurons. Overall, this study provides a simple method for culturing low-density cerebellar granule neurons and insights in to the neuronal-type dependent function of GSK-3 in neuronal morphogenesis. © 2013 Elsevier B.V. All rights reserved.

Neurotoxic effects of indocyanine green -cerebellar granule cell culture viability study

PubMed Central

Toczylowska, Beata; Zieminska, Elzbieta; Goch, Grazyna; Milej, Daniel; Gerega, Anna; Liebert, Adam

2014-01-01

The aim of this study was to examine neurotoxicity indocyanine green (ICG). We assessed viability of primary cerebellar granule cell culture (CGC) exposed to ICG to test two mechanisms that could be the first triggers causing neuronal toxicity: imbalance in calcium homeostasis and the degree of oligomerization of ICG molecules. We have observed this imbalance in CGC after exposure to 75-125μΜ ICG and dose and application sequence dependent protective effect of Gadovist on surviving neurons in vitro when used with ICG. Spectroscopic studies suggest the major cause of toxicity of the ICG is connected with oligomers formation. ICG at concentration of 25 μM (which is about 4 times higher than the highest concentration of ICG in the brain applied in in-vivo human studies) is not neurotoxic in the cell culture. PMID:24688815

Aryl Hydrocarbon Receptor (AhR) Deletion in Cerebellar Granule Neuron Precursors Impairs Neurogenesis

PubMed Central

Dever, Daniel P.; Adham, Zachariah O.; Thompson, Bryan; Genestine, Matthieu; Cherry, Jonathan; Olschowka, John A.; DiCicco-Bloom, Emanuel; Opanashuk, Lisa A.

2015-01-01

The aryl hydrocarbon receptor (AhR) is a ligand-activated member of the basic-helix-loop-helix (bHLH)/PER-ARNT-SIM(PAS) transcription factor superfamily that also mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increasing evidence suggests that AhR influences the development of many tissues, including the central nervous system. Our previous studies suggest that sustained AhR activation by TCDD and/or AhR deletion disrupts cerebellar granule neuron precursor (GNP) development. In the current study, to determine whether endogenous AhR controls GNP development in a cell autonomous manner, we created a GNP-specific AhR deletion mouse, AhRfx/fx/Math1CRE/+ (AhR CKO). Selective AhR deletion in GNPs produced abnormalities in proliferation and differentiation. Specifically, fewer GNPs were engaged in S-phase, as demonstrated by ~25% reductions in thymidine (in vitro) and BrdU (in vivo) incorporation. Furthermore, total granule neuron numbers in the IGL at PND21 and PND60 were diminished in AhR CKO mice compared to controls. On the other hand, differentiation was enhanced, including ~40% increase in neurite outgrowth and 50% increase in GABARα6 receptor expression in deletion mutants. Our results suggest that AhR activity plays a role in regulating granule neuron number and differentiation, possibly by coordinating this GNP developmental transition. These studies provide novel insights for understanding the normal roles of AhR signaling during cerebellar granule cell neurogenesis, and may have important implications for the effects of environmental factors in cerebellar dysgenesis. PMID:26243376

LXR agonist rescued the deficit in the proliferation of the cerebellar granule cells induced by dexamethasone

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

Bian, Xuting; Zhong, Hongyu; Li, Fen

Dexamethasone (DEX) exposure during early postnatal life produces permanent neuromotor and intellectual deficits and stunts cerebellar growth. The liver X receptor (LXR) plays important roles in CNS development. However, the effects of LXR on the DEX-mediated impairment of cerebellar development remain undetermined. Thus, mice were pretreated with LXR agonist TO901317 (TO) and were later exposed to DEX to evaluate its protective effects on DEX-mediated deficit during cerebellar development. The results showed that an acute exposure of DEX on postnatal day 7 resulted in a significant impairment in cerebellar development and decreased the proliferation of granule neuron precursors in the externalmore » granule layer of cerebellum. This effect was attenuated by pretreatment with TO. We further found that the decrease in the proliferation caused by DEX occurred via up-regulation of glucocorticoid receptor and p27kip1, which could be partially prevented by LXR agonist pretreatment. Overall, our results suggest that LXR agonist pretreatment could protect against DEX-induced deficits in cerebellar development in postnatal mice and may thus be perspective recruited to counteract such GC side effects.« less

Persistent Nav1.6 current at axon initial segments tunes spike timing of cerebellar granule cells

PubMed Central

Osorio, Nancy; Cathala, Laurence; Meisler, Miriam H; Crest, Marcel; Magistretti, Jacopo; Delmas, Patrick

2010-01-01

Cerebellar granule (CG) cells generate high-frequency action potentials that have been proposed to depend on the unique properties of their voltage-gated ion channels. To address the in vivo function of Nav1.6 channels in developing and mature CG cells, we combined the study of the developmental expression of Nav subunits with recording of acute cerebellar slices from young and adult granule-specific Scn8a KO mice. Nav1.2 accumulated rapidly at early-formed axon initial segments (AISs). In contrast, Nav1.6 was absent at early postnatal stages but accumulated at AISs of CG cells from P21 to P40. By P40–P65, both Nav1.6 and Nav1.2 co-localized at CG cell AISs. By comparing Na+ currents in mature CG cells (P66–P74) from wild-type and CG-specific Scn8a KO mice, we found that transient and resurgent Na+ currents were not modified in the absence of Nav1.6 whereas persistent Na+ current was strongly reduced. Action potentials in conditional Scn8a KO CG cells showed no alteration in threshold and overshoot, but had a faster repolarization phase and larger post-spike hyperpolarization. In addition, although Scn8a KO CG cells kept their ability to fire action potentials at very high frequency, they displayed increased interspike-interval variability and firing irregularity in response to sustained depolarization. We conclude that Nav1.6 channels at axon initial segments contribute to persistent Na+ current and ensure a high degree of temporal precision in repetitive firing of CG cells. PMID:20173079

AMPK is activated early in cerebellar granule cells undergoing apoptosis and influences VADC1 phosphorylation status and activity.

PubMed

Bobba, A; Casalino, E; Amadoro, G; Petragallo, V A; Atlante, A

2017-09-01

The neurodegeneration of cerebellar granule cells, after low potassium induced apoptosis, is known to be temporally divided into an early and a late phase. Voltage-dependent anion channel-1 (VDAC1) protein, changing from the closed inactive state to the active open state, is central to the switch between the early and late phase. It is also known that: (i) VDAC1 can undergo phosphorylation events and (ii) AMP-activated protein kinase (AMPK), the sensor of cellular stress, may have a role in neuronal homeostasis. In the view of this, the involvement of AMPK activation and its correlation with VDAC1 status and activity has been investigated in the course of cerebellar granule cells apoptosis. The results reported in this study show that an increased level of the phosphorylated, active, isoform of AMPK occurs in the early phase, peaks at 3 h and guarantees an increase in the phosphorylation status of VDCA1, resulting in a reduced activity of this latter. However this situation is transient in nature, since, in the late phase, AMPK activation decreases as well as the level of phosphorylated VDAC1. In a less phosphorylated status, VDAC1 fully recovers its gating activity and drives cells along the death route.

Control of cerebellar granule cell output by sensory-evoked Golgi cell inhibition

PubMed Central

Duguid, Ian; Branco, Tiago; Chadderton, Paul; Arlt, Charlotte; Powell, Kate; Häusser, Michael

2015-01-01

Classical feed-forward inhibition involves an excitation–inhibition sequence that enhances the temporal precision of neuronal responses by narrowing the window for synaptic integration. In the input layer of the cerebellum, feed-forward inhibition is thought to preserve the temporal fidelity of granule cell spikes during mossy fiber stimulation. Although this classical feed-forward inhibitory circuit has been demonstrated in vitro, the extent to which inhibition shapes granule cell sensory responses in vivo remains unresolved. Here we combined whole-cell patch-clamp recordings in vivo and dynamic clamp recordings in vitro to directly assess the impact of Golgi cell inhibition on sensory information transmission in the granule cell layer of the cerebellum. We show that the majority of granule cells in Crus II of the cerebrocerebellum receive sensory-evoked phasic and spillover inhibition prior to mossy fiber excitation. This preceding inhibition reduces granule cell excitability and sensory-evoked spike precision, but enhances sensory response reproducibility across the granule cell population. Our findings suggest that neighboring granule cells and Golgi cells can receive segregated and functionally distinct mossy fiber inputs, enabling Golgi cells to regulate the size and reproducibility of sensory responses. PMID:26432880

Shp2 Acts Downstream of SDF-1α/CXCR4 in Guiding Granule Cell Migration During Cerebellar Development

PubMed Central

Hagihara, Kazuki; Zhang, Eric E.; Ke, Yue-Hai; Liu, Guofa; Liu, Jan-Jan; Rao, Yi; Feng, Gen-Sheng

2009-01-01

Shp2 is a non-receptor protein tyrosine phosphatase containing two Src homology 2 (SH2) domains that is implicated in intracellular signaling events controlling cell proliferation, differentiation and migration. To examine the role of Shp2 in brain development, we created mice with Shp2 selectively deleted in neural stem/progenitor cells. Homozygous mutant mice exhibited early postnatal lethality with defects in neural stem cell self-renewal and neuronal/glial cell fate specification. Here we report a critical role of Shp2 in guiding neuronal cell migration in the cerebellum. In homozygous mutants, we observed reduced and less foliated cerebellum, ectopic presence of external granule cells and mispositioned Purkinje cells, a phenotype very similar to that of mutant mice lacking either SDF-1α or CXCR4. Consistently, Shp2-deficient granule cells failed to migrate toward SDF-1α in an in vitro cell migration assay, and SDF-1α treatment triggered a robust induction of tyrosyl phosphorylation on Shp2. Together, these results suggest that although Shp2 is involved in multiple signaling events during brain development, a prominent role of the phosphatase is to mediate SDF-1α/CXCR4 signal in guiding cerebellar granule cell migration. PMID:19635473

The natural scorpion peptide, BmK NT1 activates voltage-gated sodium channels and produces neurotoxicity in primary cultured cerebellar granule cells.

PubMed

Zou, Xiaohan; He, Yuwei; Qiao, Jinping; Zhang, Chunlei; Cao, Zhengyu

2016-01-01

The scorpion Buthus martensii Karsch has been used in Traditional Chinese Medicine to treat neuronal diseases such as neuropathic pain, paralysis and epilepsy for thousands of years. Studies have demonstrated that scorpion venom is the primary active component. Although scorpion venom can effectively attenuate pain in the clinic, it also produces neurotoxic response. In this study, toxicity guided purification led to identify a mammalian toxin termed BmK NT1 comprising of 65 amino acid residues and an amidated C-terminus, a mature peptide encoded by the nucleotide sequence (GenBank No. AF464898). In contract to the recombinant product of the same nucleotide sequence, BmK AGAP, which displayed analgesic and anti-tumor effect, intravenous injection (i.v.) of BmK NT1 produced acute toxicity in mice with an LD50 value of 1.36 mg/kg. In primary cultured cerebellar granule cells, BmK NT1 produced a concentration-dependent cell death with an IC50 value of 0.65 μM (0.41-1.03 μM, 95% Confidence Intervals, 95% CI) which was abolished by TTX, a voltage-gated sodium channel (VGSC) blocker. We also demonstrated that BmK NT1 produced modest sodium influx in cerebellar granule cell cultures with an EC50 value of 2.19 μM (0.76-6.40 μM, 95% CI), an effect similar to VGSC agonist, veratridine. The sodium influx response was abolished by TTX suggesting that BmK NT1-induced sodium influx is solely through activation of VGSC. Considered these data together, we demonstrated that BmK NT1 activated VGSC and produced neurotoxicity in cerebellar granule cell cultures. Copyright © 2015 Elsevier Ltd. All rights reserved.

Somato-synaptic variation of GABA(A) receptors in cultured murine cerebellar granule cells: investigation of the role of the alpha6 subunit.

PubMed

Mellor, J R; Wisden, W; Randall, A D

2000-07-10

Electrophysiological investigation of cultured cerebellar murine granule cells revealed differences between the GABA(A) receptors at inhibitory synapses and those on the cell body. Specifically, mIPSCs decayed more rapidly than cell body receptors deactivated, the mean single channel conductance at the synapse (32 pS) was greater than that at cell body (21 pS) and only cell body receptors were sensitive to Zn(2+) (150 microM), which depressed response amplitude by 82+/-5% and almost doubled the rate of channel deactivation. The GABA(A) receptor alpha6 subunit is selectively expressed in cerebellar granule cells. Although concentrated at synapses, it is also found on extrasynaptic membranes. Using a mouse line (Deltaalpha6lacZ) lacking this subunit, we investigated its role in the somato-synaptic differences in GABA(A) receptor function. All differences between cell body and synaptic GABA(A) receptors observed in wild-type (WT) granule cells persisted in Deltaalpha6lacZ cells, thus demonstrating that they are not specifically due to the cellular distribution of the alpha6 subunit. However, mIPSCs from WT and Deltaalpha6lacZ cells differed in both their kinetics (faster decay in WT cells) and underlying single channel conductance (32 pS WT, 25 pS Deltaalpha6lacZ). This provides good evidence for a functional contribution of the alpha6 subunit to postsynaptic GABA(A) receptors in these cells. Despite this, deactivation kinetics of mIPSCs in WT and Deltaalpha6lacZ granule cells exhibited similar benzodiazepene (BDZ) sensitivity. This suggests that the enhanced BDZ-induced ataxia seen in Deltaalpha6lacZ mice may reflect physiological activity at extrasynaptic receptors which, unlike those at synapses, display differential BDZ-sensitivity in WT and Deltaalpha6lacZ granule cells (Jones, A.M., Korpi, E.R., McKernan, R.M., Nusser, Z., Pelz, R., Makela, R., Mellor, J.R., Pollard, S., Bahn, S., Stephenson, F.A., Randall, A.D., Sieghart, W., Somogyi, P., Smith, A.J.H., Wisden

Characterization of Ca2+ channel currents in cultured rat cerebellar granule neurones.

PubMed

Pearson, H A; Sutton, K G; Scott, R H; Dolphin, A C

1995-02-01

1. High-threshold voltage-gated calcium channel currents (IBa) were studied in cultured rat cerebellar granule neurones using the whole-cell patch clamp technique with 10 mM Ba2+ as the charge carrier. The putative P-type component of whole-cell current was characterized by utilizing the toxin omega-agatoxin IVA (omega-Aga IVA) in combination with other blockers. 2. omega-Aga IVA (100 nM) inhibited the high voltage-activated (HVA) IBa by 40.9 +/- 3.4% (n = 27), and the dissociation constant Kd was 2.7 nM. Maximal inhibition occurred within a 2-3 min time course, and was irreversible. The isolated omega-Aga IVA-sensitive current was non-inactivating. 3. omega-Aga IVA exhibited overlapping selectivity with both N- and L-channel blockers; omega-conotoxin GVIA (omega-CTX GVIA) (1 microM) and the dihydropyridine (-)-202-709 (1 microM), respectively. Together these toxins reduced the omega-Aga IVA-sensitive component to just 4.5 +/- 1.4% (n = 3). Thus only a small proportion of the current can be unequivocally attributed to P-type current. Inhibition of the HVA IBa by omega-Aga IA also reduced the proportion of omega-Aga IVA-sensitive current to 28.0 +/- 3.2% (n = 3). 4. Application of omega-Aga IVA and a synthetic form of funnel-web toxin, N-(7-amino-4-azaheptyl)-L-argininamide (sFTX-3.3; 10 microM), produced an additive block of the HVA IBa. Consequently these two toxins do not act on the same channel in cerebellar granule neurones. 5. omega-Aga IVA inhibition of low voltage-activated (LVA) IBa was studied in the ND7-23 neuronal cell line. omega-Aga IVA (100 nM) reduced the LVA current by 41.3 +/- 3.2% (n = 17) in a fully reversible manner with no shift in the steady-state inactivation of the channel. 6. A component of current insensitive to N-, L- and P-channel blockers remained unclassified in all our studies. This component, and also that remaining following block by omega-Aga IVA and omega-Aga IA, exhibited relatively rapid, although incomplete, inactivation

MicroRNAs Promote Granule Cell Expansion in the Cerebellum Through Gli2.

PubMed

Constantin, Lena; Wainwright, Brandon J

2015-12-01

MicroRNAs (miRNAs) are important regulators of cerebellar function and homeostasis. Their deregulation results in cerebellar neuronal degeneration and spinocerebellar ataxia type 1 and contributes to medulloblastoma. Canonical miRNA processing involves Dicer, which cleaves precursor miRNAs into mature double-stranded RNA duplexes. In order to address the role of miRNAs in cerebellar granule cell precursor development, loxP-flanked exons of Dicer1 were conditionally inactivated using the granule cell precursor-specific Atoh1-Cre recombinase. A reduction of 87% in Dicer1 transcript was achieved in this conditional Dicer knockdown model. Although knockdown resulted in normal survival, mice had disruptions to the cortical layering of the anterior cerebellum, which resulted from the premature differentiation of granule cell precursors in this region during neonatal development. This defect manifested as a thinner external granular layer with ectopic mature granule cells, and a depleted internal granular layer. We found that expression of the activator components of the Hedgehog-Patched pathway, the Gli family of transcription factors, was perturbed in conditional Dicer knockdown mice. We propose that loss of Gli2 mRNA mediated the anterior-restricted defect in conditional Dicer knockdown mice and, as proof of principle, were able to show that miR-106b positively regulated Gli2 mRNA expression. These findings confirm the importance of miRNAs as positive mediators of Hedgehog-Patched signalling during granule cell precursor development.

Different Molecular Mechanisms Mediate Direct or Glia-Dependent Prion Protein Fragment 90-231 Neurotoxic Effects in Cerebellar Granule Neurons.

PubMed

Thellung, Stefano; Gatta, Elena; Pellistri, Francesca; Villa, Valentina; Corsaro, Alessandro; Nizzari, Mario; Robello, Mauro; Florio, Tullio

2017-10-01

Glia over-stimulation associates with amyloid deposition contributing to the progression of central nervous system neurodegenerative disorders. Here we analyze the molecular mechanisms mediating microglia-dependent neurotoxicity induced by prion protein (PrP)90-231, an amyloidogenic polypeptide corresponding to the protease-resistant portion of the pathological prion protein scrapie (PrP Sc ). PrP90-231 neurotoxicity is enhanced by the presence of microglia within neuronal culture, and associated to a rapid neuronal [Ca ++ ] i increase. Indeed, while in "pure" cerebellar granule neuron cultures, PrP90-231 causes a delayed intracellular Ca ++ entry mediated by the activation of NMDA receptors; when neuron and glia are co-cultured, a transient increase of [Ca ++ ] i occurs within seconds after treatment in both granule neurons and glial cells, then followed by a delayed and sustained [Ca ++ ] i raise, associated with the induction of the expression of inducible nitric oxide synthase and phagocytic NADPH oxidase. [Ca ++ ] i fast increase in neurons is dependent on the activation of multiple pathways since it is not only inhibited by the blockade of voltage-gated channel activity and NMDA receptors but also prevented by the inhibition of nitric oxide and PGE 2 release from glial cells. Thus, Ca ++ homeostasis alteration, directly induced by PrP90-231 in cerebellar granule cells, requires the activation of NMDA receptors, but is greatly enhanced by soluble molecules released by activated glia. In glia-enriched cerebellar granule cultures, the activation of inducible nitric oxide (iNOS) and NADPH oxidase represents the main mechanism of toxicity since their pharmacological inhibition prevented PrP90-231 neurotoxicity, whereas NMDA blockade by D(-)-2-amino-5-phosphonopentanoic acid is ineffective; conversely, in pure cerebellar granule cultures, NMDA blockade but not iNOS inhibition strongly reduced PrP90-231 neurotoxicity. These data indicate that amyloidogenic peptides

The cell adhesion molecule CHL1 interacts with patched-1 to regulate apoptosis during postnatal cerebellar development.

PubMed

Katic, Jelena; Loers, Gabriele; Tosic, Jelena; Schachner, Melitta; Kleene, Ralf

2017-08-01

The immunoglobulin superfamily adhesion molecule close homolog of L1 (CHL1) plays important roles during nervous system development. Here, we identified the hedgehog receptor patched-1 (PTCH1) as a novel CHL1-binding protein and showed that CHL1 interacts with the first extracellular loop of PTCH1 via its extracellular domain. Colocalization and co-immunoprecipitation of CHL1 with PTCH1 suggest an association of CHL1 with this major component of the hedgehog signaling pathway. The trans -interaction of CHL1 with PTCH1 promotes neuronal survival in cultures of dissociated cerebellar granule cells and of organotypic cerebellar slices. An inhibitor of the PTCH1-regulated hedgehog signal transducer, smoothened (SMO), and inhibitors of RhoA and Rho-associated kinase (ROCK) 1 and 2 prevent CHL1-dependent survival of cultured cerebellar granule cells and survival of cerebellar granule and Purkinje cells in organotypic cultures. In histological sections from 10- and 14-day-old CHL1-deficient mice, enhanced apoptosis of granule, but not Purkinje, cells was observed. The results of the present study indicate that CHL1 triggers PTCH1-, SMO-, RhoA- and ROCK-dependent signal transduction pathways to promote neuronal survival after cessation of the major morphogenetic events during mouse cerebellar development. © 2017. Published by The Company of Biologists Ltd.

Profilin1 activity in cerebellar granule neurons is required for radial migration in vivo

PubMed Central

Kullmann, Jan A; Wickertsheim, Ines; Minnerup, Lara; Costell, Mercedes; Friauf, Eckhard; Rust, Marco B

2015-01-01

Neuron migration defects are an important aspect of human neuropathies. The underlying molecular mechanisms of such migration defects are largely unknown. Actin dynamics has been recognized as an important determinant of neuronal migration, and we recently found that the actin-binding protein profilin1 is relevant for radial migration of cerebellar granule neurons (CGN). As the exploited brain-specific mutants lacked profilin1 in both neurons and glial cells, it remained unknown whether profilin1 activity in CGN is relevant for CGN migration in vivo. To test this, we capitalized on a transgenic mouse line that expresses a tamoxifen-inducible Cre variant in CGN, but no other cerebellar cell type. In these profilin1 mutants, the cell density was elevated in the molecular layer, and ectopic CGN occurred. Moreover, 5-bromo-2′-deoxyuridine tracing experiments revealed impaired CGN radial migration. Hence, our data demonstrate the cell autonomous role of profilin1 activity in CGN for radial migration. PMID:25495756

Utilization of alpha-ketoglutarate as a precursor for transmitter glutamate in cultured cerebellar granule cells.

PubMed

Peng, L A; Schousboe, A; Hertz, L

1991-01-01

Alpha-ketoglutarate together with an amino group donor (alanine) was shown to be able to serve as a precursor for the glutamate pool which is released by potassium-induced depolarization (i.e., transmitter glutamate) in cerebellar granule cells. However, these compounds could not be utilized as precursors for intracellular glutamate or for release of transmitter aspartate. The formation of transmitter glutamate was inhibited by the transamination inhibitor aminooxyacetic acid but not by phenylsuccinate, an inhibitor of the dicarboxylate carrier in the mitochondrial membrane. Both of these inhibitors have previously been found to inhibit synthesis of transmitter glutamate from glutamine. The results support the hypothesis that alpha-ketoglutarate and alanine undergo transmination in the cytosol to form pyruvate and glutamate, and that this glutamate pool is available for transmitter release of glutamate but does not constitute the major intracellular pool of glutamate.

The cerebellar Golgi cell and spatiotemporal organization of granular layer activity

PubMed Central

D'Angelo, Egidio; Solinas, Sergio; Mapelli, Jonathan; Gandolfi, Daniela; Mapelli, Lisa; Prestori, Francesca

2013-01-01

The cerebellar granular layer has been suggested to perform a complex spatiotemporal reconfiguration of incoming mossy fiber signals. Central to this role is the inhibitory action exerted by Golgi cells over granule cells: Golgi cells inhibit granule cells through both feedforward and feedback inhibitory loops and generate a broad lateral inhibition that extends beyond the afferent synaptic field. This characteristic connectivity has recently been investigated in great detail and been correlated with specific functional properties of these neurons. These include theta-frequency pacemaking, network entrainment into coherent oscillations and phase resetting. Important advances have also been made in terms of determining the membrane and synaptic properties of the neuron, and clarifying the mechanisms of activation by input bursts. Moreover, voltage sensitive dye imaging and multi-electrode array (MEA) recordings, combined with mathematical simulations based on realistic computational models, have improved our understanding of the impact of Golgi cell activity on granular layer circuit computations. These investigations have highlighted the critical role of Golgi cells in: generating dense clusters of granule cell activity organized in center-surround structures, implementing combinatorial operations on multiple mossy fiber inputs, regulating transmission gain, and cut-off frequency, controlling spike timing and burst transmission, and determining the sign, intensity and duration of long-term synaptic plasticity at the mossy fiber-granule cell relay. This review considers recent advances in the field, highlighting the functional implications of Golgi cells for granular layer network computation and indicating new challenges for cerebellar research. PMID:23730271

In vitro study of uptake and synthesis of creatine and its precursors by cerebellar granule cells and astrocytes suggests some hypotheses on the physiopathology of the inherited disorders of creatine metabolism

PubMed Central

2012-01-01

Background The discovery of the inherited disorders of creatine (Cr) synthesis and transport in the last few years disclosed the importance of blood Cr supply for the normal functioning of the brain. These putatively rare diseases share a common pathogenetic mechanism (the depletion of brain Cr) and similar phenotypes characterized by mental retardation, language disturbances, seizures and movement disorders. In the effort to improve our knowledge on the mechanisms regulating Cr pool inside the nervous tissue, Cr transport and synthesis and related gene transcripts were explored in primary cultures of rat cerebellar granule cells and astrocytes. Methods Cr uptake and synthesis were explored in vitro by incubating monotypic primary cultures of rat type I astrocytes and cerebellar granule cells with: a) D3-Creatine (D3Cr) and D3Cr plus β-guanidinopropionate (GPA, an inhibitor of Cr transporter), and b) labelled precursors of Guanidinoacetate (GAA) and Cr (Arginine, Arg; Glycine, Gly). Intracellular D3Cr and labelled GAA and Cr were assessed by ESI-MS/MS. Creatine transporter (CT1), L-arginine:glycine amidinotransferase (AGAT), and S-adenosylmethionine:guanidinoacetate N-methyltransferase (GAMT) gene expression was assessed in the same cells by real time PCR. Results D3Cr signal was extremely high in cells incubated with this isotope (labelled/unlabelled Cr ratio reached about 10 and 122, respectively in cerebellar granule cells and astrocytes) and was reduced by GPA. Labelled Arg and Gly were taken up by the cells and incorporated in GAA, whose concentration paralleled that of these precursors both in the extracellular medium and inside the cells (astrocytes). In contrast, the increase of labelled Cr was relatively much more limited since labelled Cr after precursors' supplementation did not exceed 2,7% (cerebellar granule cells) and 21% (astrocytes) of unlabelled Cr. Finally, AGAT, GAMT and SLC6A8 were expressed in both kind of cells. Conclusions Our results

In vitro study of uptake and synthesis of creatine and its precursors by cerebellar granule cells and astrocytes suggests some hypotheses on the physiopathology of the inherited disorders of creatine metabolism.

PubMed

Carducci, Claudia; Carducci, Carla; Santagata, Silvia; Adriano, Enrico; Artiola, Cristiana; Thellung, Stefano; Gatta, Elena; Robello, Mauro; Florio, Tullio; Antonozzi, Italo; Leuzzi, Vincenzo; Balestrino, Maurizio

2012-04-26

The discovery of the inherited disorders of creatine (Cr) synthesis and transport in the last few years disclosed the importance of blood Cr supply for the normal functioning of the brain. These putatively rare diseases share a common pathogenetic mechanism (the depletion of brain Cr) and similar phenotypes characterized by mental retardation, language disturbances, seizures and movement disorders. In the effort to improve our knowledge on the mechanisms regulating Cr pool inside the nervous tissue, Cr transport and synthesis and related gene transcripts were explored in primary cultures of rat cerebellar granule cells and astrocytes. Cr uptake and synthesis were explored in vitro by incubating monotypic primary cultures of rat type I astrocytes and cerebellar granule cells with: a) D3-Creatine (D3Cr) and D3Cr plus β-guanidinopropionate (GPA, an inhibitor of Cr transporter), and b) labelled precursors of Guanidinoacetate (GAA) and Cr (Arginine, Arg; Glycine, Gly). Intracellular D3Cr and labelled GAA and Cr were assessed by ESI-MS/MS. Creatine transporter (CT1), L-arginine:glycine amidinotransferase (AGAT), and S-adenosylmethionine:guanidinoacetate N-methyltransferase (GAMT) gene expression was assessed in the same cells by real time PCR. D3Cr signal was extremely high in cells incubated with this isotope (labelled/unlabelled Cr ratio reached about 10 and 122, respectively in cerebellar granule cells and astrocytes) and was reduced by GPA. Labelled Arg and Gly were taken up by the cells and incorporated in GAA, whose concentration paralleled that of these precursors both in the extracellular medium and inside the cells (astrocytes). In contrast, the increase of labelled Cr was relatively much more limited since labelled Cr after precursors' supplementation did not exceed 2,7% (cerebellar granule cells) and 21% (astrocytes) of unlabelled Cr. Finally, AGAT, GAMT and SLC6A8 were expressed in both kind of cells. Our results confirm that both neurons and astrocytes have

Cerebellar Granule Cell Replenishment Post-Injury by Adaptive Reprogramming of Nestin+ Progenitors

PubMed Central

Wojcinski, Alexandre; Lawton, Andrew K.; Bayin, N Sumru.; Lao, Zhimin; Stephen, Daniel N.; Joyner, Alexandra L.

2017-01-01

Regeneration of several organs involves adaptive reprogramming of progenitors, however, the intrinsic capacity of the developing brain to replenish lost cells remains largely unknown. In this study, we discovered that the developing cerebellum has unappreciated progenitor plasticity, since it undergoes near full growth and functional recovery following acute depletion of granule cells, the most plentiful neuron population in the brain. We demonstrate that following postnatal ablation of granule cell progenitors, Nestin-expressing progenitors (NEPs) specified during mid-embryogenesis to produce astroglia and interneurons, switch their fate and generate granule neurons in mice. Moreover, Hedgehog-signaling in two NEP populations is crucial not only for the compensatory replenishment of granule neurons but also to scale interneuron and astrocyte numbers. Thus we provide insights into the mechanisms underlying robustness of circuit formation in the cerebellum, and speculate that adaptive reprogramming of progenitors in other brain regions plays a greater role than appreciated in developmental regeneration. PMID:28805814

Electrophysiological evidence for glial-subtype glutamate transporter functional expression in rat cerebellar granule neurons.

PubMed

Mafra, R A; Leão, R M; Beirão, P S L; Cruz, J S

2003-07-01

A glutamate-sensitive inward current (Iglu) is described in rat cerebellar granule neurons and related to a glutamate transport mechanism. We examined the features of Iglu using the patch-clamp technique. In steady-state conditions the Iglu measured 8.14 1.9 pA. Iglu was identified as a voltage-dependent inward current showing a strong rectification at positive potentials. L-Glutamate activated the inward current in a dose-dependent manner, with a half-maximal effect at about 18 M and a maximum increase of 51.2 4.4%. The inward current was blocked by the presence of dihydrokainate (0.5 mM), shown by others to readily block the GLT1 isoform. We thus speculate that Iglu could be attributed to the presence of a native glutamate transporter in cerebellar granule neurons.

Bax-inhibiting peptide protects glutamate-induced cerebellar granule cell death by blocking Bax translocation.

PubMed

Iriyama, Takayuki; Kamei, Yoshimasa; Kozuma, Shiro; Taketani, Yuji

2009-02-13

Glutamate-induced excitotoxicity has been implicated in the pathogenesis of various neurological damages and disorders. In the brain damage of immature animals such as neonatal hypoxic-ischemic brain injury, the excitotoxicity appears to be more intimately involved through apoptosis. Bax, a member of the Bcl-2 family proteins, plays a key role in the promotion of apoptosis by translocation from the cytosol to the mitochondria and the release of apoptogenic factors such as cytochrome c. Recently, Bax-inhibiting peptide (BIP), a novel membrane-permeable peptide which can bind Bax in the cytosol and inhibit its translocation to the mitochondria, was developed. To investigate the possibility of a new neuroprotection strategy targeting Bax translocation in glutamate-induced neuronal cell death, cerebellar granule neurons (CGNs) were exposed to glutamate with or without BIP. Pretreatment of CGNs with BIP elicited a dose-dependent reduction of glutamate-induced neuronal cell death as measured by MTT assay. BIP significantly suppressed both the number of TUNEL-positive cells and the increase in caspases 3 and 9 activities induced by glutamate. In addition, immunoblotting after subcellular fractionation revealed that BIP prevented the glutamate-induced Bax translocation to the mitochondria and the release of cytochrome c from the mitochondria. These results suggest that agents capable of inhibiting Bax activity such as BIP might lead to new drugs for glutamate-related diseases in the future.

Cell proliferation and apoptosis during histogenesis of the guinea pig and rabbit cerebellar cortex.

PubMed

Lossi, Laura; Coli, Alessandra; Giannessi, Elisabetta; Stornelli, Maria Rita; Marroni, Paolo

2002-01-01

Cell proliferation and apoptosis are essential for development of the nervous system. In this study we have investigated the histogenesis of the cerebellar cortex in guinea pig (a precocial species) and rabbit (an altricial species) at different stages of pregnancy and postnatal life. Proliferating cells were identified after labeling with antibodies against the proliferating cell nuclear antigen (PCNA) and/or the Ki-67 antigen. Apoptotic cells were visualized in situ by the TUNEL method and by immunodetection of cleaved caspase 3 and 9. In guinea pigs, both proliferating and apoptotic cells were detected during pre-natal life (E0-E40). Conversely, cell proliferation and apoptosis in rabbits were temporally restricted to early postnatal weeks (P0-P20). In both species cell proliferation was mainly linked to differentiation and migration of the granule cells. In both species, the majority of cells undergoing programmed cell death likely corresponded to granule cells. They were mainly detected in the external granular layer, and were by far more common than previously reported in other locations of the postnatal brain. This study shows that apoptosis is a shared process of cell death during cerebellar development in both altricial and precocial animals, and that there is a direct spatial and temporal correlation between cell proliferation and death in two mammals with different time tables in cerebellar maturation.

YB-1 is elevated in medulloblastoma and drives proliferation in Sonic hedgehog-dependent cerebellar granule neuron progenitor cells and medulloblastoma cells.

PubMed

Dey, A; Robitaille, M; Remke, M; Maier, C; Malhotra, A; Gregorieff, A; Wrana, J L; Taylor, M D; Angers, S; Kenney, A M

2016-08-11

Postnatal proliferation of cerebellar granule neuron precursors (CGNPs), proposed cells of origin for the SHH-associated subgroup of medulloblastoma, is driven by Sonic hedgehog (Shh) and insulin-like growth factor (IGF) in the developing cerebellum. Shh induces the oncogene Yes-associated protein (YAP), which drives IGF2 expression in CGNPs and mouse Shh-associated medulloblastomas. To determine how IGF2 expression is regulated downstream of YAP, we carried out an unbiased screen for transcriptional regulators bound to IGF2 promoters. We report that Y-box binding protein-1 (YB-1), an onco-protein regulating transcription and translation, binds to IGF2 promoter P3. We observed that YB-1 is upregulated across human medulloblastoma subclasses as well as in other varieties of pediatric brain tumors. Utilizing the cerebellar progenitor model for the Shh subgroup of medulloblastoma in mice, we show for the first time that YB-1 is induced by Shh in CGNPs. Its expression is YAP-dependent and it is required for IGF2 expression in CGNPs. Finally, both gain-of function and loss-of-function experiments reveal that YB-1 activity is required for sustaining CGNP and medulloblastoma cell (MBC) proliferation. Collectively, our findings describe a novel role for YB-1 in driving proliferation in the developing cerebellum and MBCs and they identify the SHH:YAP:YB1:IGF2 axis as a powerful target for therapeutic intervention in medulloblastomas.

Mice deficient in carbonic anhydrase type 8 exhibit motor dysfunctions and abnormal calcium dynamics in the somatic region of cerebellar granule cells.

PubMed

Lamont, Matthew G; Weber, John T

2015-06-01

The waddles (wdl) mouse is characterized by a namesake "side-to-side" waddling gait due to a homozygous mutation of the Car8 gene. This mutation results in non-functional copies of the protein carbonic anhydrase type 8. Rota-rod testing was conducted to characterize the wdl mutations' effect on motor output. Results indicated that younger homozygotes outperformed their older cohorts, an effect not seen in previous studies. Heterozygotes, which were thought to be free of motor impairment, displayed motor learning deficiencies when compared with wild type performance. Acute cerebellar slices were then utilized for fluorescent calcium imaging experiments, which revealed significant alterations in cerebellar granule cell somatic calcium signaling when exposed to glutamate. The contribution of GABAergic signaling to these alterations was also verified using bath application of bicuculline. Changes in somatic calcium signals were found to be applicable to an in vivo scenario by comparing group responses to electrical stimulation of afferent mossy fiber projections. Finally, intracellular calcium store function was also found to be altered by the wdl mutation when slices were treated with thapsigargin. These findings, taken together with previous work on the wdl mouse, indicate a widespread disruption in cerebellar circuitry hampering proper neuronal communication. Copyright © 2015 Elsevier B.V. All rights reserved.

Endogenous bax translocation in SH-SY5Y human neuroblastoma cells and cerebellar granule neurons undergoing apoptosis.

PubMed

McGinnis, K M; Gnegy, M E; Wang, K K

1999-05-01

Changes at the mitochondria are an early, required step in apoptosis in various cell types. We used western blot analysis to demonstrate that the proapoptotic protein Bax translocated from the cytosolic to the mitochondrial fraction in SH-SY5Y human neuroblastoma cells undergoing staurosporine- or EGTA-mediated apoptosis. Levels of mitochondrial Bax increased 15 min after staurosporine treatment. In EGTA-treated cells, increased levels of mitochondrial Bax were seen at 4 h, consistent with a slower onset of apoptosis in EGTA versus staurosporine treatments. We also demonstrate the concomitant translocation of cytochrome c from the mitochondrial to the cytosolic fractions. We correlated these translocations with changes in caspase-3-like activity. An increase in caspase-3-like activity was evident 2 h after staurosporine treatment. Inhibition of the mitochondrial permeability transition had no effect on Bax translocation or caspase-3-like activity in staurosporine-treated SH-SY5Y cells. In primary cultures of cerebellar granule neurons undergoing low K(+)-mediated apoptosis, Bax translocation to the mitochondrial fraction was evident at 3 h. Cytochrome c release into the cytosol was not significant until 8 h after treatment. These data support a model of apoptosis in which Bax acts directly at the mitochondria to allow the release of cytochrome c.

The effect of cyclic phosphatidic acid on the proliferation and differentiation of mouse cerebellar granule precursor cells during cerebellar development.

PubMed

Konakazawa, Misa; Gotoh, Mari; Murakami-Murofushi, Kimiko; Hamano, Ayana; Miyamoto, Yasunori

2015-07-21

The proliferation and differentiation of cerebellar granule cell precursors (GCPs) are highly regulated spatiotemporally during development. We focused on cyclic phosphatidic acid (cPA) as a lipid mediator with a cyclic phosphate group as a regulatory factor of GCPs. While its structure is similar to that of lysophosphatidic acid (LPA), its function is very unique. cPA is known to be present in the cerebellum at high levels, but its function has not been fully elucidated. In this study, we examined the role of cPA on the proliferation and differentiation of GCPs. A cell cycle analysis of GCPs revealed that cPA reduced the number of phospho-histone H3 (Phh3)-positive cells and bromodeoxy uridine (BrdU)-incorporated cells and increased an index of the cell cycle exit. We next analyzed the effect of cPA on GCP differentiation using Tuj1 as a neuronal marker of final differentiation. The results show that cPA increased the number of Tuj1-positive cells. Further analysis of the proliferation of GCPs showed that cPA suppressed Sonic hedgehog (Shh)-dependent proliferation, but did not suppress insulin-like growth factor-1 (IGF-1)-dependent proliferation. P2Y5 (LPA6), an LPA receptor, is highly expressed in GCPs. The knockdown of P2Y5 suppressed the inhibitory effect of cPA on the proliferation of GCPs, suggesting that P2Y5 is a candidate receptor for cPA. Thus, cPA suppresses the Shh-dependent proliferation of GCPs and promotes the differentiation of GCPs through P2Y5. These results demonstrate that cPA plays a critical role in the development of GCPs. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Kinetic and functional analysis of transient, persistent and resurgent sodium currents in rat cerebellar granule cells in situ: an electrophysiological and modelling study

PubMed Central

Magistretti, Jacopo; Castelli, Loretta; Forti, Lia; D'Angelo, Egidio

2006-01-01

Cerebellar neurones show complex and differentiated mechanisms of action potential generation that have been proposed to depend on peculiar properties of their voltage-dependent Na+ currents. In this study we analysed voltage-dependent Na+ currents of rat cerebellar granule cells (GCs) by performing whole-cell, patch-clamp experiments in acute rat cerebellar slices. A transient Na+ current (INaT) was always present and had the properties of a typical fast-activating/inactivating Na+ current. In addition to INaT, robust persistent (INaP) and resurgent (INaR) Na+ currents were observed. INaP peaked at ∼−40 mV, showed half-maximal activation at ∼−55 mV, and its maximal amplitude was about 1.5% of that of INaT. INaR was elicited by repolarizing pulses applied following step depolarizations able to activate/inactivate INaT, and showed voltage- and time-dependent activation and voltage-dependent decay kinetics. The conductance underlying INaR showed a bell-shaped voltage dependence, with peak at −35 mV. A significant correlation was found between GC INaR and INaT peak amplitudes; however, GCs expressing INaT of similar size showed marked variability in terms of INaR amplitude, and in a fraction of cells INaR was undetectable. INaT, INaP and INaR could be accounted for by a 13-state kinetic scheme comprising closed, open, inactivated and blocked states. Current-clamp experiments carried out to identify possible functional correlates of INaP and/or INaR revealed that in GCs single action potentials were followed by depolarizing afterpotentials (DAPs). In a majority of cells, DAPs showed properties consistent with INaR playing a role in their generation. Computer modelling showed that INaR promotes DAP generation and enhances high-frequency firing, whereas INaP boosts near-threshold firing activity. Our findings suggest that special properties of voltage-dependent Na+ currents provides GCs with mechanisms suitable for shaping activity patterns, with potentially

Single Neuron Optimization as a Basis for Accurate Biophysical Modeling: The Case of Cerebellar Granule Cells.

PubMed

Masoli, Stefano; Rizza, Martina F; Sgritta, Martina; Van Geit, Werner; Schürmann, Felix; D'Angelo, Egidio

2017-01-01

In realistic neuronal modeling, once the ionic channel complement has been defined, the maximum ionic conductance (G i-max ) values need to be tuned in order to match the firing pattern revealed by electrophysiological recordings. Recently, selection/mutation genetic algorithms have been proposed to efficiently and automatically tune these parameters. Nonetheless, since similar firing patterns can be achieved through different combinations of G i-max values, it is not clear how well these algorithms approximate the corresponding properties of real cells. Here we have evaluated the issue by exploiting a unique opportunity offered by the cerebellar granule cell (GrC), which is electrotonically compact and has therefore allowed the direct experimental measurement of ionic currents. Previous models were constructed using empirical tuning of G i-max values to match the original data set. Here, by using repetitive discharge patterns as a template, the optimization procedure yielded models that closely approximated the experimental G i-max values. These models, in addition to repetitive firing, captured additional features, including inward rectification, near-threshold oscillations, and resonance, which were not used as features. Thus, parameter optimization using genetic algorithms provided an efficient modeling strategy for reconstructing the biophysical properties of neurons and for the subsequent reconstruction of large-scale neuronal network models.

Attenuation of excitatory amino acid toxicity by metabotropic glutamate receptor agonists and aniracetam in primary cultures of cerebellar granule cells.

PubMed

Pizzi, M; Fallacara, C; Arrighi, V; Memo, M; Spano, P F

1993-08-01

Activation of glutamate ionotropic receptors represents the primary event in the neurotoxicity process triggered by excitatory amino acids. We demonstrate here that the concentration-dependent stimulation of metabotropic glutamate receptor (mGluR) by the selective agonist trans-1-aminocyclopentane-1,3-dicarboxylate or by quisqualate counteracts both glutamate- and kainate-induced neurotoxicity in primary cultures of rat cerebellar granule cells. The mGluR-evoked responses are potentiated by aniracetam, which per se also elicits neuroprotection. Aniracetam concentration-dependently counteracted glutamate-, kainate-, or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced cell death and greatly facilitated neuroprotective response achieved by different concentrations of both quisqualate and trans-1-aminocyclopentane-1,3-dicarboxylate. In addition, aniracetam potentiated the mGluR-coupled stimulation of phospholipase C, as revealed by the measurement of 3H-inositol phosphate formation. Thus, mGluRs could be a suitable target for novel pharmacological strategies pointing to the treatment of neurodegenerative diseases.

Clostridium perfringens epsilon toxin targets granule cells in the mouse cerebellum and stimulates glutamate release.

PubMed

Lonchamp, Etienne; Dupont, Jean-Luc; Wioland, Laetitia; Courjaret, Raphaël; Mbebi-Liegeois, Corinne; Jover, Emmanuel; Doussau, Frédéric; Popoff, Michel R; Bossu, Jean-Louis; de Barry, Jean; Poulain, Bernard

2010-09-30

Epsilon toxin (ET) produced by C. perfringens types B and D is a highly potent pore-forming toxin. ET-intoxicated animals express severe neurological disorders that are thought to result from the formation of vasogenic brain edemas and indirect neuronal excitotoxicity. The cerebellum is a predilection site for ET damage. ET has been proposed to bind to glial cells such as astrocytes and oligodendrocytes. However, the possibility that ET binds and attacks the neurons remains an open question. Using specific anti-ET mouse polyclonal antibodies and mouse brain slices preincubated with ET, we found that several brain structures were labeled, the cerebellum being a prominent one. In cerebellar slices, we analyzed the co-staining of ET with specific cell markers, and found that ET binds to the cell body of granule cells, oligodendrocytes, but not astrocytes or nerve endings. Identification of granule cells as neuronal ET targets was confirmed by the observation that ET induced intracellular Ca(2+) rises and glutamate release in primary cultures of granule cells. In cultured cerebellar slices, whole cell patch-clamp recordings of synaptic currents in Purkinje cells revealed that ET greatly stimulates both spontaneous excitatory and inhibitory activities. However, pharmacological dissection of these effects indicated that they were only a result of an increased granule cell firing activity and did not involve a direct action of the toxin on glutamatergic nerve terminals or inhibitory interneurons. Patch-clamp recordings of granule cell somata showed that ET causes a decrease in neuronal membrane resistance associated with pore-opening and depolarization of the neuronal membrane, which subsequently lead to the firing of the neuronal network and stimulation of glutamate release. This work demonstrates that a subset of neurons can be directly targeted by ET, suggesting that part of ET-induced neuronal damage observed in neuronal tissue is due to a direct effect of ET on

Clostridium perfringens Epsilon Toxin Targets Granule Cells in the Mouse Cerebellum and Stimulates Glutamate Release

PubMed Central

Lonchamp, Etienne; Dupont, Jean-Luc; Wioland, Laetitia; Courjaret, Raphaël; Mbebi-Liegeois, Corinne; Jover, Emmanuel; Doussau, Frédéric; Popoff, Michel R.; Bossu, Jean-Louis; de Barry, Jean; Poulain, Bernard

2010-01-01

Epsilon toxin (ET) produced by C. perfringens types B and D is a highly potent pore-forming toxin. ET-intoxicated animals express severe neurological disorders that are thought to result from the formation of vasogenic brain edemas and indirect neuronal excitotoxicity. The cerebellum is a predilection site for ET damage. ET has been proposed to bind to glial cells such as astrocytes and oligodendrocytes. However, the possibility that ET binds and attacks the neurons remains an open question. Using specific anti-ET mouse polyclonal antibodies and mouse brain slices preincubated with ET, we found that several brain structures were labeled, the cerebellum being a prominent one. In cerebellar slices, we analyzed the co-staining of ET with specific cell markers, and found that ET binds to the cell body of granule cells, oligodendrocytes, but not astrocytes or nerve endings. Identification of granule cells as neuronal ET targets was confirmed by the observation that ET induced intracellular Ca2+ rises and glutamate release in primary cultures of granule cells. In cultured cerebellar slices, whole cell patch-clamp recordings of synaptic currents in Purkinje cells revealed that ET greatly stimulates both spontaneous excitatory and inhibitory activities. However, pharmacological dissection of these effects indicated that they were only a result of an increased granule cell firing activity and did not involve a direct action of the toxin on glutamatergic nerve terminals or inhibitory interneurons. Patch-clamp recordings of granule cell somata showed that ET causes a decrease in neuronal membrane resistance associated with pore-opening and depolarization of the neuronal membrane, which subsequently lead to the firing of the neuronal network and stimulation of glutamate release. This work demonstrates that a subset of neurons can be directly targeted by ET, suggesting that part of ET-induced neuronal damage observed in neuronal tissue is due to a direct effect of ET on neurons

Abnormal cerebellar development and Purkinje cell defects in Lgl1-Pax2 conditional knockout mice.

PubMed

Hou, Congzhe; Ding, Lingcui; Zhang, Jian; Jin, Yecheng; Sun, Chen; Li, Zhenzu; Sun, Xiaoyang; Zhang, Tingting; Zhang, Aizhen; Li, Huashun; Gao, Jiangang

2014-11-01

Lgl1 was initially identified as a tumour suppressor in flies and is characterised as a key regulator of epithelial polarity and asymmetric cell division. A previous study indicated that More-Cre-mediated Lgl1 knockout mice exhibited significant brain dysplasia and died within 24h after birth. To overcome early neonatal lethality, we generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in almost all cells in the cerebellum, and we examined the functions of Lgl1 in the cerebellum. Impaired motor coordination was detected in the mutant mice. Consistent with this abnormal behaviour, homozygous mice possessed a smaller cerebellum with fewer lobes, reduced granule precursor cell (GPC) proliferation, decreased Purkinje cell (PC) quantity and dendritic dysplasia. Loss of Lgl1 in the cerebellum led to hyperproliferation and impaired differentiation of neural progenitors in ventricular zone. Based on the TUNEL assay, we observed increased apoptosis in the cerebellum of mutant mice. We proposed that impaired differentiation and increased apoptosis may contribute to decreased PC quantity. To clarify the effect of Lgl1 on cerebellar granule cells, we used Math1-Cre to specifically delete Lgl1 in granule cells. Interestingly, the Lgl1-Math1 conditional knockout mice exhibited normal proliferation of GPCs and cerebellar development. Thus, we speculated that the reduction in the proliferation of GPCs in Lgl1-Pax2 conditional knockout mice may be secondary to the decreased number of PCs, which secrete the mitogenic factor Sonic hedgehog to regulate GPC proliferation. Taken together, these findings suggest that Lgl1 plays a key role in cerebellar development and folia formation by regulating the development of PCs. Copyright © 2014. Published by Elsevier Inc.

Neurotoxicity of amphetamine derivatives is mediated by caspase pathway activation in rat cerebellar granule cells.

PubMed

Jiménez, Andrés; Jordà, Elvira G; Verdaguer, Ester; Pubill, David; Sureda, Francesc X; Canudas, Anna M; Escubedo, Elena; Camarasa, Jordi; Camins, Antoni; Pallàs, Mercè

2004-04-15

The neurotoxic action of the abuse drugs methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA) on cerebellar granule neurones (CGNs) culture was examined. Treatment for 48 h with METH or MDMA (1-5 mM) induced a higher decrease in viability than 24 h treatment. z.VAD.fmk (100 microM) but not MK-801 nor NBQX recovered control viability values. In both cases, cell death was characterised as apoptotic rather than necrotic by morphology cell observation. Apoptosis measured by flow cytometry indicated an increase in the hypodiploid population after 48 h treatment with METH and MDMA. Apoptosis was reverted by the presence of z.VAD.fmk (100 microM) but not by 10 microM MK-801 or NBQX. Similar results were obtained by analysing nuclear chromatine condensation. These results ruled out excitotoxic participation in amphetamine derivative-induced neurotoxicity in CGNs. Participation of radical oxygen species (ROS) was evaluated using alpha-tocopherol (1-15 microM) and cytometric studies. The co-treatment with 4 mM METH or MDMA for 48 h partially reverted neurotoxic action and apoptotic features, indicating ROS implication in CGNs death by amphetamine derivatives. Alteration of mitochondrial function induced cytochrome C (Cyt C) release after 48-h treatment with METH and MDMA (4 mM). There was also indication of caspase-3-like activation, measured by immunoanalysis and biochemically. Finally, neurodegenerative action caused by amphetamine derivatives may be prevented by using caspase inhibitors.

Hydroxylated polychlorinated biphenyls increase reactive oxygen species formation and induce cell death in cultured cerebellar granule cells

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

Dreiem, Anne; Norwegian Defence Research Establishment, Department for Protection, Kjeller; Rykken, Sidsel

2009-10-15

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that bioaccumulate in the body, however, they can be metabolized to more water-soluble products. Although they are more readily excreted than the parent compounds, some of the metabolites are still hydrophobic and may be more available to target tissues, such as the brain. They can also cross the placenta and reach a developing foetus. Much less is known about the toxicity of PCB metabolites than about the parent compounds. In the present study, we have investigated the effects of eight hydroxylated (OH) PCB congeners (2'-OH PCB 3, 4-OH PCB 14, 4-OH PCB 34,more » 4'-OH PCB 35, 4-OH PCB 36, 4'-OH PCB 36, 4-OH PCB 39, and 4'-OH PCB 68) on reactive oxygen species (ROS) formation and cell viability in rat cerebellar granule cells. We found that, similar to their parent compounds, OH-PCBs are potent ROS inducers with potency 4-OH PCB 14 < 4-OH PCB 36 < 4-OH PCB 34 < 4'-OH PCB 36 < 4'-OH PCB 68 < 4-OH PCB 39 < 4'-OH PCB 35. 4-OH PCB 36 was the most potent cell death inducer, and caused apoptotic or necrotic morphology depending on concentration. Inhibition of ERK1/2 kinase with U0126 reduced both cell death and ROS formation, suggesting that ERK1/2 activation is involved in OH-PCB toxicity. The results indicate that the hydroxylation of PCBs may not constitute a detoxification reaction. Since OH-PCBs like their parent compounds are retained in the body and may be more widely distributed to sensitive tissues, it is important that not only the levels of the parent compounds but also the levels of their metabolites are taken into account during risk assessment of PCBs and related compounds.« less

The Cellular State Determines the Effect of Melatonin on the Survival of Mixed Cerebellar Cell Culture

PubMed Central

Franco, Daiane Gil; Markus, Regina P.

2014-01-01

The constitutive activation of nuclear factor-κB (NF-κB), a key transcription factor involved in neuroinflammation, is essential for the survival of neurons in situ and of cerebellar granule cells in culture. Melatonin is known to inhibit the activation of NF-κB and has a cytoprotective function. In this study, we evaluated whether the cytoprotective effect of melatonin depends on the state of activation of a mixed cerebellar culture that is composed predominantly of granule cells; we tested the effect of melatonin on cultured rat cerebellar cells stimulated or not with lipopolysaccharide (LPS). The addition of melatonin (0.1 nM–1 µM) reduced the survival of naïve cells while inhibiting LPS-induced cell death. Melatonin (100 nM) transiently (15 min) inhibited the nuclear translocation of both NF-κB dimers (p50/p50, p50/RelA) and, after 60 min, increased the activation of p50/RelA. Melatonin-induced p50/RelA activity in naïve cells resulted in the transcription of inducible nitric oxide synthase (iNOS) and the production of NO. Otherwise, in cultures treated with LPS, melatonin blocked the LPS-induced activation of p50/RelA and the reduction in p50/p50 levels and inhibited iNOS expression and NO synthesis. Therefore, melatonin in vehicle-treated cells induces cell death, while it protects against LPS-induced cytotoxicity. In summary, we confirmed that melatonin is a neuroprotective drug when cerebellar cells are challenged; however, melatonin can also lead to cell death when the normal balance of the NF-κB pathway is disturbed. Our data provide a mechanistic basis for understanding the influence of cell context on the final output response of melatonin. PMID:25184316

Pseudomonas fluorescens lipopolysaccharide inhibits both delayed rectifier and transient A-type K+ channels of cultured rat cerebellar granule neurons.

PubMed

Mezghani-Abdelmoula, Sana; Chevalier, Sylvie; Lesouhaitier, Olivier; Orange, Nicole; Feuilloley, Marc G J; Cazin, Lionel

2003-09-05

Pseudomonas fluorescens is a Gram-negative bacillus closely related to the pathogen P. aeruginosa known to provoke infectious disorders in the central nervous system (CNS). The endotoxin lipopolysaccharide (LPS) expressed by the bacteria is the first infectious factor that can interact with the plasma membrane of host cells. In the present study, LPS extracted from P. fluorescens MF37 was examined for its actions on delayed rectifier and A-type K(+) channels, two of the main types of voltage-activated K(+) channels involved in the action potential firing. Current recordings were performed in cultured rat cerebellar granule neurons at days 7 or 8, using the whole-cell patch-clamp technique. A 3-h incubation with LPS (200 ng/ml) markedly depressed both the delayed rectifier (I(KV)) and transient A-type (I(A)) K(+) currents evoked by depolarizations above 0 and -40 mV, respectively. The percent decrease of I(KV) and I(A) ( approximately 30%) did not vary with membrane potential, suggesting that inhibition of both types of K(+) channels by LPS was voltage-insensitive. The endotoxin did neither modify the steady-state voltage-dependent activation properties of I(KV) and I(A) nor the steady-state inactivation of I(A). The present results suggest that, by inhibiting I(KV) and I(A), LPS applied extracellulary increases the action potential firing in cerebellar granule neurons. It is concluded that P. fluorescens MF37 may provoke in the CNS disorders associated with sever alterations of membrane ionic channel functions.

4-aminopyridine, a Kv channel antagonist, prevents apoptosis of rat cerebellar granule neurons.

PubMed

Hu, Chang-Long; Liu, Zheng; Zeng, Xi-Min; Liu, Zi-Qiang; Chen, Xian-Hua; Zhang, Zhi-Hong; Mei, Yan-Ai

2006-09-01

Compelling evidence indicates that excessive potassium (K+) efflux and intracellular K+ depletion are the key early steps in apoptosis. Previously, we reported that apoptosis of cerebellar granule neurons induced by incubation in low-K+ (5 mM) and serum-free medium was associated with an increase in A-type transient inactivation of K+ channel current (IA) amplitude and modulation of channels' gating properties. Here, we showed that a classic K+ channel blocker, 4-aminopyradine (4-AP), significantly inhibited IA amplitude in a concentration-dependent manner (reduction of current by 10 microM and 10 mM 4-AP was 11.4+/-1.3% and 72.2+/-3.3%, respectively). Moreover, 4-AP modified the steady-state activation and inactivation kinetics of IA channels, such that the activation and inactivation curves were shifted to the right about 20 mV and 17 mV, respectively. Fluorescence staining showed that 4-AP dramatically increased the viability of cells undergoing apoptosis in a dose-dependent manner. That is, while 5 mM 4-AP was present, cell viability was 84.9+/-5.2%. Consistent with the cell viability analysis, internucleosomal DNA fragmentation by gel electrophoresis analysis showed that 5 mM 4-AP also protected against neuronal apoptosis. Furthermore, 4-AP significantly inhibited cytochrome c release and caspase-3 activity induced by low-K+/serum-free incubation. Finally, current-clamp analysis indicated that 5 mM 4-AP did not significantly depolarize the membrane potential. These results suggest that 4-AP has robust neuroprotective effects on apoptotic granule cells. The neuroprotective effect of 4-AP is likely not due to membrane depolarization, but rather that 4-AP may modulate the gating properties of IA channels in an anti-apoptotic manner.

The Changeable Nervous System: Studies On Neuroplasticity In Cerebellar Cultures

PubMed Central

Seil, Fredrick J.

2014-01-01

Circuit reorganization after injury was studied in a cerebellar culture model. When cerebellar cultures derived from newborn mice were exposed at explantation to a preparation of cytosine arabinoside that destroyed granule cells and oligodendrocytes and compromised astrocytes, Purkinje cells surviving in greater than usual numbers were unensheathed by astrocytic processes and received twice the control number of inhibitory axosomatic synapses. Purkinje cell axon collaterals sprouted and many of their terminals formed heterotypical synapses with other Purkinje cell dendritic spines. The resulting circuit reorganization preserved inhibition in the cerebellar cortex. Following this reorganization, replacement of the missing granule cells and glia was followed by a restitution of the normal circuitry. Most of these developmental and reconstructive changes were not dependent on neuronal activity, the major exception being inhibitory synaptogenesis. The full complement of inhibitory synapses did not develop in the absence of neuronal activity, which could be mitigated by application of exogenous TrkB receptor ligands. Inhibitory synaptogenesis could also be promoted by activity-induced release of endogenous TrkB receptor ligands or by antibody activation of the TrkB receptor. PMID:24933693

Oligodendrocyte ablation affects the coordinated interaction between granule and Purkinje neurons during cerebellum development

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

Collin, Ludovic; Doretto, Sandrine; Department of Psychiatry and Human Behavior, University of California Irvine, 3226 Gillespie Neuroscience Research Facility, Irvine CA 92697

2007-08-01

Oligodendrocytes (OLs) are the glial cells of the central nervous system (CNS) classically known to be devoted to the formation of myelin sheaths around most axons of the vertebrate brain. We have addressed the role of these cells during cerebellar development, by ablating OLs in vivo. Previous analyses had indicated that OL ablation during the first six postnatal days results into a striking cerebellar phenotype, whose major features are a strong reduction of granule neurons and aberrant Purkinje cells development. These two cell types are highly interconnected during cerebellar development through the production of molecules that help their proliferation, differentiationmore » and maintenance. In this article, we present data showing that OL ablation has major effects on the physiology of Purkinje (PC) and granule cells (GC). In particular, OL ablation results into a reduction of sonic hedgehog (Shh), Brain Derived Neurotrophic Factor (BDNF), and Reelin (Rln) expression. These results indicate that absence of OLs profoundly alters the normal cerebellar developmental program.« less

The organization of plasticity in the cerebellar cortex: from synapses to control.

PubMed

D'Angelo, Egidio

2014-01-01

The cerebellum is thought to play a critical role in procedural learning, but the relationship between this function and the underlying cellular and synaptic mechanisms remains largely speculative. At present, at least nine forms of long-term synaptic and nonsynaptic plasticity (some of which are bidirectional) have been reported in the cerebellar cortex and deep cerebellar nuclei. These include long-term potentiation (LTP) and long-term depression at the mossy fiber-granule cell synapse, at the synapses formed by parallel fibers, climbing fibers, and molecular layer interneurons on Purkinje cells, and at the synapses formed by mossy fibers and Purkinje cells on deep cerebellar nuclear cells, as well as LTP of intrinsic excitability in granule cells, Purkinje cells, and deep cerebellar nuclear cells. It is suggested that the complex properties of cerebellar learning would emerge from the distribution of plasticity in the network and from its dynamic remodeling during the different phases of learning. Intrinsic and extrinsic factors may hold the key to explain how the different forms of plasticity cooperate to select specific transmission channels and to regulate the signal-to-noise ratio through the cerebellar cortex. These factors include regulation of neuronal excitation by local inhibitory networks, engagement of specific molecular mechanisms by spike bursts and theta-frequency oscillations, and gating by external neuromodulators. Therefore, a new and more complex view of cerebellar plasticity is emerging with respect to that predicted by the original "Motor Learning Theory," opening issues that will require experimental and computational testing. © 2014 Elsevier B.V. All rights reserved.

Cannabinoid Type 1 Receptors Transiently Silence Glutamatergic Nerve Terminals of Cultured Cerebellar Granule Cells

PubMed Central

Ramírez-Franco, Jorge; Bartolomé-Martín, David; Alonso, Beatris; Torres, Magdalena; Sánchez-Prieto, José

2014-01-01

Cannabinoid receptors are the most abundant G protein-coupled receptors in the brain and they mediate retrograde short-term inhibition of neurotransmitter release, as well as long-term depression of synaptic transmission at many excitatory synapses. The induction of presynaptically silent synapses is a means of modulating synaptic strength, which is important for synaptic plasticity. Persistent activation of cannabinoid type 1 receptors (CB1Rs) mutes GABAergic terminals, although it is unclear if CB1Rs can also induce silencing at glutamatergic synapses. Cerebellar granule cells were transfected with VGLUT1-pHluorin to visualise the exo-endocytotic cycle. We found that prolonged stimulation (10 min) of cannabinoid receptors with the agonist HU-210 induces the silencing of previously active synapses. However, the presynaptic silencing induced by HU-210 is transient as it reverses after 20 min. cAMP with forskolin prevented CB1R-induced synaptic silencing, via activation of the Exchange Protein directly Activated by cAMP (Epac). Furthermore, Epac activation accelerated awakening of already silent boutons. Electron microscopy revealed that silencing was associated with synaptic vesicle (SV) redistribution within the nerve terminal, which diminished the number of vesicles close to the active zone of the plasma membrane. Finally, by combining functional and immunocytochemical approaches, we observed a strong correlation between the release capacity of the nerve terminals and RIM1α protein content, but not that of Munc13-1 protein. These results suggest that prolonged stimulation of cannabinoid receptors can transiently silence glutamatergic nerve terminals. PMID:24533119

Acquisition of granule neuron precursor identity is a critical determinant of progenitor cell competence to form Hedgehog-induced medulloblastoma

PubMed Central

Schüller, Ulrich; Heine, Vivi M.; Mao, Junhao; Kho, Alvin T.; Dillon, Allison K.; Han, Young-Goo; Huillard, Emmanuelle; Sun, Tao; Ligon, Azra H.; Qian, Ying; Ma, Qiufu; Alvarez-Buylla, Arturo; McMahon, Andrew P.; Rowitch, David H.; Ligon, Keith L.

2008-01-01

Origins of the brain tumor, medulloblastoma, from stem cells or restricted progenitor cells are unclear. To investigate this, we activated oncogenic Hedgehog (Hh) signaling in multipotent and lineage-restricted CNS progenitors. We observed that normal unipotent cerebellar granule neuron precursors (CGNP) derive from hGFAP+ and Olig2+ RL progenitors. Hh activation in a spectrum of early and late stage CNS progenitors generated similar medulloblastomas, but not other brain cancers, indicating that acquisition of CGNP identity is essential for tumorigenesis. We show in human and mouse medulloblastoma that cells expressing the glia-associated markers Gfap and Olig2 are neoplastic and that they retain features of embryonic-type granule lineage progenitors. Thus, oncogenic Hh signaling promotes medulloblastoma from lineage-restricted granule cell progenitors. PMID:18691547

Mefenamic acid bi-directionally modulates the transient outward K{sup +} current in rat cerebellar granule cells

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

Zhang Man; Shi Wenjie; Fei Xiaowei

2008-02-01

The effect of non-steroidal anti-inflammatory drugs (NSAIDs) on ion channels has been widely studied in several cell models, but less is known about their modulatory mechanisms. In this report, the effect of mefenamic acid on voltage-activated transient outward K{sup +} current (I{sub A}) in cultured rat cerebellar granule cells was investigated. At a concentration of 5 {mu}M to 100 {mu}M, mefenamic acid reversibly inhibited I{sub A} in a dose-dependent manner. However, mefenamic acid at a concentration of 1 {mu}M significantly increased the amplitude of I{sub A} to 113 {+-} 1.5% of the control. At more than 10 {mu}M, mefenamic acidmore » inhibited the amplitude of I{sub A} without any effect on activation or inactivation. In addition, a higher concentration of mefenamic acid induced a significant acceleration of recovery from inactivation with an increase of the peak amplitude elicited by the second test pulse. Intracellular application of mefenamic acid could significantly increase the amplitude of I{sub A}, but had no effect on the inhibition induced by extracellular mefenamic acid, implying that mefenamic acid may exert its effect from both inside and outside the ion channel. Furthermore, the activation of current induced by intracellular application of mefenamic acid was mimicked by other cyclooxygenase inhibitors and arachidonic acid. Our data demonstrate that mefenamic acid is able to bi-directionally modulate I{sub A} channels in neurons at different concentrations and by different methods of application, and two different mechanisms may be involved.« less

Comparative sensitivity of rat cerebellar neurons to dysregulation of divalent cation homeostasis and cytotoxicity caused by methylmercury

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

Edwards, Joshua R.; Marty, M. Sue; Atchison, William D.

2005-11-01

The objective of the present study was to determine the relative effectiveness of methylmercury (MeHg) to alter divalent cation homeostasis and cause cell death in MeHg-resistant cerebellar Purkinje and MeHg-sensitive granule neurons. Application of 0.5-5 {mu}M MeHg to Purkinje and granule cells grown in culture caused a concentration- and time-dependent biphasic increase in fura-2 fluorescence. At 0.5 and 1 {mu}M MeHg, the elevations of fura-2 fluorescence induced by MeHg were biphasic in both cell types, but significantly delayed in Purkinje as compared to granule cells. Application of the heavy-metal chelator, TPEN, to Purkinje cells caused a precipitous decline in amore » proportion of the fura-2 fluorescence signal, indicating that MeHg causes release of Ca{sup 2+} and non-Ca{sup 2+} divalent cations. Purkinje cells were also more resistant than granule cells to the neurotoxic effects of MeHg. At 24.5 h after-application of 5 {mu}M MeHg, 97.7% of Purkinje cells were viable. At 3 {mu}M MeHg there was no detectable loss of Purkinje cell viability. In contrast, only 40.6% of cerebellar granule cells were alive 24.5 h after application of 3 {mu}M MeHg. In conclusion, Purkinje neurons in primary cultures appear to be more resistant to MeHg-induced dysregulation of divalent cation homeostasis and subsequent cell death when compared to cerebellar granule cells. There is a significant component of non-Ca{sup 2+} divalent cation released by MeHg in Purkinje neurons.« less

The effect of methylmercury exposure on behavior and cerebellar granule cell physiology in aged mice.

PubMed

Bellum, Sairam; Thuett, Kerry A; Bawa, Bhupinder; Abbott, Louise C

2013-09-01

Epidemiology studies have clearly documented that the central nervous system is highly susceptible to methylmercury toxicity, and exposure to this neurotoxicant in humans primarily results from consumption of contaminated fish. While the effects of methylmercury exposure have been studied in great detail, comparatively little is known about the effects of moderate to low dose methylmercury toxicity in the aging central nervous system. We examined the toxic effects of a moderate dose of methylmercury on the aging mouse cerebellum. Male and female C57BL/6 mice at 16-20 months of age were exposed to methylmercury by feeding a total dose of 5.0 mg kg(-1) body weight and assessed using four behavioral tests. Methylmercury-treated aged mice performed significantly worse in open field, footprint analysis and the vertical pole test compared with age-matched control mice. Isolated cerebellar granule cells from methylmercury-treated aged mice exhibited higher levels of reactive oxygen species and reduced mitochondrial membrane potentials, but no differences in basal intracellular calcium ion levels compared with age-matched control mice. When aged mice were exposed to a moderate dose of methylmercury, they exhibited a similar degree of impairment when compared with young adult mice exposed to the same moderate dose of methylmercury, as reported in earlier studies from this laboratory. Thus, at least in mice, exposure of the aged brain to moderate concentrations methylmercury does not pose greater risk compared with the young adult brain exposed to similar concentrations of methylmercury. Copyright © 2012 John Wiley & Sons, Ltd.

Leading-process actomyosin coordinates organelle positioning and adhesion receptor dynamics in radially migrating cerebellar granule neurons

DOE PAGES

Trivedi, Niraj; Ramahi, Joseph S.; Karakaya, Mahmut; ...

2014-12-02

During brain development, neurons migrate from germinal zones to their final positions to assemble neural circuits. A unique saltatory cadence involving cyclical organelle movement (e.g., centrosome motility) and leading-process actomyosin enrichment prior to nucleokinesis organizes neuronal migration. While functional evidence suggests that leading-process actomyosin is essential for centrosome motility, the role of the actin-enriched leading process in globally organizing organelle transport or traction forces remains unexplored. Our results show that myosin ii motors and F-actin dynamics are required for Golgi apparatus positioning before nucleokinesis in cerebellar granule neurons (CGNs) migrating along glial fibers. Moreover, we show that primary cilia aremore » motile organelles, localized to the leading-process F-actin-rich domain and immobilized by pharmacological inhibition of myosin ii and F-actin dynamics. Finally, leading process adhesion dynamics are dependent on myosin ii and F-actin. In conclusion, we propose that actomyosin coordinates the overall polarity of migrating CGNs by controlling asymmetric organelle positioning and cell-cell contacts as these cells move along their glial guides.« less

Leading-process actomyosin coordinates organelle positioning and adhesion receptor dynamics in radially migrating cerebellar granule neurons

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

Trivedi, Niraj; Ramahi, Joseph S.; Karakaya, Mahmut

During brain development, neurons migrate from germinal zones to their final positions to assemble neural circuits. A unique saltatory cadence involving cyclical organelle movement (e.g., centrosome motility) and leading-process actomyosin enrichment prior to nucleokinesis organizes neuronal migration. While functional evidence suggests that leading-process actomyosin is essential for centrosome motility, the role of the actin-enriched leading process in globally organizing organelle transport or traction forces remains unexplored. Our results show that myosin ii motors and F-actin dynamics are required for Golgi apparatus positioning before nucleokinesis in cerebellar granule neurons (CGNs) migrating along glial fibers. Moreover, we show that primary cilia aremore » motile organelles, localized to the leading-process F-actin-rich domain and immobilized by pharmacological inhibition of myosin ii and F-actin dynamics. Finally, leading process adhesion dynamics are dependent on myosin ii and F-actin. In conclusion, we propose that actomyosin coordinates the overall polarity of migrating CGNs by controlling asymmetric organelle positioning and cell-cell contacts as these cells move along their glial guides.« less

Postnatal Migration of Cerebellar Interneurons

PubMed Central

Galas, Ludovic; Bénard, Magalie; Lebon, Alexis; Komuro, Yutaro; Schapman, Damien; Vaudry, Hubert; Vaudry, David; Komuro, Hitoshi

2017-01-01

Due to its continuing development after birth, the cerebellum represents a unique model for studying the postnatal orchestration of interneuron migration. The combination of fluorescent labeling and ex/in vivo imaging revealed a cellular highway network within cerebellar cortical layers (the external granular layer, the molecular layer, the Purkinje cell layer, and the internal granular layer). During the first two postnatal weeks, saltatory movements, transient stop phases, cell-cell interaction/contact, and degradation of the extracellular matrix mark out the route of cerebellar interneurons, notably granule cells and basket/stellate cells, to their final location. In addition, cortical-layer specific regulatory factors such as neuropeptides (pituitary adenylate cyclase-activating polypeptide (PACAP), somatostatin) or proteins (tissue-type plasminogen activator (tPA), insulin growth factor-1 (IGF-1)) have been shown to inhibit or stimulate the migratory process of interneurons. These factors show further complexity because somatostatin, PACAP, or tPA have opposite or no effect on interneuron migration depending on which layer or cell type they act upon. External factors originating from environmental conditions (light stimuli, pollutants), nutrients or drug of abuse (alcohol) also alter normal cell migration, leading to cerebellar disorders. PMID:28587295

Exposure to 50 Hz electromagnetic radiation promote early maturation and differentiation in newborn rat cerebellar granule neurons.

PubMed

Lisi, A; Ciotti, M T; Ledda, M; Pieri, M; Zona, C; Mercanti, D; Rieti, S; Giuliani, L; Grimaldi, S

2005-08-01

The wish of this work is the study of the effect of electromagnetic (EMF) radiations at a frequency of 50 Hz on the development of cerebellar granule neurons (CGN). Granule neurons, prepared from newborn rat cerebellum (8 days after birth), were cultured after plate-seeding in the presence of EMF radiations, with the plan of characterizing their cellular and molecular biochemistry, after exposure to the electromagnetic stimulus. Five days challenge to EMF radiations showed, by the cytotoxic glutamate (Glu) pulse test, a 30% decrease of cells survival, while only 5% of mortality was reported for unexposed sample. Moreover, blocking the glutamate receptor (GluR) with the Glu competitor MK-801, no toxicity effect after CGN challenge to EMF radiations and Glu was detected. By patch-clamp recording technique, the Kainate-induced currents from 6 days old exposed CGN exhibited a significant increase with respect to control cells. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses show that EMF exposure of rats CGN, induces a change in both GluRs proteins and mRNAs expression with respect to control. In addition, the use of monoclonal antibody raised against neurofilament protein (NF-200) reveals an increase in NF-200 synthesis in the exposed CGN. All these results indicate that exposure to non-ionizing radiations contribute to a premature expression of GluRs reducing the life span of CGN, leading to a more rapid cell maturation. (c) 2005 Wiley-Liss, Inc.

Cell Signaling and Neurotoxicity: 3H-Arachidonic acid release (Phospholipase A2) in cerebellar granule neurons

EPA Science Inventory

Cell signaling is a complex process which controls basic cellular activities and coordinates actions to maintain normal cellular homeostasis. Alterations in signaling processes have been associated with neurological diseases such as Alzheimer's and cerebellar ataxia, as well as, ...

Local and long-range circuit elements for cerebellar function.

PubMed

Xiao, Le; Scheiffele, Peter

2018-02-01

The view of cerebellar functions has been extended from controlling sensorimotor processes to processing 'contextual' information and generating predictions for a diverse range of behaviors. These functions rely on the computation of the local cerebellar microcircuits and long-range connectivity that relays cerebellar output to various brain areas. In this review, we discuss recent work on two of the circuit elements, which are thought to be fundamental for a wide range of non-sensorimotor behaviors: The role for cerebellar granule cells in multimodal integration in the cerebellar cortex and the long-range connectivity between the deep cerebellar nuclei and the basal ganglia. Lastly, we discuss how studies on synapses and circuits of the cerebellum in rodent models of autism-spectrum disorders might contribute to our understanding of the pathophysiology of this class of neurodevelopmental disorders. Copyright © 2017. Published by Elsevier Ltd.

Evidence of two populations of GABA(A) receptors in cerebellar granule cells in culture: different desensitization kinetics, pharmacology, serine/threonine kinase sensitivity, and localization.

PubMed

Robello, M; Amico, C; Cupello, A

1999-12-20

GABA(A) receptors of rat cerebellar granule cells in culture have been studied by the whole cell patch clamp technique. The biphasic desensitization kinetic observed could be due either to different desensitization mechanisms of a single receptor population or to different receptor populations. The overall data indicate that the latter hypothesis is most probably the correct one. In fact, the fast desensitizing component was selectively potentiated by a benzodiazepine agonist and preferentially down-regulated by activation of the protein serine/threonine kinases A and G, as a consequence of the latter characteristic that receptor population was preferentially down-regulated by previous activation of N-methyl-d-aspartate glutamate receptors, via production of nitric oxide and PKG activation, most probably in dendrites. The other population is benzodiazepine insensitive and not influenced by activation of PKA or PKG. This slowly desensitizing population may correspond to the extrasynaptic delta subunit containing GABA(A) receptors described by other authors. Instead, the rapidly desensitizing population appears to represent dendritic synaptic GABA(A) receptors. Copyright 1999 Academic Press.

Tactile responses in the granule cell layer of cerebellar folium crus IIa of freely behaving rats

NASA Technical Reports Server (NTRS)

Hartmann, M. J.; Bower, J. M.

2001-01-01

We recorded activity from the granule cell layer (GCL) of cerebellar folium Crus IIa as freely moving rats engaged in a variety of natural behaviors, including grooming, eating, and free tactile exploration. Multiunit responses in the 1000-4500 Hz range were found to be strongly correlated with tactile stimulation of lip and whisker (perioral) regions. These responses occurred regardless of whether the stimulus was externally or self-generated and during both active and passive touch. In contrast, perioral movements that did not tactually stimulate this region of the face (e.g., chewing) produced no detectable increases in GCL activity. In addition, GCL responses were not correlated with movement extremes. When rats used their lips actively for palpation and exploration, the tactile responses in the GCL were not detectably modulated by ongoing jaw movements. However, active palpation and exploratory behaviors did result in the largest and most continuous bursts of GCL activity: responses were on average 10% larger and 50% longer during palpation and exploration than during grooming or passive stimulation. Although activity levels differed between behaviors, the position and spatial extent of the peripheral receptive field was similar over all behaviors that resulted in tactile input. Overall, our data suggest that the 1000-4500 Hz multiunit responses in the Crus IIa GCL of awake rats are correlated with tactile input rather than with movement or any movement parameter and that these responses are likely to be of particular importance during the acquisition of sensory information by perioral structures.

Nicotinic receptor abnormalities in the cerebellar cortex in autism.

PubMed

Lee, M; Martin-Ruiz, C; Graham, A; Court, J; Jaros, E; Perry, R; Iversen, P; Bauman, M; Perry, E

2002-07-01

Autism is a common developmental disorder associated with structural and inferred neurochemical abnormalities of the brain. Cerebellar abnormalities frequently have been identified, based on neuroimaging or neuropathology. Recently, the cholinergic neurotransmitter system has been implicated on the basis of nicotinic receptor loss in the cerebral cortex. Cerebellar cholinergic activities were therefore investigated in autopsy tissue from a series of autistic individuals. The presynaptic cholinergic enzyme, choline acetyltransferase, together with nicotinic and muscarinic receptor subtypes were compared in the cerebellum from age-matched mentally retarded autistic (eight), normal control (10) and non-autistic mentally retarded individuals (11). The nicotinic receptor binding the agonist epibatidine (the high affinity receptor subtype, consisting primarily of alpha3 and alpha4, together with beta2 receptor subunits) was significantly reduced by 40-50% in the granule cell, Purkinje and molecular layers in the autistic compared with the normal group (P < 0.05). There was an opposite increase (3-fold) in the nicotinic receptor binding alpha-bungarotoxin (to the alpha7 subunit) which reached significance in the granule cell layer (P < 0.05). These receptor changes were paralleled by a significant reduction (P < 0.05) and non-significant increase, respectively, of alpha4 and alpha7 receptor subunit immunoreactivity measured using western blotting. Immunohistochemically loss of alpha(4 )reactivity was apparent from Purkinje and the other cell layers, with increased alpha7 reactivity in the granule cell layer. There were no significant changes in choline acetyltransferase activity, or in muscarinic M1 and M2 receptor subtypes in autism. In the non-autistic mentally retarded group, the only significant abnormality was a reduction in epibatidine binding in the granule cell and Purkinje layers. In two autistic cases examined histologically, Purkinje cell loss was observed in

Gestational lead exposure induces developmental abnormalities and up-regulates apoptosis of fetal cerebellar cells in rats.

PubMed

Mousa, Alyaa M; Al-Fadhli, Ameera S; Rao, Muddanna S; Kilarkaje, Narayana

2015-01-01

Lead (Pb), a known environmental toxicant, adversely affects almost all organ systems. In this study, we investigated the effects of maternal lead exposure on fetal rat cerebellum. Female Sprague-Dawley rats were given lead nitrate in drinking water (0, 0.5, and 1%) for two weeks before conception, and during pregnancy. Fetuses were collected by caesarian section on gestational day 21 and observed for developmental abnormalities. The fetal cerebellar sections from control and 1% lead group were stained with cresyl violet. Immunohistochemical expressions of p53, Bax, Bcl-2, and caspase 3 were quantified by AnalySIS image analyzer (Life Science, Germany). Lead exposure induced developmental abnormalities of eyes, ear, limbs, neck and ventral abdominal wall; however, these abnormalities were commonly seen in the 1% lead-treated group. In addition, lead also caused fetal mortality and reduced body growth in both dose groups and reduced brain weight in the 1% lead-treated group. The fetal cerebella from the 1% lead-treated group showed unorganized cerebellar cortical layers, and degenerative changes in granule and Purkinje cells such as the formation of clumps of Nissl granules. An increase in Bax and caspase 3, and a decrease in Bcl-2 (p < 0.05), but not in p53, showed apoptosis of the neurons. In conclusion, gestational lead exposure in rats induces fetal toxicity and developmental abnormalities. The lead exposure also impairs development of cerebellar layers, induces structural changes, and apoptosis in the fetal cerebellar cortex. These results suggest that lead exposure during gestation is extremely toxic to developing cerebellum in rats.

Frequency-dependent actions of benzodiazepines on GABAA receptors in cultured murine cerebellar granule cells.

PubMed Central

Mellor, J R; Randall, A D

1997-01-01

1. Miniature IPSCs recorded from cultured murine cerebellar granule cells increased in half-width and amplitude following application of the benzodiazepine (BDZ) Flunitrazepam (Flu, 1 microM). The increase in the half-width was much greater than that in the amplitude. 2. Five-millisecond applications of 1 mM GABA to nucleated outside-out patches elicited rapidly rising biexponentially decaying responses that resembled IPSCs. Flu had no effect on the amplitude of such responses, but consistently slowed their deactivation by approximately 50%. This effect was reversed by Flu washout or application of the BDZ antagonist Ro15-1788. The partial inverse agonist. Ro15-4513 speeded deactivation and depressed peak current amplitude by 23 +/- 12%. 3. The EC50 for GABA was between 45 and 50 microM. At submaximally effective agonist concentrations, Flu increased response amplitude and slowed response deactivation. Both effects were present in all cells taken from young cultures (4-7 days in vitro) but the latter was absent in 55% of the neurones obtained from older cultures (14-27 days in vitro). 4. With 120 ms applications of 20 microM GABA, responses activated monoexponentially (time constant, 39.8 +/- 2.8 ms) and deactivated biexponentially (time constants, 40.4 +/- 2.1 and 251 +/- 15 ms). Application of Flu slowed both activation and deactivation. The latter effect arose from an increased contribution of the slower component of decay. 5. Desensitization of responses to 1 mM GABA was biexponential, with time constants of 47 +/- 11 and 479 +/- 49 ms. Flu speeded desensitization by decreasing both fast and slow time constants. GABAA receptor desensitization consistently slowed subsequent deactivation. No significant relationship between the level of desensitization and the amount of slowing of deactivation produced by Flu was found. 6. Responses to paired 5 ms applications of 1 mM GABA indicated that the slowing of deactivation and the speeding of desensitization produced by

Inhibition of glutamate-induced intensification of free radical reactions by gangliosides: possible role in their protective effect in rat cerebellar granule cells and brain synaptosomes.

PubMed

Avrova, N F; Victorov, I V; Tyurin, V A; Zakharova, I O; Sokolova, T V; Andreeva, N A; Stelmaschuk, E V; Tyurina, Y Y; Gonchar, V S

1998-07-01

The neurotoxic effect of exposure of rat cerebellar granule cells to glutamate (100 microM) is to a large extent prevented by incubation of neurons not only with micromolar, but even with nanomolar concentrations of gangliosides GM1, GD1b, and GT1b. GM1 was also shown to decrease significantly the per cent of dead neurons in culture after induction of lipid peroxidation. Exposure to glutamate was found to cause a significant decrease of the activity of Na+, K+-ATP-ase in rat brain cortex synaptosomes, but superoxide dismutase, alpha-tocopherol, or 10-100 nM GM1 practically prevented its action. Other data showing the ability of gangliosides to inhibit the intensification of free radical reactions by glutamate (based on the estimation of methemoglobin formation, SH group content, etc.) have been obtained. The results suggest that gangliosides are able to decrease the glutamate-induced activation of free radical reactions in nerve cells. This effect appears to contribute to their protective action against glutamate neurotoxicity.

Robustness effect of gap junctions between Golgi cells on cerebellar cortex oscillations

PubMed Central

2011-01-01

Background Previous one-dimensional network modeling of the cerebellar granular layer has been successfully linked with a range of cerebellar cortex oscillations observed in vivo. However, the recent discovery of gap junctions between Golgi cells (GoCs), which may cause oscillations by themselves, has raised the question of how gap-junction coupling affects GoC and granular-layer oscillations. To investigate this question, we developed a novel two-dimensional computational model of the GoC-granule cell (GC) circuit with and without gap junctions between GoCs. Results Isolated GoCs coupled by gap junctions had a strong tendency to generate spontaneous oscillations without affecting their mean firing frequencies in response to distributed mossy fiber input. Conversely, when GoCs were synaptically connected in the granular layer, gap junctions increased the power of the oscillations, but the oscillations were primarily driven by the synaptic feedback loop between GoCs and GCs, and the gap junctions did not change oscillation frequency or the mean firing rate of either GoCs or GCs. Conclusion Our modeling results suggest that gap junctions between GoCs increase the robustness of cerebellar cortex oscillations that are primarily driven by the feedback loop between GoCs and GCs. The robustness effect of gap junctions on synaptically driven oscillations observed in our model may be a general mechanism, also present in other regions of the brain. PMID:22330240

Potential mechanisms of cerebellar hypoplasia in prematurity.

PubMed

Tam, Emily W Y

2013-09-01

The cerebellum undergoes dramatic growth and maturation over the neonatal period after preterm birth and is thus particularly sensitive to impaired development due to various clinical factors. Impairments in growth can occur independent of cerebellar parenchymal damage, such as from local hemorrhage, resulting from reduced expression of sonic hedgehog signaling to trigger the appropriate expansion of the granule precursor cells. The primary risk factors for impaired cerebellar development include postnatal glucocorticoid exposure, which has direct effects on the sonic hedgehog pathway, and supratentorial brain injury, including intraventricular hemorrhage and white matter injury, which may result in crossed cerebellar diaschisis and local toxic effects of blood products on the external granular layer. Other cardiorespiratory and nutritional factors may also exist. Impaired cerebellar development is associated with adverse outcomes in motor and cognitive development. New approaches to care to counteract these risk factors may help improve long-term outcome after preterm birth.

Sex-specific activation of cell death signalling pathways in cerebellar granule neurons exposed to oxygen glucose deprivation followed by reoxygenation

PubMed Central

Sharma, Jaswinder; Nelluru, Geetha; Ann Wilson, Mary; Johnston, Michael V; Ahamed Hossain, Mir

2011-01-01

Neuronal death pathways following hypoxia–ischaemia are sexually dimorphic, but the underlying mechanisms are unclear. We examined cell death mechanisms during OGD (oxygen-glucose deprivation) followed by Reox (reoxygenation) in segregated male (XY) and female (XX) mouse primary CGNs (cerebellar granule neurons) that are WT (wild-type) or Parp-1 [poly(ADP-ribose) polymerase 1] KO (knockout). Exposure of CGNs to OGD (1.5 h)/Reox (7 h) caused cell death in XY and XX neurons, but cell death during Reox was greater in XX neurons. ATP levels were significantly lower after OGD/Reox in WT-XX neurons than in XY neurons; this difference was eliminated in Parp-1 KO-XX neurons. AIF (apoptosis-inducing factor) was released from mitochondria and translocated to the nucleus by 1 h exclusively in WT-XY neurons. In contrast, there was a release of Cyt C (cytochrome C) from mitochondria in WT-XX and Parp-1 KO neurons of both sexes; delayed activation of caspase 3 was observed in the same three groups. Thus deletion of Parp-1 shunted cell death towards caspase 3-dependent apoptosis. Delayed activation of caspase 8 was also observed in all groups after OGD/Reox, but was much greater in XX neurons, and caspase 8 translocated to the nucleus in XX neurons only. Caspase 8 activation may contribute to increased XX neuronal death during Reox, via caspase 3 activation. Thus, OGD/Reox induces death of XY neurons via a PARP-1-AIF-dependent mechanism, but blockade of PARP-1-AIF pathway shifts neuronal death towards a caspase-dependent mechanism. In XX neurons, OGD/Reox caused prolonged depletion of ATP and delayed activation of caspase 8 and caspase 3, culminating in greater cell death during Reox. PMID:21382016

CYP2E1 induction leads to oxidative stress and cytotoxicity in glutathione-depleted cerebellar granule neurons.

PubMed

Valencia-Olvera, Ana Carolina; Morán, Julio; Camacho-Carranza, Rafael; Prospéro-García, Oscar; Espinosa-Aguirre, Jesús Javier

2014-10-01

Increasing evidence suggests that brain cytochrome P450 (CYP) can contribute to the in situ metabolism of xenobiotics. In the liver, some xenobiotics can be metabolized by CYPs into more reactive products that can damage hepatocytes and induce cell death. In addition, normal CYP activity may produce reactive oxygen species (ROS) that contribute to cell damage through oxidative mechanisms. CYP2E1 is a CYP isoform that can generate ROS leading to cytotoxicity in multiple tissue types. The aim of this study was to determine whether CYP2E1 induction may lead to significant brain cell impairment. Immunological analysis revealed that exposure of primary cerebellar granule neuronal cultures to the CYP inducer isoniazid, increased CYP2E1 expression. In the presence of buthionine sulfoximine, an agent that reduces glutathione levels, isoniazid treatment also resulted in reactive oxygen species (ROS) production, DNA oxidation and cell death. These effects were attenuated by simultaneous exposure to diallyl sulfide, a CYP2E1 inhibitor, or to a mimetic of superoxide dismutase/catalase, (Euka). These results suggest that in cases of reduced antioxidant levels, the induction of brain CYP2E1 could represent a risk of in situ neuronal damage. Copyright © 2014 Elsevier Ltd. All rights reserved.

Phenotypic outcomes in Mouse and Human Foxc1 dependent Dandy-Walker cerebellar malformation suggest shared mechanisms.

PubMed

Haldipur, Parthiv; Dang, Derek; Aldinger, Kimberly A; Janson, Olivia K; Guimiot, Fabien; Adle-Biasette, Homa; Dobyns, William B; Siebert, Joseph R; Russo, Rosa; Millen, Kathleen J

2017-01-16

FOXC1 loss contributes to Dandy-Walker malformation (DWM), a common human cerebellar malformation. Previously, we found that complete Foxc1 loss leads to aberrations in proliferation, neuronal differentiation and migration in the embryonic mouse cerebellum (Haldipur et al., 2014). We now demonstrate that hypomorphic Foxc1 mutant mice have granule and Purkinje cell abnormalities causing subsequent disruptions in postnatal cerebellar foliation and lamination. Particularly striking is the presence of a partially formed posterior lobule which echoes the posterior vermis DW 'tail sign' observed in human imaging studies. Lineage tracing experiments in Foxc1 mutant mouse cerebella indicate that aberrant migration of granule cell progenitors destined to form the posterior-most lobule causes this unique phenotype. Analyses of rare human del chr 6p25 fetal cerebella demonstrate extensive phenotypic overlap with our Foxc1 mutant mouse models, validating our DWM models and demonstrating that many key mechanisms controlling cerebellar development are likely conserved between mouse and human.

Select putative neurodevelopmental toxins modify SNAP-25 expression in primary cultures of rat cerebellar granule cells.

PubMed

Zieminska, Elzbieta; Lenart, Jacek; Lazarewicz, Jerzy W

2016-08-31

A presynaptic protein SNAP-25 belonging to SNARE complex which is instrumental in intracellular vesicular trafficking and exocytosis, has been implicated in hyperactivity and cognitive abilities in some neuropsychiatric disorders. The unclear etiology of the behavior disrupting neurodevelopmental disabilities in addition to genetic causes most likely involves environmental factors. The aim of this in vitro study was to test if various suspected developmental neurotoxins can alter SNAP-25 mRNA and protein expression in neurons. Real-time PCR and Western blotting analyses were used to assess SNAP-25 mRNA and protein levels in primary cultures of rat cerebellar granule cells (CGCs). The test substances: tetrabromobisphenol-A (TBBPA), thimerosal (TH), silver nanoparticles (NAg), valproic acid (VPA) and thalidomide (THAL), were administered to CGC cultures at subtoxic concentrations for 24h. The results demonstrated that SNAP-25 mRNA levels were increased by 49 and 66% by TBBPA and THAL, respectively, whereas VPA and NAg reduced these levels to 48 and 64% of the control, respectively. The SNAP-25 protein content in CGCs was increased by 79% by TBBPA, 25% by THAL and 21% by NAg; VPA and TH reduced these levels to 73 and 69% of the control, respectively. The variety of changes in SNAP-25 expression on mRNA and protein level suggests the diversity of the mechanism of action of the test substances. This initial study provided no data on concentration-effect relations and on functional changes in CGCs. However it is the first to demonstrate the effect of different compounds that are suspected of causing neurodevelopmental disabilities on SNAP-25 expression. These results suggest that this protein may be a common target for not only inherited but also environmental modifications linked to behavioral deficits in neurodevelopmental disabilities. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Stimulus-Dependent State Transition between Synchronized Oscillation and Randomly Repetitive Burst in a Model Cerebellar Granular Layer

PubMed Central

Tanaka, Shigeru; Nagao, Soichi; Nishino, Tetsuro

2011-01-01

Information processing of the cerebellar granular layer composed of granule and Golgi cells is regarded as an important first step toward the cerebellar computation. Our previous theoretical studies have shown that granule cells can exhibit random alternation between burst and silent modes, which provides a basis of population representation of the passage-of-time (POT) from the onset of external input stimuli. On the other hand, another computational study has reported that granule cells can exhibit synchronized oscillation of activity, as consistent with observed oscillation in local field potential recorded from the granular layer while animals keep still. Here we have a question of whether an identical network model can explain these distinct dynamics. In the present study, we carried out computer simulations based on a spiking network model of the granular layer varying two parameters: the strength of a current injected to granule cells and the concentration of Mg2+ which controls the conductance of NMDA channels assumed on the Golgi cell dendrites. The simulations showed that cells in the granular layer can switch activity states between synchronized oscillation and random burst-silent alternation depending on the two parameters. For higher Mg2+ concentration and a weaker injected current, granule and Golgi cells elicited spikes synchronously (synchronized oscillation state). In contrast, for lower Mg2+ concentration and a stronger injected current, those cells showed the random burst-silent alternation (POT-representing state). It is suggested that NMDA channels on the Golgi cell dendrites play an important role for determining how the granular layer works in response to external input. PMID:21779155

Control of a simulated arm using a novel combination of Cerebellar learning mechanisms

NASA Technical Reports Server (NTRS)

Assad, C.; Hartmann, M.; Paulin, M. G.

2001-01-01

We present a model of cerebellar cortex that combines two types of learning: feedforward predicitve association based on local Hebbian-type learning between granule cell ascending branch and parallel fiber inputs, and reinforcement learning with feedback error correction based on climbing fiber activity.

Integrated plasticity at inhibitory and excitatory synapses in the cerebellar circuit.

PubMed

Mapelli, Lisa; Pagani, Martina; Garrido, Jesus A; D'Angelo, Egidio

2015-01-01

The way long-term potentiation (LTP) and depression (LTD) are integrated within the different synapses of brain neuronal circuits is poorly understood. In order to progress beyond the identification of specific molecular mechanisms, a system in which multiple forms of plasticity can be correlated with large-scale neural processing is required. In this paper we take as an example the cerebellar network, in which extensive investigations have revealed LTP and LTD at several excitatory and inhibitory synapses. Cerebellar LTP and LTD occur in all three main cerebellar subcircuits (granular layer, molecular layer, deep cerebellar nuclei) and correspondingly regulate the function of their three main neurons: granule cells (GrCs), Purkinje cells (PCs) and deep cerebellar nuclear (DCN) cells. All these neurons, in addition to be excited, are reached by feed-forward and feed-back inhibitory connections, in which LTP and LTD may either operate synergistically or homeostatically in order to control information flow through the circuit. Although the investigation of individual synaptic plasticities in vitro is essential to prove their existence and mechanisms, it is insufficient to generate a coherent view of their impact on network functioning in vivo. Recent computational models and cell-specific genetic mutations in mice are shedding light on how plasticity at multiple excitatory and inhibitory synapses might regulate neuronal activities in the cerebellar circuit and contribute to learning and memory and behavioral control.

Modulation of p53 and met expression by Krüppel-like factor 8 regulates zebrafish cerebellar development.

PubMed

Tsai, Ming-Yuan; Lu, Yu-Fen; Liu, Yu-Hsiu; Lien, Huang-Wei; Huang, Chang-Jen; Wu, Jen-Leih; Hwang, Sheng-Ping L

2015-09-01

Krüppel-like factor 8 (Klf8) is a zinc-finger transcription factor implicated in cell proliferation, and cancer cell survival and invasion; however, little is known about its role in normal embryonic development. Here, we show that Klf8 is required for normal cerebellar development in zebrafish embryos. Morpholino knockdown of klf8 resulted in abnormal cerebellar primordium morphology and the induction of p53 in the brain region at 24 hours post-fertilization (hpf). Both p53-dependent reduction of cell proliferation and augmentation of apoptosis were observed in the cerebellar anlage of 24 hpf-klf8 morphants. In klf8 morphants, expression of ptf1a in the ventricular zone was decreased from 48 to 72 hpf; on the other hand, expression of atohla in the upper rhombic lip was unaffected. Consistent with this finding, Purkinje cell development was perturbed and granule cell number was reduced in 72 hpf-klf8 morphants; co-injection of p53 MO(sp) or klf8 mRNA substantially rescued development of cerebellar Purkinje cells in klf8 morphants. Hepatocyte growth factor/Met signaling is known to regulate cerebellar development in zebrafish and mouse. We observed decreased met expression in the tectum and rhombomere 1 of 24 hpf-klf8 morphants, which was largely rescued by co-injection with klf8 mRNA. Moreover, co-injection of met mRNA substantially rescued formation of Purkinje cells in klf8 morphants at 72 hpf. Together, these results demonstrate that Klf8 modulates expression of p53 and met to maintain ptf1a-expressing neuronal progenitors, which are required for the appropriate development of cerebellar Purkinje and granule cells in zebrafish embryos. © 2014 Wiley Periodicals, Inc.

Cerebellar Structure and Function in Male Wistar-Kyoto Hyperactive Rats

PubMed Central

Thanellou, Alexandra; Green, John T.

2014-01-01

Previous research has suggested that the Wistar-Kyoto Hyperactive (WKHA) rat strain may model some of the behavioral features associated with attention-deficit/hyperactivity disorder (ADHD). We have shown that, in cerebellar-dependent eyeblink conditioning, WKHA emit eyeblink CRs with shortened onset latencies. To further characterize the shortened CR onset latencies seen in WKHA rats, we examined 750-ms delay conditioning with either a tone CS or a light CS, we extended acquisition training, and we included Wistar rats as an additional, outbred control strain. Our results indicated that WKHAs learned more quickly and showed a shortened CR onset latency to a tone CS compared to both Wistar-Kyoto Hypertensive (WKHT) and Wistars. WKHAs and Wistars show a lengthening of CR onset latency over conditioning with a tone CS and an increasing confinement of CRs to the later part of the tone CS (inhibition of delay). WKHAs learned more quickly to a light CS only in comparison to WKHTs and showed a shortened CR onset latency only in comparison to Wistars. Wistars showed an increasing confinement of CRs to the late part of the light CS over conditioning. We used unbiased stereology to estimate the number of Purkinje and granule cells in the cerebellar cortex of the three strains. Our results indicated that WKHAs have more granule cells than Wistars and WKHTs and more Purkinje cells than Wistars. Results are discussed in terms of CS processing and cerebellar cortical contributions to EBC. PMID:23398437

Multiple developmental programs are altered by loss of Zic1 and Zic4 to cause Dandy-Walker malformation cerebellar pathogenesis

PubMed Central

Blank, Marissa C.; Grinberg, Inessa; Aryee, Emmanuel; Laliberte, Christine; Chizhikov, Victor V.; Henkelman, R. Mark; Millen, Kathleen J.

2011-01-01

Heterozygous deletions encompassing the ZIC1;ZIC4 locus have been identified in a subset of individuals with the common cerebellar birth defect Dandy-Walker malformation (DWM). Deletion of Zic1 and Zic4 in mice produces both cerebellar size and foliation defects similar to human DWM, confirming a requirement for these genes in cerebellar development and providing a model to delineate the developmental basis of this clinically important congenital malformation. Here, we show that reduced cerebellar size in Zic1 and Zic4 mutants results from decreased postnatal granule cell progenitor proliferation. Through genetic and molecular analyses, we show that Zic1 and Zic4 have Shh-dependent function promoting proliferation of granule cell progenitors. Expression of the Shh-downstream genes Ptch1, Gli1 and Mycn was downregulated in Zic1/4 mutants, although Shh production and Purkinje cell gene expression were normal. Reduction of Shh dose on the Zic1+/−;Zic4+/− background also resulted in cerebellar size reductions and gene expression changes comparable with those observed in Zic1−/−;Zic4−/− mice. Zic1 and Zic4 are additionally required to pattern anterior vermis foliation. Zic mutant folial patterning abnormalities correlated with disrupted cerebellar anlage gene expression and Purkinje cell topography during late embryonic stages; however, this phenotype was Shh independent. In Zic1+/−;Zic4+/−;Shh+/−, we observed normal cerebellar anlage patterning and foliation. Furthermore, cerebellar patterning was normal in both Gli2-cko and Smo-cko mutant mice, where all Shh function was removed from the developing cerebellum. Thus, our data demonstrate that Zic1 and Zic4 have both Shh-dependent and -independent roles during cerebellar development and that multiple developmental disruptions underlie Zic1/4-related DWM. PMID:21307096

Delayed reverberation through time windows as a key to cerebellar function.

PubMed

Kistler, W M; Leo van Hemmen, J

1999-11-01

We present a functional model of the cerebellum comprising cerebellar cortex, inferior olive, deep cerebellar nuclei, and brain stem nuclei. The discerning feature of the model being time coding, we consistently describe the system in terms of postsynaptic potentials, synchronous action potentials, and propagation delays. We show by means of detailed single-neuron modeling that (i) Golgi cells can fulfill a gating task in that they form short and well-defined time windows within which granule cells can reach firing threshold, thus organizing neuronal activity in discrete 'time slices', and that (ii) rebound firing in cerebellar nuclei cells is a robust mechanism leading to a delayed reverberation of Purkinje cell activity through cerebellar-reticular projections back to the cerebellar cortex. Computer simulations of the whole cerebellar network consisting of several thousand neurons reveal that reverberation in conjunction with long-term plasticity at the parallel fiber-Purkinje cell synapses enables the system to learn, store, and recall spatio-temporal patterns of neuronal activity. Climbing fiber spikes act both as a synchronization and as a teacher signal, not as an error signal. They are due to intrinsic oscillatory properties of inferior olivary neurons and to delayed reverberation within the network. In addition to clear experimental predictions the present theory sheds new light on a number of experimental observation such as the synchronicity of climbing fiber spikes and provides a novel explanation of how the cerebellum solves timing tasks on a time scale of several hundreds of milliseconds.

Phenotypic outcomes in Mouse and Human Foxc1 dependent Dandy-Walker cerebellar malformation suggest shared mechanisms

PubMed Central

Haldipur, Parthiv; Dang, Derek; Aldinger, Kimberly A; Janson, Olivia K; Guimiot, Fabien; Adle-Biasette, Homa; Dobyns, William B; Siebert, Joseph R; Russo, Rosa; Millen, Kathleen J

2017-01-01

FOXC1 loss contributes to Dandy-Walker malformation (DWM), a common human cerebellar malformation. Previously, we found that complete Foxc1 loss leads to aberrations in proliferation, neuronal differentiation and migration in the embryonic mouse cerebellum (Haldipur et al., 2014). We now demonstrate that hypomorphic Foxc1 mutant mice have granule and Purkinje cell abnormalities causing subsequent disruptions in postnatal cerebellar foliation and lamination. Particularly striking is the presence of a partially formed posterior lobule which echoes the posterior vermis DW 'tail sign' observed in human imaging studies. Lineage tracing experiments in Foxc1 mutant mouse cerebella indicate that aberrant migration of granule cell progenitors destined to form the posterior-most lobule causes this unique phenotype. Analyses of rare human del chr 6p25 fetal cerebella demonstrate extensive phenotypic overlap with our Foxc1 mutant mouse models, validating our DWM models and demonstrating that many key mechanisms controlling cerebellar development are likely conserved between mouse and human. DOI: http://dx.doi.org/10.7554/eLife.20898.001 PMID:28092268

A Novel and Multivalent Role of Pax6 in Cerebellar Development

PubMed Central

Yeung, Joanna; Ha, Thomas J.; Swanson, Douglas J.

2016-01-01

Pax6 is a prominent gene in brain development. The deletion of Pax6 results in devastated development of eye, olfactory bulb, and cortex. However, it has been reported that the Pax6-null Sey cerebellum only has minor defects involving granule cells despite Pax6 being expressed throughout cerebellar development. The present work has uncovered a requirement of Pax6 in the development of all rhombic lip (RL) lineages. A significant downregulation of Tbr1 and Tbr2 expression is found in the Sey cerebellum, these are cell-specific markers of cerebellar nuclear (CN) neurons and unipolar brush cells (UBCs), respectively. The examination of Tbr1 and Lmx1a immunolabeling and Nissl staining confirmed the loss of CN neurons from the Sey cerebellum. CN neuron progenitors are produced in the mutant but there is an enhanced death of these neurons as shown by increased presence of caspase-3-positive cells. These data indicate that Pax6 regulates the survival of CN neuron progenitors. Furthermore, the analysis of experimental mouse chimeras suggests a cell-extrinsic role of Pax6 in CN neuron survival. For UBCs, using Tbr2 immunolabeling, these cells are significantly reduced in the Sey cerebellum. The loss of UBCs in the mutant is due partly to cell death in the RL and also to the reduced production of progenitors from the RL. These results demonstrate a critical role for Pax6 in regulating the generation and survival of UBCs. This and previous work from our laboratory demonstrate a seminal role of Pax6 in the development of all cerebellar glutamatergic neurons. SIGNIFICANCE STATEMENT Pax6 is a key molecule in development. Pax6 is best known as the master control gene in eye development with mutations causing aniridia in humans. Pax6 also plays important developmental roles in the cortex and olfactory bulb. During cerebellar development, Pax6 is robustly expressed in the germinal zone of all glutamatergic neurons [cerebellar nuclear (CN) neurons, granule cells, and unipolar brush

Excitatory Cerebellar Nucleocortical Circuit Provides Internal Amplification during Associative Conditioning.

PubMed

Gao, Zhenyu; Proietti-Onori, Martina; Lin, Zhanmin; Ten Brinke, Michiel M; Boele, Henk-Jan; Potters, Jan-Willem; Ruigrok, Tom J H; Hoebeek, Freek E; De Zeeuw, Chris I

2016-02-03

Closed-loop circuitries between cortical and subcortical regions can facilitate precision of output patterns, but the role of such networks in the cerebellum remains to be elucidated. Here, we characterize the role of internal feedback from the cerebellar nuclei to the cerebellar cortex in classical eyeblink conditioning. We find that excitatory output neurons in the interposed nucleus provide efference-copy signals via mossy fibers to the cerebellar cortical zones that belong to the same module, triggering monosynaptic responses in granule and Golgi cells and indirectly inhibiting Purkinje cells. Upon conditioning, the local density of nucleocortical mossy fiber terminals significantly increases. Optogenetic activation and inhibition of nucleocortical fibers in conditioned animals increases and decreases the amplitude of learned eyeblink responses, respectively. Our data show that the excitatory nucleocortical closed-loop circuitry of the cerebellum relays a corollary discharge of premotor signals and suggests an amplifying role of this circuitry in controlling associative motor learning. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Cadherins in cerebellar development: translation of embryonic patterning into mature functional compartmentalization.

PubMed

Redies, Christoph; Neudert, Franziska; Lin, Juntang

2011-09-01

Cadherins are cell adhesion molecules with multiple morphogenic functions in brain development, for example, in neuroblast migration and aggregation, axon navigation, neural circuit formation, and synaptogenesis. More than 100 members of the cadherin superfamily are expressed in the developing and mature brain. Most of the cadherins investigated, in particular classic cadherins and δ-protocadherins, are expressed in the cerebellum. For several cadherin subtypes, expression begins at early embryonic stages and persists until mature stages of cerebellar development. At intermediate stages, distinct Purkinje cell clusters exhibit unique rostrocaudal and mediolateral expression profiles for each cadherin. In the chicken, mouse, and other species, the Purkinje cell clusters are separated by intervening raphes of migrating granule cells. This pattern of Purkinje cell clusters/raphes is, at least in part, continuous with the parasagittal striping pattern that is apparent in the mature cerebellar cortex, for example, for zebrin II/aldolase C. Moreover, subregions of the deep cerebellar nuclei, vestibular nuclei and the olivary complex also express cadherins differentially. Neuroanatomical evidence suggests that the nuclear subregions and cortical domains that express the same cadherin subtype are connected to each other, to form neural subcircuits of the cerebellar system. Cadherins thus provide a molecular code that specifies not only embryonic structures but also functional cerebellar compartmentalization. By following the implementation of this code, it can be revealed how mature functional architecture emerges from embryonic patterning during cerebellar development. Dysfunction of some cadherins is associated with psychiatric diseases and developmental impairments and may also affect cerebellar function.

ROS Produced by NOX2 Controls In Vitro Development of Cerebellar Granule Neurons Development

PubMed Central

Olguín-Albuerne, Mauricio

2015-01-01

Reactive oxygen species (ROS) act as signaling molecules that regulate nervous system physiology. ROS have been related to neural differentiation, neuritogenesis, and programmed cell death. Nevertheless, little is known about the mechanisms involved in the regulation of ROS during neuronal development. In this study, we evaluated the mechanisms by which ROS are regulated during neuronal development and the implications of these molecules in this process. Primary cultures of cerebellar granule neurons (CGN) were used to address these issues. Our results show that during the first 3 days of CGN development in vitro (days in vitro; DIV), the levels of ROS increased, reaching a peak at 2 and 3 DIV under depolarizing (25 mM KCl) and nondepolarizing (5 mM KCl) conditions. Subsequently, under depolarizing conditions, the ROS levels markedly decreased, but in nondepolarizing conditions, the ROS levels increased gradually. This correlated with the extent of CGN maturation. Also, antioxidants and NADPH-oxidases (NOX) inhibitors reduced the expression of Tau and MAP2. On the other hand, the levels of glutathione markedly increased at 1 DIV. We inferred that the ROS increase at this time is critical for cell survival because glutathione depletion leads to axonal degeneration and CGN death only at 2 DIV. During the first 3 DIV, NOX2 was upregulated and expressed in filopodia and growth cones, which correlated with the hydrogen peroxide (H2O2) distribution in the cell. Finally, NOX2 KO CGN showed shorter neurites than wild-type CGN. Taken together, these results suggest that the regulation of ROS is critical during the early stages of CGN development. PMID:25873309

Substance P provides neuroprotection in cerebellar granule cells through Akt and MAPK/Erk activation: evidence for the involvement of the delayed rectifier potassium current.

PubMed

Amadoro, G; Pieri, M; Ciotti, M T; Carunchio, I; Canu, N; Calissano, P; Zona, C; Severini, C

2007-05-01

In the current study, we have evaluated the ability of substance P (SP) and other neurokinin 1 receptor (NK1) agonists to protect, in a dose- and time-dependent manner, primary cultures of rat cerebellar granule cells (CGCs) from serum and potassium deprivation-induced cell death (S-K5). We also established the presence of SP high affinity NK1 transcripts and the NK1 protein localization in the membrane of a sub-population of CGCs. Moreover, SP significantly and dose-dependently reduced the Akt 1/2 and Erk1/2 dephosphorylation induced by S-K5 conditions, as demonstrated by Western blot analysis. Surprisingly, in SP-treated CGCs caspase-3 activity was not inhibited, while the calpain-1 activity was moderately reduced. Corroborating this result, SP blocked calpain-mediated cleavage of tau protein, as demonstrated by the reduced appearance of a diagnostic fragment of 17 kDa by Western blot analysis. In addition, SP induced a significant reduction of the delayed rectifier K+ currents (Ik) in about 42% of the patched neurons, when these were evoked with depolarizing potential steps. Taken together, the present results demonstrate that the activation of NK1 receptors expressed in CGCs promote the neuronal survival via pathways involving Akt and Erk activation and by inhibition of Ik which can contribute to the neuroprotective effect of the peptide.

Acute Ethanol Exposure Prevents PMA-mediated Augmentation of N-methyl-d-aspartate Receptor Function in Primary Cultured Cerebellar Granule Cells

PubMed Central

Reneau, Jason; Reyland, Mary E.; Popp, R. Lisa

2011-01-01

Many intracellular proteins and signaling cascades contribute to the ethanol sensitivity of native N-methyl-d-aspartate receptors (NMDARs). One putative protein is the serine / threonine kinase, Protein kinase C (PKC). The purpose of this study was to assess if PKC modulates the ethanol sensitivity of native NMDARs expressed in primary cultured cerebellar granule cells (CGCs). With the whole-cell patch-clamp technique, we assessed if ethanol inhibition of NMDA-induced currents (INMDA) (100 μM NMDA plus 10 μM glycine) were altered in CGCs in which the novel and classical PKC isoforms were activated by phorbol-12-myristate-13-acetate (PMA). Percent inhibition by 10, 50 or 100 mM ethanol of NMDA-induced steady-state (ISS) or peak current amplitudes (IPk) of NMDARs expressed in CGCs in which PKC was activated by a 12.5 min, 100 nM PMA exposure at 37° C did not differ from currents obtained from receptors contained in control cells. However, PMA-mediated augmentation of IPk in the absence of ethanol was abolished after brief applications of 10 or 1 mM ethanol co-applied with agonists, and this suppression of enhanced receptor function was observed for up to eight minutes post-ethanol exposure. Because we had previously shown that PMA-mediated augmentation of INMDA of NMDARs expressed in these cells is by activation of PKCα, we assessed the effect of ethanol (1, 10, 50 and 100 mM) on PKCα activity. Ethanol decreased PKCα activity by 18% for 1 mM ethanol and activity decreased with increasing ethanol concentrations with a 50% inhibition observed with 100 mM ethanol. The data suggest that ethanol disruption of PMA-mediated augmentation of INMDA may be due to a decrease in PKCα activity by ethanol. However, given the incomplete blockade of PKCα activity and the low concentration of ethanol at which this phenomenon is observed, other ethanol-sensitive signaling cascades must also be involved. PMID:21624785

Arachidonic acid enhances intracellular [Ca2+]i increase and mitochondrial depolarization induced by glutamate in cerebellar granule cells.

PubMed

Surin, A M; Bolshakov, A P; Mikhailova, M M; Sorokina, E G; Senilova, Ya E; Pinelis, V G; Khodorov, B I

2006-08-01

Maturation of primary neuronal cultures is accompanied by an increase in the proportion of cells that exhibit biphasic increase in free cytoplasmic Ca2+ ([Ca2+]i) followed by synchronic decrease in electrical potential difference across the inner mitochondrial membrane (DeltaPsim) in response to stimulation of glutamate receptors. In the present study we have examined whether the appearance of the second phase of [Ca2+]i change can be attributed to arachidonic acid (AA) release in response to the effect of glutamate (Glu) on neurons. Using primary culture of rat cerebellar granule cells we have investigated the effect of AA (1-20 microM) on [Ca2+]i, DeltaPsim, and [ATP] and changes in these parameters induced by neurotoxic concentrations of Glu (100 microM, 10-40 min). At =10 microM, AA caused insignificant decrease in DeltaPsim without any influence on [Ca2+]i. The mitochondrial ATPase inhibitor oligomycin enhanced AA-induced decrease in DeltaPsim; this suggests that AA may inhibit mitochondrial respiration. Addition of AA during the treatment with Glu resulted in more pronounced augmentation of [Ca2+]i and the decrease in DeltaPsim than the changes in these parameters observed during independent action of AA; removal of Glu did not abolish these changes. An inhibitor of the cyclooxygenase and lipoxygenase pathways of AA metabolism, 5,8,11,14-eicosatetraynoic acid, increased the proportion of neurons characterized by Glu-induced biphasic increase in [Ca2+]i and the decrease in DeltaPsim. Palmitic acid (30 microM) did not increase the percentage of neurons exhibiting biphasic response to Glu. Co-administration of AA and Glu caused 2-3 times more pronounced decrease in ATP concentrations than that observed during the independent effect of AA and Glu. The data suggest that AA may influence the functional state of mitochondria, and these changes may promote biphasic [Ca2+]i and DeltaPsim responses of neurons to the neurotoxic effect of Glu.

A realistic bi-hemispheric model of the cerebellum uncovers the purpose of the abundant granule cells during motor control.

PubMed

Pinzon-Morales, Ruben-Dario; Hirata, Yutaka

2015-01-01

The cerebellar granule cells (GCs) have been proposed to perform lossless, adaptive spatio-temporal coding of incoming sensory/motor information required by downstream cerebellar circuits to support motor learning, motor coordination, and cognition. Here we use a physio-anatomically inspired bi-hemispheric cerebellar neuronal network (biCNN) to selectively enable/disable the output of GCs and evaluate the behavioral and neural consequences during three different control scenarios. The control scenarios are a simple direct current motor (1 degree of freedom: DOF), an unstable two-wheel balancing robot (2 DOFs), and a simulation model of a quadcopter (6 DOFs). Results showed that adequate control was maintained with a relatively small number of GCs (< 200) in all the control scenarios. However, the minimum number of GCs required to successfully govern each control plant increased with their complexity (i.e., DOFs). It was also shown that increasing the number of GCs resulted in higher robustness against changes in the initialization parameters of the biCNN model (i.e., synaptic connections and synaptic weights). Therefore, we suggest that the abundant GCs in the cerebellar cortex provide the computational power during the large repertoire of motor activities and motor plants the cerebellum is involved with, and bring robustness against changes in the cerebellar microcircuit (e.g., neuronal connections).

Glucose deprivation stimulates Cu(2+) toxicity in cultured cerebellar granule neurons and Cu(2+)-dependent zinc release.

PubMed

Isaev, Nickolay K; Genrikhs, Elisaveta E; Aleksandrova, Olga P; Zelenova, Elena A; Stelmashook, Elena V

2016-05-27

Copper chloride (0.01mM, 2h) did not have significant influence on the survival of cerebellar granule neurons (CGNs) incubated in balanced salt solution. However, CuCl2 caused severe neuronal damage by glucose deprivation (GD). The glutamate NMDA-receptors blocker MK-801 partially and antioxidant N-acetyl-l-cysteine (NAC) or Zn(2+) chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) almost entirely protected CGNs from this toxic effect. Measurements of intracellular calcium ions using Fluo-4 AM, or zinc ions with FluoZin-3 AM demonstrated that 1 h-exposure to GD induced intensive increase of Fluo-4 but not FluoZin-3 fluorescence in neurons. The supplementation of solution with CuCl2 caused an increase of FluoZin-3, Fluo-4 and CellROX Green (reactive oxygen species probe) fluorescence by GD. The stimulation of Fluo-4 but not FluoZin-3 fluorescence by copper could be prevented partially by MK-801 and as well as CellROX Green fluorescence by NAC at GD. This data imply that during GD copper ions induce intense displacement zinc ions from intracellular stores, in addition free radical production, glutamate release and Ca(2+) overload of CGNs, that causes death of neurons as a result. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Drebrin-mediated microtubule–actomyosin coupling steers cerebellar granule neuron nucleokinesis and migration pathway selection

DOE PAGES

Trivedi, Niraj; Stabley, Daniel R.; Cain, Blake; ...

2017-02-23

Neuronal migration from a germinal zone to a final laminar position is essential for the morphogenesis of neuronal circuits. While it is hypothesized that microtubule–actomyosin crosstalk is required for a neuron’s ‘two-stroke’ nucleokinesis cycle, the molecular mechanisms controlling such crosstalk are not defined. By using the drebrin microtubule–actin crosslinking protein as an entry point into the cerebellar granule neuron system in combination with super-resolution microscopy, we investigate how these cytoskeletal systems interface during migration. Lattice light-sheet and structured illumination microscopy reveal a proximal leading process nanoscale architecture wherein f-actin and drebrin intervene between microtubules and the plasma membrane. Functional perturbationsmore » of drebrin demonstrate that proximal leading process microtubule–actomyosin coupling steers the direction of centrosome and somal migration, as well as the switch from tangential to radial migration. Finally, the Siah2 E3 ubiquitin ligase antagonizes drebrin function, suggesting a model for control of the microtubule–actomyosin interfaces during neuronal differentiation.« less

Drebrin-mediated microtubule–actomyosin coupling steers cerebellar granule neuron nucleokinesis and migration pathway selection

PubMed Central

Trivedi, Niraj; Stabley, Daniel R.; Cain, Blake; Howell, Danielle; Laumonnerie, Christophe; Ramahi, Joseph S.; Temirov, Jamshid; Kerekes, Ryan A.; Gordon-Weeks, Phillip R.; Solecki, David J.

2017-01-01

Neuronal migration from a germinal zone to a final laminar position is essential for the morphogenesis of neuronal circuits. While it is hypothesized that microtubule–actomyosin crosstalk is required for a neuron's ‘two-stroke' nucleokinesis cycle, the molecular mechanisms controlling such crosstalk are not defined. By using the drebrin microtubule–actin crosslinking protein as an entry point into the cerebellar granule neuron system in combination with super-resolution microscopy, we investigate how these cytoskeletal systems interface during migration. Lattice light-sheet and structured illumination microscopy reveal a proximal leading process nanoscale architecture wherein f-actin and drebrin intervene between microtubules and the plasma membrane. Functional perturbations of drebrin demonstrate that proximal leading process microtubule–actomyosin coupling steers the direction of centrosome and somal migration, as well as the switch from tangential to radial migration. Finally, the Siah2 E3 ubiquitin ligase antagonizes drebrin function, suggesting a model for control of the microtubule–actomyosin interfaces during neuronal differentiation. PMID:28230156

Inverse Stochastic Resonance in Cerebellar Purkinje Cells

PubMed Central

Häusser, Michael; Gutkin, Boris S.; Roth, Arnd

2016-01-01

Purkinje neurons play an important role in cerebellar computation since their axons are the only projection from the cerebellar cortex to deeper cerebellar structures. They have complex internal dynamics, which allow them to fire spontaneously, display bistability, and also to be involved in network phenomena such as high frequency oscillations and travelling waves. Purkinje cells exhibit type II excitability, which can be revealed by a discontinuity in their f-I curves. We show that this excitability mechanism allows Purkinje cells to be efficiently inhibited by noise of a particular variance, a phenomenon known as inverse stochastic resonance (ISR). While ISR has been described in theoretical models of single neurons, here we provide the first experimental evidence for this effect. We find that an adaptive exponential integrate-and-fire model fitted to the basic Purkinje cell characteristics using a modified dynamic IV method displays ISR and bistability between the resting state and a repetitive activity limit cycle. ISR allows the Purkinje cell to operate in different functional regimes: the all-or-none toggle or the linear filter mode, depending on the variance of the synaptic input. We propose that synaptic noise allows Purkinje cells to quickly switch between these functional regimes. Using mutual information analysis, we demonstrate that ISR can lead to a locally optimal information transfer between the input and output spike train of the Purkinje cell. These results provide the first experimental evidence for ISR and suggest a functional role for ISR in cerebellar information processing. PMID:27541958

Protection by imidazol(ine) drugs and agmatine of glutamate-induced neurotoxicity in cultured cerebellar granule cells through blockade of NMDA receptor

PubMed Central

Olmos, Gabriel; DeGregorio-Rocasolano, Nuria; Regalado, M Paz; Gasull, Teresa; Boronat, M Assumpció; Trullas, Ramón; Villarroel, Alvaro; Lerma, Juan; García-Sevilla, Jesús A

1999-01-01

This study was designed to assess the potential neuroprotective effect of several imidazol(ine) drugs and agmatine on glutamate-induced necrosis and on apoptosis induced by low extracellular K+ in cultured cerebellar granule cells.Exposure (30 min) of energy deprived cells to L-glutamate (1–100 μM) caused a concentration-dependent neurotoxicity, as determined 24 h later by a decrease in the ability of the cells to metabolize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) into a reduced formazan product. L-glutamate-induced neurotoxicity (EC50=5 μM) was blocked by the specific NMDA receptor antagonist MK-801 (dizocilpine).Imidazol(ine) drugs and agmatine fully prevented neurotoxicity induced by 20 μM (EC100) L-glutamate with the rank order (EC50 in μM): antazoline (13)>cirazoline (44)>LSL 61122 [2-styryl-2-imidazoline] (54)>LSL 60101 [2-(2-benzofuranyl) imidazole] (75)>idazoxan (90)>LSL 60129 [2-(1,4-benzodioxan-6-yl)-4,5-dihydroimidazole] (101)>RX821002 (2-methoxy idazoxan) (106)>agmatine (196). No neuroprotective effect of these drugs was observed in a model of apoptotic neuronal cell death (reduction of extracellular K+) which does not involve stimulation of NMDA receptors.Imidazol(ine) drugs and agmatine fully inhibited [3H]-(+)-MK-801 binding to the phencyclidine site of NMDA receptors in rat brain. The profile of drug potency protecting against L-glutamate neurotoxicity correlated well (r=0.90) with the potency of the same compounds competing against [3H]-(+)-MK-801 binding.In HEK-293 cells transfected to express the NR1-1a and NR2C subunits of the NMDA receptor, antazoline and agmatine produced a voltage- and concentration-dependent block of glutamate-induced currents. Analysis of the voltage dependence of the block was consistent with the presence of a binding site for antazoline located within the NMDA channel pore with an IC50 of 10–12 μM at 0 mV.It is concluded that imidazol(ine) drugs and agmatine are

Early Disruption of Extracellular Pleiotrophin Distribution Alters Cerebellar Neuronal Circuit Development and Function.

PubMed

Hamza, M M; Rey, S A; Hilber, P; Arabo, A; Collin, T; Vaudry, D; Burel, D

2016-10-01

The cerebellum is a structure of the central nervous system involved in balance, motor coordination, and voluntary movements. The elementary circuit implicated in the control of locomotion involves Purkinje cells, which receive excitatory inputs from parallel and climbing fibers, and are regulated by cerebellar interneurons. In mice as in human, the cerebellar cortex completes its development mainly after birth with the migration, differentiation, and synaptogenesis of granule cells. These cellular events are under the control of numerous extracellular matrix molecules including pleiotrophin (PTN). This cytokine has been shown to regulate the morphogenesis of Purkinje cells ex vivo and in vivo via its receptor PTPζ. Since Purkinje cells are the unique output of the cerebellar cortex, we explored the consequences of their PTN-induced atrophy on the function of the cerebellar neuronal circuit in mice. Behavioral experiments revealed that, despite a normal overall development, PTN-treated mice present a delay in the maturation of their flexion reflex. Moreover, patch clamp recording of Purkinje cells revealed a significant increase in the frequency of spontaneous excitatory postsynaptic currents in PTN-treated mice, associated with a decrease of climbing fiber innervations and an abnormal perisomatic localization of the parallel fiber contacts. At adulthood, PTN-treated mice exhibit coordination impairment on the rotarod test associated with an alteration of the synchronization gait. Altogether these histological, electrophysiological, and behavior data reveal that an early ECM disruption of PTN composition induces short- and long-term defaults in the establishment of proper functional cerebellar circuit.

A severe form of epidermal nevus syndrome associated with brainstem and cerebellar malformations and neonatal medulloblastoma.

PubMed

Okumura, Akihisa; Lee, Tsubasa; Ikeno, Mitsuru; Shimojima, Keiko; Kajino, Kazunori; Inoue, Yuka; Yoshikawa, Naomi; Suganuma, Hiroki; Suzuki, Mitsuyoshi; Hisata, Ken; Shoji, Hiromichi; Takanashi, Jun-ichi; Barkovich, A James; Shimizu, Toshiaki; Yamamoto, Toshiyuki; Hayashi, Masaharu

2012-11-01

Here we report a boy with epidermal nevus syndrome associated with brainstem and cerebellar malformations and neonatal medulloblastoma. The patient had epidermal nevi and complicated brain malformations including macrocephaly with polymicrogyria, dysmorphic and enlarged midbrain tectum, enlarged cerebellar hemispheres with small and maloriented folia. The patient died after surgical resection of medulloblastoma which was newly recognized on MRI at 51 days of age. Postmortem pathological examinations showed very unique and bizarre malformation of the midbrain and hindbrain. The cerebellar cortex exhibited a coarse, irregular and bumpy surface, blurred border between the Purkinje cell layer and internal granule cell layer, and many foci of heterotopia in the cerebellar white matter. The brainstem showed multiple anomalies, including enlargement of superior colliculi, hypoplasia of pyramidal tracts and dysplasia of inferior olivary nuclei. The unusual constellation of brain malformations of our patient will widen the spectrum of epidermal nevus syndrome. Copyright © 2012 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Vaccine adjuvants: Tailor-made mast-cell granules

NASA Astrophysics Data System (ADS)

Gunzer, Matthias

2012-03-01

Mast cells induce protective immune responses through secretion of stimulatory granules. Microparticles modelled after mast-cell granules are now shown to replicate and enhance the functions of their natural counterparts and to direct the character of the resulting immunity.

Identification of polypeptides with selective affinity to intact mouse cerebellar granule neurons from a random peptide-presenting phage library.

PubMed

Hou, Sheng T; Dove, Mike; Anderson, Erica; Zhang, Jiangbing; MacKenzie, C Roger

2004-09-30

Targeting of postmitotic neurons selectively for gene delivery poses a challenge. One way to achieve such a selective targeting is to link the gene delivery vector with small ligand-binding polypeptides which have selective affinity to intact neurons. In order to identify such novel neuron selective polypeptides, we screened a phage-display library displaying random 12-mer polypeptides and subtractively bio-panned for clones having selectivity towards cultured mouse cerebellar granule neurons. The selected phage clones were amplified and sequenced. Affinities of these clones to neurons were determined by the visible presence or absence of fluorescence of phage particles as detected by immunocytochemistry using an antibody to M-13 phage. This affinity was further qualified by how much phage was bound, and where in or on the cell it tended to accumulate. The selectivity of binding to neurons was determined by the negative binding of these clones to several cultured non-neuronal cells, including, primary glial cells, NT2 cells, human embryonic kidney 293 cells, neuroblastoma cells, and mouse 3T3 cells. Among the 46 clones that we have sequenced and characterized, four clones appeared to have excellent selectivity in binding to neurons. Homology comparison of these polypeptides revealed that three of them contained a consensus D(E)-W(F)-I(N)-D-W motif. This motif was also present in the Bdm1 gene product which was predominantly expressed in postnatal brains. Further characterizations of these polypeptides are required to reveal the utilities of these peptides to function as an effective linker to facilitate gene transfer selectively to neurons.

Depolarization- and transmitter-induced changes in intracellular Ca2+ of rat cerebellar granule cells in explant cultures.

PubMed

Connor, J A; Tseng, H Y; Hockberger, P E

1987-05-01

Digital imaging of the Ca indicator fura-2 has been used to study the responses of developing granule cells in culture to depolarization and transmitter action. Unstimulated cells bathed in Krebs saline exhibited cytoplasmic Ca ion concentrations, [Ca2+], that were generally in the 30-60 nM range. Exposure of cells to high-potassium (25 mM) saline depolarized the membrane potential and produced an immediate rise in [Ca2+] that recovered within 2-3 min in normal saline. The response grew progressively larger over the first 20 d in culture. Transient increases in [Ca2+] to levels greater than 1 microM were observed after 12-14 d in vitro, at which time the cells displayed intense electrical activity when exposed to high K. At this stage, the increases were attenuated by blocking action potential activity with TTX. In TTX-treated or immature cells, in which the transient phase of the Ca change was relatively small, a second exposure to high K typically produced a much larger Ca response that the initial exposure. The duration of this facilitation of the response persisted for periods longer than 5 min. Application of the neurotransmitter GABA induced a transient increase in membrane conductance, with a reversal potential near resting potential (approx. -60 mV), and caused an intracellular Ca2+ increase that outlasted the exposure to GABA by several minutes. Glutamate, or kainate, induced an increase in membrane conductance but with a reversal potential more positive than spike threshold. These agents also elevated intracellular Ca2+, but unlike the case with GABA, this Ca response reversed rapidly upon removal of the transmitter. The facilitatory effect of repeated exposures to high-K saline, as well as the persistent Ca elevation following a brief GABA application, suggests that granule cells possess the capability of displaying activity-dependent changes in Ca levels in culture.

Cellular and genetic regulation of the development of the cerebellar system.

PubMed

Sotelo, Constantino

2004-04-01

Recent advances in molecular biology have drastically changed our vision on the development of the nervous system, the cerebellum in particular. After a classical descriptive period, we are now in a modern mechanistic epoch as we begin to answer crucial questions in our quest to understand the mechanisms underlying the emergence of brain complexity. This review begins with an analysis of the role of the "isthmic organizer" in the induction and specification of the cerebellar territory and progresses through cerebellar development to the formation of cerebellar maps. It gathers information about the control of the proliferation of granule cell precursors by Purkinje cells and the role of Shh/Gli-patched signaling. The migratory routes for cerebellar and precerebellar neurons, together with the long-range and short-range cues guiding gliophilic and, particularly, neurophilic migrations, are also discussed. Because these cues are similar to those involved in axon guidance, both processes are under the same molecular constraints. Finally, using primarily the olivocerebellar projection as a model, the cellular and molecular mechanisms involved in the formation of cerebellar maps are discussed. During embryonic development, Purkinje cells in the cerebellum and neurons in the inferior olive follow a simultaneous, but independent, process of intrinsic parcellation, giving rise to subsets of biochemically different cortical compartments. The occurrence of positional information shared between olivary axons and their postsynaptic targets, the Purkinje cells, provides a molecular code for the formation of coarse-grained maps. Activity-dependent mechanisms are required for the transition from crude to fine-grained maps. This important refinement, which confers ultimate specificity to the maps, is under the regulation of parallel fiber-Purkinje cell synaptic activity.

Functional expression of calcium-permeable canonical transient receptor potential 4-containing channels promotes migration of medulloblastoma cells.

PubMed

Wei, Wei-Chun; Huang, Wan-Chen; Lin, Yu-Ping; Becker, Esther B E; Ansorge, Olaf; Flockerzi, Veit; Conti, Daniele; Cenacchi, Giovanna; Glitsch, Maike D

2017-08-15

The proton sensing ovarian cancer G protein coupled receptor 1 (OGR1, aka GPR68) promotes expression of the canonical transient receptor potential channel subunit TRPC4 in normal and transformed cerebellar granule precursor (DAOY) cells. OGR1 and TRPC4 are prominently expressed in healthy cerebellar tissue throughout postnatal development and in primary cerebellar medulloblastoma tissues. Activation of TRPC4-containing channels in DAOY cells, but not non-transformed granule precursor cells, results in prominent increases in [Ca 2+ ] i and promotes cell motility in wound healing and transwell migration assays. Medulloblastoma cells not arising from granule precursor cells show neither prominent rises in [Ca 2+ ] i nor enhanced motility in response to TRPC4 activation unless they overexpressTRPC4. Our results suggest that OGR1 enhances expression of TRPC4-containing channels that contribute to enhanced invasion and metastasis of granule precursor-derived human medulloblastoma. Aberrant intracellular Ca 2+ signalling contributes to the formation and progression of a range of distinct pathologies including cancers. Rises in intracellular Ca 2+ concentration occur in response to Ca 2+ influx through plasma membrane channels and Ca 2+ release from intracellular Ca 2+ stores, which can be mobilized in response to activation of cell surface receptors. Ovarian cancer G protein coupled receptor 1 (OGR1, aka GPR68) is a proton-sensing G q -coupled receptor that is most highly expressed in cerebellum. Medulloblastoma (MB) is the most common paediatric brain tumour that arises from cerebellar precursor cells. We found that nine distinct human MB samples all expressed OGR1. In both normal granule cells and the transformed human cerebellar granule cell line DAOY, OGR1 promoted expression of the proton-potentiated member of the canonical transient receptor potential (TRPC) channel family, TRPC4. Consistent with a role for TRPC4 in MB, we found that all MB samples also expressed

Distorted secretory granule composition in mast cells with multiple protease deficiency.

PubMed

Grujic, Mirjana; Calounova, Gabriela; Eriksson, Inger; Feyerabend, Thorsten; Rodewald, Hans-Reimer; Tchougounova, Elena; Kjellén, Lena; Pejler, Gunnar

2013-10-01

Mast cells are characterized by an abundance of secretory granules densely packed with inflammatory mediators such as bioactive amines, cytokines, serglycin proteoglycans with negatively charged glycosaminoglycan side chains of either heparin or chondroitin sulfate type, and large amounts of positively charged proteases. Despite the large biological impact of mast cell granules and their contents on various pathologies, the mechanisms that regulate granule composition are incompletely understood. In this study, we hypothesized that granule composition is dependent on a dynamic electrostatic interrelationship between different granule compounds. As a tool to evaluate this possibility, we generated mice in which mast cells are multideficient in a panel of positively charged proteases: the chymase mouse mast cell protease-4, the tryptase mouse mast cell protease-6, and carboxypeptidase A3. Through a posttranslational effect, mast cells from these mice additionally lack mouse mast cell protease-5 protein. Mast cells from mice deficient in individual proteases showed normal morphology. In contrast, mast cells with combined protease deficiency displayed a profound distortion of granule integrity, as seen both by conventional morphological criteria and by transmission electron microscopy. An assessment of granule content revealed that the distorted granule integrity in multiprotease-deficient mast cells was associated with a profound reduction of highly negatively charged heparin, whereas no reduction in chondroitin sulfate storage was observed. Taken together with previous findings showing that the storage of basic proteases conversely is regulated by anionic proteoglycans, these data suggest that secretory granule composition in mast cells is dependent on a dynamic interrelationship between granule compounds of opposite electrical charge.

Hydroxyurea Treatment and Development of the Rat Cerebellum: Effects on the Neurogenetic Profiles and Settled Patterns of Purkinje Cells and Deep Cerebellar Nuclei Neurons.

PubMed

Martí, Joaquín; Santa-Cruz, M C; Serra, Roger; Hervás, José P

2016-11-01

The current paper analyzes the development of the male and female rat cerebellum exposed to hydroxyurea (HU) (300 or 600 mg/kg) as embryo and collected at postnatal day 90. Our study reveals that the administration of this drug compromises neither the cytoarchitecture of the cerebellar cortex nor deep nuclei (DCN). However, in comparison with the saline group, we observed that several cerebellar parameters were lower in the HU injected groups. These parameters included area of the cerebellum, cerebellar cortex length, molecular layer area, Purkinje cell number, granule cell counts, internal granular layer, white matter and cerebellar nuclei areas, and number of deep cerebellar nuclei neurons. These features were larger in the rats injected with saline, smaller in those exposed to 300 mg/kg of HU and smallest in the group receiving 600 mg/kg of this agent. No sex differences in the effect of the HU were observed. In addition, we infer the neurogenetic timetables and the neurogenetic gradients of PCs and DCN neurons in rats exposed to either saline or HU as embryos. For this purpose, 5-bromo-2'-deoxyuridine was injected into pregnant rats previously administered with saline or HU. This thymidine analog was administered following a progressively delayed cumulative labeling method. The data presented here show that systematic differences exist in the pattern of neurogenesis and in the spatial location of cerebellar neurons between rats injected with saline or HU. No sex differences in the effect of the HU were observed. These findings have implications for the administration of this compound to women in gestation as the effects of HU on the development of the cerebellum might persist throughout their offsprings' life.

The chromatin remodeling factor CHD7 controls cerebellar development by regulating reelin expression

PubMed Central

Whittaker, Danielle E.; Riegman, Kimberley L.H.; Kasah, Sahrunizam; Mohan, Conor; Yu, Tian; Sala, Blanca Pijuan; Hebaishi, Husam; Caruso, Angela; Marques, Ana Claudia; Michetti, Caterina; Smachetti, María Eugenia Sanz; Shah, Apar; Sabbioni, Mara; Kulhanci, Omer; Tee, Wee-Wei; Reinberg, Danny; Scattoni, Maria Luisa; McGonnell, Imelda; Wardle, Fiona C.; Fernandes, Cathy

2017-01-01

The mechanisms underlying the neurodevelopmental deficits associated with CHARGE syndrome, which include cerebellar hypoplasia, developmental delay, coordination problems, and autistic features, have not been identified. CHARGE syndrome has been associated with mutations in the gene encoding the ATP-dependent chromatin remodeler CHD7. CHD7 is expressed in neural stem and progenitor cells, but its role in neurogenesis during brain development remains unknown. Here we have shown that deletion of Chd7 from cerebellar granule cell progenitors (GCps) results in reduced GCp proliferation, cerebellar hypoplasia, developmental delay, and motor deficits in mice. Genome-wide expression profiling revealed downregulated expression of the gene encoding the glycoprotein reelin (Reln) in Chd7-deficient GCps. Recessive RELN mutations have been associated with severe cerebellar hypoplasia in humans. We found molecular and genetic evidence that reductions in Reln expression contribute to GCp proliferative defects and cerebellar hypoplasia in GCp-specific Chd7 mouse mutants. Finally, we showed that CHD7 is necessary for maintaining an open, accessible chromatin state at the Reln locus. Taken together, this study shows that Reln gene expression is regulated by chromatin remodeling, identifies CHD7 as a previously unrecognized upstream regulator of Reln, and provides direct in vivo evidence that a mammalian CHD protein can control brain development by modulating chromatin accessibility in neuronal progenitors. PMID:28165338

Gene expression as a sensitive endpoint to evaluate cell differentiation and maturation of the developing central nervous system in primary cultures of rat cerebellar granule cells (CGCs) exposed to pesticides

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

Hogberg, Helena T.; Department of Physiology, Wenner-Gren Institute, Stockholm University; Kinsner-Ovaskainen, Agnieszka

The major advantage of primary neuronal cultures for developmental neurotoxicity (DNT) testing is their ability to replicate the crucial stages of neurodevelopment. In our studies using primary culture of cerebellar granule cells (CGCs) we have evaluated whether the gene expression relevant to the most critical developmental processes such as neuronal differentiation (NF-68 and NF-200) and functional maturation (NMDA and GABA{sub A} receptors), proliferation and differentiation of astrocytes (GFAP and S100{beta}) as well as the presence of neural precursor cells (nestin and Sox10) could be used as an endpoint for in vitro DNT. The expression of these genes was assessed aftermore » exposure to various pesticides (paraquat parathion, dichlorvos, pentachlorophenol and cycloheximide) that could induce developmental neurotoxicity through different mechanisms. All studied pesticides significantly modified the expression of selected genes, related to the different stages of neuronal and/or glial cell development and maturation. The most significant changes were observed after exposure to paraquat and parathion (i.e. down-regulation of mRNA expression of NF-68 and NF-200, NMDA and GABA{sub A} receptors). Similarly, dichlorvos affected mainly neurons (decreased mRNA expression of NF-68 and GABA{sub A} receptors) whereas cycloheximide had an effect on neurons and astrocytes, as significant decreases in the mRNA expression of both neurofilaments (NF-68 and NF-200) and the astrocyte marker (S100{beta}) were observed. Our results suggest that toxicity induced by pesticides that target multiple pathways of neurodevelopment can be identified by studying expression of genes that are involved in different stages of cell development and maturation, and that gene expression could be used as a sensitive endpoint for initial screening to identify the compounds with the potential to cause developmental neurotoxicity.« less

Cerebellar neuronal loss in amyotrophic lateral sclerosis cases with ATXN2 intermediate repeat expansions.

PubMed

Tan, Rachel H; Kril, Jillian J; McGinley, Ciara; Hassani, Mohammad; Masuda-Suzukake, Masami; Hasegawa, Masato; Mito, Remika; Kiernan, Matthew C; Halliday, Glenda M

2016-02-01

Despite evidence suggesting that the cerebellum may be targeted in amyotrophic lateral sclerosis (ALS), particularly in cases with repeat expansions in the ATXN2 and C9ORF72 genes, the integrity of cerebellar neurons has yet to be examined. The present study undertakes a histopathological analysis to assess the impact of these repeat expansions on cerebellar neurons and determine whether similar cerebellar pathology occurs in sporadic disease. Purkinje and granule cells were quantified in the vermis and lateral cerebellar hemispheres of ALS cases with repeat expansions in the ATXN2 and C9ORF72 genes, sporadic disease, and sporadic progressive muscular atrophy with only lower motor neuron degeneration. ALS cases with intermediate repeat expansions in the ATXN2 gene demonstrate a significant loss in Purkinje cells in the cerebellar vermis only. Despite ALS cases with expansions in the C9ORF72 gene having the highest burden of inclusion pathology, no neuronal loss was observed in this group. Neuronal numbers were also unchanged in sporadic ALS and sporadic PMA cases. The present study has established a selective loss of Purkinje cells in the cerebellar vermis of ALS cases with intermediate repeat expansions in the ATXN2 gene, suggesting a divergent pathogenic mechanism independent of upper and lower motor neuron degeneration in ALS. We discuss these findings in the context of large repeat expansions in ATXN2 and spinocerebellar ataxia type 2, providing evidence that intermediate repeats in ATXN2 cause significant, albeit less substantial, spinocerebellar damage compared with longer repeats in ATXN2. © 2016 American Neurological Association.

Weaker control of the electrical properties of cerebellar granule cells by tonically active GABAA receptors in the Ts65Dn mouse model of Down’s syndrome

PubMed Central

2013-01-01

Background Down’s syndrome (DS) is caused by triplication of all or part of human chromosome 21 and is characterized by a decrease in the overall size of the brain. One of the brain regions most affected is the cerebellum, in which the number of granule cells (GCs) is markedly decreased. GCs process sensory information entering the cerebellum via mossy fibres and pass it on to Purkinje cells and inhibitory interneurons. How GCs transform incoming signals depends on their input–output relationship, which is adjusted by tonically active GABAA receptor channels. Results We report that in the Ts65Dn mouse model of DS, in which cerebellar volume and GC number are decreased as in DS, the tonic GABAA receptor current in GCs is smaller than in wild-type mice and is less effective in moderating input resistance and raising the minimum current required for action potential firing. We also find that tonically active GABAA receptors curb the height and broaden the width of action potentials in wild-type GCs but not in Ts65Dn GCs. Single-cell real-time quantitative PCR reveals that these electrical differences are accompanied by decreased expression of the gene encoding the GABAA receptor β3 subunit but not genes coding for some of the other GABAA receptor subunits expressed in GCs (α1, α6, β2 and δ). Conclusions Weaker moderation of excitability and action potential waveform in GCs of the Ts65Dn mouse by tonically active GABAA receptors is likely to contribute to atypical transfer of information through the cerebellum. Similar changes may occur in DS. PMID:23870245

Cerebellar Output in Zebrafish: An Analysis of Spatial Patterns and Topography in Eurydendroid Cell Projections

PubMed Central

Heap, Lucy A.; Goh, Chi Ching; Kassahn, Karin S.; Scott, Ethan K.

2013-01-01

The cerebellum is a brain region responsible for motor coordination and for refining motor programs. While a great deal is known about the structure and connectivity of the mammalian cerebellum, fundamental questions regarding its function in behavior remain unanswered. Recently, the zebrafish has emerged as a useful model organism for cerebellar studies, owing in part to the similarity in cerebellar circuits between zebrafish and mammals. While the cell types composing their cerebellar cortical circuits are generally conserved with mammals, zebrafish lack deep cerebellar nuclei, and instead a majority of cerebellar output comes from a single type of neuron: the eurydendroid cell. To describe spatial patterns of cerebellar output in zebrafish, we have used genetic techniques to label and trace eurydendroid cells individually and en masse. We have found that cerebellar output targets the thalamus and optic tectum, and have confirmed the presence of pre-synaptic terminals from eurydendroid cells in these structures using a synaptically targeted GFP. By observing individual eurydendroid cells, we have shown that different medial-lateral regions of the cerebellum have eurydendroid cells projecting to different targets. Finally, we found topographic organization in the connectivity between the cerebellum and the optic tectum, where more medial eurydendroid cells project to the rostral tectum while lateral cells project to the caudal tectum. These findings indicate that there is spatial logic underpinning cerebellar output in zebrafish with likely implications for cerebellar function. PMID:23554587

The autism susceptibility gene met regulates zebrafish cerebellar development and facial motor neuron migration

PubMed Central

Elsen, Gina E.; Choi, Louis Y.; Prince, Victoria E.; Ho, Robert K.

2009-01-01

During development, Met signaling regulates a range of cellular processes including growth, differentiation, survival and migration. The Met gene encodes a tyrosine kinase receptor, which is activated by Hgf (hepatocyte growth factor) ligand. Altered regulation of human MET expression has been implicated in autism. In mouse, Met signaling has been shown to regulate cerebellum development. Since abnormalities in cerebellar structure have been reported in some autistic patients, we have used the zebrafish to address the role of Met signaling during cerebellar development and thus further our understanding of the molecular basis of autism. We find that zebrafish met is expressed in the cerebellar primordium, later localizing to the ventricular zone (VZ), with the hgf1 and hgf2 ligand genes expressed in surrounding tissues. Morpholino knockdown of either Met or its Hgf ligands leads to a significant reduction in the size of the cerebellum, primarily as a consequence of reduced proliferation. Met signaling knockdown disrupts specification of VZ-derived cell types, and also reduces granule cell numbers, due to an early effect on cerebellar proliferation and/or as an indirect consequence of loss of signals from VZ-derived cells later in development. These patterning defects preclude analysis of cerebellar neuronal migration, but we have found that Met signaling is necessary for migration of hindbrain facial motor neurons. In summary, we have described roles for Met signaling in coordinating growth and cell type specification within the developing cerebellum, and in migration of hindbrain neurons. These functions may underlie the correlation between altered MET regulation and Autism Spectrum Disorders. PMID:19732764

Constitutively polarized granules prime KHYG-1 NK cells.

PubMed

Suck, Garnet; Branch, Donald R; Aravena, Paola; Mathieson, Mark; Helke, Simone; Keating, Armand

2006-09-01

The major mechanism for NK cell lysis of tumor cells is granule-mediated cytotoxicity. Polarization of granules is a prelude to the release of their cytotoxic contents in response to target-cell binding. We describe the novel observation of constitutive granule polarization in the cytotoxic NK cell line, KHYG-1. Continuous degranulation of KHYG-1 cells, however, does not occur and still requires target-cell contact. Disruption of microtubules with colcemid is sufficient to disperse the granules in KHYG-1 and significantly decreases cytotoxicity. A similar effect is not obtained by inhibiting extracellular signal-related kinase 2 (ERK2), the most distal kinase investigated in the cytolytic pathway. Disruption of microtubules significantly down-regulates activation receptors, NKp44 and NKG2D, implicating them as potential microtubule-trafficking receptors. Such changes in upstream receptor expression may have caused deactivation of ERK2, since NKG2D cross-linking also leads to receptor down-regulation and diminished ERK phosphorylation. Thus, a functional role for NKG2D in KHYG-1 cytotoxicity is demonstrated. Moreover, the novel primed state may contribute to the high cytotoxicity exhibited by KHYG-1.

Optogenetic Modulation and Multi-Electrode Analysis of Cerebellar Networks In Vivo

PubMed Central

Kruse, Wolfgang; Krause, Martin; Aarse, Janna; Mark, Melanie D.; Manahan-Vaughan, Denise; Herlitze, Stefan

2014-01-01

The firing patterns of cerebellar Purkinje cells (PCs), as the sole output of the cerebellar cortex, determine and tune motor behavior. PC firing is modulated by various inputs from different brain regions and by cell-types including granule cells (GCs), climbing fibers and inhibitory interneurons. To understand how signal integration in PCs occurs and how subtle changes in the modulation of PC firing lead to adjustment of motor behaviors, it is important to precisely record PC firing in vivo and to control modulatory pathways in a spatio-temporal manner. Combining optogenetic and multi-electrode approaches, we established a new method to integrate light-guides into a multi-electrode system. With this method we are able to variably position the light-guide in defined regions relative to the recording electrode with micrometer precision. We show that PC firing can be precisely monitored and modulated by light-activation of channelrhodopsin-2 (ChR2) expressed in PCs, GCs and interneurons. Thus, this method is ideally suited to investigate the spatio/temporal modulation of PCs in anesthetized and in behaving mice. PMID:25144735

Gray-level co-occurrence matrix analysis of several cell types in mouse brain using resolution-enhanced photothermal microscopy

NASA Astrophysics Data System (ADS)

Kobayashi, Takayoshi; Sundaram, Durga; Nakata, Kazuaki; Tsurui, Hiromichi

2017-03-01

Qualifications of intracellular structure were performed for the first time using the gray-level co-occurrence matrix (GLCM) method for images of cells obtained by resolution-enhanced photothermal imaging. The GLCM method has been used to extract five parameters of texture features for five different types of cells in mouse brain; pyramidal neurons and glial cells in the basal nucleus (BGl), dentate gyrus granule cells, cerebellar Purkinje cells, and cerebellar granule cells. The parameters are correlation, contrast, angular second moment (ASM), inverse difference moment (IDM), and entropy for the images of cells of interest in a mouse brain. The parameters vary depending on the pixel distance taken in the analysis method. Based on the obtained results, we identified that the most suitable GLCM parameter is IDM for pyramidal neurons and BGI, granule cells in the dentate gyrus, Purkinje cells and granule cells in the cerebellum. It was also found that the ASM is the most appropriate for neurons in the basal nucleus.

Cerebellar stem cells do not produce neurons and astrocytes in adult mouse

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

Su, Xin; Guan, Wuqiang; Yu, Yong-Chun

Highlights: • No new neurons and astrocytes are generated in adult mouse cerebellum. • Very few mash1{sup +} or nestin{sup +} stem cells exist, and most of them are quiescent. • Cell proliferation rate is diversified among cerebellar regions and decreases over time. - Abstract: Although previous studies implied that cerebellar stem cells exist in some adult mammals, little is known about whether these stem cells can produce new neurons and astrocytes. In this study by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection, we found that there are abundant BrdU{sup +} cells in adult mouse cerebellum, and their quantity and density decreasesmore » significantly over time. We also found cell proliferation rate is diversified in different cerebellar regions. Among these BrdU{sup +} cells, very few are mash1{sup +} or nestin{sup +} stem cells, and the vast majority of cerebellar stem cells are quiescent. Data obtained by in vivo retrovirus injection indicate that stem cells do not produce neurons and astrocytes in adult mouse cerebellum. Instead, some cells labeled by retrovirus are Iba1{sup +} microglia. These results indicate that very few stem cells exist in adult mouse cerebellum, and none of these stem cells contribute to neurogenesis and astrogenesis under physiological condition.« less

Onset of Tlx-3 expression in the chick cerebellar cortex correlates with the morphological development of fissures and delineates a posterior transverse boundary.

PubMed

Logan, Cairine; Millar, Cassie; Bharadia, Vinay; Rouleau, Katherine

2002-06-24

Recent studies have shown that the mammalian cerebellar cortex can be subdivided into a reproducible array of zones and stripes. In particular, discontinuous patterns of gene expression together with mutational analysis suggest that there are at least four distinct transverse zones along the rostrocaudal axis in mouse: the anterior zone (lobules I-V), the central zone (lobules VI and VII), the posterior zone (lobules VIII and IX), and the nodular zone (lobule X). Here we show that the divergent homeobox-containing transcription factor, Tlx- 3 (also known as Hox11L2 or Rnx) is transiently expressed in external granule cells in a distinct transverse domain of the developing chick cerebellar cortex. Expression is first detected at Hamburger and Hamilton (HH) stage 35. Interestingly, Tlx-3 mRNA expression is initially confined to, and coincident with, the morphological development of fissures. Slightly later, at HH stage 38, expression extends throughout the developing external granular layer (EGL) of lobules I-IXab. Notably, no Tlx-3 expression was detected in lobules IXc and X at any developmental time point examined. Expression is noticeably stronger in nonproliferating cells located in the deep layer of the EGL. Tlx-3 expression is downregulated as granule cells migrate inward to form the internal granule layer and is undetectable shortly after birth. These results suggest that Tlx-3 is expressed as granule cells become postmitotic and suggest that Tlx-3 may play a role in the differentiation of distinct neuronal populations in the cerebellum. Copyright 2002 Wiley-Liss, Inc.

GABAergic excitation after febrile seizures induces ectopic granule cells and adult epilepsy.

PubMed

Koyama, Ryuta; Tao, Kentaro; Sasaki, Takuya; Ichikawa, Junya; Miyamoto, Daisuke; Muramatsu, Rieko; Matsuki, Norio; Ikegaya, Yuji

2012-08-01

Temporal lobe epilepsy (TLE) is accompanied by an abnormal location of granule cells in the dentate gyrus. Using a rat model of complex febrile seizures, which are thought to be a precipitating insult of TLE later in life, we report that aberrant migration of neonatal-generated granule cells results in granule cell ectopia that persists into adulthood. Febrile seizures induced an upregulation of GABA(A) receptors (GABA(A)-Rs) in neonatally generated granule cells, and hyperactivation of excitatory GABA(A)-Rs caused a reversal in the direction of granule cell migration. This abnormal migration was prevented by RNAi-mediated knockdown of the Na(+)K(+)2Cl(-) co-transporter (NKCC1), which regulates the excitatory action of GABA. NKCC1 inhibition with bumetanide after febrile seizures rescued the granule cell ectopia, susceptibility to limbic seizures and development of epilepsy. Thus, this work identifies a previously unknown pathogenic role of excitatory GABA(A)-R signaling and highlights NKCC1 as a potential therapeutic target for preventing granule cell ectopia and the development of epilepsy after febrile seizures.

BNN27, a 17-Spiroepoxy Steroid Derivative, Interacts With and Activates p75 Neurotrophin Receptor, Rescuing Cerebellar Granule Neurons from Apoptosis.

PubMed

Pediaditakis, Iosif; Kourgiantaki, Alexandra; Prousis, Kyriakos C; Potamitis, Constantinos; Xanthopoulos, Kleanthis P; Zervou, Maria; Calogeropoulou, Theodora; Charalampopoulos, Ioannis; Gravanis, Achille

2016-01-01

Neurotrophin receptors mediate a plethora of signals affecting neuronal survival. The p75 pan-neurotrophin receptor controls neuronal cell fate after its selective activation by immature and mature isoforms of all neurotrophins. It also exerts pleiotropic effects interacting with a variety of ligands in different neuronal or non-neuronal cells. In the present study, we explored the biophysical and functional interactions of a blood-brain-barrier (BBB) permeable, C17-spiroepoxy steroid derivative, BNN27, with p75 NTR receptor. BNN27 was recently shown to bind to NGF high-affinity receptor, TrkA. We now tested the p75 NTR -mediated effects of BNN27 in mouse Cerebellar Granule Neurons (CGNs), expressing p75 NTR , but not TrkA receptors. Our findings show that BNN27 physically interacts with p75 NTR receptors in specific amino-residues of its extracellular domain, inducing the recruitment of p75 NTR receptor to its effector protein RIP2 and the simultaneous release of RhoGDI in primary neuronal cells. Activation of the p75 NTR receptor by BNN27 reverses serum deprivation-induced apoptosis of CGNs resulting in the decrease of the phosphorylation of pro-apoptotic JNK kinase and of the cleavage of Caspase-3, effects completely abolished in CGNs, isolated from p75 NTR null mice. In conclusion, BNN27 represents a lead molecule for the development of novel p75 NTR ligands, controlling specific p75 NTR -mediated signaling of neuronal cell fate, with potential applications in therapeutics of neurodegenerative diseases and brain trauma.

BNN27, a 17-Spiroepoxy Steroid Derivative, Interacts With and Activates p75 Neurotrophin Receptor, Rescuing Cerebellar Granule Neurons from Apoptosis

PubMed Central

Pediaditakis, Iosif; Kourgiantaki, Alexandra; Prousis, Kyriakos C.; Potamitis, Constantinos; Xanthopoulos, Kleanthis P.; Zervou, Maria; Calogeropoulou, Theodora; Charalampopoulos, Ioannis; Gravanis, Achille

2016-01-01

Neurotrophin receptors mediate a plethora of signals affecting neuronal survival. The p75 pan-neurotrophin receptor controls neuronal cell fate after its selective activation by immature and mature isoforms of all neurotrophins. It also exerts pleiotropic effects interacting with a variety of ligands in different neuronal or non-neuronal cells. In the present study, we explored the biophysical and functional interactions of a blood-brain-barrier (BBB) permeable, C17-spiroepoxy steroid derivative, BNN27, with p75NTR receptor. BNN27 was recently shown to bind to NGF high-affinity receptor, TrkA. We now tested the p75NTR-mediated effects of BNN27 in mouse Cerebellar Granule Neurons (CGNs), expressing p75NTR, but not TrkA receptors. Our findings show that BNN27 physically interacts with p75NTR receptors in specific amino-residues of its extracellular domain, inducing the recruitment of p75NTR receptor to its effector protein RIP2 and the simultaneous release of RhoGDI in primary neuronal cells. Activation of the p75NTR receptor by BNN27 reverses serum deprivation-induced apoptosis of CGNs resulting in the decrease of the phosphorylation of pro-apoptotic JNK kinase and of the cleavage of Caspase-3, effects completely abolished in CGNs, isolated from p75NTR null mice. In conclusion, BNN27 represents a lead molecule for the development of novel p75NTR ligands, controlling specific p75NTR-mediated signaling of neuronal cell fate, with potential applications in therapeutics of neurodegenerative diseases and brain trauma. PMID:28082899

Purkinje Cell Activity in the Cerebellar Anterior Lobe after Rabbit Eyeblink Conditioning

ERIC Educational Resources Information Center

Green, John T.; Steinmetz, Joseph E.

2005-01-01

The cerebellar anterior lobe may play a critical role in the execution and proper timing of learned responses. The current study was designed to monitor Purkinje cell activity in the rabbit cerebellar anterior lobe after eyeblink conditioning, and to assess whether Purkinje cells in recording locations may project to the interpositus nucleus.…

NK cells converge lytic granules to promote cytotoxicity and prevent bystander killing

PubMed Central

Hsu, Hsiang-Ting; Viswanath, Dixita I.; Önfelt, Björn

2016-01-01

Natural killer (NK) cell activation triggers sequential cellular events leading to destruction of diseased cells. We previously identified lytic granule convergence, a dynein- and integrin signal–dependent movement of lysosome-related organelles to the microtubule-organizing center, as an early step in the cell biological process underlying NK cell cytotoxicity. Why lytic granules converge during NK cell cytotoxicity, however, remains unclear. We experimentally controlled the availability of human ligands to regulate NK cell signaling and promote granule convergence with either directed or nondirected degranulation. By the use of acoustic trap microscopy, we generated specific effector–target cell arrangements to define the impact of the two modes of degranulation. NK cells with converged granules had greater targeted and less nonspecific “bystander” killing. Additionally, NK cells in which dynein was inhibited or integrin blocked under physiological conditions demonstrated increased nondirected degranulation and bystander killing. Thus, NK cells converge lytic granules and thereby improve the efficiency of targeted killing and prevent collateral damage to neighboring healthy cells. PMID:27903610

Activation of sodium channels by α-scorpion toxin, BmK NT1, produced neurotoxicity in cerebellar granule cells: an association with intracellular Ca2+ overloading.

PubMed

He, Yuwei; Zou, Xiaohan; Li, Xichun; Chen, Juan; Jin, Liang; Zhang, Fan; Yu, Boyang; Cao, Zhengyu

2017-02-01

Voltage-gated sodium channels (VGSCs) are responsible for the action potential generation in excitable cells including neurons and involved in many physiological and pathological processes. Scorpion toxins are invaluable tools to explore the structure and function of ion channels. BmK NT1, a scorpion toxin from Buthus martensii Karsch, stimulates sodium influx in cerebellar granule cells (CGCs). In this study, we characterized the mode of action of BmK NT1 on the VGSCs and explored the cellular response in CGC cultures. BmK NT1 delayed the fast inactivation of VGSCs, increased the Na + currents, and shifted the steady-state activation and inactivation to more hyperpolarized membrane potential, which was similar to the mode of action of α-scorpion toxins. BmK NT1 stimulated neuron death (EC 50  = 0.68 µM) and produced massive intracellular Ca 2+ overloading (EC 50  = 0.98 µM). TTX abrogated these responses, suggesting that both responses were subsequent to the activation of VGSCs. The Ca 2+ response of BmK NT1 was primary through extracellular Ca 2+ influx since reducing the extracellular Ca 2+ concentration suppressed the Ca 2+ response. Further pharmacological evaluation demonstrated that BmK NT1-induced Ca 2+ influx and neurotoxicity were partially blocked either by MK-801, an NMDA receptor blocker, or by KB-R7943, an inhibitor of Na + /Ca 2+ exchangers. Nifedipine, an L-type Ca 2+ channel inhibitor, slightly suppressed both Ca 2+ response and neurotoxicity. A combination of these three inhibitors abrogated both responses. Considered together, these data ambiguously demonstrated that activation of VGSCs by an α-scorpion toxin was sufficient to produce neurotoxicity which was associated with intracellular Ca 2+ overloading through both NMDA receptor- and Na + /Ca 2+ exchanger-mediated Ca 2+ influx.

The Role of Ca2+ Imbalance in the Induction of Acute Oxidative Stress and Cytotoxicity in Cultured Rat Cerebellar Granule Cells Challenged with Tetrabromobisphenol A.

PubMed

Zieminska, Elzbieta; Lenart, Jacek; Diamandakis, Dominik; Lazarewicz, Jerzy W

2017-03-01

Using primary cultures of rat cerebellar granule cells (CGC) we examined the role of calcium transients induced by tetrabromobisphenol A (TBBPA) in triggering oxidative stress and cytotoxicity. CGC were exposed for 30 min to 10 or 25 µM TBBPA. Changes in intracellular calcium concentration ([Ca 2+ ] i ), in the production of reactive oxygen species (ROS), and in the potential of mitochondria (∆Ψm) were measured fluorometrically during the exposure. The intracellular glutathione (GSH) and catalase activity were determined after the incubation; cell viability was evaluated 24 h later. TBBPA concentration-dependently increased [Ca 2+ ] i and ROS production, and reduced GSH content, catalase activity, ∆Ψm and neuronal viability. The combination of NMDA and ryanodine receptor antagonists, MK-801 and bastadin 12 with ryanodine, respectively, prevented Ca 2+ transients and partially reduced cytotoxicity induced by TBBPA at both concentrations. The antagonists also completely inhibited oxidative stress and depolarization of mitochondria evoked by 10 µM TBBPA, whereas these effects were only partially reduced in the 25 µM TBBPA treatment. Free radical scavengers prevented TBBPA-induced development of oxidative stress and improved CGC viability without having any effect on the rises in Ca 2+ and drop in ∆Ψm. The co-administration of scavengers with NMDA and ryanodine receptor antagonists provided almost complete neuroprotection. These results indicate that Ca 2+ imbalance and oxidative stress both mediate acute toxicity of TBBPA in CGC. At 10 µM TBBPA Ca 2+ imbalance is a primary event, inducing oxidative stress, depolarization of mitochondria and cytotoxicity, whilst at a concentration of 25 µM TBBPA an additional Ca 2+ -independent portion of oxidative stress and cytotoxicity emerges.

Laminin α1 is essential for mouse cerebellar development

PubMed Central

Ichikawa-Tomikawa, Naoki; Ogawa, Junko; Douet, Vanessa; Xu, Zhuo; Kamikubo, Yuji; Sakurai, Takashi; Kohsaka, Shinichi; Chiba, Hideki; Hattori, Nobutaka; Yamada, Yoshihiko; Arikawa-Hirasawa, Eri

2011-01-01

Laminin α1 (Lama1), which is a subunit of laminin-1 (laminin-111), a heterotrimeric ECM protein, is essential for embryonic development and promotes neurite outgrowth in culture. Because the deletion of Lama1 causes lethality at early embryonic stages in mice, the in vivo role of Lama1 in neural development and functions has not yet been possible to determine. In this study, we generated conditional Lama1 knockout (Lama1CKO) mice in the epiblast lineage using Sox2-Cre mice. These Lama1CKO mice survived, but displayed behavioral disorders and impaired formation of the cerebellum. Deficiency of Lama1 in the pial basement membrane of the meninges resulted in defects in the conformation of the meninges. During cerebellar development, Lama1 deficiency also caused a decrease in the proliferation and migration of granule cell precursors, disorganization of Bergmann glial fibers and endfeet, and a transient reduction in the activity of Akt. A marked reduction in numbers of dendritic processes in Purkinje cells was observed in Lama1CKO mice. Together, these results indicate that Lama1 is required for cerebellar development and functions. PMID:21983115

A theory of cerebellar cortex and adaptive motor control based on two types of universal function approximation capability.

PubMed

Fujita, Masahiko

2016-03-01

Lesions of the cerebellum result in large errors in movements. The cerebellum adaptively controls the strength and timing of motor command signals depending on the internal and external environments of movements. The present theory describes how the cerebellar cortex can control signals for accurate and timed movements. A model network of the cerebellar Golgi and granule cells is shown to be equivalent to a multiple-input (from mossy fibers) hierarchical neural network with a single hidden layer of threshold units (granule cells) that receive a common recurrent inhibition (from a Golgi cell). The weighted sum of the hidden unit signals (Purkinje cell output) is theoretically analyzed regarding the capability of the network to perform two types of universal function approximation. The hidden units begin firing as the excitatory inputs exceed the recurrent inhibition. This simple threshold feature leads to the first approximation theory, and the network final output can be any continuous function of the multiple inputs. When the input is constant, this output becomes stationary. However, when the recurrent unit activity is triggered to decrease or the recurrent inhibition is triggered to increase through a certain mechanism (metabotropic modulation or extrasynaptic spillover), the network can generate any continuous signals for a prolonged period of change in the activity of recurrent signals, as the second approximation theory shows. By incorporating the cerebellar capability of two such types of approximations to a motor system, in which learning proceeds through repeated movement trials with accompanying corrections, accurate and timed responses for reaching the target can be adaptively acquired. Simple models of motor control can solve the motor error vs. sensory error problem, as well as the structural aspects of credit (or error) assignment problem. Two physiological experiments are proposed for examining the delay and trace conditioning of eyelid responses, as

Rapid Feedforward Inhibition and Asynchronous Excitation Regulate Granule Cell Activity in the Mammalian Main Olfactory Bulb

PubMed Central

Burton, Shawn D.

2015-01-01

Granule cell-mediated inhibition is critical to patterning principal neuron activity in the olfactory bulb, and perturbation of synaptic input to granule cells significantly alters olfactory-guided behavior. Despite the critical role of granule cells in olfaction, little is known about how sensory input recruits granule cells. Here, we combined whole-cell patch-clamp electrophysiology in acute mouse olfactory bulb slices with biophysical multicompartmental modeling to investigate the synaptic basis of granule cell recruitment. Physiological activation of sensory afferents within single glomeruli evoked diverse modes of granule cell activity, including subthreshold depolarization, spikelets, and suprathreshold responses with widely distributed spike latencies. The generation of these diverse activity modes depended, in part, on the asynchronous time course of synaptic excitation onto granule cells, which lasted several hundred milliseconds. In addition to asynchronous excitation, each granule cell also received synchronous feedforward inhibition. This inhibition targeted both proximal somatodendritic and distal apical dendritic domains of granule cells, was reliably recruited across sniff rhythms, and scaled in strength with excitation as more glomeruli were activated. Feedforward inhibition onto granule cells originated from deep short-axon cells, which responded to glomerular activation with highly reliable, short-latency firing consistent with tufted cell-mediated excitation. Simulations showed that feedforward inhibition interacts with asynchronous excitation to broaden granule cell spike latency distributions and significantly attenuates granule cell depolarization within local subcellular compartments. Collectively, our results thus identify feedforward inhibition onto granule cells as a core feature of olfactory bulb circuitry and establish asynchronous excitation and feedforward inhibition as critical regulators of granule cell activity. SIGNIFICANCE

Comparative Analysis of Zearalenone Effects on Thyroid Receptor Alpha (TRα) and Beta (TRβ) Expression in Rat Primary Cerebellar Cell Cultures.

PubMed

Kiss, David Sandor; Ioja, Eniko; Toth, Istvan; Barany, Zoltan; Jocsak, Gergely; Bartha, Tibor; Horvath, Tamas L; Zsarnovszky, Attila

2018-05-11

Thyroid receptors play an important role in postnatal brain development. Zearalenone (ZEN), a major mycotoxin of Fusarium fungi, is well known to cause serious health problems in animals and humans through various mechanisms, including the physiological pathways of thyroid hormone (TH). In the present study, we aimed to investigate the expression of thyroid receptors α (TRα) and β (TRβ) in primary cerebellar neurons in the presence or absence of glia and following ZEN treatment, using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Primary cerebellar granule cells were treated with low doses of ZEN (0.1 nM) in combination with physiologically relevant concentrations of l-thyroxine (T4), 3,3',5-triiodo-l-thyronine (T3) and 17β-estradiol (E2). Expression levels of TRα and TRβ at mRNA and protein levels were slightly modified by ZEN administered alone; however, along with thyroid and steroid hormones, modelling the physiological conditions, expression levels of TRs varied highly depending on the given treatment. Gene expression levels were also highly modulated by the presence or absence of glial cells, with mostly contrasting effects. Our results demonstrate divergent transcriptional and translational mechanisms involved in the expression of TRs implied by ZEN and hormonal milieu, as well as culturing conditions.

Effects of Transforming Growth Factor Beta 1 in Cerebellar Development: Role in Synapse Formation

PubMed Central

Araujo, Ana P. B.; Diniz, Luan P.; Eller, Cristiane M.; de Matos, Beatriz G.; Martinez, Rodrigo; Gomes, Flávia C. A.

2016-01-01

Granule cells (GC) are the most numerous glutamatergic neurons in the cerebellar cortex and represent almost half of the neurons of the central nervous system. Despite recent advances, the mechanisms of how the glutamatergic synapses are formed in the cerebellum remain unclear. Among the TGF-β family, TGF-beta 1 (TGF-β1) has been described as a synaptogenic molecule in invertebrates and in the vertebrate peripheral nervous system. A recent paper from our group demonstrated that TGF-β1 increases the excitatory synapse formation in cortical neurons. Here, we investigated the role of TGF-β1 in glutamatergic cerebellar neurons. We showed that the expression profile of TGF-β1 and its receptor, TβRII, in the cerebellum is consistent with a role in synapse formation in vitro and in vivo. It is low in the early postnatal days (P1–P9), increases after postnatal day 12 (P12), and remains high until adulthood (P30). We also found that granule neurons express the TGF-β receptor mRNA and protein, suggesting that they may be responsive to the synaptogenic effect of TGF-β1. Treatment of granular cell cultures with TGF-β1 increased the number of glutamatergic excitatory synapses by 100%, as shown by immunocytochemistry assays for presynaptic (synaptophysin) and post-synaptic (PSD-95) proteins. This effect was dependent on TβRI activation because addition of a pharmacological inhibitor of TGF-β, SB-431542, impaired the formation of synapses between granular neurons. Together, these findings suggest that TGF-β1 has a specific key function in the cerebellum through regulation of excitatory synapse formation between granule neurons. PMID:27199658

The type II cGMP dependent protein kinase regulates GluA1 levels at the plasma membrane of developing cerebellar granule cells

PubMed Central

Incontro, Salvatore; Ciruela, Francisco; Ziff, Edward; Hofmann, Franz; Sánchez-Prieto, José; Torres, Magdalena

2014-01-01

Trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is regulated by specific interactions with other proteins and by post-translational mechanisms, such as phosphorylation. We have found that the type II cGMP-dependent protein kinase (cGKII) phosphorylates GluA1 (formerly GluR1) at S845, augmenting the surface expression of AMPARs at both synaptic and extrasynaptic sites. Activation of cGKII by 8-Br-cGMP enhances the surface expression of GluA1, whereas its inhibition or suppression effectively diminished the expression of this protein at the cell surface. In granule cells, NMDA receptor activation (NMDAR) stimulates nitric oxide and cGMP production, which in turn activates cGKII and induces the phosphorylation of GluA1, promoting its accumulation in the plasma membrane. GluA1 is mainly incorporated into calcium permeable AMPARs as exposure to 8-Br-cGMP or NMDA activation enhanced AMPA-elicited calcium responses that are sensitive to NASPM inhibition. We summarize evidence for an increase of calcium permeable AMPA receptors downstream of NMDA receptor activation that might be relevant for granule cell development and plasticity. PMID:23545413

Cerebellar defects in a mouse model of juvenile neuronal ceroid lipofuscinosis.

PubMed

Weimer, Jill M; Benedict, Jared W; Getty, Amanda L; Pontikis, Charlie C; Lim, Ming J; Cooper, Jonathan D; Pearce, David A

2009-04-17

Juvenile neuronal ceroid lipofuscinosis (JNCL), or Batten disease, is a neurodegenerative disease resulting from a mutation in CLN3, which presents clinically with visual deterioration, seizures, motor impairments, cognitive decline, hallucinations, loss of circadian rhythm, and premature death in the late-twenties to early-thirties. Using a Cln3 null (Cln3(-/-)) mouse, we report here several deficits in the cerebellum in the absence of Cln3, including cell loss and early onset motor deficits. Surprisingly, early onset glial activation and selective neuronal loss within the mature fastigial pathway of the deep cerebellar nuclei (DCN), a region critical for balance and coordination, are seen in many regions of the Cln3(-/-) cerebellum. Additionally, there is a loss of Purkinje cells (PC) in regions of robust Bergmann glia activation in Cln3(-/-) mice and human JNCL post-mortem cerebellum. Moreover, the Cln3(-/-) cerebellum had a mis-regulation in granule cell proliferation and maintenance of PC dendritic arborization and spine density. Overall, this study defines a novel multi-faceted, early-onset cerebellar disruption in the Cln3 null brain, including glial activation, cell loss, and aberrant cell proliferation and differentiation. These early alterations in the maturation of the cerebellum could underlie some of the motor deficits and pathological changes seen in JNCL patients.

Cerebellar Development and Disease

PubMed Central

Gleeson, Joseph G.

2008-01-01

Recent Advances The molecular control of cell type specification within the developing cerebellum as well as the genetic causes of the most common human developmental cerebellar disorders have long remained mysterious. Recent genetic lineage and loss-of-function data from mice have revealed unique and non-overlapping anatomical origins for GABAergic neurons from ventricular zone precursors and glutamatergic cell from rhombic lip precursors, mirroring distinct origins for these neurotransmitter-specific cell types in the cerebral cortex. Mouse studies elucidating the role of Ptf1a as a cerebellar ventricular zone GABerigic fate switch were actually preceded by the recognition that PTF1A mutations in humans cause cerebellar agenesis, a birth defect of the human cerebellum. Indeed, several genes for congenital human cerebellar malformations have recently been identified, including genes causing Joubert syndrome, Dandy-Walker malformation and Ponto-cerebellar hypoplasia. These studies have pointed to surprisingly complex roles for transcriptional regulation, mitochondrial function and neuronal cilia in patterning, homeostasis and cell proliferation during cerebellar development. Together mouse and human studies are synergistically advancing our understanding of the developmental mechanisms that generate the uniquely complex mature cerebellum. PMID:18513948

Current source density correlates of cerebellar Golgi and Purkinje cell responses to tactile input

PubMed Central

Tahon, Koen; Wijnants, Mike; De Schutter, Erik

2011-01-01

The overall circuitry of the cerebellar cortex has been known for over a century, but the function of many synaptic connections remains poorly characterized in vivo. We used a one-dimensional multielectrode probe to estimate the current source density (CSD) of Crus IIa in response to perioral tactile stimuli in anesthetized rats and to correlate current sinks and sources to changes in the spike rate of corecorded Golgi and Purkinje cells. The punctate stimuli evoked two distinct early waves of excitation (at <10 and ∼20 ms) associated with current sinks in the granular layer. The second wave was putatively of corticopontine origin, and its associated sink was located higher in the granular layer than the first trigeminal sink. The distinctive patterns of granular-layer sinks correlated with the spike responses of corecorded Golgi cells. In general, Golgi cell spike responses could be linearly reconstructed from the CSD profile. A dip in simple-spike activity of coregistered Purkinje cells correlated with a current source deep in the molecular layer, probably generated by basket cell synapses, interspersed between sparse early sinks presumably generated by synapses from granule cells. The late (>30 ms) enhancement of simple-spike activity in Purkinje cells was characterized by the absence of simultaneous sinks in the granular layer and by the suppression of corecorded Golgi cell activity, pointing at inhibition of Golgi cells by Purkinje axon collaterals as a likely mechanism of late Purkinje cell excitation. PMID:21228303

Sigma-1 Receptor Enhances Neurite Elongation of Cerebellar Granule Neurons via TrkB Signaling

PubMed Central

Kimura, Yuriko; Fujita, Yuki; Shibata, Kumi; Mori, Megumi; Yamashita, Toshihide

2013-01-01

Sigma-1 receptor (Sig-1R) is an integral membrane protein predominantly expressed in the endoplasmic reticulum. Sig-1R demonstrates a high affinity to various synthetic compounds including well-known psychotherapeutic drugs in the central nervous system (CNS). For that, it is considered as an alternative target for psychotherapeutic drugs. On the cellular level, when Sig-1R is activated, it is known to play a role in neuroprotection and neurite elongation. These effects are suggested to be mediated by its ligand-operated molecular chaperone activity, and/or upregulation of various Ca2+ signaling. In addition, recent studies show that Sig-1R activation induces neurite outgrowth via neurotrophin signaling. Here, we tested the hypothesis that Sig-1R activation promotes neurite elongation through activation of tropomyosin receptor kinase (Trk), a family of neurotrophin receptors. We found that 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate (PRE-084), a selective Sig-1R agonist, significantly promoted neurite outgrowth, and K252a, a Trk inhibitor, attenuated Sig-1R-mediated neurite elongation in cerebellar granule neurons (CGNs). Moreover, we revealed that Sig-1R interacts with TrkB, and PRE-084 treatment enhances phosphorylation of Y515, but not Y706. Thus, our results indicate that Sig-1R activation promotes neurite outgrowth in CGNs through Y515 phosphorylation of TrkB. PMID:24116072

Sigma-1 receptor enhances neurite elongation of cerebellar granule neurons via TrkB signaling.

PubMed

Kimura, Yuriko; Fujita, Yuki; Shibata, Kumi; Mori, Megumi; Yamashita, Toshihide

2013-01-01

Sigma-1 receptor (Sig-1R) is an integral membrane protein predominantly expressed in the endoplasmic reticulum. Sig-1R demonstrates a high affinity to various synthetic compounds including well-known psychotherapeutic drugs in the central nervous system (CNS). For that, it is considered as an alternative target for psychotherapeutic drugs. On the cellular level, when Sig-1R is activated, it is known to play a role in neuroprotection and neurite elongation. These effects are suggested to be mediated by its ligand-operated molecular chaperone activity, and/or upregulation of various Ca(2+) signaling. In addition, recent studies show that Sig-1R activation induces neurite outgrowth via neurotrophin signaling. Here, we tested the hypothesis that Sig-1R activation promotes neurite elongation through activation of tropomyosin receptor kinase (Trk), a family of neurotrophin receptors. We found that 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate (PRE-084), a selective Sig-1R agonist, significantly promoted neurite outgrowth, and K252a, a Trk inhibitor, attenuated Sig-1R-mediated neurite elongation in cerebellar granule neurons (CGNs). Moreover, we revealed that Sig-1R interacts with TrkB, and PRE-084 treatment enhances phosphorylation of Y515, but not Y706. Thus, our results indicate that Sig-1R activation promotes neurite outgrowth in CGNs through Y515 phosphorylation of TrkB.

Repeated intermittent alcohol exposure during the third trimester-equivalent increases expression of the GABA(A) receptor δ subunit in cerebellar granule neurons and delays motor development in rats.

PubMed

Diaz, Marvin R; Vollmer, Cyndel C; Zamudio-Bulcock, Paula A; Vollmer, William; Blomquist, Samantha L; Morton, Russell A; Everett, Julie C; Zurek, Agnieszka A; Yu, Jieying; Orser, Beverley A; Valenzuela, C Fernando

2014-04-01

Exposure to ethanol (EtOH) during fetal development can lead to long-lasting alterations, including deficits in fine motor skills and motor learning. Studies suggest that these are, in part, a consequence of cerebellar damage. Cerebellar granule neurons (CGNs) are the gateway of information into the cerebellar cortex. Functionally, CGNs are heavily regulated by phasic and tonic GABAergic inhibition from Golgi cell interneurons; however, the effect of EtOH exposure on the development of GABAergic transmission in immature CGNs has not been investigated. To model EtOH exposure during the 3rd trimester-equivalent of human pregnancy, neonatal pups were exposed intermittently to high levels of vaporized EtOH from postnatal day (P) 2 to P12. This exposure gradually increased pup serum EtOH concentrations (SECs) to ∼60 mM (∼0.28 g/dl) during the 4 h of exposure. EtOH levels gradually decreased to baseline 8 h after the end of exposure. Surprisingly, basal tonic and phasic GABAergic currents in CGNs were not significantly affected by postnatal alcohol exposure (PAE). However, PAE increased δ subunit expression at P28 as detected by immunohistochemical and western blot analyses. Also, electrophysiological studies with an agonist that is highly selective for δ-containing GABA(A) receptors, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine-3-ol (THIP), showed an increase in THIP-induced tonic current. Behavioral studies of PAE rats did not reveal any deficits in motor coordination, except for a delay in the acquisition of the mid-air righting reflex that was apparent at P15 to P18. These findings demonstrate that repeated intermittent exposure to high levels of EtOH during the equivalent of the last trimester of human pregnancy has significant but relatively subtle effects on motor coordination and GABAergic transmission in CGNs in rats. Copyright © 2013 Elsevier Ltd. All rights reserved.

Differential timing of granule cell production during cerebellum development underlies generation of the foliation pattern.

PubMed

Legué, Emilie; Gottshall, Jackie L; Jaumouillé, Edouard; Roselló-Díez, Alberto; Shi, Wei; Barraza, Luis Humberto; Washington, Senna; Grant, Rachel L; Joyner, Alexandra L

2016-09-08

The mouse cerebellum (Cb) has a remarkably complex foliated three-dimensional (3D) structure, but a stereotypical cytoarchitecture and local circuitry. Little is known of the cellular behaviors and genes that function during development to determine the foliation pattern. In the anteroposterior axis the mammalian cerebellum is divided by lobules with distinct sizes, and the foliation pattern differs along the mediolateral axis defining a medial vermis and two lateral hemispheres. In the vermis, lobules are further grouped into four anteroposterior zones (anterior, central, posterior and nodular zones) based on genetic criteria, and each has distinct lobules. Since each cerebellar afferent group projects to particular lobules and zones, it is critical to understand how the 3D structure of the Cb is acquired. During cerebellar development, the production of granule cells (gcs), the most numerous cell type in the brain, is required for foliation. We hypothesized that the timing of gc accumulation is different in the four vermal zones during development and contributes to the distinct lobule morphologies. In order to test this idea, we used genetic inducible fate mapping to quantify accumulation of gcs in each lobule during the first two postnatal weeks in mice. The timing of gc production was found to be particular to each lobule, and delayed in the central zone lobules relative to the other zones. Quantification of gc proliferation and differentiation at three time-points in lobules representing different zones, revealed the delay involves a later onset of maximum differentiation and prolonged proliferation of gc progenitors in the central zone. Similar experiments in Engrailed mutants (En1 (-/+) ;En2 (-/-) ), which have a smaller Cb and altered foliation pattern preferentially outside the central zone, showed that gc production, proliferation and differentiation are altered such that the differences between zones are attenuated compared to wild-type mice. Our

Epidermal growth factor receptor expression is related to post-mitotic events in cerebellar development: regulation by thyroid hormone.

PubMed

Carrasco, Emilce; Blum, Mariann; Weickert, Cynthia Shannon; Casper, Diana

2003-01-10

It has been established that thyroid hormone and neurotrophic factors both orchestrate developmental events in the brain. However, it is not clear how these two influences are related. In this study, we investigated the effects of thyroid hormone on cerebellar development and the coincident expression of transforming growth factor-alpha (TGF-alpha), a ligand in the epidermal growth factor (EGF) family, and the epidermal growth factor receptor (EGFR). Profiles of thyroid hormone expression were measured in postnatal animals and were found to peak at postnatal day 15 (P15). These levels dropped below detectable levels when mice were made hypothyroid with propylthiouracil (PTU). TGF-alpha and EGFR expression, as determined by RNAse protection assay, was maximal at P6 in normal animals, but remained low in hypothyroid animals, suggesting that thyroid hormone was responsible for their induction. In situ hybridization and immunohistochemical analysis of EGFR expression revealed that this receptor was present on granule cells within the inner zone of the external granule cell layer (EGL), suggesting that EGFR-ligands were not inducing granule cell proliferation. The persistence of EGFR expression on migrating granule cells and subsequent down-regulation of expression in the internal granule cell layer (IGL) implicates a role for EGFR-ligands in differentiation and/or migration. In hypothyroid animals, we observed a delayed progression of granule cell migration, consistent with the persistence of EGFR labeling in the EGL, and in the 'pile-up' of labeled cells at the interface between the molecular layer and the Purkinje cell layer. Taken together, these results implicate thyroid hormone in the coordinated expression of TGF-alpha and EGFR, which are positioned to play a role in post-mitotic developmental events in the cerebellum.

Dendrites of cerebellar granule cells correctly recognize their target axons for synaptogenesis in vitro.

PubMed

Ito, Shoko; Takeichi, Masatoshi

2009-08-04

Neural circuits are generated by precisely ordered synaptic connections among neurons, and this process is thought to rely on the ability of neurons to recognize specific partners. However, it is also known that neurons promiscuously form synapses with nonspecific partners, in particular when cultured in vitro, causing controversies about neural recognition mechanisms. Here we reexamined whether neurons can or cannot select particular partners in vitro. In the cerebellum, granule cell (GC) dendrites form synaptic connections specifically with mossy fibers, but not with climbing fibers. We cocultured GC neurons with pontine or inferior olivary axons, the major sources for mossy and climbing fibers, respectively, as well as with hippocampal axons as a control. The GC neurons formed synapses with pontine axons predominantly at the distal ends of their dendrites, reproducing the characteristic morphology of their synapses observed in vivo, whereas they failed to do so when combined with other axons. In the latter case, synaptic proteins could accumulate between axons and dendrites, but these synapses were randomly distributed throughout the contact sites, and also their synaptic vesicle recycling was anomalous. These observations suggest that GC dendrites can select their authentic partners for synaptogenesis even in vitro, forming the synapses with a GC-specific nature only with them.

Neurotoxicological effects of nicotine on the embryonic development of cerebellar cortex of chick embryo during various stages of incubation.

PubMed

El-Beltagy, Abd El-Fattah B M; Abou-El-Naga, Amoura M; Sabry, Dalia M

2015-10-01

Long-acting nicotine is known to exert pathological effects on almost all tissues including the cerebellar cortex. The present work was designed to elucidate the effect of nicotine on the development of cerebellar cortex of chick embryo during incubation period. The fertilized eggs of hen (Gallus gallus domesticus) were injected into the air space by a single dose of long acting nicotine (1.6 mg/kg/egg) at the 4th day of incubation. The embryos were taken out of the eggs on days 8, 12 and 16 of incubation. The cerebellum of the control and treated embryos at above ages were processed for histopathological examination. The TEM were examined at 16th day of incubation. The results of the present study revealed that, exposure to long-acting nicotine markedly influence the histogenesis of cerebellar cortex of chick embryo during the incubation period. At 8th day of incubation, nicotine delayed the differentiation of the cerebellar analge; especially the external granular layer (EGL) and inner cortical layer (ICL). Furthermore, at 12th day of incubation, the cerebellar foliation was irregular and the Purkinje cells not recognized. By 16th day of incubation, the cerebellar foliations were irregular with interrupted cerebellar cortex and irregular arrangement of Purkinje cells. Immunohistochemical analysis for antibody P53 protein revealed that the cerebellar cortex in all stages of nicotine treated groups possessed a moderate to weak reaction for P53 protein however; this reaction was markedly stronger in the cerebellar cortex of control groups. Moreover, the flow cytometric analysis confirmed that the percentage of apoptosis in control group was significantly higher compared with that of nicotine treated group. At the TEM level, the cerebellar Purkinje cells of 16th day of treated groups showed multiple subcellular alterations in compared with those of the corresponding control group. Such changes represented by appearing of vacuolated mitochondria, cisternal

Olfactory granule cell development in normal and hyperthyroid rats.

PubMed

Brunjes, P C; Schwark, H D; Greenough, W T

1982-10-01

Dendritic development was examined in olfactory bulbs of both normal 7-, 14-, 21- and 60-day-old rats and littermates treated on postnatal days 1-4 with 1 microgram/g body weight of L-thyroxine sodium. Tissue was processed via the Golgi-Cox technique and subjected to quantitative analyses of mitral and internal layer granule cell development. These populations of granule cells were selected because their pattern of late proliferation suggested potentially greater susceptibility to postnatal hormonal alterations. Although neonatal hyperthyroidism induces widespread acceleration of maturation, including precocious chemosensitivity, granule cell development was unaffected relative to littermate controls. Both normal and hyperthyroid groups exhibited an inverted U-shaped pattern of cellular development, with rapid dendritic dendritic growth and expansion occurring during the earliest ages tested, but with loss of processes and dendritic field size occurring after day 21.

Hilar Mossy Cell Degeneration Causes Transient Dentate Granule Cell Hyperexcitability and Impaired Pattern Separation

PubMed Central

Jinde, Seiichiro; Zsiros, Veronika; Jiang, Zhihong; Nakao, Kazuhito; Pickel, James; Kohno, Kenji; Belforte, Juan E.; Nakazawa, Kazu

2012-01-01

Summary Although excitatory mossy cells of the hippocampal hilar region are known to project both to dentate granule cells and to interneurons, it is as yet unclear whether mossy cell activity’s net effect on granule cells is excitatory or inhibitory. To explore their influence on dentate excitability and hippocampal function, we generated a conditional transgenic mouse line, using the Cre/loxP system, in which diphtheria toxin receptor was selectively expressed in mossy cells. One week after injecting toxin into this line, mossy cells throughout the longitudinal axis were degenerated extensively, theta wave power of dentate local field potentials increased during exploration, and deficits occurred in contextual discrimination. By contrast, we detected no epileptiform activity, spontaneous behavioral seizures, or mossy-fiber sprouting 5–6 weeks after mossy cell degeneration. These results indicate that the net effect of mossy cell excitation is to inhibit granule cell activity and enable dentate pattern separation. PMID:23259953

Impaired eye-blink conditioning in waggler, a mutant mouse with cerebellar BDNF deficiency.

PubMed

Bao, S; Chen, L; Qiao, X; Knusel, B; Thompson, R F

1998-01-01

In addition to their trophic functions, neurotrophins are also implicated in synaptic modulation and learning and memory. Although gene knockout techniques have been used widely in studying the roles of neurotrophins at molecular and cellular levels, behavioral studies using neurotrophin knockouts are limited by the early-onset lethality and various sensory deficits associated with the gene knockout mice. In the present study, we found that in a spontaneous mutant mouse, waggler, the expression of brain-derived neurotrophic factor (BDNF) was selectively absent in the cerebellar granule cells. The cytoarchitecture of the waggler cerebellum appeared to be normal at the light microscope level. The mutant mice exhibited no sensory deficits to auditory stimuli or heat-induced pain. However, they were massively impaired in classic eye-blink conditioning. These results suggest that BDNF may have a role in normal cerebellar neuronal function, which, in turn, is essential for classic eye-blink conditioning.

Cerebellar defects in a mouse model of juvenile neuronal ceroid lipofuscinosis

PubMed Central

Weimer, Jill M.; Benedict, Jared W.; Getty, Amanda L.; Pontikis, Charlie C.; Lim, Ming J.; Cooper, Jonathan D.; Pearce, David A.

2013-01-01

Juvenile neuronal ceroid lipofuscinosis (JNCL), or Batten disease, is a neurodegenerative disease resulting from a mutation in CLN3, which presents clinically with visual deterioration, seizures, motor impairments, cognitive decline, hallucinations, loss of circadian rhythm, and premature death in the late-twenties to early-thirties. Using a Cln3 null (Cln3−/−) mouse, we report here several deficits in the cerebellum in the absence of Cln3, including cell loss and early onset motor deficits. Surprisingly, early onset glial activation and selective neuronal loss within the mature fastigial pathway of the deep cerebellar nuclei (DCN), a region critical for balance and coordination, are seen in many regions of the Cln3−/− cerebellum. Additionally, there is a loss of Purkinje cells (PC) in regions of robust Bergmann glia activation in Cln3−/− mice and human JNCL post-mortem cerebellum. Moreover, the Cln3−/− cerebellum had a mis-regulation in granule cell proliferation and maintenance of PC dendritic arborization and spine density. Overall, this study defines a novel multi-faceted, early-onset cerebellar disruption in the Cln3 null brain, including glial activation, cell loss, and aberrant cell proliferation and differentiation. These early alterations in the maturation of the cerebellum could underlie some of the motor deficits and pathological changes seen in JNCL patients. PMID:19230832

The Cerebellar Mossy Fiber Synapse as a Model for High-Frequency Transmission in the Mammalian CNS.

PubMed

Delvendahl, Igor; Hallermann, Stefan

2016-11-01

The speed of neuronal information processing depends on neuronal firing frequency. Here, we describe the evolutionary advantages and ubiquitous occurrence of high-frequency firing within the mammalian nervous system in general. The highest firing frequencies so far have been observed at the cerebellar mossy fiber to granule cell synapse. The mechanisms enabling high-frequency transmission at this synapse are reviewed and compared with other synapses. Finally, information coding of high-frequency signals at the mossy fiber synapse is discussed. The exceptionally high firing frequencies and amenability to high-resolution technical approaches both in vitro and in vivo establish the cerebellar mossy fiber synapse as an attractive model to investigate high-frequency signaling from the molecular up to the network level. Copyright © 2016 Elsevier Ltd. All rights reserved.

Transient inhibition of the ERK pathway prevents cerebellar developmental defects and improves long-term motor functions in murine models of neurofibromatosis type 1.

PubMed

Kim, Edward; Wang, Yuan; Kim, Sun-Jung; Bornhorst, Miriam; Jecrois, Emmanuelle S; Anthony, Todd E; Wang, Chenran; Li, Yi E; Guan, Jun-Lin; Murphy, Geoffrey G; Zhu, Yuan

2014-12-23

Individuals with neurofibromatosis type 1 (NF1) frequently exhibit cognitive and motor impairments and characteristics of autism. The cerebellum plays a critical role in motor control, cognition, and social interaction, suggesting that cerebellar defects likely contribute to NF1-associated neurodevelopmental disorders. Here we show that Nf1 inactivation during early, but not late stages of cerebellar development, disrupts neuronal lamination, which is partially caused by overproduction of glia and subsequent disruption of the Bergmann glia (BG) scaffold. Specific Nf1 inactivation in glutamatergic neuronal precursors causes premature differentiation of granule cell (GC) precursors and ectopic production of unipolar brush cells (UBCs), indirectly disrupting neuronal migration. Transient MEK inhibition during a neonatal window prevents cerebellar developmental defects and improves long-term motor performance of Nf1-deficient mice. This study reveals essential roles of Nf1 in GC/UBC migration by generating correct numbers of glia and controlling GC/UBC fate-specification/differentiation, identifying a therapeutic prevention strategy for multiple NF1-associcated developmental abnormalities.

Pten Knockdown in vivo Increases Excitatory Drive onto Dentate Granule Cells

PubMed Central

Luikart, Bryan W.; Schnell, Eric; Washburn, Eric K.; Bensen, AeSoon L.; Tovar, Kenneth R.; Westbrook, Gary L.

2011-01-01

Some cases of autism spectrum disorder (ASD) have mutations in the lipid phosphatase, Pten (phosphatase and tensin homolog on chromosome 10). Tissue specific deletion of Pten in the hippocampus and cortex of mice causes anatomical and behavioral abnormalities similar to human autism. However, the impact of reductions in Pten on synaptic and circuit function remains unexplored. We used in vivo stereotaxic injections of lentivirus expressing an shRNA to knockdown Pten in mouse neonatal and young adult dentate granule cells. We then assessed the morphology and synaptic physiology between two weeks and four months later. Confocal imaging of the hippocampus revealed a marked increase in granule cell size and an increase in dendritic spine density. The onset of morphological changes occurred earlier in neonatal mice than in young adults. We used whole-cell recordings from granule cells in acute slices to assess synaptic function following Pten knockdown. Consistent with the increase in dendritic spines, the frequency of excitatory miniature and spontaneous postsynaptic currents increased. However, there was little or no effect on inhibitory postsynaptic currents. Thus Pten knockdown results in an imbalance between excitatory and inhibitory synaptic activity. Because reductions in Pten affected mature granule cells as well as developing granule cells, we suggest that the disruption of circuit function by Pten hypofunction may be ongoing well beyond early development. PMID:21411674

Ternary Kv4.2 channels recapitulate voltage-dependent inactivation kinetics of A-type K+ channels in cerebellar granule neurons.

PubMed

Amarillo, Yimy; De Santiago-Castillo, Jose A; Dougherty, Kevin; Maffie, Jonathon; Kwon, Elaine; Covarrubias, Manuel; Rudy, Bernardo

2008-04-15

Kv4 channels mediate most of the somatodendritic subthreshold operating A-type current (I(SA)) in neurons. This current plays essential roles in the regulation of spike timing, repetitive firing, dendritic integration and plasticity. Neuronal Kv4 channels are thought to be ternary complexes of Kv4 pore-forming subunits and two types of accessory proteins, Kv channel interacting proteins (KChIPs) and the dipeptidyl-peptidase-like proteins (DPPLs) DPPX (DPP6) and DPP10. In heterologous cells, ternary Kv4 channels exhibit inactivation that slows down with increasing depolarization. Here, we compared the voltage dependence of the inactivation rate of channels expressed in heterologous mammalian cells by Kv4.2 proteins with that of channels containing Kv4.2 and KChIP1, Kv4.2 and DPPX-S, or Kv4.2, KChIP1 and DPPX-S, and found that the relation between inactivation rate and membrane potential is distinct for these four conditions. Moreover, recordings from native neurons showed that the inactivation kinetics of the I(SA) in cerebellar granule neurons has voltage dependence that is remarkably similar to that of ternary Kv4 channels containing KChIP1 and DPPX-S proteins in heterologous cells. The fact that this complex and unique behaviour (among A-type K(+) currents) is observed in both the native current and the current expressed in heterologous cells by the ternary complex containing Kv4, DPPX and KChIP proteins supports the hypothesis that somatically recorded native Kv4 channels in neurons include both types of accessory protein. Furthermore, quantitative global kinetic modelling showed that preferential closed-state inactivation and a weakly voltage-dependent opening step can explain the slowing of the inactivation rate with increasing depolarization. Therefore, it is likely that preferential closed-state inactivation is the physiological mechanism that regulates the activity of both ternary Kv4 channel complexes and native I(SA)-mediating channels.

Questioning the cerebellar doctrine.

PubMed

Galliano, Elisa; De Zeeuw, Chris I

2014-01-01

The basic principles of cerebellar function were originally described by Flourens, Cajal, and Marr/Albus/Ito, and they constitute the pillars of what can be considered to be the classic cerebellar doctrine. In their concepts, the main cerebellar function is to control motor behavior, Purkinje cells are the only cortical neuron receiving and integrating inputs from climbing fiber and mossy-parallel fiber pathways, and plastic modification at the parallel fiber synapses onto Purkinje cells constitutes the substrate of motor learning. Yet, because of recent technical advances and new angles of investigation, all pillars of the cerebellar doctrine now face regular re-examination. In this review, after summarizing the classic concepts and recent disputes, we attempt to synthesize an integrated view and propose a revisited version of the cerebellar doctrine. © 2014 Elsevier B.V. All rights reserved.

The biological significance of storage granules in rat parathyroid cells.

PubMed

Setoguti, T; Inoue, Y; Wild, P

1995-10-01

Both prosecretory and storage granules are concomitantly formed at the trans Golgi network including the innermost Golgi cisterna. Prosecretory granules develop into small secretory granules that release their contents by exocytosis finely regulated by a complex mechanism for maintaining calcium homeostasis. In the rat parathyroid cells, storage granules are large secretory granules storing parathyroid hormone for an emergency supply. The hormone is rapidly discharged by exocytosis when serum calcium concentration is decreased. The granules are constantly produced even under conditions of low serum calcium concentration in the regions of 8 mg/dl. The granule content is constantly hydrolyzed when not discharged, leading to a decreased core and finally to the formation of vacuolar bodies. The fate of the vacuolar bodies is unknown. Hypercalcemic conditions accelerate hydrolysis. The threshold value of calcium concentration required for the release of storage granule contents is between 8.0 and 7.5 mg/dl and that of calcium concentration for accelerating degradation of storage granules is about 11.5 mg/dl. Sympathetic stimulation causes storage granules to be discharged regardless of hypercalcemia or hypocalcemia. Parasympathetic stimulation accelerates hydrolysis. The degradation of storage granules seems to be closely associated with an intracellular regulatory mechanism for parathyroid hormone secretion.

Dendritic excitation–inhibition balance shapes cerebellar output during motor behaviour

PubMed Central

Jelitai, Marta; Puggioni, Paolo; Ishikawa, Taro; Rinaldi, Arianna; Duguid, Ian

2016-01-01

Feedforward excitatory and inhibitory circuits regulate cerebellar output, but how these circuits interact to shape the somatodendritic excitability of Purkinje cells during motor behaviour remains unresolved. Here we perform dendritic and somatic patch-clamp recordings in vivo combined with optogenetic silencing of interneurons to investigate how dendritic excitation and inhibition generates bidirectional (that is, increased or decreased) Purkinje cell output during self-paced locomotion. We find that granule cells generate a sustained depolarization of Purkinje cell dendrites during movement, which is counterbalanced by variable levels of feedforward inhibition from local interneurons. Subtle differences in the dendritic excitation–inhibition balance generate robust, bidirectional changes in simple spike (SSp) output. Disrupting this balance by selectively silencing molecular layer interneurons results in unidirectional firing rate changes, increased SSp regularity and disrupted locomotor behaviour. Our findings provide a mechanistic understanding of how feedforward excitatory and inhibitory circuits shape Purkinje cell output during motor behaviour. PMID:27976716

GDF15 regulates Kv2.1-mediated outward K+ current through the Akt/mTOR signalling pathway in rat cerebellar granule cells.

PubMed

Wang, Chang-Ying; Huang, An-Qi; Zhou, Meng-Hua; Mei, Yan-Ai

2014-05-15

GDF15 (growth/differentiation factor 15), a novel member of the TGFβ (transforming growth factor β) superfamily, plays critical roles in the central and peripheral nervous systems, but the signal transduction pathways and receptor subtypes involved are not well understood. In the present paper, we report that GDF15 specifically increases the IK (delayed-rectifier outward K+ current) in rat CGNs (cerebellar granule neurons) in time- and concentration-dependent manners. The GDF15-induced amplification of the IK is mediated by the increased expression and reduced lysosome-dependent degradation of the Kv2.1 protein, the main α-subunit of the IK channel. Exposure of CGNs to GDF15 markedly induced the phosphorylation of ERK (extracellular-signal-regulated kinase), Akt and mTOR (mammalian target of rapamycin), but the GDF15-induced IK densities and increased expression of Kv2.1 were attenuated only by Akt and mTOR, and not ERK, inhibitors. Pharmacological inhibition of the Src-mediated phosphorylation of TGFβR2 (TGFβ receptor 2), not TGFβR1, abrogated the effect of GDF15 on IK amplification and Kv2.1 induction. Immunoprecipitation assays showed that GDF15 increased the tyrosine phosphorylation of TGFβRII in the CGN lysate. The results of the present study reveal a novel regulation of Kv2.1 by GDF15 mediated through the TGFβRII-activated Akt/mTOR pathway, which is a previously uncharacterized Smad-independent mechanism of GDF15 signalling.

Fast insulin secretion reflects exocytosis of docked granules in mouse pancreatic B-cells.

PubMed

Olofsson, Charlotta S; Göpel, Sven O; Barg, Sebastian; Galvanovskis, Juris; Ma, Xiaosong; Salehi, Albert; Rorsman, Patrik; Eliasson, Lena

2002-05-01

A readily releasable pool (RRP) of granules has been proposed to underlie the first phase of insulin secretion. In the present study we combined electron microscopy, insulin secretion measurements and recordings of cell capacitance in an attempt to define this pool ultrastructurally. Mouse pancreatic B-cells contain approximately 9,000 granules, of which 7% are docked below the plasma membrane. The number of docked granules was reduced by 30% (200 granules) during 10 min stimulation with high K+. This stimulus depolarized the cell to -10 mV, elevated cytosolic [Ca2+] ([Ca2+](i)) from a basal concentration of 130 nM to a peak of 1.3 microM and released 0.5 ng insulin/islet, corresponding to 200-300 granules/cell. The Ca2+ transient decayed towards the prestimulatory concentration within approximately 200 s, presumably reflecting Ca2+ channel inactivation. Renewed stimulation with high K+ failed to stimulate insulin secretion when applied in the absence of glucose. The size of the RRP, derived from the insulin measurements, is similar to that estimated from the increase in cell capacitance elicited by photolytic release of caged Ca2+. We propose that the RRP represents a subset of the docked pool of granules and that replenishment of RRP can be accounted for largely by chemical modification of granules already in place or situated close to the plasma membrane.

Distinct roles of neuroepithelial-like and radial glia-like progenitor cells in cerebellar regeneration.

PubMed

Kaslin, Jan; Kroehne, Volker; Ganz, Julia; Hans, Stefan; Brand, Michael

2017-04-15

Zebrafish can regenerate after brain injury, and the regenerative process is driven by resident stem cells. Stem cells are heterogeneous in the vertebrate brain, but the significance of having heterogeneous stem cells in regeneration is not understood. Limited availability of specific stem cells might impair the regeneration of particular cell lineages. We studied regeneration of the adult zebrafish cerebellum, which contains two major stem and progenitor cell types: ventricular zone and neuroepithelial cells. Using conditional lineage tracing we demonstrate that cerebellar regeneration depends on the availability of specific stem cells. Radial glia-like cells are thought to be the predominant stem cell type in homeostasis and after injury. However, we find that radial glia-like cells play a minor role in adult cerebellar neurogenesis and in recovery after injury. Instead, we find that neuroepithelial cells are the predominant stem cell type supporting cerebellar regeneration after injury. Zebrafish are able to regenerate many, but not all, cell types in the cerebellum, which emphasizes the need to understand the contribution of different adult neural stem and progenitor cell subtypes in the vertebrate central nervous system. © 2017. Published by The Company of Biologists Ltd.

FGF-2 signal promotes proliferation of cerebellar progenitor cells and their oligodendrocytic differentiation at early postnatal stage

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

Naruse, Masae; Shibasaki, Koji; Ishizaki, Yasuki, E-mail: yasukiishizaki@gunma-u.ac.jp

The origins and developmental regulation of cerebellar oligodendrocytes are largely unknown, although some hypotheses of embryonic origins have been suggested. Neural stem cells exist in the white matter of postnatal cerebellum, but it is unclear whether these neural stem cells generate oligodendrocytes at postnatal stages. We previously showed that cerebellar progenitor cells, including neural stem cells, widely express CD44 at around postnatal day 3. In the present study, we showed that CD44-positive cells prepared from the postnatal day 3 cerebellum gave rise to neurospheres, while CD44-negative cells prepared from the same cerebellum did not. These neurospheres differentiated mainly into oligodendrocytesmore » and astrocytes, suggesting that CD44-positive neural stem/progenitor cells might generate oligodendrocytes in postnatal cerebellum. We cultured CD44-positive cells from the postnatal day 3 cerebellum in the presence of signaling molecules known as mitogens or inductive differentiation factors for oligodendrocyte progenitor cells. Of these, only FGF-2 promoted survival and proliferation of CD44-positive cells, and these cells differentiated into O4+ oligodendrocytes. Furthermore, we examined the effect of FGF-2 on cerebellar oligodendrocyte development ex vivo. FGF-2 enhanced proliferation of oligodendrocyte progenitor cells and increased the number of O4+ and CC1+ oligodendrocytes in slice cultures. These results suggest that CD44-positive cells might be a source of cerebellar oligodendrocytes and that FGF-2 plays important roles in their development at an early postnatal stage. - Highlights: • CD44 is expressed in cerebellar neural stem/progenitor cells at postnatal day 3 (P3). • FGF-2 promoted proliferation of CD44-positive progenitor cells from P3 cerebellum. • FGF-2 promoted oligodendrocytic differentiation of CD44-positive progenitor cells. • FGF-2 increased the number of oligodendrocytes in P3 cerebellar slice culture.« less

Nos2 inactivation promotes the development of medulloblastoma in Ptch1(+/-) mice by deregulation of Gap43-dependent granule cell precursor migration.

PubMed

Haag, Daniel; Zipper, Petra; Westrich, Viola; Karra, Daniela; Pfleger, Karin; Toedt, Grischa; Blond, Frederik; Delhomme, Nicolas; Hahn, Meinhard; Reifenberger, Julia; Reifenberger, Guido; Lichter, Peter

2012-01-01

Medulloblastoma is the most common malignant brain tumor in children. A subset of medulloblastoma originates from granule cell precursors (GCPs) of the developing cerebellum and demonstrates aberrant hedgehog signaling, typically due to inactivating mutations in the receptor PTCH1, a pathomechanism recapitulated in Ptch1(+/-) mice. As nitric oxide may regulate GCP proliferation and differentiation, we crossed Ptch1(+/-) mice with mice lacking inducible nitric oxide synthase (Nos2) to investigate a possible influence on tumorigenesis. We observed a two-fold higher medulloblastoma rate in Ptch1(+/-) Nos2(-/-) mice compared to Ptch1(+/-) Nos2(+/+) mice. To identify the molecular mechanisms underlying this finding, we performed gene expression profiling of medulloblastomas from both genotypes, as well as normal cerebellar tissue samples of different developmental stages and genotypes. Downregulation of hedgehog target genes was observed in postnatal cerebellum from Ptch1(+/+) Nos2(-/-) mice but not from Ptch1(+/-) Nos2(-/-) mice. The most consistent effect of Nos2 deficiency was downregulation of growth-associated protein 43 (Gap43). Functional studies in neuronal progenitor cells demonstrated nitric oxide dependence of Gap43 expression and impaired migration upon Gap43 knock-down. Both effects were confirmed in situ by immunofluorescence analyses on tissue sections of the developing cerebellum. Finally, the number of proliferating GCPs at the cerebellar periphery was decreased in Ptch1(+/+) Nos2(-/-) mice but increased in Ptch1(+/-) Nos2(-/) (-) mice relative to Ptch1(+/-) Nos2(+/+) mice. Taken together, these results indicate that Nos2 deficiency promotes medulloblastoma development in Ptch1(+/-) mice through retention of proliferating GCPs in the external granular layer due to reduced Gap43 expression. This study illustrates a new role of nitric oxide signaling in cerebellar development and demonstrates that the localization of pre-neoplastic cells during

Heterogeneous integration of adult-generated granule cells into the epileptic brain

PubMed Central

Murphy, Brian L.; Pun, Raymund Y.K.; Yin, Hulian; Faulkner, Christian R.; Loepke, Andreas W.; Danzer, Steve C.

2011-01-01

The functional impact of adult-generated granule cells in the epileptic brain is unclear, with data supporting both protective and maladaptive roles. These conflicting findings could be explained if new granule cells integrate heterogeneously, with some cells taking neutral or adaptive roles, while others contribute to recurrent circuitry supporting seizures. Here, we tested this hypothesis by completing detailed morphological characterizations of age- and experience-defined cohorts of adult-generated granule cells from transgenic mice. The majority of newborn cells exposed to an epileptogenic insult exhibited reductions in dendritic spine number, suggesting reduced excitatory input to these cells. A significant subset, however, exhibited higher spine numbers. These latter cells tended to have enlarged cell bodies, long basal dendrites or both. Moreover, cells with basal dendrites received significantly more recurrent mossy fiber input through their apical dendrites, indicating that these cells are robustly integrated into the pathological circuitry of the epileptic brain. These data imply that newborn cells play complex – and potentially conflicting – roles in epilepsy. PMID:21209195

Colchicine induced intraneuronal free zinc accumulation and dentate granule cell degeneration.

PubMed

Choi, Bo Young; Lee, Bo Eun; Kim, Jin Hee; Kim, Hyun Jung; Sohn, Min; Song, Hong Ki; Chung, Tae Nyoung; Suh, Sang Won

2014-08-01

Colchicine has been discovered to inhibit many inflammatory processes such as gout, familial Mediterranean fever, pericarditis and Behcet disease. Other than these beneficial anti-inflammatory effects, colchicine blocks microtubule-assisted axonal transport, which results in the selective loss of dentate granule cells of the hippocampus. The mechanism of the colchicine-induced dentate granule cell death and depletion of mossy fiber terminals still remains unclear. In the present study, we hypothesized that colchicine-induced dentate granule cell death may be caused by accumulation of labile intracellular zinc. 10 μg kg(-1) of colchicine was injected into the adult rat hippocampus and then brain sections were evaluated at 1 day or 1 week later. Neuronal cell death was evaluated by H&E staining or Fluoro-Jade B. Zinc accumulation and vesicular zinc were detected by N-(6-methoxy-8-quinolyl)-para-toluene sulfonamide (TSQ) staining. To test whether an extracellular zinc chelator can prevent this process, CaEDTA was injected into the hippocampus over a 5 min period with colchicine. To test whether other microtubule toxins also produce similar effects as colchicine, vincristine was injected into the hippocampus. The present study found that colchicine injection induced intracellular zinc accumulation in the dentate granule cells and depleted vesicular zinc from mossy fiber terminals. Injection of a zinc chelator, CaEDTA, did not block the zinc accumulation and neuronal death. Vincristine also produced intracellular zinc accumulation and neuronal death. These results suggest that colchicine-induced dentate granule cell death is caused by blocking axonal zinc flow and accumulation of intracellular labile zinc.

NMDA-receptor dependent synaptic activation of TRPC channels in olfactory bulb granule cells

PubMed Central

Stroh, Olga; Freichel, Marc; Kretz, Oliver; Birnbaumer, Lutz; Hartmann, Jana; Egger, Veronica

2012-01-01

TRPC channels are widely expressed throughout the nervous system including the olfactory bulb where their function is largely unknown. Here we describe their contribution to central synaptic processing at the reciprocal mitral and tufted cell - granule cell microcircuit, the most abundant synapse of the mammalian olfactory bulb. Suprathreshold activation of the synapse causes sodium action potentials in mouse granule cells and a subsequent long-lasting depolarization (LLD) linked to a global dendritic postsynaptic calcium signal recorded with two-photon laser scanning microscopy. These signals are not observed after action potentials evoked by current injection in the same cells. The LLD persists in the presence of group I metabotropic glutamate receptor antagonists but is entirely absent from granule cells deficient for the NMDA receptor subunit NR1. Moreover, both depolarization and Ca2+ rise are sensitive to the blockade of NMDA receptors. The LLD and the accompanying Ca2+ rise are also absent in granule cells from mice deficient for both TRPC channel subtypes 1 and 4, whereas the deletion of either TRPC1 or TRPC4 results in only a partial reduction of the LLD. Recordings from mitral cells in the absence of both subunits reveal a reduction of asynchronous neurotransmitter release from the granule cells during recurrent inhibition. We conclude that TRPC1 and TRPC4 can be activated downstream of NMDA receptor activation and contribute to slow synaptic transmission in the olfactory bulb, including the calcium dynamics required for asynchronous release from the granule cell spine. PMID:22539836

Distributed Cerebellar Motor Learning: A Spike-Timing-Dependent Plasticity Model

PubMed Central

Luque, Niceto R.; Garrido, Jesús A.; Naveros, Francisco; Carrillo, Richard R.; D'Angelo, Egidio; Ros, Eduardo

2016-01-01

Deep cerebellar nuclei neurons receive both inhibitory (GABAergic) synaptic currents from Purkinje cells (within the cerebellar cortex) and excitatory (glutamatergic) synaptic currents from mossy fibers. Those two deep cerebellar nucleus inputs are thought to be also adaptive, embedding interesting properties in the framework of accurate movements. We show that distributed spike-timing-dependent plasticity mechanisms (STDP) located at different cerebellar sites (parallel fibers to Purkinje cells, mossy fibers to deep cerebellar nucleus cells, and Purkinje cells to deep cerebellar nucleus cells) in close-loop simulations provide an explanation for the complex learning properties of the cerebellum in motor learning. Concretely, we propose a new mechanistic cerebellar spiking model. In this new model, deep cerebellar nuclei embed a dual functionality: deep cerebellar nuclei acting as a gain adaptation mechanism and as a facilitator for the slow memory consolidation at mossy fibers to deep cerebellar nucleus synapses. Equipping the cerebellum with excitatory (e-STDP) and inhibitory (i-STDP) mechanisms at deep cerebellar nuclei afferents allows the accommodation of synaptic memories that were formed at parallel fibers to Purkinje cells synapses and then transferred to mossy fibers to deep cerebellar nucleus synapses. These adaptive mechanisms also contribute to modulate the deep-cerebellar-nucleus-output firing rate (output gain modulation toward optimizing its working range). PMID:26973504

Functional properties of granule cells with hilar basal dendrites in the epileptic dentate gyrus.

PubMed

Kelly, Tony; Beck, Heinz

2017-01-01

The maturation of adult-born granule cells and their functional integration into the network is thought to play a key role in the proper functioning of the dentate gyrus. In temporal lobe epilepsy, adult-born granule cells in the dentate gyrus develop abnormally and possess a hilar basal dendrite (HBD). Although morphological studies have shown that these HBDs have synapses, little is known about the functional properties of these HBDs or the intrinsic and network properties of the granule cells that possess these aberrant dendrites. We performed patch-clamp recordings of granule cells within the granule cell layer "normotopic" from sham-control and status epilepticus (SE) animals. Normotopic granule cells from SE animals possessed an HBD (SE + HBD + cells) or not (SE + HBD - cells). Apical and basal dendrites were stimulated using multiphoton uncaging of glutamate. Two-photon Ca 2+ imaging was used to measure Ca 2+ transients associated with back-propagating action potentials (bAPs). Near-synchronous synaptic input integrated linearly in apical dendrites from sham-control animals and was not significantly different in apical dendrites of SE + HBD - cells. The majority of HBDs integrated input linearly, similar to apical dendrites. However, 2 of 11 HBDs were capable of supralinear integration mediated by a dendritic spike. Furthermore, the bAP-evoked Ca 2+ transients were relatively well maintained along HBDs, compared with apical dendrites. This further suggests an enhanced electrogenesis in HBDs. In addition, the output of granule cells from epileptic tissue was enhanced, with both SE + HBD - and SE + HBD + cells displaying increased high-frequency (>100 Hz) burst-firing. Finally, both SE + HBD - and SE + HBD + cells received recurrent excitatory input that was capable of generating APs, especially in the absence of feedback inhibition. Taken together, these data suggest that the enhanced excitability of HBDs combined with the altered intrinsic and network

Cerebellar Purkinje Cells Generate Highly Correlated Spontaneous Slow-Rate Fluctuations.

PubMed

Cao, Ying; Liu, Yu; Jaeger, Dieter; Heck, Detlef H

2017-01-01

Cerebellar Purkinje cells (PC) fire action potentials at high, sustained rates. Changes in spike rate that last a few tens of milliseconds encode sensory and behavioral events. Here we investigated spontaneous fluctuations of PC simple spike rate at a slow time scale of the order of 1 s. Simultaneous recordings from pairs of PCs that were aligned either along the sagittal or transversal axis of the cerebellar cortex revealed that simple spike rate fluctuations at the 1 s time scale were highly correlated. Each pair of PCs had either a predominantly positive or negative slow-rate correlation, with negative correlations observed only in PC pairs aligned along the transversal axis. Slow-rate correlations were independent of faster rate changes that were correlated with fluid licking behavior. Simultaneous recordings from PCs and cerebellar nuclear (CN) neurons showed that slow-rate fluctuations in PC and CN activity were also highly correlated, but their correlations continually alternated between periods of positive and negative correlation. The functional significance of this new aspect of cerebellar spike activity remains to be determined. Correlated slow-rate fluctuations seem too slow to be involved in the real-time control of ongoing behavior. However, slow-rate fluctuations of PCs converging on the same CN neuron are likely to modulate the excitability of the CN neuron, thus introduce a possible slow modulation of cerebellar output activity.

Transplantation of cerebellar neural stem cells improves motor coordination and neuropathology in Machado-Joseph disease mice.

PubMed

Mendonça, Liliana S; Nóbrega, Clévio; Hirai, Hirokazu; Kaspar, Brian K; Pereira de Almeida, Luís

2015-02-01

Machado-Joseph disease is a neurodegenerative disease without effective treatment. Patients with Machado-Joseph disease exhibit significant motor impairments such as gait ataxia, associated with multiple neuropathological changes including mutant ATXN3 inclusions, marked neuronal loss and atrophy of the cerebellum. Thus, an effective treatment of symptomatic patients with Machado-Joseph disease may require cell replacement, which we investigated in this study. For this purpose, we injected cerebellar neural stem cells into the cerebellum of adult Machado-Joseph disease transgenic mice and assessed the effect on the neuropathology, neuroinflammation mediators and neurotrophic factor levels and motor coordination. We found that upon transplantation into the cerebellum of adult Machado-Joseph disease mice, cerebellar neural stem cells differentiate into neurons, astrocytes and oligodendrocytes. Importantly, cerebellar neural stem cell transplantation mediated a significant and robust alleviation of the motor behaviour impairments, which correlated with preservation from Machado-Joseph disease-associated neuropathology, namely reduction of Purkinje cell loss, reduction of cellular layer shrinkage and mutant ATXN3 aggregates. Additionally, a significant reduction of neuroinflammation and an increase of neurotrophic factors levels was observed, indicating that transplantation of cerebellar neural stem cells also triggers important neuroprotective effects. Thus, cerebellar neural stem cells have the potential to be used as a cell replacement and neuroprotective approach for Machado-Joseph disease therapy. © The Author (2014). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The Prohormone VGF Regulates β Cell Function via Insulin Secretory Granule Biogenesis.

PubMed

Stephens, Samuel B; Edwards, Robert J; Sadahiro, Masato; Lin, Wei-Jye; Jiang, Cheng; Salton, Stephen R; Newgard, Christopher B

2017-09-05

The prohormone VGF is expressed in neuroendocrine and endocrine tissues and regulates nutrient and energy status both centrally and peripherally. We and others have shown that VGF-derived peptides have direct action on the islet β cell as secretagogues and cytoprotective agents; however, the endogenous function of VGF in the β cell has not been described. Here, we demonstrate that VGF regulates secretory granule formation. VGF loss-of-function studies in both isolated islets and conditional knockout mice reveal a profound decrease in stimulus-coupled insulin secretion. Moreover, VGF is necessary to facilitate efficient exit of granule cargo from the trans-Golgi network and proinsulin processing. It also functions to replenish insulin granule stores following nutrient stimulation. Our data support a model in which VGF operates at a critical node of granule biogenesis in the islet β cell to coordinate insulin biosynthesis with β cell secretory capacity. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Turnover of pigment granules: cyclic catabolism and anabolism of ommochromes within epidermal cells.

PubMed

Insausti, T C; Casas, J

2009-12-01

Ommochromes are end products of the tryptophan metabolism in arthropods. While the anabolism of ommochromes has been well studied, the catabolism is totally unknown. In order to study it, we used the crab-spider Misumena vatia, which is able to change color reversibly in a few days, from yellow to white and back. Ommochromes is the only pigment class responsible for the body coloration in this animal. The aim of this study was to analyze the fine structure of the epidermal cells in bleaching spiders, in an attempt to correlate morphological changes with the fate of the pigment granules. Central to the process of bleaching is the lysis of the ommochrome granules. In the same cell, intact granules and granules in different degradation stages are found. The degradation begins with granule autolysis. Some components are extruded in the extracellular space and others are recycled via autophagy. Abundant glycogen appears associated to granulolysis. In a later stage of bleaching, ommochrome progranules, typical of white spiders, appear in the distal zone of the same epidermal cell. Catabolism and anabolism of pigment granules thus take place simultaneously in spider epidermal cells. A cyclic pathway of pigment granules formation and degradation, throughout a complete cycle of color change is proposed, together with an explanation for this turnover, involving photoprotection against UV by ommochromes metabolites. The presence of this turnover for melanins is discussed.

Incorporation of DPP6a and DPP6K variants in ternary Kv4 channel complex reconstitutes properties of A-type K current in rat cerebellar granule cells.

PubMed

Jerng, Henry H; Pfaffinger, Paul J

2012-01-01

Dipeptidyl peptidase-like protein 6 (DPP6) proteins co-assemble with Kv4 channel α-subunits and Kv channel-interacting proteins (KChIPs) to form channel protein complexes underlying neuronal somatodendritic A-type potassium current (I(SA)). DPP6 proteins are expressed as N-terminal variants (DPP6a, DPP6K, DPP6S, DPP6L) that result from alternative mRNA initiation and exhibit overlapping expression patterns. Here, we study the role DPP6 variants play in shaping the functional properties of I(SA) found in cerebellar granule (CG) cells using quantitative RT-PCR and voltage-clamp recordings of whole-cell currents from reconstituted channel complexes and native I(SA) channels. Differential expression of DPP6 variants was detected in rat CG cells, with DPP6K (41 ± 3%)>DPP6a (33 ± 3%)>DPP6S (18 ± 2%)>DPP6L (8 ± 3%). To better understand how DPP6 variants shape native neuronal I(SA), we focused on studying interactions between the two dominant variants, DPP6K and DPP6a. Although previous studies did not identify unique functional effects of DPP6K, we find that the unique N-terminus of DPP6K modulates the effects of KChIP proteins, slowing recovery and producing a negative shift in the steady-state inactivation curve. By contrast, DPP6a uses its distinct N-terminus to directly confer rapid N-type inactivation independently of KChIP3a. When DPP6a and DPP6K are co-expressed in ratios similar to those found in CG cells, their distinct effects compete in modulating channel function. The more rapid inactivation from DPP6a dominates during strong depolarization; however, DPP6K produces a negative shift in the steady-state inactivation curve and introduces a slow phase of recovery from inactivation. A direct comparison to the native CG cell I(SA) shows that these mixed effects are present in the native channels. Our results support the hypothesis that the precise expression and co-assembly of different auxiliary subunit variants are important factors in shaping the I

Formation of tRNA granules in the nucleus of heat-induced human cells

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

Miyagawa, Ryu; Department of Biological Science, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654; Mizuno, Rie

Highlights: Black-Right-Pointing-Pointer tRNAs are tranlocated into the nucleus in heat-induced HeLa cells. Black-Right-Pointing-Pointer tRNAs form the unique granules in the nucleus. Black-Right-Pointing-Pointer tRNA ganules overlap with nuclear stress granules. -- Abstract: The stress response, which can trigger various physiological phenomena, is important for living organisms. For instance, a number of stress-induced granules such as P-body and stress granule have been identified. These granules are formed in the cytoplasm under stress conditions and are associated with translational inhibition and mRNA decay. In the nucleus, there is a focus named nuclear stress body (nSB) that distinguishes these structures from cytoplasmic stress granules.more » Many splicing factors and long non-coding RNA species localize in nSBs as a result of stress. Indeed, tRNAs respond to several kinds of stress such as heat, oxidation or starvation. Although nuclear accumulation of tRNAs occurs in starved Saccharomyces cerevisiae, this phenomenon is not found in mammalian cells. We observed that initiator tRNA{sup Met} (Meti) is actively translocated into the nucleus of human cells under heat stress. During this study, we identified unique granules of Meti that overlapped with nSBs. Similarly, elongator tRNA{sup Met} was translocated into the nucleus and formed granules during heat stress. Formation of tRNA granules is closely related to the translocation ratio. Then, all tRNAs may form the specific granules.« less

Neuroligin-1 overexpression in newborn granule cells in vivo.

PubMed

Schnell, Eric; Bensen, Aesoon L; Washburn, Eric K; Westbrook, Gary L

2012-01-01

Adult-born dentate granule cells integrate into the hippocampal network, extend neurites and form synapses in otherwise mature tissue. Excitatory and inhibitory inputs innervate these new granule cells in a stereotyped, temporally segregated manner, which presents a unique opportunity to study synapse development in the adult brain. To examine the role of neuroligins as synapse-inducing molecules in vivo, we infected dividing neural precursors in adult mice with a retroviral construct that increased neuroligin-1 levels during granule cell differentiation. By 21 days post-mitosis, exogenous neuroligin-1 was expressed at the tips of dendritic spines and increased the number of dendritic spines. Neuroligin-1-overexpressing cells showed a selective increase in functional excitatory synapses and connection multiplicity by single afferent fibers, as well as an increase in the synaptic AMPA/NMDA receptor ratio. In contrast to its synapse-inducing ability in vitro, neuroligin-1 overexpression did not induce precocious synapse formation in adult-born neurons. However, the dendrites of neuroligin-1-overexpressing cells did have more thin protrusions during an early period of dendritic outgrowth, suggesting enhanced filopodium formation or stabilization. Our results indicate that neuroligin-1 expression selectively increases the degree, but not the onset, of excitatory synapse formation in adult-born neurons.

The effects of exercise and stress on the survival and maturation of adult-generated granule cells

PubMed Central

Snyder, Jason S.; Glover, Lucas R.; Sanzone, Kaitlin M.; Kamhi, J. Frances; Cameron, Heather A.

2009-01-01

Stress strongly inhibits proliferation of granule cell precursors in the dentate gyrus, while voluntary running has the opposite effect. Few studies, however, have examined the possible effects of these environmental manipulations on the maturation and survival of young granule cells. We examined number of surviving granule cells and the proportion of young neurons that were functionally mature, as defined by seizure-induced immediate-early gene expression, in 14 and 21 day-old granule cells in mice that were given access to a running wheel, restrained daily for 2 hours, or given no treatment during this period. Importantly, treatments began two days after BrdU injection, to isolate effects on survival from those on cell proliferation. We found a large increase in granule cell survival in running mice compared with controls at both time points. In addition, running increased the proportion of granule cells expressing the immediate-early gene Arc in response to seizures, suggesting that it speeds incorporation into circuits, i.e., functional maturation. Stressed mice showed no change in Arc expression, compared to control animals, but, surprisingly, showed a transient increase in survival of 14-day-old granule cells, which was gone 7 days later. Examination of cell proliferation, using the endogenous mitotic marker proliferating cell nuclear antigen (PCNA) showed an increase in cell proliferation after 12 days of running but not after 19 days of running. The number of proliferating cells was unchanged 24 hours after the 12th or 19th episode of daily restraint stress. These findings demonstrate that running has strong effects on survival and maturation of young granule cells as well as their birth and that stress can have positive but short-lived effects on granule cell survival. PMID:19156854

Transient inhibition of the ERK pathway prevents cerebellar developmental defects and improves long-term motor functions in murine models of neurofibromatosis type 1

PubMed Central

Kim, Edward; Wang, Yuan; Kim, Sun-Jung; Bornhorst, Miriam; Jecrois, Emmanuelle S; Anthony, Todd E; Wang, Chenran; Li, Yi E; Guan, Jun-Lin; Murphy, Geoffrey G; Zhu, Yuan

2014-01-01

Individuals with neurofibromatosis type 1 (NF1) frequently exhibit cognitive and motor impairments and characteristics of autism. The cerebellum plays a critical role in motor control, cognition, and social interaction, suggesting that cerebellar defects likely contribute to NF1-associated neurodevelopmental disorders. Here we show that Nf1 inactivation during early, but not late stages of cerebellar development, disrupts neuronal lamination, which is partially caused by overproduction of glia and subsequent disruption of the Bergmann glia (BG) scaffold. Specific Nf1 inactivation in glutamatergic neuronal precursors causes premature differentiation of granule cell (GC) precursors and ectopic production of unipolar brush cells (UBCs), indirectly disrupting neuronal migration. Transient MEK inhibition during a neonatal window prevents cerebellar developmental defects and improves long-term motor performance of Nf1-deficient mice. This study reveals essential roles of Nf1 in GC/UBC migration by generating correct numbers of glia and controlling GC/UBC fate-specification/differentiation, identifying a therapeutic prevention strategy for multiple NF1-associcated developmental abnormalities. DOI: http://dx.doi.org/10.7554/eLife.05151.001 PMID:25535838

Cerebellar associative sensory learning defects in five mouse autism models

PubMed Central

Kloth, Alexander D; Badura, Aleksandra; Li, Amy; Cherskov, Adriana; Connolly, Sara G; Giovannucci, Andrea; Bangash, M Ali; Grasselli, Giorgio; Peñagarikano, Olga; Piochon, Claire; Tsai, Peter T; Geschwind, Daniel H; Hansel, Christian; Sahin, Mustafa; Takumi, Toru; Worley, Paul F; Wang, Samuel S-H

2015-01-01

Sensory integration difficulties have been reported in autism, but their underlying brain-circuit mechanisms are underexplored. Using five autism-related mouse models, Shank3+/ΔC, Mecp2R308/Y, Cntnap2−/−, L7-Tsc1 (L7/Pcp2Cre::Tsc1flox/+), and patDp(15q11-13)/+, we report specific perturbations in delay eyeblink conditioning, a form of associative sensory learning requiring cerebellar plasticity. By distinguishing perturbations in the probability and characteristics of learned responses, we found that probability was reduced in Cntnap2−/−, patDp(15q11-13)/+, and L7/Pcp2Cre::Tsc1flox/+, which are associated with Purkinje-cell/deep-nuclear gene expression, along with Shank3+/ΔC. Amplitudes were smaller in L7/Pcp2Cre::Tsc1flox/+ as well as Shank3+/ΔC and Mecp2R308/Y, which are associated with granule cell pathway expression. Shank3+/ΔC and Mecp2R308/Y also showed aberrant response timing and reduced Purkinje-cell dendritic spine density. Overall, our observations are potentially accounted for by defects in instructed learning in the olivocerebellar loop and response representation in the granule cell pathway. Our findings indicate that defects in associative temporal binding of sensory events are widespread in autism mouse models. DOI: http://dx.doi.org/10.7554/eLife.06085.001 PMID:26158416

Cerebellar learning properties are modulated by the CRF receptor in granular cells.

PubMed

Ezra-Nevo, Gili; Prestori, Francesca; Locatelli, Francesca; Soda, Teresa; Ten Brinke, Michiel M; Engel, Mareen; Boele, Henk-Jan; Botta, Laura; Leshkowitz, Dena; Ramot, Assaf; Tsoory, Michael; Biton, Inbal E; Deussing, Jan; D'Angelo, Egidio; De Zeeuw, Chris I; Chen, Alon

2018-06-22

Corticotropin-releasing factor (CRF) and its type 1 receptor (CRFR 1 ) play an important role in the responses to stressful challenges. Despite the well-established expression of CRFR 1 in granular cells (GrCs), its role in procedural motor performance and memory formation remains elusive. To investigate the role of CRFR 1 expression in cerebellar GrCs, we used a mouse model depleted of CRFR 1 in these cells. We detected changes in the cellular learning mechanisms in GrCs depleted of CRFR 1 in that they showed changes in intrinsic excitability and long-term synaptic plasticity. Moreover, male mice depleted of CRFR 1 specifically in GrCs showed accelerated Pavlovian associative eye-blink conditioning, but no differences in baseline motor performance, locomotion or fear and anxiety-related behaviors. Last, we analyzed cerebella transcriptome of KO and control mice and detected prominent alterations in the expression of calcium signaling pathways components. Our findings shed light on the interplay between stress-related central mechanisms and cerebellar motor conditioning, highlighting the role of the CRF system in regulating particular forms of cerebellar learning. SIGNIFICANCE STATEMENT Although it is known that CRFR 1 is highly expressed in the cerebellum, little attention has been given to its role in cerebellar functions in the behaving animal. Moreover, most of the attention was directed to the effect of CRF on Purkinje cells at the cellular level, and to this date, almost no data exist on the role of this stress-related receptor in other cerebellar structures. Here, we explored the behavioral and cellular effect of GrCs specific ablation of CRFR 1 We found a profound effect on learning, both at the cellular and behavioral levels, without affecting baseline motor skills. Copyright © 2018 the authors.

Aspm sustains postnatal cerebellar neurogenesis and medulloblastoma growth in mice

PubMed Central

Williams, Scott E.; Garcia, Idoia; Crowther, Andrew J.; Li, Shiyi; Stewart, Alyssa; Liu, Hedi; Lough, Kendall J.; O'Neill, Sean; Veleta, Katherine; Oyarzabal, Esteban A.; Merrill, Joseph R.; Shih, Yen-Yu Ian; Gershon, Timothy R.

2015-01-01

Alterations in genes that regulate brain size may contribute to both microcephaly and brain tumor formation. Here, we report that Aspm, a gene that is mutated in familial microcephaly, regulates postnatal neurogenesis in the cerebellum and supports the growth of medulloblastoma, the most common malignant pediatric brain tumor. Cerebellar granule neuron progenitors (CGNPs) express Aspm when maintained in a proliferative state by sonic hedgehog (Shh) signaling, and Aspm is expressed in Shh-driven medulloblastoma in mice. Genetic deletion of Aspm reduces cerebellar growth, while paradoxically increasing the mitotic rate of CGNPs. Aspm-deficient CGNPs show impaired mitotic progression, altered patterns of division orientation and differentiation, and increased DNA damage, which causes progenitor attrition through apoptosis. Deletion of Aspm in mice with Smo-induced medulloblastoma reduces tumor growth and increases DNA damage. Co-deletion of Aspm and either of the apoptosis regulators Bax or Trp53 (also known as p53) rescues the survival of neural progenitors and reduces the growth restriction imposed by Aspm deletion. Our data show that Aspm functions to regulate mitosis and to mitigate DNA damage during CGNP cell division, causes microcephaly through progenitor apoptosis when mutated, and sustains tumor growth in medulloblastoma. PMID:26450969

Dystonia and Cerebellar Degeneration in the Leaner Mouse Mutant

PubMed Central

Raike, Robert S.; Hess, Ellen J.; Jinnah, H.A.

2015-01-01

Cerebellar degeneration is traditionally associated with ataxia. Yet, there are examples of both ataxia and dystonia occurring in individuals with cerebellar degeneration. There is also substantial evidence suggesting that cerebellar dysfunction alone may cause dystonia. The types of cerebellar defects that may cause ataxia, dystonia, or both have not been delineated. In the current study, we explored the relationship between cerebellar degeneration and dystonia using the leaner mouse mutant. Leaner mice have severe dystonia that is associated with dysfunctional and degenerating cerebellar Purkinje cells. Whereas the density of Purkinje cells was not significantly reduced in 4 week-old leaner mice, approximately 50% of the neurons were lost by 34 weeks of age. On the other hand, the dystonia and associated functional disability became significantly less severe during this same interval. In other words, dystonia improved as Purkinje cells were lost, suggesting that dysfunctional Purkinje cells, rather than Purkinje cell loss, contribute to the dystonia. These results provide evidence that distorted cerebellar function may cause dystonia and support the concept that different types of cerebellar defects can have different functional consequences. PMID:25791619

Autistic-like behaviour and cerebellar dysfunction in Purkinje cell Tsc1 mutant mice.

PubMed

Tsai, Peter T; Hull, Court; Chu, YunXiang; Greene-Colozzi, Emily; Sadowski, Abbey R; Leech, Jarrett M; Steinberg, Jason; Crawley, Jacqueline N; Regehr, Wade G; Sahin, Mustafa

2012-08-30

Autism spectrum disorders (ASDs) are highly prevalent neurodevelopmental disorders, but the underlying pathogenesis remains poorly understood. Recent studies have implicated the cerebellum in these disorders, with post-mortem studies in ASD patients showing cerebellar Purkinje cell (PC) loss, and isolated cerebellar injury has been associated with a higher incidence of ASDs. However, the extent of cerebellar contribution to the pathogenesis of ASDs remains unclear. Tuberous sclerosis complex (TSC) is a genetic disorder with high rates of comorbid ASDs that result from mutation of either TSC1 or TSC2, whose protein products dimerize and negatively regulate mammalian target of rapamycin (mTOR) signalling. TSC is an intriguing model to investigate the cerebellar contribution to the underlying pathogenesis of ASDs, as recent studies in TSC patients demonstrate cerebellar pathology and correlate cerebellar pathology with increased ASD symptomatology. Functional imaging also shows that TSC patients with ASDs display hypermetabolism in deep cerebellar structures, compared to TSC patients without ASDs. However, the roles of Tsc1 and the sequelae of Tsc1 dysfunction in the cerebellum have not been investigated so far. Here we show that both heterozygous and homozygous loss of Tsc1 in mouse cerebellar PCs results in autistic-like behaviours, including abnormal social interaction, repetitive behaviour and vocalizations, in addition to decreased PC excitability. Treatment of mutant mice with the mTOR inhibitor, rapamycin, prevented the pathological and behavioural deficits. These findings demonstrate new roles for Tsc1 in PC function and define a molecular basis for a cerebellar contribution to cognitive disorders such as autism.

Bergmann glia modulate cerebellar Purkinje cell bistability via Ca2+-dependent K+ uptake

PubMed Central

Wang, Fushun; Xu, Qiwu; Wang, Weishan; Takano, Takahiro; Nedergaard, Maiken

2012-01-01

Recent studies have shown that cerebellar Bergmann glia display coordinated Ca2+ transients in live mice. However, the functional significance of Bergmann glial Ca2+ signaling remains poorly understood. Using transgenic mice that allow selective stimulation of glial cells, we report here that cytosolic Ca2+ regulates uptake of K+ by Bergmann glia, thus providing a powerful mechanism for control of Purkinje cell-membrane potential. The decline in extracellular K+ evoked by agonist-induced Ca2+ in Bergmann glia transiently increased spike activity of Purkinje cells in cerebellar slices as well as in live anesthetized mice. Thus, Bergmann glia play a previously unappreciated role in controlling the membrane potential and thereby the activity of adjacent Purkinje cells. PMID:22547829

Origin, lineage and function of cerebellar glia.

PubMed

Buffo, Annalisa; Rossi, Ferdinando

2013-10-01

The glial cells of the cerebellum, and particularly astrocytes and oligodendrocytes, are characterized by a remarkable phenotypic variety, in which highly peculiar morphological features are associated with specific functional features, unique among the glial cells of the entire CNS. Here, we provide a critical report about the present knowledge of the development of cerebellar glia, including lineage relationships between cerebellar neurons, astrocytes and oligodendrocytes, the origins and the genesis of the repertoire of glial types, and the processes underlying their acquisition of mature morphological and functional traits. In parallel, we describe and discuss some fundamental roles played by specific categories of glial cells during cerebellar development. In particular, we propose that Bergmann glia exerts a crucial scaffolding activity that, together with the organizing function of Purkinje cells, is necessary to achieve the normal pattern of foliation and layering of the cerebellar cortex. Moreover, we discuss some of the functional tasks of cerebellar astrocytes and oligodendrocytes that are distinctive of cerebellar glia throughout the CNS. Notably, we report about the regulation of synaptic signalling in the molecular and granular layer mediated by Bergmann glia and parenchymal astrocytes, and the functional interaction between oligodendrocyte precursor cells and neurons. On the whole, this review provides an extensive overview of the available literature and some novel insights about the origin and differentiation of the variety of cerebellar glial cells and their function in the developing and mature cerebellum. Copyright © 2013 Elsevier Ltd. All rights reserved.

Environmental enrichment alters dentate granule cell morphology in oldest-old rat.

PubMed

Darmopil, Sanja; Petanjek, Zdravko; Mohammed, Abdul H; Bogdanović, Nenad

2009-08-01

The hippocampus of aged rats shows marked age-related morphological changes that could cause memory deficits. Experimental evidence has established that environmental enrichment attenuates memory deficits in aged rats. We therefore studied whether environmental enrichment produces morphological changes on the dentate granule cells of aged rats. Fifteen male Sprague-Dawley rats, 24 months of age, were randomly distributed in two groups that were housed under standard (n = 7) or enriched (n = 8) environmental conditions for 26 days. Quantitative data of dendritic morphology from dentate gyrus granule cells were obtained on Golgi-Cox stained sections. Environmental enrichment significantly increased the complexity and size of dendritic tree (total number of segments increased by 61% and length by 116%), and spine density (88% increase). There were large interindividual differences within the enriched group, indicating differential individual responses to environmental stimulation. Previous studies in young animals have shown changes produced by environmental enrichment in the morphology of dentate gyrus granule cells. The results of the present study show that environmental enrichment can also produce changes in dentate granule cell morphology in the senescent brain. In conclusion, the hippocampus retains its neuroplastic capacity during aging, and enriched environmental housing conditions can attenuate age-related dendritic regression and synaptic loss, thus preserving memory functions.

Genome-wide analysis of alternative splicing in medulloblastoma identifies splicing patterns characteristic of normal cerebellar development

PubMed Central

Menghi, Francesca; Jacques, Thomas S.; Barenco, Martino; Schwalbe, Ed C.; Clifford, Steven C.; Hubank, Mike; Ham, Jonathan

2011-01-01

Alternative splicing is an important mechanism for the generation of protein diversity at a post-transcriptional level. Modifications in the splicing patterns of several genes have been shown to contribute to the malignant transformation of different tissue types. In this study, we used the Affymetrix Exon arrays to investigate patterns of differential splicing between paediatric medulloblastomas and normal cerebellum on a genome-wide scale. Of the 1262 genes identified as potentially generating tumour-associated splice forms, we selected 14 examples of differential splicing of known cassette exons and successfully validated 11 of them by RT-PCR. The pattern of differential splicing of three validated events was characteristic for the molecular subset of Sonic Hedgehog (Shh)-driven medulloblastomas, suggesting that their unique gene signature includes the expression of distinctive transcript variants. Generally, we observed that tumour and normal fetal cerebellar samples shared significantly lower exon inclusion rates compared to normal adult cerebellum. We investigated whether tumour-associated splice forms were expressed in primary cultures of Shh-dependent mouse cerebellar granule cell precursors (GCPs) and found that Shh caused a decrease in the cassette exon inclusion rate of five out of the seven tested genes. Furthermore, we observed a significant increase in exon inclusion between post-natal days 7 and 14 of mouse cerebellar development, at the time when GCPs mature into post-mitotic neurons. We conclude that inappropriate splicing frequently occurs in human medulloblastomas and may be linked to the activation of developmental signalling pathways and a failure of cerebellar precursor cells to differentiate. PMID:21248070

New supervised learning theory applied to cerebellar modeling for suppression of variability of saccade end points.

PubMed

Fujita, Masahiko

2013-06-01

A new supervised learning theory is proposed for a hierarchical neural network with a single hidden layer of threshold units, which can approximate any continuous transformation, and applied to a cerebellar function to suppress the end-point variability of saccades. In motor systems, feedback control can reduce noise effects if the noise is added in a pathway from a motor center to a peripheral effector; however, it cannot reduce noise effects if the noise is generated in the motor center itself: a new control scheme is necessary for such noise. The cerebellar cortex is well known as a supervised learning system, and a novel theory of cerebellar cortical function developed in this study can explain the capability of the cerebellum to feedforwardly reduce noise effects, such as end-point variability of saccades. This theory assumes that a Golgi-granule cell system can encode the strength of a mossy fiber input as the state of neuronal activity of parallel fibers. By combining these parallel fiber signals with appropriate connection weights to produce a Purkinje cell output, an arbitrary continuous input-output relationship can be obtained. By incorporating such flexible computation and learning ability in a process of saccadic gain adaptation, a new control scheme in which the cerebellar cortex feedforwardly suppresses the end-point variability when it detects a variation in saccadic commands can be devised. Computer simulation confirmed the efficiency of such learning and showed a reduction in the variability of saccadic end points, similar to results obtained from experimental data.

Nitric oxide inhibits exocytosis of cytolytic granules from lymphokine-activated killer cells

PubMed Central

Ferlito, Marcella; Irani, Kaikobad; Faraday, Nauder; Lowenstein, Charles J.

2006-01-01

NO inhibits cytotoxic T lymphocyte killing of target cells, although the precise mechanism is unknown. We hypothesized that NO decreases exocytosis of cytotoxic granules from activated lymphocytes. We now show that NO inhibits lymphokine-activated killer cell killing of K562 target cells. Exogenous and endogenous NO decreases the release of granzyme B, granzyme A, and perforin: all contents of cytotoxic granules. NO inhibits the signal transduction cascade initiated by cross-linking of the T cell receptor that leads to granule exocytosis. In particular, we found that NO decreases the expression of Ras, a critical signaling component within the exocytic pathway. Ectopic expression of Ras prevents NO inhibition of exocytosis. Our data suggest that Ras mediates NO inhibition of lymphocyte cytotoxicity and emphasize that alterations in the cellular redox state may regulate the exocytic signaling pathway. PMID:16857739

Light scattering on PHA granules protects bacterial cells against the harmful effects of UV radiation.

PubMed

Slaninova, Eva; Sedlacek, Petr; Mravec, Filip; Mullerova, Lucie; Samek, Ota; Koller, Martin; Hesko, Ondrej; Kucera, Dan; Marova, Ivana; Obruca, Stanislav

2018-02-01

Numerous prokaryotes accumulate polyhydroxyalkanoates (PHA) in the form of intracellular granules. The primary function of PHA is the storage of carbon and energy. Nevertheless, there are numerous reports that the presence of PHA granules in microbial cells enhances their stress resistance and fitness when exposed to various stress factors. In this work, we studied the protective mechanism of PHA granules against UV irradiation employing Cupriavidus necator as a model bacterial strain. The PHA-accumulating wild type strain showed substantially higher UV radiation resistance than the PHA non-accumulating mutant. Furthermore, the differences in UV-Vis radiation interactions with both cell types were studied using various spectroscopic approaches (turbidimetry, absorption spectroscopy, and nephelometry). Our results clearly demonstrate that intracellular PHA granules efficiently scatter UV radiation, which provides a substantial UV-protective effect for bacterial cells and, moreover, decreases the intracellular level of reactive oxygen species in UV-challenged cells. The protective properties of the PHA granules are enhanced by the fact that granules specifically bind to DNA, which in turn provides shield-like protection of DNA as the most UV-sensitive molecule. To conclude, the UV-protective action of PHA granules adds considerable value to their primary storage function, which can be beneficial in numerous environments.

Protein kinases are associated with multiple, distinct cytoplasmic granules in quiescent yeast cells.

PubMed

Shah, Khyati H; Nostramo, Regina; Zhang, Bo; Varia, Sapna N; Klett, Bethany M; Herman, Paul K

2014-12-01

The cytoplasm of the eukaryotic cell is subdivided into distinct functional domains by the presence of a variety of membrane-bound organelles. The remaining aqueous space may be further partitioned by the regulated assembly of discrete ribonucleoprotein (RNP) complexes that contain particular proteins and messenger RNAs. These RNP granules are conserved structures whose importance is highlighted by studies linking them to human disorders like amyotrophic lateral sclerosis. However, relatively little is known about the diversity, composition, and physiological roles of these cytoplasmic structures. To begin to address these issues, we examined the cytoplasmic granules formed by a key set of signaling molecules, the protein kinases of the budding yeast Saccharomyces cerevisiae. Interestingly, a significant fraction of these proteins, almost 20%, was recruited to cytoplasmic foci specifically as cells entered into the G0-like quiescent state, stationary phase. Colocalization studies demonstrated that these foci corresponded to eight different granules, including four that had not been reported previously. All of these granules were found to rapidly disassemble upon the resumption of growth, and the presence of each was correlated with cell viability in the quiescent cultures. Finally, this work also identified new constituents of known RNP granules, including the well-characterized processing body and stress granule. The composition of these latter structures is therefore more varied than previously thought and could be an indicator of additional biological activities being associated with these complexes. Altogether, these observations indicate that quiescent yeast cells contain multiple distinct cytoplasmic granules that may make important contributions to their long-term survival. Copyright © 2014 by the Genetics Society of America.

CCL11 elicits secretion of RNases from mouse eosinophils and their cell-free granules

PubMed Central

Shamri, Revital; Melo, Rossana C. N.; Young, Kristen M.; Bivas-Benita, Maytal; Xenakis, Jason J.; Spencer, Lisa A.; Weller, Peter F.

2012-01-01

Rapid secretion of eosinophil-associated RNases (EARs), such as the human eosinophilic cationic protein (ECP), from intracellular granules is central to the role of eosinophils in allergic diseases and host immunity. Our knowledge regarding allergic inflammation has advanced based on mouse experimental models. However, unlike human eosinophils, capacities of mouse eosinophils to secrete granule proteins have been controversial. To study mechanisms of mouse eosinophil secretion and EAR release, we combined an RNase assay of mouse EARs with ultrastructural studies. In vitro, mouse eosinophils stimulated with the chemokine eotaxin-1 (CCL11) secreted enzymatically active EARs (EC50 5 nM) by piecemeal degranulation. In vivo, in a mouse model of allergic airway inflammation, increased airway eosinophil infiltration (24-fold) correlated with secretion of active RNases (3-fold). Moreover, we found that eosinophilic inflammation in mice can involve eosinophil cytolysis and release of cell-free granules. Cell-free mouse eosinophil granules expressed functional CCR3 receptors and secreted their granule proteins, including EAR and eosinophil peroxidase in response to CCL11. Collectively, these data demonstrate chemokine-dependent secretion of EARs from both intact mouse eosinophils and their cell-free granules, findings pertinent to understanding the pathogenesis of eosinophil-associated diseases, in which EARs are key factors.—Shamri, R., Melo, R. C. N., Young, K. M., B.-B, M., Xenakis, J. J., Spencer, L. A., Weller, P. F. CCL11 elicits secretion of RNases from mouse eosinophils and their cell-free granules. PMID:22294786

A Role for Serglycin Proteoglycan in Mast Cell Apoptosis Induced by a Secretory Granule-mediated Pathway*

PubMed Central

Melo, Fabio Rabelo; Waern, Ida; Rönnberg, Elin; Åbrink, Magnus; Lee, David M.; Schlenner, Susan M.; Feyerabend, Thorsten B.; Rodewald, Hans-Reimer; Turk, Boris; Wernersson, Sara; Pejler, Gunnar

2011-01-01

Mast cell secretory granules (secretory lysosomes) contain large amounts of fully active proteases bound to serglycin proteoglycan. Damage to the granule membrane will thus lead to the release of serglycin and serglycin-bound proteases into the cytosol, which potentially could lead to proteolytic activation of cytosolic pro-apoptotic compounds. We therefore hypothesized that mast cells are susceptible to apoptosis induced by permeabilization of the granule membrane and that this process is serglycin-dependent. Indeed, we show that wild-type mast cells are highly sensitive to apoptosis induced by granule permeabilization, whereas serglycin-deficient cells are largely resistant. The reduced sensitivity of serglycin−/− cells to apoptosis was accompanied by reduced granule damage, reduced release of proteases into the cytosol, and defective caspase-3 activation. Mechanistically, the apoptosis-promoting effect of serglycin involved serglycin-dependent proteases, as indicated by reduced sensitivity to apoptosis and reduced caspase-3 activation in cells lacking individual mast cell-specific proteases. Together, these findings implicate serglycin proteoglycan as a novel player in mast cell apoptosis. PMID:21123167

Morphologic Integration of Hilar Ectopic Granule Cells into Dentate Gyrus Circuitry in the Pilocarpine Model of Temporal Lobe Epilepsy

PubMed Central

Cameron, Michael C.; Zhan, Ren-Zhi; Nadler, J. Victor

2014-01-01

After pilocarpine-induced status epilepticus, many granule cells born into the postseizure environment migrate aberrantly into the dentate hilus. Hilar ectopic granule cells (HEGCs) are hyperexcitable and may therefore increase circuit excitability. This study determined the distribution of their axons and dendrites. HEGCs and normotopic granule cells were filled with biocytin during whole-cell patch clamp recording in hippocampal slices from pilocarpine-treated rats. The apical dendrite of 86% of the biocytin-labeled HEGCs extended to the outer edge of the dentate molecular layer. The total length and branching of HEGC apical dendrites that penetrated the molecular layer were significantly reduced compared with apical dendrites of normotopic granule cells. HEGCs were much more likely to have a hilar basal dendrite than normotopic granule cells. They were about as likely as normotopic granule cells to project to CA3 pyramidal cells within the slice, but were much more likely to send at least one recurrent mossy fiber into the molecular layer. HEGCs with burst capability had less well-branched apical dendrites than nonbursting HEGCs, their dendrites were more likely to be confined to the hilus, and some exhibited dendritic features similar to those of immature granule cells. HEGCs thus have many paths along which to receive synchronized activity from normotopic granule cells and to transmit their own hyperactivity to both normotopic granule cells and CA3 pyramidal cells. They may therefore contribute to the highly interconnected granule cell hubs that have been proposed as crucial to development of a hyperexcitable, potentially seizure-prone circuit. PMID:21455997

The Low-Threshold Calcium Channel Cav3.2 Mediates Burst Firing of Mature Dentate Granule Cells

PubMed Central

Dumenieu, Mael; Senkov, Oleg; Mironov, Andrey; Bourinet, Emmanuel; Kreutz, Michael R; Dityatev, Alexander; Heine, Martin; Bikbaev, Arthur

2018-01-01

Abstract Mature granule cells are poorly excitable neurons that were recently shown to fire action potentials, preferentially in bursts. It is believed that the particularly pronounced short-term facilitation of mossy fiber synapses makes granule cell bursting a very effective means of properly transferring information to CA3. However, the mechanism underlying the unique bursting behavior of mature granule cells is currently unknown. Here, we show that Cav3.2 T-type channels at the axon initial segment are responsible for burst firing of mature granule cells in rats and mice. Accordingly, Cav3.2 knockout mice fire tonic spikes and exhibit impaired bursting, synaptic plasticity and dentate-to-CA3 communication. The data show that Cav3.2 channels are strong modulators of bursting and can be considered a critical molecular switch that enables effective information transfer from mature granule cells to the CA3 pyramids. PMID:29790938

Regular Patterns in Cerebellar Purkinje Cell Simple Spike Trains

PubMed Central

Shin, Soon-Lim; Hoebeek, Freek E.; Schonewille, Martijn; De Zeeuw, Chris I.; Aertsen, Ad; De Schutter, Erik

2007-01-01

Background Cerebellar Purkinje cells (PC) in vivo are commonly reported to generate irregular spike trains, documented by high coefficients of variation of interspike-intervals (ISI). In strong contrast, they fire very regularly in the in vitro slice preparation. We studied the nature of this difference in firing properties by focusing on short-term variability and its dependence on behavioral state. Methodology/Principal Findings Using an analysis based on CV2 values, we could isolate precise regular spiking patterns, lasting up to hundreds of milliseconds, in PC simple spike trains recorded in both anesthetized and awake rodents. Regular spike patterns, defined by low variability of successive ISIs, comprised over half of the spikes, showed a wide range of mean ISIs, and were affected by behavioral state and tactile stimulation. Interestingly, regular patterns often coincided in nearby Purkinje cells without precise synchronization of individual spikes. Regular patterns exclusively appeared during the up state of the PC membrane potential, while single ISIs occurred both during up and down states. Possible functional consequences of regular spike patterns were investigated by modeling the synaptic conductance in neurons of the deep cerebellar nuclei (DCN). Simulations showed that these regular patterns caused epochs of relatively constant synaptic conductance in DCN neurons. Conclusions/Significance Our findings indicate that the apparent irregularity in cerebellar PC simple spike trains in vivo is most likely caused by mixing of different regular spike patterns, separated by single long intervals, over time. We propose that PCs may signal information, at least in part, in regular spike patterns to downstream DCN neurons. PMID:17534435

Cerebellar Ataxia, Seizures, Premature Death, and Cardiac Abnormalities in Mice with Targeted Disruption of the Cacna2d2 Gene

PubMed Central

Ivanov, Sergey V.; Ward, Jerrold M.; Tessarollo, Lino; McAreavey, Dorothea; Sachdev, Vandana; Fananapazir, Lameh; Banks, Melissa K.; Morris, Nicole; Djurickovic, Draginja; Devor-Henneman, Deborah E.; Wei, Ming-Hui; Alvord, Gregory W.; Gao, Boning; Richardson, James A.; Minna, John D.; Rogawski, Michael A.; Lerman, Michael I.

2004-01-01

CACNA2D2 is a putative tumor suppressor gene located in the human chromosome 3p21.3 region that shows frequent allelic imbalances in lung, breast, and other cancers. The α2δ-2 protein encoded by the gene is a regulatory subunit of voltage-dependent calcium channels and is expressed in brain, heart, and other tissues. Here we report that mice homozygous for targeted disruption of the Cacna2d2 gene exhibit growth retardation, reduced life span, ataxic gait with apoptosis of cerebellar granule cells followed by Purkinje cell depletion, enhanced susceptibility to seizures, and cardiac abnormalities. The Cacna2d2tm1NCIF null phenotype has much in common with that of Cacna1a mutants, such as cerebellar neuro-degeneration associated with ataxia, seizures, and premature death. A tendency to bradycardia and limited response of null mutants to isoflurane implicate α2δ-2 in sympathetic regulation of cardiac function. In summary, our findings provide genetic evidence that the α2δ-2 subunit serves in vivo as a component of P/Q-type calcium channels, is indispensable for the central nervous system function, and may be involved in hereditary cerebellar ataxias and epileptic disorders in humans. PMID:15331424

Cerebellar learning mechanisms

PubMed Central

Freeman, John H.

2014-01-01

The mechanisms underlying cerebellar learning are reviewed with an emphasis on old arguments and new perspectives on eyeblink conditioning. Eyeblink conditioning has been used for decades a model system for elucidating cerebellar learning mechanisms. The standard model of the mechanisms underlying eyeblink conditioning is that there two synaptic plasticity processes within the cerebellum that are necessary for acquisition of the conditioned response: 1) long-term depression (LTD) at parallel fiber-Purkinje cell synapses and 2) long-term potentiation (LTP) at mossy fiber-interpositus nucleus synapses. Additional Purkinje cell plasticity mechanisms may also contribute to eyeblink conditioning including LTP, excitability, and entrainment of deep nucleus activity. Recent analyses of the sensory input pathways necessary for eyeblink conditioning indicate that the cerebellum regulates its inputs to facilitate learning and maintain plasticity. Cerebellar learning during eyeblink conditioning is therefore a dynamic interactive process which maximizes responding to significant stimuli and suppresses responding to irrelevant or redundant stimuli. PMID:25289586

Snapshot 3D tracking of insulin granules in live cells

NASA Astrophysics Data System (ADS)

Wang, Xiaolei; Huang, Xiang; Gdor, Itay; Daddysman, Matthew; Yi, Hannah; Selewa, Alan; Haunold, Theresa; Hereld, Mark; Scherer, Norbert F.

2018-02-01

Rapid and accurate volumetric imaging remains a challenge, yet has the potential to enhance understanding of cell function. We developed and used a multifocal microscope (MFM) for 3D snapshot imaging to allow 3D tracking of insulin granules labeled with mCherry in MIN6 cells. MFM employs a special diffractive optical element (DOE) to simultaneously image multiple focal planes. This simultaneous acquisition of information determines the 3D location of single objects at a speed only limited by the array detector's frame rate. We validated the accuracy of MFM imaging/tracking with fluorescence beads; the 3D positions and trajectories of single fluorescence beads can be determined accurately over a wide range of spatial and temporal scales. The 3D positions and trajectories of single insulin granules in a 3.2um deep volume were determined with imaging processing that combines 3D decovolution, shift correction, and finally tracking using the Imaris software package. We find that the motion of the granules is superdiffusive, but less so in 3D than 2D for cells grown on coverslip surfaces, suggesting an anisotropy in the cytoskeleton (e.g. microtubules and action).

Odontogenic Differentiation of Human Dental Pulp Stem Cells Stimulated by the Calcium Phosphate Porous Granules

PubMed Central

Nam, Sunyoung; Won, Jong-Eun; Kim, Cheol-Hwan; Kim, Hae-Won

2011-01-01

Effects of three-dimensional (3D) calcium phosphate (CaP) porous granules on the growth and odontogenic differentiation of human dental pulp stem cells (hDPSCs) were examined for dental tissue engineering. hDPSCs isolated from adult human dental pulps were cultured for 3-4 passages, and populated on porous granules. Cell growth on the culture dish showed an ongoing increase for up to 21 days, whereas the growth on the 3D granules decreased after 14 days. This reduction in proliferative potential on the 3D granules was more conspicuous under the osteogenic medium conditions, indicating that the 3D granules may induce the odontogenic differentiation of hDPSCs. Differentiation behavior on the 3D granules was confirmed by the increased alkaline phosphatase activity, up-regulation of odontoblast-specific genes, including dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) by quantitative polymerase chain reaction, and greater level of dentin sialoprotein synthesis by western blot. Moreover, the cellular mineralization, as assessed by Alizarin red S and calcium quantification, was significantly higher in the 3D CaP granules than in the culture dish. Taken all, the 3D CaP porous granules should be useful for dental tissue engineering in combination with hDPSCs by providing favorable 3D substrate conditions for cell growth and odontogenic development. PMID:21772958

Developmental Injury to the Cerebellar Cortex Following Hydroxyurea Treatment in Early Postnatal Life: An Immunohistochemical and Electron Microscopic Study.

PubMed

Martí, Joaquín; Molina, Vanesa; Santa-Cruz, M C; Hervás, José P

2017-02-01

Postnatal development of the cerebellar cortex was studied in rats administered with a single dose (2 mg/g) of the cytotoxic agent hydroxyurea (HU) on postnatal day (P) 9 and collected at appropriate times ranging from 6 h to 45 days. Quantification of several parameters such as the density of pyknotic, mitotic, BrdU-positive, and vimentin-stained cells revealed that HU compromises the survival of the external granular layer (EGL) cells. Moreover, vimentin immunocytochemistry revealed overexpression and thicker immunoreactive glial processes in HU-treated rats. On the other hand, we also show that HU leads to the activation of apoptotic cellular events, resulting in a substantial number of dying EGL cells, as revealed by TUNEL staining and at the electron microscope level. Additionally, we quantified several features of the cerebellar cortex of rats exposed to HU in early postnatal life and collected in adulthood. Data analysis indicated that the analyzed parameters were less pronounced in rats administered with this agent. Moreover, we observed several alterations in the cerebellar cortex cytoarchitecture of rats injected with HU. Anomalies included ectopic placement of Purkinje cells and abnormities in the dendritic arbor of these macroneurons. Ectopic granule cells were also found in the molecular layer. These findings provide a clue for investigating the mechanisms of HU-induced toxicity during the development of the central nervous system. Our results also suggest that it is essential to avoid underestimating the adverse effects of this hydroxylated analog of urea when administered during early postnatal life.

DEPS-1 promotes P-granule assembly and RNA interference in C. elegans germ cells

PubMed Central

Spike, Caroline A.; Bader, Jason; Reinke, Valerie; Strome, Susan

2008-01-01

P granules are germ-cell-specific cytoplasmic structures containing RNA and protein, and required for proper germ cell development in C. elegans. PGL-1 and GLH-1 were previously identified as critical components of P granules. We have identified a new P-granule-associated protein, DEPS-1, the loss of which disrupts P-granule structure and function. DEPS-1 is required for the proper localization of PGL-1 to P granules, the accumulation of glh-1 mRNA and protein, and germ cell proliferation and fertility at elevated temperatures. In addition, DEPS-1 is required for RNA interference (RNAi) of germline-expressed genes, possibly because DEPS-1 promotes the accumulation of RDE-4, a dsRNA-binding protein required for RNAi. A genome wide analysis of gene expression in deps-1 mutant germ lines identified additional targets of DEPS-1 regulation, many of which are also regulated by the RNAi factor RDE-3. Our studies suggest that DEPS-1 is a key component of the P-granule assembly pathway and that its roles include promoting accumulation of some mRNAs, such as glh-1 and rde-4, and reducing accumulation of other mRNAs, perhaps by collaborating with RDE-3 to generate endogenous short interfering RNAs (endo-siRNAs). PMID:18234720

DEPS-1 promotes P-granule assembly and RNA interference in C. elegans germ cells.

PubMed

Spike, Caroline A; Bader, Jason; Reinke, Valerie; Strome, Susan

2008-03-01

P granules are germ-cell-specific cytoplasmic structures containing RNA and protein, and required for proper germ cell development in C. elegans. PGL-1 and GLH-1 were previously identified as critical components of P granules. We have identified a new P-granule-associated protein, DEPS-1, the loss of which disrupts P-granule structure and function. DEPS-1 is required for the proper localization of PGL-1 to P granules, the accumulation of glh-1 mRNA and protein, and germ cell proliferation and fertility at elevated temperatures. In addition, DEPS-1 is required for RNA interference (RNAi) of germline-expressed genes, possibly because DEPS-1 promotes the accumulation of RDE-4, a dsRNA-binding protein required for RNAi. A genome wide analysis of gene expression in deps-1 mutant germ lines identified additional targets of DEPS-1 regulation, many of which are also regulated by the RNAi factor RDE-3. Our studies suggest that DEPS-1 is a key component of the P-granule assembly pathway and that its roles include promoting accumulation of some mRNAs, such as glh-1 and rde-4, and reducing accumulation of other mRNAs, perhaps by collaborating with RDE-3 to generate endogenous short interfering RNAs (endo-siRNAs).

Direct conversion of injury-site myeloid cells to fibroblast-like cells of granulation tissue.

PubMed

Sinha, Mithun; Sen, Chandan K; Singh, Kanhaiya; Das, Amitava; Ghatak, Subhadip; Rhea, Brian; Blackstone, Britani; Powell, Heather M; Khanna, Savita; Roy, Sashwati

2018-03-05

Inflammation, following injury, induces cellular plasticity as an inherent component of physiological tissue repair. The dominant fate of wound macrophages is unclear and debated. Here we show that two-thirds of all granulation tissue fibroblasts, otherwise known to be of mesenchymal origin, are derived from myeloid cells which are likely to be wound macrophages. Conversion of myeloid to fibroblast-like cells is impaired in diabetic wounds. In cross-talk between keratinocytes and myeloid cells, miR-21 packaged in extracellular vesicles (EV) is required for cell conversion. EV from wound fluid of healing chronic wound patients is rich in miR-21 and causes cell conversion more effectively compared to that by fluid from non-healing patients. Impaired conversion in diabetic wound tissue is rescued by targeted nanoparticle-based delivery of miR-21 to macrophages. This work introduces a paradigm wherein myeloid cells are recognized as a major source of fibroblast-like cells in the granulation tissue.

Voltage-gated calcium channel autoimmune cerebellar degeneration

PubMed Central

McKasson, Marilyn; Clawson, Susan A.; Hill, Kenneth E.; Wood, Blair; Carlson, Noel; Bromberg, Mark; Greenlee, John E.

2016-01-01

Objectives: To describe response to treatment in a patient with autoantibodies against voltage-gated calcium channels (VGCCs) who presented with autoimmune cerebellar degeneration and subsequently developed Lambert-Eaton myasthenic syndrome (LEMS), and to study the effect of the patient's autoantibodies on Purkinje cells in rat cerebellar slice cultures. Methods: Case report and study of rat cerebellar slice cultures incubated with patient VGCC autoantibodies. Results: A 53-year-old man developed progressive incoordination with ataxic speech. Laboratory evaluation revealed VGCC autoantibodies without other antineuronal autoantibodies. Whole-body PET scans 6 and 12 months after presentation detected no malignancy. The patient improved significantly with IV immunoglobulin G (IgG), prednisone, and mycophenolate mofetil, but worsened after IV IgG was halted secondary to aseptic meningitis. He subsequently developed weakness with electrodiagnostic evidence of LEMS. The patient's IgG bound to Purkinje cells in rat cerebellar slice cultures, followed by neuronal death. Reactivity of the patient's autoantibodies with VGCCs was confirmed by blocking studies with defined VGCC antibodies. Conclusions: Autoimmune cerebellar degeneration associated with VGCC autoantibodies may precede onset of LEMS and may improve with immunosuppressive treatment. Binding of anti-VGCC antibodies to Purkinje cells in cerebellar slice cultures may be followed by cell death. Patients with anti-VGCC autoantibodies may be at risk of irreversible neurologic injury over time, and treatment should be initiated early. PMID:27088118

Cytoplasmic RNA Granules in Somatic Maintenance.

PubMed

Moujaber, Ossama; Stochaj, Ursula

2018-05-30

Cytoplasmic RNA granules represent subcellular compartments that are enriched in protein-bound RNA species. RNA granules are produced by evolutionary divergent eukaryotes, including yeast, mammals, and plants. The functions of cytoplasmic RNA granules differ widely. They are dictated by the cell type and physiological state, which in turn is determined by intrinsic cell properties and environmental factors. RNA granules provide diverse cellular functions. However, all of the granules contribute to aspects of RNA metabolism. This is exemplified by transcription, RNA storage, silencing, and degradation, as well as mRNP remodeling and regulated translation. Several forms of cytoplasmic mRNA granules are linked to normal physiological processes. For instance, they may coordinate protein synthesis and thereby serve as posttranscriptional "operons". RNA granules also participate in cytoplasmic mRNA trafficking, a process particularly well understood for neurons. Many forms of RNA granules support the preservation of somatic cell performance under normal and stress conditions. On the other hand, severe insults or disease can cause the formation and persistence of RNA granules that contribute to cellular dysfunction, especially in the nervous system. Neurodegeneration and many other diseases linked to RNA granules are associated with aging. Nevertheless, information related to the impact of aging on the various types of RNA granules is presently very limited. This review concentrates on cytoplasmic RNA granules and their role in somatic cell maintenance. We summarize the current knowledge on different types of RNA granules in the cytoplasm, their assembly and function under normal, stress, or disease conditions. Specifically, we discuss processing bodies, neuronal granules, stress granules, and other less characterized cytoplasmic RNA granules. Our focus is primarily on mammalian and yeast models, because they have been critical to unravel the physiological role of various

Synthetic regulators of the 2-oxoglutarate oxidative decarboxylation alleviate the glutamate excitotoxicity in cerebellar granule neurons.

PubMed

Kabysheva, Maria S; Storozhevykh, Tatiana P; Pinelis, Vsevolod G; Bunik, Victoria I

2009-05-01

Impairment of the 2-oxoglutarate oxidative decarboxylation by the 2-oxoglutarate dehydrogenase complex (OGDHC) is associated with the glutamate accumulation, ROS production and neuropathologies. We hypothesized that correct function of OGDHC under metabolic stress is essential to overcome the glutamate excitotoxic action on neurons. We show that synthetic phosphono analogs of 2-oxoglutarate, succinyl phosphonate and its phosphono ethyl ester, improve the catalysis by brain OGDHC through inhibiting the side reaction of irreversible inactivation of its first component, 2-oxoglutarate dehydrogenase. Under the substrate and cofactor saturation, the component and complex undergo the inactivation during catalysis with the apparent rate constant 0.2 min(-1). The inactivation rate is reduced by 90% and 60% in the presence of 50 microM succinyl phosphonate and its phosphono ethyl ester, correspondingly. In cultured cerebellar granule neurons exposed to excitotoxic glutamate, the phosphonates (100 microM) protect from the irreversible impairment of mitochondrial function and delayed calcium deregulation. The deregulation amplitude is decreased by succinyl phosphonate and its phosphono ethyl ester by 50% and 30%, correspondingly. Thus, succinyl phosphonate is more potent than its phosphono ethyl ester in protecting both the isolated brain OGDHC from inactivation and cultured neurons from the glutamate-induced calcium deregulation. The correlation of the relative efficiency of the phosphonates in vitro and in situ indicates that their cellular effects are due to targeting OGDHC, which is in accord with independent studies. We conclude that the compounds preserving the 2-oxoglutarate dehydrogenase activity are of neuroprotective value upon metabolic disbalance induced by glutamate excess.

Infantile onset progressive cerebellar atrophy and anterior horn cell degeneration--a late onset variant of PCH-1?

PubMed

Lev, Dorit; Michelson-Kerman, Marina; Vinkler, Chana; Blumkin, Lubov; Shalev, Stavit A; Lerman-Sagie, Tally

2008-03-01

Despite major recent advances in our understanding of developmental cerebellar disorders, classification and delineation of these disorders remains difficult. The term pontocerebellar hypoplasia is used when there is a structural defect, originating in utero of both pons and cerebellar hemispheres. The term olivopontocerebellar atrophy is used when the disorder starts later in life and the process is a primary degeneration of cerebellar neurons. Pontocerebellar hypoplasia type 1 is associated with spinal anterior horn cell degeneration, congenital contractures, microcephaly, polyhydramnion and respiratory insufficiency leading to early death. However, anterior horn cell degeneration has also been described in cases with later onset pontocerebellar atrophy and recently the spectrum has even been further extended to include the association of anterior horn cell degeneration and cerebellar atrophy without pontine involvement. We describe two siblings from a consanguineous Moslem Arabic family who presented with progressive degeneration of both the cerebellum and the anterior horn cells. The patients presented after 1 year of age with a slow neurodegenerative course that included both cognitive and motor functions. There is considerable phenotypic variability; the sister shows a much milder course. Both children are still alive at 6 and 9 years. The sister could still crawl and speak two word sentences at the age of 3 years while the brother was bedridden and only uttered guttural sounds at the same age. Our cases further extend the phenotype of the cerebellar syndromes with anterior horn cell involvement to include a childhood onset and protracted course and further prove that this neurodegenerative disorder may start in utero or later in life.

Protein Biomarkers Associated With Growth And Synaptogenesis In a cell culture model of neuronal development

EPA Science Inventory

Cerebellar granule cells (CGC) provide a homogenous population of cells which can be used as an in vitro model for studying the cellular processes involved in the normal development of the CNS. They may also be useful for hazard identification as in vitro screens fo...

Synchrony and neural coding in cerebellar circuits

PubMed Central

Person, Abigail L.; Raman, Indira M.

2012-01-01

The cerebellum regulates complex movements and is also implicated in cognitive tasks, and cerebellar dysfunction is consequently associated not only with movement disorders, but also with conditions like autism and dyslexia. How information is encoded by specific cerebellar firing patterns remains debated, however. A central question is how the cerebellar cortex transmits its integrated output to the cerebellar nuclei via GABAergic synapses from Purkinje neurons. Possible answers come from accumulating evidence that subsets of Purkinje cells synchronize their firing during behaviors that require the cerebellum. Consistent with models predicting that coherent activity of inhibitory networks has the capacity to dictate firing patterns of target neurons, recent experimental work supports the idea that inhibitory synchrony may regulate the response of cerebellar nuclear cells to Purkinje inputs, owing to the interplay between unusually fast inhibitory synaptic responses and high rates of intrinsic activity. Data from multiple laboratories lead to a working hypothesis that synchronous inhibitory input from Purkinje cells can set the timing and rate of action potentials produced by cerebellar nuclear cells, thereby relaying information out of the cerebellum. If so, then changing spatiotemporal patterns of Purkinje activity would allow different subsets of inhibitory neurons to control cerebellar output at different times. Here we explore the evidence for and against the idea that a synchrony code defines, at least in part, the input–output function between the cerebellar cortex and nuclei. We consider the literature on the existence of simple spike synchrony, convergence of Purkinje neurons onto nuclear neurons, and intrinsic properties of nuclear neurons that contribute to responses to inhibition. Finally, we discuss factors that may disrupt or modulate a synchrony code and describe the potential contributions of inhibitory synchrony to other motor circuits. PMID

Effects of pentylenetetrazole and glutamate on metabolism of [U-(13)C]glucose in cultured cerebellar granule neurons.

PubMed

Eloqayli, Haytham; Qu, Hong; Unsgård, Geirmund; Sletvold, Olav; Hadidi, Hakam; Sonnewald, Ursula

2002-02-01

This study was performed to analyze the effects of glutamate and the epileptogenic agent pentylenetetrazole (PTZ) on neuronal glucose metabolism. Cerebellar granule neurons were incubated for 2 h in medium containing 3 mM [U-(13)C]glucose, with and without 0.25 mM glutamate and/or 10 mM PTZ. In the presence of PTZ, decreased glucose consumption with unchanged lactate release was observed, indicating decreased glucose oxidation. PTZ also slowed down tricarboxylic acid (TCA) cycle activity as evidenced by the decreased amounts of labeled aspartate and [1,2-(13)C]glutamate. When glutamate was present, glucose consumption was also decreased. However, the amount of glutamate, derived from [U-(13)C]glucose via the first turn of the TCA cycle, was increased. The decreased amount of [1,2-(13)C]glutamate, derived from the second turn in the TCA cycle, and increased amount of aspartate indicated the dilution of label due to the entrance of unlabeled glutamate into TCA cycle. In the presence of glutamate plus PTZ, the effect of PTZ was enhanced by glutamate. Labeled alanine was detected only in the presence of glutamate plus PTZ, which indicated that oxaloacetate was a better amino acid acceptor than pyruvate. Furthermore, there was also evidence for intracellular compartmentation of oxaloacetate metabolism. Glutamate and PTZ caused similar metabolic changes, however, via different mechanisms. Glutamate substituted for glucose as energy substrate in the TCA cycle, whereas, PTZ appeared to decrease mitochondrial activity.

The dynamic relationship between cerebellar Purkinje cell simple spikes and the spikelet number of complex spikes.

PubMed

Burroughs, Amelia; Wise, Andrew K; Xiao, Jianqiang; Houghton, Conor; Tang, Tianyu; Suh, Colleen Y; Lang, Eric J; Apps, Richard; Cerminara, Nadia L

2017-01-01

Purkinje cells are the sole output of the cerebellar cortex and fire two distinct types of action potential: simple spikes and complex spikes. Previous studies have mainly considered complex spikes as unitary events, even though the waveform is composed of varying numbers of spikelets. The extent to which differences in spikelet number affect simple spike activity (and vice versa) remains unclear. We found that complex spikes with greater numbers of spikelets are preceded by higher simple spike firing rates but, following the complex spike, simple spikes are reduced in a manner that is graded with spikelet number. This dynamic interaction has important implications for cerebellar information processing, and suggests that complex spike spikelet number may maintain Purkinje cells within their operational range. Purkinje cells are central to cerebellar function because they form the sole output of the cerebellar cortex. They exhibit two distinct types of action potential: simple spikes and complex spikes. It is widely accepted that interaction between these two types of impulse is central to cerebellar cortical information processing. Previous investigations of the interactions between simple spikes and complex spikes have mainly considered complex spikes as unitary events. However, complex spikes are composed of an initial large spike followed by a number of secondary components, termed spikelets. The number of spikelets within individual complex spikes is highly variable and the extent to which differences in complex spike spikelet number affects simple spike activity (and vice versa) remains poorly understood. In anaesthetized adult rats, we have found that Purkinje cells recorded from the posterior lobe vermis and hemisphere have high simple spike firing frequencies that precede complex spikes with greater numbers of spikelets. This finding was also evident in a small sample of Purkinje cells recorded from the posterior lobe hemisphere in awake cats. In addition

The dynamic relationship between cerebellar Purkinje cell simple spikes and the spikelet number of complex spikes

PubMed Central

Burroughs, Amelia; Wise, Andrew K.; Xiao, Jianqiang; Houghton, Conor; Tang, Tianyu; Suh, Colleen Y.; Lang, Eric J.

2016-01-01

Key points Purkinje cells are the sole output of the cerebellar cortex and fire two distinct types of action potential: simple spikes and complex spikes.Previous studies have mainly considered complex spikes as unitary events, even though the waveform is composed of varying numbers of spikelets.The extent to which differences in spikelet number affect simple spike activity (and vice versa) remains unclear.We found that complex spikes with greater numbers of spikelets are preceded by higher simple spike firing rates but, following the complex spike, simple spikes are reduced in a manner that is graded with spikelet number.This dynamic interaction has important implications for cerebellar information processing, and suggests that complex spike spikelet number may maintain Purkinje cells within their operational range. Abstract Purkinje cells are central to cerebellar function because they form the sole output of the cerebellar cortex. They exhibit two distinct types of action potential: simple spikes and complex spikes. It is widely accepted that interaction between these two types of impulse is central to cerebellar cortical information processing. Previous investigations of the interactions between simple spikes and complex spikes have mainly considered complex spikes as unitary events. However, complex spikes are composed of an initial large spike followed by a number of secondary components, termed spikelets. The number of spikelets within individual complex spikes is highly variable and the extent to which differences in complex spike spikelet number affects simple spike activity (and vice versa) remains poorly understood. In anaesthetized adult rats, we have found that Purkinje cells recorded from the posterior lobe vermis and hemisphere have high simple spike firing frequencies that precede complex spikes with greater numbers of spikelets. This finding was also evident in a small sample of Purkinje cells recorded from the posterior lobe hemisphere in awake

Reevaluation of the Beam and Radial Hypotheses of Parallel Fiber Action in the Cerebellar Cortex

PubMed Central

Cramer, Samuel W.; Gao, Wangcai; Chen, Gang

2013-01-01

The role of parallel fibers (PFs) in cerebellar physiology remains controversial. Early studies inspired the “beam” hypothesis whereby granule cell (GC) activation results in PF-driven, postsynaptic excitation of beams of Purkinje cells (PCs). However, the “radial” hypothesis postulates that the ascending limb of the GC axon provides the dominant input to PCs and generates patch-like responses. Using optical imaging and single-cell recordings in the mouse cerebellar cortex in vivo, this study reexamines the beam versus radial controversy. Electrical stimulation of mossy fibers (MFs) as well as microinjection of NMDA in the granular layer generates beam-like responses with a centrally located patch-like response. Remarkably, ipsilateral forepaw stimulation evokes a beam-like response in Crus I. Discrete molecular layer lesions demonstrate that PFs contribute to the peripherally generated responses in Crus I. In contrast, vibrissal stimulation induces patch-like activation of Crus II and GABAA antagonists fail to convert this patch-like activity into a beam-like response, implying that molecular layer inhibition does not prevent beam-like responses. However, blocking excitatory amino acid transporters (EAATs) generates beam-like responses in Crus II. These beam-like responses are suppressed by focal inhibition of MF-GC synaptic transmission. Using EAAT4 reporter transgenic mice, we show that peripherally evoked patch-like responses in Crus II are aligned between parasagittal bands of EAAT4. This is the first study to demonstrate beam-like responses in the cerebellar cortex to peripheral, MF, and GC stimulation in vivo. Furthermore, the spatial pattern of the responses depends on extracellular glutamate and its local regulation by EAATs. PMID:23843513

Status epilepticus increases mature granule cells in the molecular layer of the dentate gyrus in rats★

PubMed Central

Liang, Zhaoliang; Gao, Fei; Wang, Fajun; Wang, Xiaochen; Song, Xinyu; Liu, Kejing; Zhan, Ren-Zhi

2013-01-01

Enhanced neurogenesis in the dentate gyrus of the hippocampus following seizure activity, especially status epilepticus, is associated with ectopic residence and aberrant integration of newborn granule cells. Hilar ectopic granule cells may be detrimental to the stability of dentate circuitry by means of their electrophysiological properties and synaptic connectivity. We hypothesized that status epilepticus also increases ectopic granule cells in the molecular layer. Status epilepticus was induced in male Sprague-Dawley rats by intraperitoneal injection of pilocarpine. Immunostaining showed that many doublecortin-positive cells were present in the molecular layer and the hilus 7 days after the induction of status epilepticus. At least 10 weeks after status epilepticus, the estimated number of cells positive for both prospero homeobox protein 1 and neuron-specific nuclear protein in the hilus was significantly increased. A similar trend was also found in the molecular layer. These findings indicate that status epilepticus can increase the numbers of mature and ectopic newborn granule cells in the molecular layer. PMID:25206705

A spiking network model of cerebellar Purkinje cells and molecular layer interneurons exhibiting irregular firing

PubMed Central

Lennon, William; Hecht-Nielsen, Robert; Yamazaki, Tadashi

2014-01-01

While the anatomy of the cerebellar microcircuit is well-studied, how it implements cerebellar function is not understood. A number of models have been proposed to describe this mechanism but few emphasize the role of the vast network Purkinje cells (PKJs) form with the molecular layer interneurons (MLIs)—the stellate and basket cells. We propose a model of the MLI-PKJ network composed of simple spiking neurons incorporating the major anatomical and physiological features. In computer simulations, the model reproduces the irregular firing patterns observed in PKJs and MLIs in vitro and a shift toward faster, more regular firing patterns when inhibitory synaptic currents are blocked. In the model, the time between PKJ spikes is shown to be proportional to the amount of feedforward inhibition from an MLI on average. The two key elements of the model are: (1) spontaneously active PKJs and MLIs due to an endogenous depolarizing current, and (2) adherence to known anatomical connectivity along a parasagittal strip of cerebellar cortex. We propose this model to extend previous spiking network models of the cerebellum and for further computational investigation into the role of irregular firing and MLIs in cerebellar learning and function. PMID:25520646

COUP-TFI mitotically regulates production and migration of dentate granule cells and modulates hippocampal Cxcr4 expression.

PubMed

Parisot, Joséphine; Flore, Gemma; Bertacchi, Michele; Studer, Michèle

2017-06-01

Development of the dentate gyrus (DG), the primary gateway for hippocampal inputs, spans embryonic and postnatal stages, and involves complex morphogenetic events. We have previously identified the nuclear receptor COUP-TFI as a novel transcriptional regulator in the postnatal organization and function of the hippocampus. Here, we dissect its role in DG morphogenesis by inactivating it in either granule cell progenitors or granule neurons. Loss of COUP-TFI function in progenitors leads to decreased granule cell proliferative activity, precocious differentiation and increased apoptosis, resulting in a severe DG growth defect in adult mice. COUP-TFI-deficient cells express high levels of the chemokine receptor Cxcr4 and migrate abnormally, forming heterotopic clusters of differentiated granule cells along their paths. Conversely, high COUP-TFI expression levels downregulate Cxcr4 expression, whereas increased Cxcr4 expression in wild-type hippocampal cells affects cell migration. Finally, loss of COUP-TFI in postmitotic cells leads to only minor and transient abnormalities, and to normal Cxcr4 expression. Together, our results indicate that COUP-TFI is required predominantly in DG progenitors for modulating expression of the Cxcr4 receptor during granule cell neurogenesis and migration. © 2017. Published by The Company of Biologists Ltd.

Wolfram syndrome 1 gene (WFS1) product localizes to secretory granules and determines granule acidification in pancreatic beta-cells.

PubMed

Hatanaka, Masayuki; Tanabe, Katsuya; Yanai, Akie; Ohta, Yasuharu; Kondo, Manabu; Akiyama, Masaru; Shinoda, Koh; Oka, Yoshitomo; Tanizawa, Yukio

2011-04-01

Wolfram syndrome is an autosomal recessive disorder characterized by juvenile-onset insulin-dependent diabetes mellitus and optic atrophy. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER) resident transmembrane protein. The Wfs1-null mouse exhibits progressive insulin deficiency causing diabetes. Previous work suggested that the function of the WFS1 protein is connected to unfolded protein response and to intracellular Ca(2+) homeostasis. However, its precise molecular function in pancreatic β-cells remains elusive. In our present study, immunofluorescent and electron-microscopic analyses revealed that WFS1 localizes not only to ER but also to secretory granules in pancreatic β-cells. Intragranular acidification was assessed by measuring intracellular fluorescence intensity raised by the acidotrophic agent, 3-[2,4-dinitroanilino]-3'-amino-N-methyldipropyramine. Compared with wild-type β-cells, there was a 32% reduction in the intensity in WFS1-deficient β-cells, indicating the impairment of granular acidification. This phenotype may, at least partly, account for the evidence that Wfs1-null islets have impaired proinsulin processing, resulting in an increased circulating proinsulin level. Morphometric analysis using electron microscopy evidenced that the density of secretory granules attached to the plasma membrane was significantly reduced in Wfs1-null β-cells relative to that in wild-type β-cells. This may be relevant to the recent finding that granular acidification is required for the priming of secretory granules preceding exocytosis and may partly explain the fact that glucose-induced insulin secretion is profoundly impaired in young prediabetic Wfs1-null mice. These results thus provide new insights into the molecular mechanisms of β-cell dysfunction in patients with Wolfram syndrome.

Metabolism of two Go alpha isoforms in neuronal cells during differentiation.

PubMed

Brabet, P; Pantaloni, C; Bockaert, J; Homburger, V

1991-07-15

We have previously shown that undifferentiated N1E-115 neuroblastoma cells express only one isoform of Go alpha (pI = 5.8), whereas differentiated neuroblastoma cells expressed, in addition to this isoform, another Go alpha with a more acidic pI (5.55). Moreover, primary cultures of cerebellar granule cells, which are extremely well differentiated cells yielding a high density of synapses, expressed only a single Go alpha isoform with a pI of 5.55 (Brabet, P., Pantaloni, C., Rodriguez Martinez, J., Bockaert, J., and Homburger, V. (1990) J. Neurochem. 54, 1310-1320). In this report, using biosynthetic labeling with [35S]methionine and specific quantitative immunoprecipitation with a polyclonal antibody raised against the purified Go alpha protein, we have determined 1) the degradation rate of total Go alpha (sum of the two isoforms) in differentiated as well as in undifferentiated neuroblastoma cells and in cerebellar granule cells, 2) the degradation rates of each isoform in differentiated neuroblastoma cells. The t 1/2 for total Go alpha protein degradation was very different in the three neuronal cell populations and was 28 +/- 5 h (n = 5), 58 +/- 9 h (n = 5), and 154 +/- 22 h (n = 6) in undifferentiated, differentiated neuroblastoma, and granule cells, respectively. Using two-dimensional gel analysis of immunoprecipitates, we have also determined the individual t 1/2 for degradation of each Go alpha isoform in differentiated neuroblastoma cells, in which the two Go alpha isoforms were expressed. Results indicated that the two Go alpha isoforms exhibit similar t1/2 for degradation (49 +/- 5 h, n = 3). Thus, the t1/2 for degradation of the more basic Go alpha isoform is higher in differentiated neuroblastoma cells (49 +/- 5 h, n = 3) than in undifferentiated neuroblastoma cells (28 +/- 5 h, n = 5) which expressed only the more basic Go alpha isoform. It can be concluded that the degradation rate of the more basic Go alpha isoform is not a characteristic of the

Contribution of Cerebellar Sensorimotor Adaptation to Hippocampal Spatial Memory

PubMed Central

Passot, Jean-Baptiste; Sheynikhovich, Denis; Duvelle, Éléonore; Arleo, Angelo

2012-01-01

Complementing its primary role in motor control, cerebellar learning has also a bottom-up influence on cognitive functions, where high-level representations build up from elementary sensorimotor memories. In this paper we examine the cerebellar contribution to both procedural and declarative components of spatial cognition. To do so, we model a functional interplay between the cerebellum and the hippocampal formation during goal-oriented navigation. We reinterpret and complete existing genetic behavioural observations by means of quantitative accounts that cross-link synaptic plasticity mechanisms, single cell and population coding properties, and behavioural responses. In contrast to earlier hypotheses positing only a purely procedural impact of cerebellar adaptation deficits, our results suggest a cerebellar involvement in high-level aspects of behaviour. In particular, we propose that cerebellar learning mechanisms may influence hippocampal place fields, by contributing to the path integration process. Our simulations predict differences in place-cell discharge properties between normal mice and L7-PKCI mutant mice lacking long-term depression at cerebellar parallel fibre-Purkinje cell synapses. On the behavioural level, these results suggest that, by influencing the accuracy of hippocampal spatial codes, cerebellar deficits may impact the exploration-exploitation balance during spatial navigation. PMID:22485133

N-acetyl-l-cysteine and Mn2+ attenuate Cd2+-induced disturbance of the intracellular free calcium homeostasis in cultured cerebellar granule neurons.

PubMed

Isaev, Nickolay K; Avilkina, Svetlana; Golyshev, Sergey A; Genrikhs, Elisaveta E; Alexandrova, Olga P; Kapkaeva, Marina R; Stelmashook, Elena V

2018-01-15

Cadmium is a highly toxic heavy metal that is capable of accumulating in the body via direct exposure or through the alimentary and respiratory tract, leading to neurodegeneration. In this article, we show that the application of CdCl 2 (0.001-0.005mM) for 48h induced high dose-dependent death rate of cultured cerebellar granule neurons (CGNs). Unlike Trolox or vitamin E, antioxidant N-acetyl-l-cysteine (NAC, 1mM) and Mn 2+ (0.0025-0.005mM) significantly protected CGNs from this toxic effect. Using Fluo-4 AM, measurements of intracellular calcium ions demonstrated that 24h-exposure to Cd 2+ induced intensive increase of Fluo-4 fluorescence in neurons accompanied by mitochondria swelling. These data imply that the cadmium-induced Ca 2+ increase is an important element in the death of neurons due to toxic effect of cadmium and the mechanism of protective action of manganese and NAC is mediated by the prevention of increase in calcium levels. Copyright © 2017 Elsevier B.V. All rights reserved.

Differential 3’ processing of specific transcripts expands regulatory and protein diversity across neuronal cell types

PubMed Central

Jereb, Saša; Hwang, Hun-Way; Van Otterloo, Eric; Govek, Eve-Ellen; Fak, John J; Yuan, Yuan; Hatten, Mary E

2018-01-01

Alternative polyadenylation (APA) regulates mRNA translation, stability, and protein localization. However, it is unclear to what extent APA regulates these processes uniquely in specific cell types. Using a new technique, cTag-PAPERCLIP, we discovered significant differences in APA between the principal types of mouse cerebellar neurons, the Purkinje and granule cells, as well as between proliferating and differentiated granule cells. Transcripts that differed in APA in these comparisons were enriched in key neuronal functions and many differed in coding sequence in addition to 3’UTR length. We characterize Memo1, a transcript that shifted from expressing a short 3’UTR isoform to a longer one during granule cell differentiation. We show that Memo1 regulates granule cell precursor proliferation and that its long 3’UTR isoform is targeted by miR-124, contributing to its downregulation during development. Our findings provide insight into roles for APA in specific cell types and establish a platform for further functional studies. PMID:29578408

Curcumin Pretreatment Induces Nrf2 and an Antioxidant Response and Prevents Hemin-Induced Toxicity in Primary Cultures of Cerebellar Granule Neurons of Rats

PubMed Central

González-Reyes, Susana; Guzmán-Beltrán, Silvia; Medina-Campos, Omar Noel; Pedraza-Chaverri, José

2013-01-01

Curcumin is a bifunctional antioxidant derived from Curcuma longa. This study identifies curcumin as a neuroprotectant against hemin-induced damage in primary cultures of cerebellar granule neurons (CGNs) of rats. Hemin, the oxidized form of heme, is a highly reactive compound that induces cellular injury. Pretreatment of CGNs with 5–30 μM curcumin effectively increased by 2.3–4.9 fold heme oxygenase-1 (HO-1) expression and by 5.6–14.3-fold glutathione (GSH) levels. Moreover, 15 μM curcumin attenuated by 55% the increase in reactive oxygen species (ROS) production, by 94% the reduction of GSH/glutathione disulfide (GSSG) ratio, and by 49% the cell death induced by hemin. The inhibition of heme oxygenase system or GSH synthesis with tin mesoporphyrin and buthionine sulfoximine, respectively, suppressed the protective effect of curcumin against hemin-induced toxicity. These data strongly suggest that HO-1 and GSH play a major role in the protective effect of curcumin. Furthermore, it was found that 24 h of incubation with curcumin increases by 1.4-, 2.3-, and 5.2-fold the activity of glutathione reductase, glutathione S-transferase and superoxide dismutase, respectively. Additionally, it was found that curcumin was capable of inducing nuclear factor (erythroid-derived 2)-like 2 (Nrf2) translocation into the nucleus. These data suggest that the pretreatment with curcumin induces Nrf2 and an antioxidant response that may play an important role in the protective effect of this antioxidant against hemin-induced neuronal death. PMID:24454990

Comparative neuronal morphology of the cerebellar cortex in afrotherians, carnivores, cetartiodactyls, and primates

PubMed Central

Jacobs, Bob; Johnson, Nicholas L.; Wahl, Devin; Schall, Matthew; Maseko, Busisiwe C.; Lewandowski, Albert; Raghanti, Mary A.; Wicinski, Bridget; Butti, Camilla; Hopkins, William D.; Bertelsen, Mads F.; Walsh, Timothy; Roberts, John R.; Reep, Roger L.; Hof, Patrick R.; Sherwood, Chet C.; Manger, Paul R.

2014-01-01

Although the basic morphological characteristics of neurons in the cerebellar cortex have been documented in several species, virtually nothing is known about the quantitative morphological characteristics of these neurons across different taxa. To that end, the present study investigated cerebellar neuronal morphology among eight different, large-brained mammalian species comprising a broad phylogenetic range: afrotherians (African elephant, Florida manatee), carnivores (Siberian tiger, clouded leopard), cetartiodactyls (humpback whale, giraffe) and primates (human, common chimpanzee). Specifically, several neuron types (e.g., stellate, basket, Lugaro, Golgi, and granule neurons; N = 317) of the cerebellar cortex were stained with a modified rapid Golgi technique and quantified on a computer-assisted microscopy system. There was a 64-fold variation in brain mass across species in our sample (from clouded leopard to the elephant) and a 103-fold variation in cerebellar volume. Most dendritic measures tended to increase with cerebellar volume. The cerebellar cortex in these species exhibited the trilaminate pattern common to all mammals. Morphologically, neuron types in the cerebellar cortex were generally consistent with those described in primates (Fox et al., 1967) and rodents (Palay and Chan-Palay, 1974), although there was substantial quantitative variation across species. In particular, Lugaro neurons in the elephant appeared to be disproportionately larger than those in other species. To explore potential quantitative differences in dendritic measures across species, MARSplines analyses were used to evaluate whether species could be differentiated from each other based on dendritic characteristics alone. Results of these analyses indicated that there were significant differences among all species in dendritic measures. PMID:24795574

Excitotoxicity through NMDA receptors mediates cerebellar granule neuron apoptosis induced by prion protein 90-231 fragment.

PubMed

Thellung, Stefano; Gatta, Elena; Pellistri, Francesca; Corsaro, Alessandro; Villa, Valentina; Vassalli, Massimo; Robello, Mauro; Florio, Tullio

2013-05-01

Prion diseases recognize, as a unique molecular trait, the misfolding of CNS-enriched prion protein (PrP(C)) into an aberrant isoform (PrP(Sc)). In this work, we characterize the in vitro toxicity of amino-terminally truncated recombinant PrP fragment (amino acids 90-231, PrP90-231), on rat cerebellar granule neurons (CGN), focusing on glutamatergic receptor activation and Ca(2+) homeostasis impairment. This recombinant fragment assumes a toxic conformation (PrP90-231(TOX)) after controlled thermal denaturation (1 h at 53 °C) acquiring structural characteristics identified in PrP(Sc) (enrichment in β-structures, increased hydrophobicity, partial resistance to proteinase K, and aggregation in amyloid fibrils). By annexin-V binding assay, and evaluation of the percentage of fragmented and condensed nuclei, we show that treatment with PrP90-231(TOX), used in pre-fibrillar aggregation state, induces CGN apoptosis. This effect was associated with a delayed, but sustained elevation of [Ca(2+)]i. Both CGN apoptosis and [Ca(2+)]i increase were not observed using PrP90-231 in PrP(C)-like conformation. PrP90-231(TOX) effects were significantly reduced in the presence of ionotropic glutamate receptor antagonists. In particular, CGN apoptosis and [Ca(2+)]i increase were largely reduced, although not fully abolished, by pre-treatment with the NMDA antagonists APV and memantine, while the AMPA antagonist CNQX produced a lower, although still significant, effect. In conclusion, we report that CGN apoptosis induced by PrP90-231(TOX) correlates with a sustained elevation of [Ca(2+)]i mediated by the activation of NMDA and AMPA receptors.

Overexpression of Lin28b in Neural Stem Cells is Insufficient for Brain Tumor Formation, but Induces Pathological Lobulation of the Developing Cerebellum.

PubMed

Wefers, Annika K; Lindner, Sven; Schulte, Johannes H; Schüller, Ulrich

2017-02-01

LIN28B is a homologue of the RNA-binding protein LIN28A and regulates gene expression during development and carcinogenesis. It is strongly upregulated in a variety of brain tumors, such as medulloblastoma, embryonal tumor with multilayered rosettes (ETMR), atypical teratoid/rhabdoid tumor (AT/RT), or glioblastoma, but the effect of an in vivo overexpression of LIN28B on the developing central nervous system is unknown. We generated transgenic mice that either overexpressed Lin28b in Math1-positive cerebellar granule neuron precursors or in a broad range of Nestin-positive neural precursors. Sections of the cerebellar vermis from adult Math1-Cre::lsl-Lin28b mice had an additional subfissure in lobule IV. Vermes from p0 and p7 Nestin-Cre::lsl-Lin28b mice appeared normal, but we found a pronounced vermal hypersublobulation at p15 and p21 in these mice. Also, the external granule cell layer (EGL) was thicker at p15 than in controls, contained more proliferating cells, and persisted up to p21. Consistently, some Pax6- and NeuN-positive cells were present in the EGL of Nestin-Cre::lsl-Lin28b mice even at p21, and we detected more NeuN-positive granule neuron precursors in the molecular layer (ML) as compared to control. Finally, we found some residual Pax2-positive precursors of inhibitory interneurons in the ML of Nestin-Cre::lsl-Lin28b mice at p21, which have already disappeared in controls. We conclude that while overexpression of LIN28B in Nestin-positive cells does not lead to tumor formation, it results in a protracted development of granule cells and inhibitory interneurons and leads to a hypersublobulation of the cerebellar vermis.

Properties of single NMDA receptor channels in human dentate gyrus granule cells

PubMed Central

Lieberman, David N; Mody, Istvan

1999-01-01

Cell-attached single-channel recordings of NMDA channels were carried out in human dentate gyrus granule cells acutely dissociated from slices prepared from hippocampi surgically removed for the treatment of temporal lobe epilepsy (TLE). The channels were activated by l-aspartate (250–500 nm) in the presence of saturating glycine (8 μm). The main conductance was 51 ± 3 pS. In ten of thirty granule cells, clear subconductance states were observed with a mean conductance of 42 ± 3 pS, representing 8 ± 2% of the total openings. The mean open times varied from cell to cell, possibly owing to differences in the epileptogenicity of the tissue of origin. The mean open time was 2.70 ± 0.95 ms (range, 1.24–4.78 ms). In 87% of the cells, three exponential components were required to fit the apparent open time distributions. In the remaining neurons, as in control rat granule cells, two exponentials were sufficient. Shut time distributions were fitted by five exponential components. The average numbers of openings in bursts (1.74 ± 0.09) and clusters (3.06 ± 0.26) were similar to values obtained in rodents. The mean burst (6.66 ± 0.9 ms), cluster (20.1 ± 3.3 ms) and supercluster lengths (116.7 ± 17.5 ms) were longer than those in control rat granule cells, but approached the values previously reported for TLE (kindled) rats. As in rat NMDA channels, adjacent open and shut intervals appeared to be inversely related to each other, but it was only the relative areas of the three open time constants that changed with adjacent shut time intervals. The long openings of human TLE NMDA channels resembled those produced by calcineurin inhibitors in control rat granule cells. Yet the calcineurin inhibitor FK-506 (500 nm) did not prolong the openings of human channels, consistent with a decreased calcineurin activity in human TLE. Many properties of the human NMDA channels resemble those recorded in rat hippocampal neurons. Both have similar slope conductances, five

Clonal Analysis of Newborn Hippocampal Dentate Granule Cell Proliferation and Development in Temporal Lobe Epilepsy1,2,3

PubMed Central

LaSarge, Candi L.; McAuliffe, John J.

2015-01-01

Abstract Hippocampal dentate granule cells are among the few neuronal cell types generated throughout adult life in mammals. In the normal brain, new granule cells are generated from progenitors in the subgranular zone and integrate in a typical fashion. During the development of epilepsy, granule cell integration is profoundly altered. The new cells migrate to ectopic locations and develop misoriented “basal” dendrites. Although it has been established that these abnormal cells are newly generated, it is not known whether they arise ubiquitously throughout the progenitor cell pool or are derived from a smaller number of “bad actor” progenitors. To explore this question, we conducted a clonal analysis study in mice expressing the Brainbow fluorescent protein reporter construct in dentate granule cell progenitors. Mice were examined 2 months after pilocarpine-induced status epilepticus, a treatment that leads to the development of epilepsy. Brain sections were rendered translucent so that entire hippocampi could be reconstructed and all fluorescently labeled cells identified. Our findings reveal that a small number of progenitors produce the majority of ectopic cells following status epilepticus, indicating that either the affected progenitors or their local microenvironments have become pathological. By contrast, granule cells with “basal” dendrites were equally distributed among clonal groups. This indicates that these progenitors can produce normal cells and suggests that global factors sporadically disrupt the dendritic development of some new cells. Together, these findings strongly predict that distinct mechanisms regulate different aspects of granule cell pathology in epilepsy. PMID:26756038

Abnormal nuclear envelope in the cerebellar Purkinje cells and impaired motor learning in DYT11 myoclonus-dystonia mouse models

PubMed Central

Yokoi, Fumiaki; Dang, Mai T.; Yang, Guang; Li, JinDong; Doroodchi, Atbin; Zhou, Tong; Li, Yuqing

2011-01-01

Myoclonus-dystonia (M-D) is a movement disorder characterized by myoclonic jerks with dystonia. DYT11 M-D is caused by mutations in SGCE which codes for ε-sarcoglycan. SGCE is maternally imprinted and paternally expressed. Abnormal nuclear envelope has been reported in mouse models of DYT1 generalized torsion dystonia. However, it is not known whether similar alterations occur in DYT11 M-D. We developed a mouse model of DYT11 M-D using paternally-inherited Sgce heterozygous knockout (Sgce KO) mice and reported that they had myoclonus and motor coordination and learning deficits in the beam-walking test. However, the specific brain regions that contribute to these phenotypes have not been identified. Since ε-sarcoglycan is highly expressed in the cerebellar Purkinje cells, here we examined the nuclear envelope in these cells using a transmission electron microscope and found that they are abnormal in Sgce KO mice. Our results put DYT11 M-D in a growing family of nuclear envelopathies. To analyze the effect of loss of ε-sarcoglycan function in the cerebellar Purkinje cells, we produced paternally-inherited cerebellar Purkinje cell-specific Sgce conditional knockout (Sgce pKO) mice. Sgce pKO mice showed motor learning deficits, while they did not show abnormal nuclear envelope in the cerebellar Purkinje cells, robust motor deficits, or myoclonus. The results suggest that ε-sarcoglycan in the cerebellar Purkinje cells contributes to the motor learning, while loss of ε-sarcoglycan in other brain regions may contribute to nuclear envelope abnormality, myoclonus and motor coordination deficits. PMID:22040906

Survival of Swiss-Webster mouse cerebellar granule neurons is promoted by a combination of potassium channel blockers.

PubMed

Collins, Anthony; Larson, Maureen K; Pfaff, Jilleen E; Ishmael, Jane E

2007-06-15

Cultured cerebellar granule neurons (CGN) are commonly used to assess neurotoxicity, but are routinely maintained in supraphysiological (25 mM) extracellular K(+) concentrations [K(+)](o). We investigated the effect of potassium channel blockade on survival of CGN derived from Swiss-Webster mice in supraphysiological (25 mM) and physiological (5.6 mM) [K(+)](o). CGN were cultured for 5 days in 25 mM K(+), then in 5.6 mM K(+) or 25 mM K(+) (control). Viability, assayed 24 h later by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) reduction and by lactate dehydrogenase (LDH) release, was approximately 50% in 5.6 mM K(+) versus 25 mM K(+) (p<.001). Potassium channel blockers, 2 mM 4-aminopyridine (4-AP), 2 mM tetraethylammonium (TEA) or 1 mM Ba(2+), individually afforded limited protection in 5.6 mM K(+). However, survival in 5.6 mM K(+) with a combination of 4-AP, TEA and Ba(2+) was similar to survival in 25 mM K(+) without blockers (p<.001 versus 5.6 mM K(+) alone). CGN survival in 25 mM K(+) was attenuated 25% by 2 microM nifedipine (p>.001), but nifedipine did not attenuate neuroprotection by K(+) channel blockers. Together, these results suggest that the survival of CGN depends on the K(+) permeability of the membrane rather than the activity of a particular type of K(+) channel, and that the mechanism of neuroprotection by K(+) channel blockers is different from that of elevated [K(+)](o).

Cerebellar abiotrophy in a family of Border Collie dogs.

PubMed

Sandy, J R; Slocombe, R E; Mitten, R W; Jedwab, D

2002-11-01

Cerebellar abiotrophies have a nonsex-linked, autosomal, recessively inherited basis in a number of species, and lesions typically reflect profound and progressive loss of Purkinje cells. In this report, an unusual form of abiotrophy is described for two sibling Border Collies. Extensive loss of the cerebellar granular cell layer was present with relative sparing of Purkinje cells of two female pups. The biochemical basis for this form of cerebellar abiotrophy is unknown, but the lack of disease in other siblings supports an autosomal recessive mode of inheritance.

Changes in mitogen-activated protein kinase in cerebellar granule neurons by polybrominated diphenyl ethers and polychlorinated biphenyls

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

Fan Chunyang; Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC 27599; Besas, Jonathan

2010-05-15

Polybrominated diphenyl ethers (PBDEs) are used as additive flame retardants and have been detected in human blood, adipose tissue, and breast milk. Both in vitro and in vivo studies have shown that the effects of PBDEs are similar to the known human developmental neurotoxicants such as polychlorinated biphenyls (PCBs) on a molar basis. Previously, we reported that PBDE mixtures and congeners, perturbed calcium homeostasis which is critical for the development and function of the nervous system. In the present study, we tested whether environmentally relevant PBDE/PCB mixtures and congeners affected mitogen-activated protein kinase (MAPK) pathways, which are down-stream events ofmore » calcium signaling in cerebellar granule neuronal cultures. In this study, phosphorylated extracellular signal-regulated kinase (pERK)1/2, a widely studied MAPK cascade and known to be involved in learning and memory, levels were quantitated using western blot technique with phospho-specific antibodies. Glutamate (a positive control) increased pERK1/2 in a time- and concentration-dependent manner reaching maximum activation at 5-30 min of exposure and at doses >= 10 muM. Both Aroclor 1254 (a commercial penta PCB mixture) and DE-71 (a commercial penta PBDE mixture) elevated phospho-ERK1/2, producing maximum stimulation at 30 min and at concentrations >= 3 mug/ml; Aroclor 1254 was more efficacious than DE-71. DE-79 (an octabrominated diphenyl ether mixture) also elevated phospho-ERK1/2, but to a lesser extent than that of DE-71. PBDE congeners 47, 77, 99, and 153 also increased phospo-ERK1/2 in a concentration-dependent manner. The data indicated that PBDE congeners are more potent than the commercial mixtures. PCB 47 also increased phospho-ERK1/2 like its structural analog PBDE 47, but to a lesser extent, suggesting that these chemicals affect similar pathways. Cytotoxicity, measured as %LDH release, data showed that higher concentrations (> 30 muM) and longer exposures (> 30 min

BarTeL, a Genetically Versatile, Bioluminescent and Granule Neuron Precursor-Targeted Mouse Model for Medulloblastoma

PubMed Central

Mahdi, Min Y.; Gonzalez-Gomez, Ignacio; Asgharzadeh, Shahab; D’Apuzzo, Massimo; Erdreich-Epstein, Anat; Moats, Rex A.

2016-01-01

Medulloblastomas are the most common malignant pediatric brain tumor and have been divided into four major molecular subgroups. Animal models that mimic the principal molecular aberrations of these subgroups will be important tools for preclinical studies and allow greater understanding of medulloblastoma biology. We report a new transgenic model of medulloblastoma that possesses a unique combination of desirable characteristics including, among others, the ability to incorporate multiple and variable genes of choice and to produce bioluminescent tumors from a limited number of somatic cells within a normal cellular environment. This model, termed BarTeL, utilizes a Barhl1 homeobox gene promoter to target expression of a bicistronic transgene encoding both the avian retroviral receptor TVA and an eGFP-Luciferase fusion protein to neonatal cerebellar granule neuron precursor (cGNP) cells, which are cells of origin for the sonic hedgehog (SHH) subgroup of human medulloblastomas. The Barhl1 promoter-driven transgene is expressed strongly in mammalian cGNPs and weakly or not at all in mature granule neurons. We efficiently induced bioluminescent medulloblastomas expressing eGFP-luciferase in BarTeL mice by infection of a limited number of somatic cGNPs with avian retroviral vectors encoding the active N-terminal fragment of SHH and a stabilized MYCN mutant. Detection and quantification of the increasing bioluminescence of growing tumors in young BarTeL mice was facilitated by the declining bioluminescence of their uninfected maturing cGNPs. Inclusion of eGFP in the transgene allowed enriched sorting of cGNPs from neonatal cerebella. Use of a single bicistronic avian vector simultaneously expressing both Shh and Mycn oncogenes increased the medulloblastoma incidence and aggressiveness compared to mixed virus infections. Bioluminescent tumors could also be produced by ex vivo transduction of neonatal BarTeL cerebellar cells by avian retroviruses and subsequent

Using a million cell simulation of the cerebellum: network scaling and task generality.

PubMed

Li, Wen-Ke; Hausknecht, Matthew J; Stone, Peter; Mauk, Michael D

2013-11-01

Several factors combine to make it feasible to build computer simulations of the cerebellum and to test them in biologically realistic ways. These simulations can be used to help understand the computational contributions of various cerebellar components, including the relevance of the enormous number of neurons in the granule cell layer. In previous work we have used a simulation containing 12000 granule cells to develop new predictions and to account for various aspects of eyelid conditioning, a form of motor learning mediated by the cerebellum. Here we demonstrate the feasibility of scaling up this simulation to over one million granule cells using parallel graphics processing unit (GPU) technology. We observe that this increase in number of granule cells requires only twice the execution time of the smaller simulation on the GPU. We demonstrate that this simulation, like its smaller predecessor, can emulate certain basic features of conditioned eyelid responses, with a slight improvement in performance in one measure. We also use this simulation to examine the generality of the computation properties that we have derived from studying eyelid conditioning. We demonstrate that this scaled up simulation can learn a high level of performance in a classic machine learning task, the cart-pole balancing task. These results suggest that this parallel GPU technology can be used to build very large-scale simulations whose connectivity ratios match those of the real cerebellum and that these simulations can be used guide future studies on cerebellar mediated tasks and on machine learning problems. Copyright © 2012 Elsevier Ltd. All rights reserved.

NAADP Activates Two-Pore Channels on T Cell Cytolytic Granules to Stimulate Exocytosis and Killing

PubMed Central

Davis, Lianne C.; Morgan, Anthony J.; Chen, Ji-Li; Snead, Charlotte M.; Bloor-Young, Duncan; Shenderov, Eugene; Stanton-Humphreys, Megan N.; Conway, Stuart J.; Churchill, Grant C.; Parrington, John; Cerundolo, Vincenzo; Galione, Antony

2012-01-01

Summary A cytotoxic T lymphocyte (CTL) kills an infected or tumorigenic cell by Ca2+-dependent exocytosis of cytolytic granules at the immunological synapse formed between the two cells. Although inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release from the endoplasmic reticulum activates the store-operated Ca2+-influx pathway that is necessary for exocytosis, it is not a sufficient stimulus [1–4]. Here we identify the Ca2+-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) and its recently identified molecular target, two-pore channels (TPCs) [5–7], as being important for T cell receptor signaling in CTLs. We demonstrate that cytolytic granules are not only reservoirs of cytolytic proteins but are also the acidic Ca2+ stores mobilized by NAADP via TPC channels on the granules themselves, so that TPCs migrate to the immunological synapse upon CTL activation. Moreover, NAADP activates TPCs to drive exocytosis in a way that is not mimicked by global Ca2+ signals induced by IP3 or ionomycin, suggesting that critical, local Ca2+ nanodomains around TPCs stimulate granule exocytosis. Hence, by virtue of the NAADP/TPC pathway, cytolytic granules generate Ca2+ signals that lead to their own exocytosis and to cell killing. This study highlights a selective role for NAADP in stimulating exocytosis crucial for immune cell function and may impact on stimulus-secretion coupling in wider cellular contexts. PMID:23177477

Effects of Ethanol on the Cerebellum: Advances and Prospects.

PubMed

Luo, Jia

2015-08-01

Alcohol abuse causes cerebellar dysfunction and cerebellar ataxia is a common feature in alcoholics. Alcohol exposure during development also impacts the cerebellum. Children with fetal alcohol spectrum disorder (FASD) show many symptoms associated specifically with cerebellar deficits. However, the cellular and molecular mechanisms are unclear. This special issue discusses the most recent advances in the study of mechanisms underlying alcoholinduced cerebellar deficits. The alteration in GABAA receptor-dependent neurotransmission is a potential mechanism for ethanol-induced cerebellar dysfunction. Recent advances indicate ethanol-induced increases in GABA release are not only in Purkinje cells (PCs), but also in molecular layer interneurons and granule cells. Ethanol is shown to disrupt the molecular events at the mossy fiber - granule cell - Golgi cell (MGG) synaptic site and granule cell parallel fibers - PCs (GPP) synaptic site, which may be responsible for ethanol-induced cerebellar ataxia. Aging and ethanol may affect the smooth endoplasmic reticulum (SER) of PC dendrites and cause dendritic regression. Ethanol withdrawal causes mitochondrial damage and aberrant gene modifications in the cerebellum. The interaction between these events may result in neuronal degeneration, thereby contributing to motoric deficit. Ethanol activates doublestranded RNA (dsRNA)-activated protein kinase (PKR) and PKR activation is involved ethanolinduced neuroinflammation and neurotoxicity in the developing cerebellum. Ethanol alters the development of cerebellar circuitry following the loss of PCs, which could result in modifications of the structure and function of other brain regions that receive cerebellar inputs. Lastly, choline, an essential nutrient is evaluated for its potential protection against ethanol-induced cerebellar damages. Choline is shown to ameliorate ethanol-induced cerebellar dysfunction when given before ethanol exposure.

Trajectory Analysis Unveils Reelin's Role in the Directed Migration of Granule Cells in the Dentate Gyrus.

PubMed

Wang, Shaobo; Brunne, Bianka; Zhao, Shanting; Chai, Xuejun; Li, Jiawei; Lau, Jeremie; Failla, Antonio Virgilio; Zobiak, Bernd; Sibbe, Mirjam; Westbrook, Gary L; Lutz, David; Frotscher, Michael

2018-01-03

Reelin controls neuronal migration and layer formation. Previous studies in reeler mice deficient in Reelin focused on the result of the developmental process in fixed tissue sections. It has remained unclear whether Reelin affects the migratory process, migration directionality, or migrating neurons guided by the radial glial scaffold. Moreover, Reelin has been regarded as an attractive signal because newly generated neurons migrate toward the Reelin-containing marginal zone. Conversely, Reelin might be a stop signal because migrating neurons in reeler , but not in wild-type mice, invade the marginal zone. Here, we monitored the migration of newly generated proopiomelanocortin-EGFP -expressing dentate granule cells in slice cultures from reeler , reeler -like mutants and wild-type mice of either sex using real-time microscopy. We discovered that not the actual migratory process and migratory speed, but migration directionality of the granule cells is controlled by Reelin. While wild-type granule cells migrated toward the marginal zone of the dentate gyrus, neurons in cultures from reeler and reeler -like mutants migrated randomly in all directions as revealed by vector analyses of migratory trajectories. Moreover, live imaging of granule cells in reeler slices cocultured to wild-type dentate gyrus showed that the reeler neurons changed their directions and migrated toward the Reelin-containing marginal zone of the wild-type culture, thus forming a compact granule cell layer. In contrast, directed migration was not observed when Reelin was ubiquitously present in the medium of reeler slices. These results indicate that topographically administered Reelin controls the formation of a granule cell layer. SIGNIFICANCE STATEMENT Neuronal migration and the various factors controlling its onset, speed, directionality, and arrest are poorly understood. Slice cultures offer a unique model to study the migration of individual neurons in an almost natural environment. In the

Abnormal nuclear envelope in the cerebellar Purkinje cells and impaired motor learning in DYT11 myoclonus-dystonia mouse models.

PubMed

Yokoi, Fumiaki; Dang, Mai T; Yang, Guang; Li, Jindong; Doroodchi, Atbin; Zhou, Tong; Li, Yuqing

2012-02-01

Myoclonus-dystonia (M-D) is a movement disorder characterized by myoclonic jerks with dystonia. DYT11 M-D is caused by mutations in SGCE which codes for ɛ-sarcoglycan. SGCE is maternally imprinted and paternally expressed. Abnormal nuclear envelope has been reported in mouse models of DYT1 generalized torsion dystonia. However, it is not known whether similar alterations occur in DYT11 M-D. We developed a mouse model of DYT11 M-D using paternally inherited Sgce heterozygous knockout (Sgce KO) mice and reported that they had myoclonus and motor coordination and learning deficits in the beam-walking test. However, the specific brain regions that contribute to these phenotypes have not been identified. Since ɛ-sarcoglycan is highly expressed in the cerebellar Purkinje cells, here we examined the nuclear envelope in these cells using a transmission electron microscope and found that they are abnormal in Sgce KO mice. Our results put DYT11 M-D in a growing family of nuclear envelopathies. To analyze the effect of loss of ɛ-sarcoglycan function in the cerebellar Purkinje cells, we produced paternally inherited cerebellar Purkinje cell-specific Sgce conditional knockout (Sgce pKO) mice. Sgce pKO mice showed motor learning deficits, while they did not show abnormal nuclear envelope in the cerebellar Purkinje cells, robust motor deficits, or myoclonus. The results suggest that ɛ-sarcoglycan in the cerebellar Purkinje cells contributes to the motor learning, while loss of ɛ-sarcoglycan in other brain regions may contribute to nuclear envelope abnormality, myoclonus and motor coordination deficits. Copyright © 2011 Elsevier B.V. All rights reserved.

3D Culture for Self-Formation of the Cerebellum from Human Pluripotent Stem Cells Through Induction of the Isthmic Organizer.

PubMed

Muguruma, Keiko

2017-01-01

Pluripotent stem cells (PSCs) possess self-organizing abilities in 3D culture. This property has been demonstrated in recent studies, including the generation of various neuroectodermal and endodermal tissues. For example, PSCs are able to differentiate into specific type of neural tissues, such as the neocortex and the optic cup, in response to local positional information brought about by signals during embryogenesis. In contrast, the generation of cerebellar tissue from PSCs requires a secondary induction by a signaling center, called the isthmic organizer, which first appears in the cell aggregate in 3D culture. Such developmental complexity of cerebellum has hampered establishment of effective differentiation culture system from PSCs, thus far.We recently reported that cerebellar neurons are generated from human PSCs (hPSCs). In this chapter, we describe an efficient protocol for differentiation of 3D cerebellar neuroepithelium from hPSCs. We also describe the protocols for further differentiation into specific neurons in the cerebellar cortex, such as Purkinje cells and the granule cells.

Short-term block of Na+/K+-ATPase in neuro-glial cell cultures of cerebellum induces glutamate dependent damage of granule cells.

PubMed

Stelmashook, E V; Weih, M; Zorov, D; Victorov, I; Dirnagl, U; Isaev, N

1999-07-30

Granule cells in a dissociated neuro-glial cell culture of cerebellum when exposed to ouabain (10(-3) M) for 25 min apparently swell, increase their [Ca2+]i with obvious depolarization of the mitochondrial membrane. In 3 h after ouabain was omitted from the solution, 62 +/- 3% of granule cells had pycnotic nuclei. The supplement of a solution with competitive specific antagonist of NMDA receptors, L-2-amino-7-phosphonoheptanoate (10(-4) M, APH) together with ouabain prevented cells from swelling, mitochondrial deenergization, neuronal death and increase of [Ca2+]i. These data suggest that cellular Na+/K+-ATPase inactivation in neuro-glial cell cultures of cerebellum leads to glutamate (Glu) accumulation, hyperstimulation of glutamate receptors, higher Ca2+ and Na+ influxes into the cells through the channels activated by Glu. This process leads to cell swelling, mitochondrial deenergization and death of granule cells. Possibly, the decrease of Na+/K+-ATPase activity in brain cells can lead to the onset of at least some chronic neurological disorders.

Three-dimensional ultrastructural analyses of anterior pituitary gland expose spatial relationships between endocrine cell secretory granule localization and capillary distribution.

PubMed

Yoshitomi, Munetake; Ohta, Keisuke; Kanazawa, Tomonoshin; Togo, Akinobu; Hirashima, Shingo; Uemura, Kei-Ichiro; Okayama, Satoko; Morioka, Motohiro; Nakamura, Kei-Ichiro

2016-10-31

Endocrine and endothelial cells of the anterior pituitary gland frequently make close appositions or contacts, and the secretory granules of each endocrine cell tend to accumulate at the perivascular regions, which is generally considered to facilitate secretory functions of these cells. However, three-dimensional relationships between the localization pattern of secretory granules and blood vessels are not fully understood. To define and characterize these spatial relationships, we used scanning electron microscopy (SEM) three-dimensional reconstruction method based on focused ion-beam slicing and scanning electron microscopy (FIB/SEM). Full three-dimensional cellular architectures of the anterior pituitary tissue at ultrastructural resolution revealed that about 70% of endocrine cells were in apposition to the endothelial cells, while almost 30% of endocrine cells were entirely isolated from perivascular space in the tissue. Our three-dimensional analyses also visualized the distribution pattern of secretory granules in individual endocrine cells, showing an accumulation of secretory granules in regions in close apposition to the blood vessels in many cases. However, secretory granules in cells isolated from the perivascular region tended to distribute uniformly in the cytoplasm of these cells. These data suggest that the cellular interactions between the endocrine and endothelial cells promote an uneven cytoplasmic distribution of the secretory granules.

Imaging exocytosis of single glucagon-like peptide-1 containing granules in a murine enteroendocrine cell line with total internal reflection fluorescent microscopy

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

Ohara-Imaizumi, Mica; Aoyagi, Kyota; Akimoto, Yoshihiro

To analyze the exocytosis of glucagon-like peptide-1 (GLP-1) granules, we imaged the motion of GLP-1 granules labeled with enhanced yellow fluorescent protein (Venus) fused to human growth hormone (hGH-Venus) in an enteroendocrine cell line, STC-1 cells, by total internal reflection fluorescent (TIRF) microscopy. We found glucose stimulation caused biphasic GLP-1 granule exocytosis: during the first phase, fusion events occurred from two types of granules (previously docked granules and newcomers), and thereafter continuous fusion was observed mostly from newcomers during the second phase. Closely similar to the insulin granule fusion from pancreatic {beta} cells, the regulated biphasic exocytosis from two typesmore » of granules may be a common mechanism in glucose-evoked hormone release from endocrine cells.« less

Cerebellar microfolia and other abnormalities of neuronal growth, migration, and lamination in the Pit1dw-J homozygote mutant mouse

NASA Technical Reports Server (NTRS)

Sekiguchi, M.; Abe, H.; Moriya, M.; Tanaka, O.; Nowakowski, R. S.

1998-01-01

The Snell dwarf mouse (Pit1dw-J homozygote) has a mutation in the Pit1 gene that prevents the normal formation of the anterior pituitary. In neonates and adults there is almost complete absence of growth hormone (GH), prolactin (PRL), thyroxin (T4), and thyroid-stimulating hormone (TSH). Since these hormones have been suggested to play a role in normal development of the central nervous system (CNS), we have investigated the effects of the Pit1dw-J mutation on the cerebellum and hippocampal formation. In the cerebellum, there were abnormalities of both foliation and lamination. The major foliation anomalies were 1) changes in the relative size of specific folia and also the proportional sizes of the anterior vs posterior cerebellum; and 2) the presence of between one and three microfolia per half cerebellum. The microfolia were all in the medial portion of the hemisphere in the caudal part of the cerebellum. Each microfolium was just rostral to a normal fissure and interposed between the fissure and a normal gyrus. Lamination abnormalities included an increase in the number of single ectopic granule cells in the molecular layer in both cerebellar vermis (86%) and hemisphere (40%) in comparison with the wild-type mouse. In the hippocampus of the Pit1dw-J homozygote mouse, the number of pyramidal cells was decreased, although the width of the pyramidal cell layer throughout areas CA1-CA3 appeared to be normal, but less densely populated than in the wild-type mouse. Moreover, the number of granule cells that form the granule cell layer was decreased from the wild-type mouse and some ectopic granule cells (occurring both as single cells and as small clusters) were observed in the innermost portion of the molecular layer. The abnormalities observed in the Pit1dw-J homozygote mouse seem to be caused by both direct and indirect effects of the deficiency of TSH (or T4), PRL, or GH rather than by a direct effect of the deletion of Pit1.

Altered cerebellar development in nuclear receptor TAK1/ TR4 null mice is associated with deficits in GLAST(+) glia, alterations in social behavior, motor learning, startle reactivity, and microglia.

PubMed

Kim, Yong-Sik; Harry, G Jean; Kang, Hong Soon; Goulding, David; Wine, Rob N; Kissling, Grace E; Liao, Grace; Jetten, Anton M

2010-09-01

Previously, deficiency in the expression of the nuclear orphan receptor TAK1 was found to be associated with delayed cerebellar granule cell migration and Purkinje cell maturation with a permanent deficit in foliation of lobules VI–VII, suggesting a role for TAK1 in cerebellum development. In this study, we confirm that TAK1-deficient (TAK1(−/−)) mice have a smaller cerebellum and exhibit a disruption of lobules VI–VII. We extended these studies and show that at postnatal day 7, TAK1(−/−) mice exhibit a delay in monolayer maturation of dysmorphic calbindin 28K-positive Purkinje cells. The astrocyte-specific glutamate transporter (GLAST) was expressed within Bergmann fibers and internal granule cell layer at significantly lower levels in the cerebellum of TAK1(−/−) mice. At PND21, Golgi-positive Purkinje cells in TAK1(−/−) mice displayed a smaller soma (18%) and shorter distance to first branch point (35%). Neuronal death was not observed in TAK1(−/−) mice at PND21; however, activated microglia were present in the cerebellum, suggestive of earlier cell death. These structural deficits in the cerebellum were not sufficient to alter motor strength, coordination, or activity levels; however, deficits in acoustic startle response, prepulse startle inhibition, and social interactions were observed. Reactions to a novel environment were inhibited in a light/dark chamber, open-field, and home-cage running wheel. TAK1(−/−) mice displayed a plateau in performance on the running wheel, suggesting a deficit in learning to coordinate performance on a motor task. These data indicate that TAK1 is an important transcriptional modulator of cerebellar development and neurodevelopmentally regulated behavior.

Bax deficiency prolongs cerebellar neurogenesis, accelerates medulloblastoma formation and paradoxically increases both malignancy and differentiation.

PubMed

Garcia, I; Crowther, A J; Gama, V; Miller, C R; Miller, C Ryan; Deshmukh, M; Gershon, T R

2013-05-02

Neurogenesis requires negative regulation through differentiation of progenitors or their programmed cell death (PCD). Growth regulation is particularly important in the postnatal cerebellum, where excessive progenitor proliferation promotes medulloblastoma, the most common malignant brain tumor in children. We present evidence that PCD operates alongside differentiation to regulate cerebellar granule neuron progenitors (CGNPs) and to prevent medulloblastoma. Here, we show that genetic deletion of pro-apoptotic Bax disrupts regulation of cerebellar neurogenesis and promotes medulloblastoma formation. In Bax(-/-) mice, the period of neurogenesis was extended into the third week of postnatal life, and ectopic neurons and progenitors collected in the molecular layer of the cerebellum and adjacent tectum. Importantly, genetic deletion of Bax in medulloblastoma-prone ND2:SmoA1 transgenic mice greatly accelerated tumorigenesis. Bax-deficient medulloblastomas exhibited strikingly distinct pathology, with reduced apoptosis, increased neural differentiation and tectal migration. Comparing Bax(+/+) and Bax(-/-) medulloblastomas, we were able to identify upregulation of Bcl-2 and nuclear exclusion of p27 as tumorigenic changes that are required to mitigate the tumor suppressive effect of Bax. Studies on human tumors confirmed the importance of modulating Bax in medulloblastoma pathogenesis. Our results demonstrate that Bax-dependent apoptosis regulates postnatal cerebellar neurogenesis, suppresses medulloblastoma formation and imposes selective pressure on tumors that form. Functional resistance to Bax-mediated apoptosis, required for medulloblastoma tumorigenesis, may be a tumor-specific vulnerability to be exploited for therapeutic benefit.

Host cell subversion by Toxoplasma GRA16, an exported dense granule protein that targets the host cell nucleus and alters gene expression.

PubMed

Bougdour, Alexandre; Durandau, Eric; Brenier-Pinchart, Marie-Pierre; Ortet, Philippe; Barakat, Mohamed; Kieffer, Sylvie; Curt-Varesano, Aurélie; Curt-Bertini, Rose-Laurence; Bastien, Olivier; Coute, Yohann; Pelloux, Hervé; Hakimi, Mohamed-Ali

2013-04-17

After invading host cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the host cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the host cell nucleus, where it positively modulates genes involved in cell-cycle progression and the p53 tumor suppressor pathway. GRA16 binds two host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate host function. Copyright © 2013 Elsevier Inc. All rights reserved.

The Stress Granule RNA-Binding Protein TIAR-1 Protects Female Germ Cells from Heat Shock in Caenorhabditis elegans.

PubMed

Huelgas-Morales, Gabriela; Silva-García, Carlos Giovanni; Salinas, Laura S; Greenstein, David; Navarro, Rosa E

2016-04-07

In response to stressful conditions, eukaryotic cells launch an arsenal of regulatory programs to protect the proteome. One major protective response involves the arrest of protein translation and the formation of stress granules, cytoplasmic ribonucleoprotein complexes containing the conserved RNA-binding proteins TIA-1 and TIAR. The stress granule response is thought to preserve mRNA for translation when conditions improve. For cells of the germline-the immortal cell lineage required for sexual reproduction-protection from stress is critically important for perpetuation of the species, yet how stress granule regulatory mechanisms are deployed in animal reproduction is incompletely understood. Here, we show that the stress granule protein TIAR-1 protects the Caenorhabditis elegans germline from the adverse effects of heat shock. Animals containing strong loss-of-function mutations in tiar-1 exhibit significantly reduced fertility compared to the wild type following heat shock. Analysis of a heat-shock protein promoter indicates that tiar-1 mutants display an impaired heat-shock response. We observed that TIAR-1 was associated with granules in the gonad core and oocytes during several stressful conditions. Both gonad core and oocyte granules are dynamic structures that depend on translation; protein synthesis inhibitors altered their formation. Nonetheless, tiar-1 was required for the formation of gonad core granules only. Interestingly, the gonad core granules did not seem to be needed for the germ cells to develop viable embryos after heat shock. This suggests that TIAR-1 is able to protect the germline from heat stress independently of these structures. Copyright © 2016 Huelgas-Morales et al.

The Stress Granule RNA-Binding Protein TIAR-1 Protects Female Germ Cells from Heat Shock in Caenorhabditis elegans

PubMed Central

Huelgas-Morales, Gabriela; Silva-García, Carlos Giovanni; Salinas, Laura S.; Greenstein, David; Navarro, Rosa E.

2016-01-01

In response to stressful conditions, eukaryotic cells launch an arsenal of regulatory programs to protect the proteome. One major protective response involves the arrest of protein translation and the formation of stress granules, cytoplasmic ribonucleoprotein complexes containing the conserved RNA-binding proteins TIA-1 and TIAR. The stress granule response is thought to preserve mRNA for translation when conditions improve. For cells of the germline—the immortal cell lineage required for sexual reproduction—protection from stress is critically important for perpetuation of the species, yet how stress granule regulatory mechanisms are deployed in animal reproduction is incompletely understood. Here, we show that the stress granule protein TIAR-1 protects the Caenorhabditis elegans germline from the adverse effects of heat shock. Animals containing strong loss-of-function mutations in tiar-1 exhibit significantly reduced fertility compared to the wild type following heat shock. Analysis of a heat-shock protein promoter indicates that tiar-1 mutants display an impaired heat-shock response. We observed that TIAR-1 was associated with granules in the gonad core and oocytes during several stressful conditions. Both gonad core and oocyte granules are dynamic structures that depend on translation; protein synthesis inhibitors altered their formation. Nonetheless, tiar-1 was required for the formation of gonad core granules only. Interestingly, the gonad core granules did not seem to be needed for the germ cells to develop viable embryos after heat shock. This suggests that TIAR-1 is able to protect the germline from heat stress independently of these structures. PMID:26865701

Bisphenol A influences oestrogen- and thyroid hormone-regulated thyroid hormone receptor expression in rat cerebellar cell culture.

PubMed

Somogyi, Virág; Horváth, Tamás L; Tóth, István; Bartha, Tibor; Frenyó, László Vilmos; Kiss, Dávid Sándor; Jócsák, Gergely; Kerti, Annamária; Naftolin, Frederick; Zsarnovszky, Attila

2016-12-01

Thyroid hormones (THs) and oestrogens are crucial in the regulation of cerebellar development. TH receptors (TRs) mediate these hormone effects and are regulated by both hormone families. We reported earlier that THs and oestradiol (E 2 ) determine TR levels in cerebellar cell culture. Here we demonstrate the effects of low concentrations (10 -10 M) of the endocrine disruptor (ED) bisphenol A (BPA) on the hormonal (THs, E 2 ) regulation of TRα,β in rat cerebellar cell culture. Primary cerebellar cell cultures, glia-containing and glia-destroyed, were treated with BPA or a combination of BPA and E 2 and/or THs. Oestrogen receptor and TH receptor mRNA and protein levels were determined by real-time qPCR and Western blot techniques. The results show that BPA alone decreases, while BPA in combination with THs and/or E 2 increases TR mRNA expression. In contrast, BPA alone increased receptor protein expressions, but did not further increase them in combination with THs and/or E 2 . The modulatory effects of BPA were mediated by the glia; however, the degree of changes also depended on the specific hormone ligand used. The results signify the importance of the regulatory mechanisms interposed between transcription and translation and raise the possibility that BPA could act to influence nuclear hormone receptor levels independently of ligand-receptor interaction.

Common pediatric cerebellar tumors: correlation between cell densities and apparent diffusion coefficient metrics.

PubMed

Koral, Korgün; Mathis, Derek; Gimi, Barjor; Gargan, Lynn; Weprin, Bradley; Bowers, Daniel C; Margraf, Linda

2013-08-01

To test whether there is correlation between cell densities and apparent diffusion coefficient (ADC) metrics of common pediatric cerebellar tumors. This study was reviewed for issues of patient safety and confidentiality and was approved by the Institutional Review Board of the University of Texas Southwestern Medical Center and was compliant with HIPAA. The need for informed consent was waived. Ninety-five patients who had preoperative magnetic resonance imaging and surgical pathologic findings available between January 2003 and June 2011 were included. There were 37 pilocytic astrocytomas, 34 medulloblastomas (23 classic, eight desmoplastic-nodular, two large cell, one anaplastic), 17 ependymomas (13 World Health Organization [WHO] grade II, four WHO grade III), and seven atypical teratoid rhabdoid tumors. ADCs of solid tumor components and normal cerebellum were measured. Tumor-to-normal brain ADC ratios (hereafter, ADC ratio) were calculated. The medulloblastomas and ependymomas were subcategorized according to the latest WHO classification, and tumor cellularity was calculated. Correlation was sought between cell densities and mean tumor ADCs, minimum tumor ADCs, and ADC ratio. When all tumors were considered together, negative correlation was found between cellularity and mean tumor ADCs (ρ = -0.737, P < .05) and minimum tumor ADCs (ρ = -0.736, P < .05) of common pediatric cerebellar tumors. There was no correlation between cellularity and ADC ratio. Negative correlation was found between cellularity and minimum tumor ADC in atypical teratoid rhabdoid tumors (ρ = -0.786, P < .05). In atypical teratoid rhabdoid tumors, no correlation was found between cellularity and mean tumor ADC and ADC ratio. There was no correlation between the ADC metrics and cellularity of the pilocytic astrocytomas, medulloblastomas, and ependymomas. Negative correlation was found between cellularity and ADC metrics of common pediatric cerebellar tumors. Although ADC metrics are

Intracisternal granules in the adipokinetic cells of locusts are not degraded and apparently function as supplementary stores of secretory material.

PubMed

Harthoorn, L F; Diederen, J H; Oudejans, R C; Verstegen, M M; Vullings, H G; Van der Horst, D J

2000-01-01

The intracisternal granules in locust adipokinetic cells appear to represent accumulations of secretory material within cisternae of the rough endoplasmic reticulum. An important question is whether these granules are destined for degradation or represent stores of (pro)hormones. Two strategies were used to answer this question. First, cytochemistry was applied to elucidate the properties of intracisternal granules. The endocytic tracers horseradish peroxidase and wheat-germ agglutinin-conjugated horseradish peroxidase were used to facilitate the identification of endocytic, autophagic, and lysosomal organelles, which may be involved in the degradation of intracisternal granules. No intracisternal granules could be found within autophagosomes, and granules fused with endocytic and lysosomal organelles were not observed, nor could tracer be found within the granules. The lysosomal enzyme acid phosphatase was absent from the granules. Second, biochemical analysis of the content of intracisternal granules revealed that these granules contain prohormones as well as hormones. Prohormones were present in relatively higher amounts compared with ordinary secretory granules. Since the intracisternal granules in locust adipokinetic cells are not degraded and contain intact (pro)hormones it is concluded that they function as supplementary stores of secretory material.

Ca2+-dependent dephosphorylation of kinesin heavy chain on beta-granules in pancreatic beta-cells. Implications for regulated beta-granule transport and insulin exocytosis

NASA Technical Reports Server (NTRS)

Donelan, Matthew J.; Morfini, Gerardo; Julyan, Richard; Sommers, Scott; Hays, Lori; Kajio, Hiroshi; Briaud, Isabelle; Easom, Richard A.; Molkentin, Jeffery D.; Brady, Scott T.;

Information processing in the hemisphere of the cerebellar cortex for control of wrist movement

PubMed Central

Tomatsu, Saeka; Ishikawa, Takahiro; Tsunoda, Yoshiaki; Lee, Jongho; Hoffman, Donna S.

2015-01-01

A region of cerebellar lobules V and VI makes strong loop connections with the primary motor (M1) and premotor (PM) cortical areas and is assumed to play essential roles in limb motor control. To examine its functional role, we compared the activities of its input, intermediate, and output elements, i.e., mossy fibers (MFs), Golgi cells (GoCs), and Purkinje cells (PCs), in three monkeys performing wrist movements in two different forearm postures. The results revealed distinct steps of information processing. First, MF activities displayed temporal and directional properties that were remarkably similar to those of M1/PM neurons, suggesting that MFs relay near copies of outputs from these motor areas. Second, all GoCs had a stereotyped pattern of activity independent of movement direction or forearm posture. Instead, GoC activity resembled an average of all MF activities. Therefore, inhibitory GoCs appear to provide a filtering function that passes only prominently modulated MF inputs to granule cells. Third, PCs displayed highly complex spatiotemporal patterns of activity, with coordinate frames distinct from those of MF inputs and directional tuning that changed abruptly before movement onset. The complexity of PC activities may reflect rapidly changing properties of the peripheral motor apparatus during movement. Overall, the cerebellar cortex appears to transform a representation of outputs from M1/PM into different movement representations in a posture-dependent manner and could work as part of a forward model that predicts the state of the peripheral motor apparatus. PMID:26467515

A novel two-chain IGF-II-derived peptide from purified β-cell granules.

PubMed

Buchanan, Christina M; Phillips, Anthony R J; Cooper, Garth J S

2010-10-01

Insulin-like growth factor II (IGF-II) is a potent mitogen that regulates prenatal growth and development in both humans and rodents. Its role in post-natal life is less clear although immunohistochemical studies have observed IGF-II-like immunoreactivity (IGF-II-LI) associated with insulin-producing pancreatic β-cells. Here we isolated secretory granules from a β-cell line, βTC6-F7, and characterized the nature of the IGF-II-LI located therein. Secretory granules were isolated from cultured mouse βTC6-F7 cells by ultracentrifugation. Granule protein content was separated by reversed-phase HPLC, and assayed for IGF-II (radioimmunoassay) prior to identification by gas-phase NH(2)-terminal sequencing and MALDI-TOF MS. Effects of glucose incorporation into muscle glycogen were determined by incubating with isolated rat soleus muscle strips. βTC6-F7 cells contained 60 ± 8 pmol of IGF-II-LI per 10⁶ cells compared to 340 ± 44 pmol insulin-LI per 10⁶ cells. IGF-II immunoreactive fractions were found to contain an IGF-II-like molecule with a molecular mass of 6847.6 Da. The protein was found to be a two-chain insulin-like product of Igf2 that corresponds to mouse des(37-40)IGF-II, which we termed 'vesiculin'. This molecule was also detectable in βTC6-F7 cells by intact-cell mass spectrometry. Mouse vesiculin evoked concentration-dependent stimulation of muscle glycogen synthesis ex vivo with an EC(50) value of 131 nM ± 1.35. Vesiculin, des(37-40)IGF-II, is a novel two-chain insulin-like hormone and the major "IGF-II-like" peptide found in purified mouse βTC6-F7 secretory granules. It stimulated ex vivo muscle glycogen synthesis with an efficacy greater than or equal to the intrinsic potency of IGF-II when compared to insulin derived from the same species. Copyright © 2010 Growth Hormone Research Society. Published by Elsevier Ltd. All rights reserved.

PLC-dependent intracellular Ca2+ release was associated with C6-ceramide-induced inhibition of Na+ current in rat granule cells.

PubMed

Liu, Zheng; Fei, Xiao-Wei; Fang, Yan-Jia; Shi, Wen-Jie; Zhang, Yu-Qiu; Mei, Yan-Ai

2008-09-01

In this report, the effects of C(6)-ceramide on the voltage-gated inward Na(+) currents (I(Na)), two types of main K(+) current [outward rectifier delayed K(+) current (I(K)) and outward transient K(+) current (I(A))], and cell death in cultured rat cerebellar granule cells were investigated. At concentrations of 0.01-100 microM, ceramide produced a dose-dependent and reversible inhibition of I(Na) without alteration of the steady-state activation and inactivation properties. Treatment with C(2)-ceramide caused a similar inhibitory effect on I(Na). However, dihydro-C(6)-ceramide failed to modulate I(Na). The effect of C(6)-ceramide on I(Na) was abolished by intracellular infusion of the Ca(2+)-chelating agent, 1,2-bis (2-aminophenoxy) ethane-N, N, N9, N9-tetraacetic acid, but was mimicked by application of caffeine. Blocking the release of Ca(2+) from the sarcoplasmic reticulum with ryanodine receptor blocker induced a gradual increase in I(Na) amplitude and eliminated the effect of ceramide on I(Na). In contrast, the blocker of the inositol 1,4,5-trisphosphate-sensitive Ca(2+) receptor did not affect the action of C(6)-ceramide. Intracellular application of GTPgammaS also induced a gradual decrease in I(Na) amplitude, while GDPbetaS eliminated the effect of C(6)-ceramide on I(Na). Furthermore, the C(6)-ceramide effect on I(Na) was abolished after application of the phospholipase C (PLC) blockers and was greatly reduced by the calmodulin inhibitors. Fluorescence staining showed that C(6)-ceramide decreased cell viability and blocking I(Na) by tetrodotoxin did not mimic the effect of C(6)-ceramide, and inhibiting intracellular Ca(2+) release by dantrolene could not decrease the C(6)-ceramide-induced cell death. We therefore suggest that increased PLC-dependent Ca(2+) release through the ryanodine-sensitive Ca(2+) receptor may be responsible for the C(6)-ceramide-induced inhibition of I(Na), which does not seem to be associated with C(6)-ceramide-induced granule

Ectopic expression of syncollin in INS-1 beta-cells sorts it into granules and impairs regulated secretion.

PubMed

Li, Jingsong; Luo, Ruihua; Hooi, Shing Chuan; Ruga, Pilar; Zhang, Jiping; Meda, Paolo; Li, GuoDong

2005-03-22

Syncollin was first demonstrated to be a protein capable of affecting granule fusion in a cell-free system, but later studies revealed its luminal localization in zymogen granules. To determine its possible role in exocytosis in the intact cell, syncollin and a truncated form of the protein (lacking the N-terminal hydrophobic domain) were stably transfected in insulin-secreting INS-1 cells since these well-studied exocytotic cells appear not to express the protein per se. Studies by subcellular fractionation analysis, double immunofluorescence staining, and electron microscopy examination revealed that transfection of syncollin produced strong signals in the insulin secretory granules, whereas the product from transfecting the truncated syncollin was predominantly associated with the Golgi apparatus and to a lesser degree with the endoplasmic reticulum. The expressed products were associated with membranes and not the soluble fractions in either cytoplasm or the lumens of organelles. Importantly, insulin release stimulated by various secretagogues was severely impaired in cells expressing syncollin, but not affected by expressing truncated syncollin. Transfection of syncollin appeared not to impede insulin biosynthesis and processing, since cellular contents of proinsulin and insulin and the number of secretory granules were not altered. In addition, the early signals (membrane depolarization and Ca(2+) responses) for regulated insulin secretion were unaffected. These findings indicate that syncollin may be targeted to insulin secretory granules specifically and impair regulated secretion at a distal stage.

Apoptosis of Purkinje and granular cells of the cerebellum following chronic ethanol intake.

PubMed

Oliveira, Suelen A; Chuffa, Luiz Gustavo A; Fioruci-Fontanelli, Beatriz Aparecida; Lizarte Neto, Fermino Sanches; Novais, Paulo Cezar; Tirapelli, Luiz Fernando; Oishi, Jorge Camargo; Takase, Luiz Fernando; Stefanini, Maira Aparecida; Martinez, Marcelo; Martinez, Francisco Eduardo

2014-12-01

Ethanol alters motricity, learning, cognition, and cellular metabolism in the cerebellum. We evaluated the effect of ethanol on apoptosis in Golgi, Purkinje, and granule cells of the cerebellum in adult rats. There were two groups of 20 rats: a control group that did not consume ethanol and an experimental group of UChA rats that consumed ethanol at 10% (<2 g ethanol/kg body weight/day). At 120 days old, rats were anesthetized and decapitated, and their cerebella were collected and fixed. Cerebellar sections were subjected to immunohistochemistry for terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL), caspase-3, X-linked inhibitor of apoptosis protein (XIAP), and insulin-like growth factor 1-receptor (IGF-1R); real-time PCR (RT-PCR) to determine caspase-3, XIAP, and IGF-1R gene expression; and transmission electron microscopy (TEM). We identified fragmentation of DNA and an increase in caspase-3 protein and XIAP in Purkinje cells, whereas granule cells exhibited increased caspase-3 and XIAP. IGF-1R expression was unchanged. There was no significant difference in gene expression of caspase-3, XIAP, and IGF-1R. There were an increase in lipid droplets, a reduction in the cellular cytoplasm in electron-dense nuclei, and changes in the myelin sheath in the cerebellar cortex. In conclusion, our data demonstrated that ethanol induced apoptosis in the Purkinje and granule cells of the cerebellum of adult UChA rats.

Induced pluripotent stem cell technology for modelling and therapy of cerebellar ataxia

PubMed Central

Watson, Lauren M.; Wong, Maggie M. K.; Becker, Esther B. E.

2015-01-01

Induced pluripotent stem cell (iPSC) technology has emerged as an important tool in understanding, and potentially reversing, disease pathology. This is particularly true in the case of neurodegenerative diseases, in which the affected cell types are not readily accessible for study. Since the first descriptions of iPSC-based disease modelling, considerable advances have been made in understanding the aetiology and progression of a diverse array of neurodegenerative conditions, including Parkinson's disease and Alzheimer's disease. To date, however, relatively few studies have succeeded in using iPSCs to model the neurodegeneration observed in cerebellar ataxia. Given the distinct neurodevelopmental phenotypes associated with certain types of ataxia, iPSC-based models are likely to provide significant insights, not only into disease progression, but also to the development of early-intervention therapies. In this review, we describe the existing iPSC-based disease models of this heterogeneous group of conditions and explore the challenges associated with generating cerebellar neurons from iPSCs, which have thus far hindered the expansion of this research. PMID:26136256

Cell-type-specific expression of NFIX in the developing and adult cerebellum.

PubMed

Fraser, James; Essebier, Alexandra; Gronostajski, Richard M; Boden, Mikael; Wainwright, Brandon J; Harvey, Tracey J; Piper, Michael

2017-07-01

Transcription factors from the nuclear factor one (NFI) family have been shown to play a central role in regulating neural progenitor cell differentiation within the embryonic and post-natal brain. NFIA and NFIB, for instance, promote the differentiation and functional maturation of granule neurons within the cerebellum. Mice lacking Nfix exhibit delays in the development of neuronal and glial lineages within the cerebellum, but the cell-type-specific expression of this transcription factor remains undefined. Here, we examined the expression of NFIX, together with various cell-type-specific markers, within the developing and adult cerebellum using both chromogenic immunohistochemistry and co-immunofluorescence labelling and confocal microscopy. In embryos, NFIX was expressed by progenitor cells within the rhombic lip and ventricular zone. After birth, progenitor cells within the external granule layer, as well as migrating and mature granule neurons, expressed NFIX. Within the adult cerebellum, NFIX displayed a broad expression profile, and was evident within granule cells, Bergmann glia, and interneurons, but not within Purkinje neurons. Furthermore, transcriptomic profiling of cerebellar granule neuron progenitor cells showed that multiple splice variants of Nfix are expressed within this germinal zone of the post-natal brain. Collectively, these data suggest that NFIX plays a role in regulating progenitor cell biology within the embryonic and post-natal cerebellum, as well as an ongoing role within multiple neuronal and glial populations within the adult cerebellum.

P2X7, NMDA and BDNF receptors converge on GSK3 phosphorylation and cooperate to promote survival in cerebellar granule neurons.

PubMed

Ortega, Felipe; Pérez-Sen, Raquel; Morente, Verónica; Delicado, Esmerilda G; Miras-Portugal, Maria Teresa

2010-05-01

Glycogen synthase kinase-3 (GSK3) is a key player in the regulation of neuronal survival. Herein, we report evidence of an interaction between P2X7 receptors with NMDA and BDNF receptors at the level of GSK3 signalling and neuroprotection. The activation of these receptors in granule neurons led to a sustained pattern of GSK3 phosphorylation that was mainly PKC-dependent. BDNF was the most potent at inducing GSK3 phosphorylation, which was also dependent on PI3K. The P2X7 agonist, BzATP, exhibited additive effects with both NMDA and BDNF to rescue granule neurons from cell death induced by PI3K inhibition. This survival effect was mediated by the PKC-dependent GSK3 pathway. In addition, ERK1/2 proteins were also involved in BDNF protective effect. These results show the function of ATP in amplifying neuroprotective actions of glutamate and neurotrophins, and support the role of GSK3 as an important convergence point for these survival promoting factors in granule neurons.

Functional recovery after cerebellar damage is related to GAP-43-mediated reactive responses of pre-cerebellar and deep cerebellar nuclei.

PubMed

Burello, Lorena; De Bartolo, Paola; Gelfo, Francesca; Foti, Francesca; Angelucci, Francesco; Petrosini, Laura

2012-01-01

Since brain injuries in adulthood are a leading cause of long-term disabilities, the development of rehabilitative strategies able to impact on functional outcomes requires detailing adaptive neurobiological responses. Functional recovery following brain insult is mainly ascribed to brain neuroplastic properties although the close linkage between neuronal plasticity and functional recovery is not yet fully clarified. The present study analyzed the reactive responses of pre-cerebellar (inferior olive, lateral reticular nucleus and pontine nuclei) and deep cerebellar nuclei after a hemicerebellectomy, considering the great plastic potential of the cerebellar system in physiological and pathological conditions. The time course of the plastic reorganization following cerebellar lesion was investigated by monitoring the Growth Associated Protein-43 (GAP-43) immunoreactivity. The time course of recovery from cerebellar symptoms was also assessed to parallel behavioral and neurobiological parameters. A key role of GAP-43 in neuronal reactive responses was evidenced. Neurons that underwent an axotomy as consequence of the right hemicerebellectomy (neurons of left inferior olive, right lateral reticular nucleus and left pontine nuclei) exhibited enhanced GAP-43 immunoreactivity and cell death. As for the not-axotomized neurons, we found enhanced GAP-43 immunoreactivity only in right pontine nuclei projecting to the spared (left) hemicerebellum. GAP-43 levels augmented also in the three deep cerebellar nuclei of the spared hemicerebellum, indicating the ponto-cerebellar circuit as crucially involved in functional recovery. Interestingly, each nucleus showed a distinct time course in GAP-43 immunoreactivity. GAP-43 levels peaked during the first post-operative week in the fastigial and interposed nuclei and after one month in the dentate nucleus. These results suggest that the earlier plastic events of the fastigial and interposed nuclei were driving compensation of the

Ultra-structural study of insulin granules in pancreatic β-cells of db/db mouse by scanning transmission electron microscopy tomography.

PubMed

Xue, Yanhong; Zhao, Wei; Du, Wen; Zhang, Xiang; Ji, Gang; Ying, Wang; Xu, Tao

2012-07-01

Insulin granule trafficking is a key step in the secretion of glucose-stimulated insulin from pancreatic β-cells. The main feature of type 2 diabetes (T2D) is the failure of pancreatic β-cells to secrete sufficient amounts of insulin to maintain normal blood glucose levels. In this work, we developed and applied tomography based on scanning transmission electron microscopy (STEM) to image intact insulin granules in the β-cells of mouse pancreatic islets. Using three-dimensional (3D) reconstruction, we found decreases in both the number and the grey level of insulin granules in db/db mouse pancreatic β-cells. Moreover, insulin granules were closer to the plasma membrane in diabetic β-cells than in control cells. Thus, 3D ultra-structural tomography may provide new insights into the pathology of insulin secretion in T2D.

Importance of genetics in fetal alcohol effects: null mutation of the nNOS gene worsens alcohol-induced cerebellar neuronal losses and behavioral deficits

PubMed Central

Bonthius, Daniel J.; Winters, Zachary; Karacay, Bahri; Bousquet, Samantha Larimer; Bonthius, Daniel J.

2014-01-01

The cerebellum is a major target of alcohol-induced damage in the developing brain. However, the cerebella of some children are much more seriously affected than others by prenatal alcohol exposure. As a consequence of in utero alcohol exposure, some children have substantial reductions in cerebellar volume and corresponding neurodevelopmental problems, including microencephaly, ataxia, and balance deficits, while other children who were exposed to similar alcohol quantities are spared. One factor that likely plays a key role in determining the impact of alcohol on the fetal cerebellum is genetics. However, no specific gene variant has yet been identified that worsens cerebellar function as a consequence of developmental alcohol exposure. Previous studies have revealed that mice carrying a homozygous mutation of the gene for neuronal nitric oxide synthase (nNOS−/− mice) have more severe acute alcohol-induced neuronal losses from the cerebellum than wild type mice. Therefore, the goals of this study were to determine whether alcohol induces more severe cerebellum-based behavioral deficits in nNOS−/− mice than in wild type mice and to determine whether these worsened behavior deficits are associated with worsened cerebellar neuronal losses. nNOS−/− mice and their wild type controls received alcohol (0.0, 2.2, or 4.4 mg/g) daily over postnatal days 4–9. In adulthood, the mice underwent behavioral testing, followed by neuronal quantification. Alcohol caused dose-related deficits in rotarod and balance beam performance in both nNOS−/− and wild type mice. However, the alcohol-induced behavioral deficits were substantially worse in the nNOS−/− mice than in wild type. Likewise, alcohol exposure led to losses of Purkinje cells and cerebellar granule cells in mice of both genotypes, but the cell losses were more severe in the nNOS−/− mice than in wild type. Behavioral performances were correlated with neuronal number in the nNOS−/− mice, but not

Alpha-SNAP functions in insulin exocytosis from mature, but not immature secretory granules in pancreatic beta cells.

PubMed

Nakamichi, Y; Nagamatsu, S

1999-06-24

To explore alpha-SNAP function in insulin exocytosis from either immature or mature secretory granules in pancreatic beta cells, we studied the effects of overexpression of adenovirus-mediated wild-type alpha-SNAP and C-terminally deleted alpha-SNAP mutant (1-285) on newly synthesized proinsulin and insulin release by rat islets and MIN6 cells. Rat islets overexpressing alpha-SNAP and mutant alpha-SNAP were pulse-chased. Exocytosis from immature and mature insulin secretory granules was measured as fractional (%) labeled-proinsulin release immediately after the pulse-labeling and percentage labeled-insulin release after a 3-h chase period, respectively. There was no difference in percentage labeled-proinsulin release between the control and alpha-SNAP or mutant alpha-SNAP-overexpressed islets. Although percentage labeled-insulin release after a 3-h chase period was significantly increased in alpha-SNAP-overexpressed islets, it was decreased in mutant alpha-SNAP-overexpressed islets. Thus, the results demonstrated that alpha-SNAP overexpression in rat islets primarily increased exocytosis from mature, but not immature insulin secretory granules. On the other hand, in MIN6 cells, alpha-SNAP overexpression scarcely affected glucose-stimulated insulin release; therefore, we examined the effect of mutant alpha-SNAP overexpression as the dominant-negative inhibitor on the newly synthesized proinsulin/insulin release using the same protocol as in the rat islet experiments. alpha-SNAP mutant (1-285) overexpression in MIN6 cells decreased the percentage labeled insulin release from mature secretory granules, but not percentage labeled proinsulin release from immature secretory granules. Thus, our data demonstrate that alpha-SNAP functions mainly in the mature insulin secretory granules in pancreatic beta cells. Copyright 1999 Academic Press.

On the origins of the universal dynamics of endogenous granules in mammalian cells.

PubMed

Vanapalli, Siva A; Li, Yixuan; Mugele, Frieder; Duits, Michel H G

2009-12-01

Endogenous granules (EGs) that consist of lipid droplets and mitochondria have been commonly used to assess intracellular mechanical properties via multiple particle tracking microrheology (MPTM). Despite their widespread use, the nature of interaction of EGs with the cytoskeletal network and the type of forces driving their dynamics--both of which are crucial for the interpretation of the results from MPTM technique--are yet to be resolved. In this report, we study the dynamics of endogenous granules in mammalian cells using particle tracking methods. We find that the ensemble dynamics of EGs is diffusive in three types of mammalian cells (endothelial cells, smooth muscle cells and fibroblasts), thereby suggesting an apparent universality in their dynamical behavior. Moreover, in a given cell, the amplitude of the mean-squared displacement for EGs is an order of magnitude larger than that of injected particles. This observation along with results from ATP depletion and temperature intervention studies suggests that cytoskeletal active forces drive the dynamics of EGs. To elucidate the dynamical origin of the diffusive-like nonthermal motion, we consider three active force generation mechanisms--molecular motor transport, actomyosin contractility and microtubule polymerization forces. We test these mechanisms using pharmacological interventions. Experimental evidence and model calculations suggest that EGs are intimately linked to microtubules and that microtubule polymerization forces drive their dynamics. Thus, endogenous granules could serve as non-invasive probes for microtubule network dynamics in mammalian cells.

Enterovirus 71 induces anti-viral stress granule-like structures in RD cells.

PubMed

Zhu, Yuanmei; Wang, Bei; Huang, He; Zhao, Zhendong

2016-08-05

Stress granules (SGs) are dynamic cytoplasmic granules formed in response to a variety of stresses, including viral infection. Several viruses can modulate the formation of SG with different effects, but the relationship between SG formation and EV71 infection is poorly understood. In this study, we report that EV71 inhibits canonical SGs formation in infected cells and induces the formation of novel RNA granules that were distinguished from canonical SGs in composition and morphology, which we termed 'SG like structures'. Our results also demonstrated that EV71 triggered formation of SG-like structures is dependent on PKR and eIF2α phosphorylation and requires ongoing cellular mRNA synthesis. Finally, we found that SG-like structures are antiviral RNA granules that promote cellular apoptosis and suppress EV71 propagation. Taken together, our findings explain the formation mechanism of SG-like structures induced by EV71 and shed light on virus-host interaction and molecular mechanism underlying EV71 pathogenesis. Copyright © 2016. Published by Elsevier Inc.

Induction of stress granule-like structures in vesicular stomatitis virus-infected cells.

PubMed

Dinh, Phat X; Beura, Lalit K; Das, Phani B; Panda, Debasis; Das, Anshuman; Pattnaik, Asit K

2013-01-01

Previous studies from our laboratory revealed that cellular poly(C) binding protein 2 (PCBP2) downregulates vesicular stomatitis virus (VSV) gene expression. We show here that VSV infection induces the formation of granular structures in the cytoplasm containing cellular RNA-binding proteins, including PCBP2, T-cell-restricted intracellular antigen 1 (TIA1), and TIA1-related protein (TIAR). Depletion of TIA1 via small interfering RNAs (siRNAs), but not depletion of TIAR, results in enhanced VSV growth and gene expression. The VSV-induced granules appear to be similar to the stress granules (SGs) generated in cells triggered by heat shock or oxidative stress but do not contain some of the bona fide SG markers, such as eukaryotic initiation factor 3 (eIF3) or eIF4A, or the processing body (PB) markers, such as mRNA-decapping enzyme 1A (DCP1a), and thus may not represent canonical SGs or PBs. Our results revealed that the VSV-induced granules, called SG-like structures here, contain the viral replicative proteins and RNAs. The formation and maintenance of the SG-like structures required viral replication and ongoing protein synthesis, but an intact cytoskeletal network was not necessary. These results suggest that cells respond to VSV infection by aggregating the antiviral proteins, such as PCBP2 and TIA1, to form SG-like structures. The functional significance of these SG-like structures in VSV-infected cells is currently under investigation.

Killing effects of Huaier Granule combined with DC-CIK on nude mice transplanted with colon carcinoma cell line.

PubMed

Sun, Wen-Wen; Dou, Jin-Xia; Zhang, Lin; Qiao, Li-Kui; Shen, Na; Zhao, Qiang; Gao, Wen-Yuan

2017-07-11

This study aims to compare the efficacy of different treatments for nude mice transplanted with HT-29 colon carcinoma cell line. BalB/C nude mice were transplanted with HT-29 colon carcinoma cell line and randomly divided into four groups, with 5 mice in each group: blank control group, DC-CIK group, Huaier Granule group, and Huaier Granule group combined with DC-CIK group (combined treatment group). For DC-CIK group and combined treatment group, 1×106 DC-CIK cells were injected via the tail vein 4 days after transplantation. The injection was performed twice weekly for a total of 2 weeks. For Huaier Granule group and combined treatment group, Huaier Granule was administered at the dose of 20 g/60 g, by dissolving 20 g of Huaier granules in 600 ml of pure water. Intragastric administration of 0.2 ml of granules was performed once daily for 3 weeks. For the blank control group, equal volume of normal saline was given. Tumor size and body weight of nude mice were measured every 2 days during the 3-week treatment. The mice were sacrificed at the end of treatment to harvest tumors. Key genes of the signaling pathway were detected by RT-PCR. At the end of treatment, mice in combined treatment group, DC-CIK group and Huaier Granule group remained stable emotionally with normal mobility and water and food intake. However, in the blank control group, the mobility was restricted starting from the third week and the mice were on the verge of dying. The expression of PI3KR1, Akt, Wnt1, CTTNB1, Notch1, Notch2 and Notch3 genes were all downregulated significantly in the combined treatment group compared with DC-CIK group and Huaier Granule group (P<0.05). Therefore, the combined treatment of Huaier Granule combined with DC-CIK achieved the best effect in nude mice transplanted with HT-29 colon carcinoma cell line.

Maternal dazap2 Regulates Germ Granules by Counteracting Dynein in Zebrafish Primordial Germ Cells.

PubMed

Forbes, Meredyth M; Rothhämel, Sophie; Jenny, Andreas; Marlow, Florence L

2015-07-07

Primordial germ cells (PGCs) are the stem cells of the germline. Generally, germline induction occurs via zygotic factors or the inheritance of maternal determinants called germ plasm (GP). GP is packaged into ribonucleoprotein complexes within oocytes and later promotes the germline fate in embryos. Once PGCs are specified by either mechanism, GP components localize to perinuclear granular-like structures. Although components of zebrafish PGC germ granules have been studied, the maternal factors regulating their assembly and contribution to germ cell development are unknown. Here, we show that the scaffold protein Dazap2 binds to Bucky ball, an essential regulator of oocyte polarity and GP assembly, and colocalizes with the GP in oocytes and in PGCs. Mutational analysis revealed a requirement for maternal Dazap2 (MDazap2) in germ-granule maintenance. Through molecular epistasis analyses, we show that MDazap2 is epistatic to Tdrd7 and maintains germ granules in the embryonic germline by counteracting Dynein activity. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

The Vps33a gene regulates behavior and cerebellar Purkinje cell number

PubMed Central

Chintala, Sreenivasulu; Novak, Edward K.; Spernyak, Joseph A.; Mazurchuk, Richard; Torres, German; Patel, Suchith; Busch, Kristie; Meeder, Beth A.; Horowitz, Judith M.; Vaughan, Mary M.; Swank, Richard T.

2015-01-01

A mutation in the Vps33a gene causes Hermansky–Pudlak Syndrome (HPS)-like-symptoms in the buff (bf) mouse mutant. The encoded product, Vps33a, is a member of the Sec1 and Class C multi-protein complex that regulates vesicle trafficking to specialized lysosome-related organelles. As Sec1 signaling pathways have been implicated in pre-synaptic function, we examined brain size, cerebellar cell number and the behavioral phenotype of bf mutants. Standardized behavioral tests (SHIRPA protocols) demonstrated significant motor deficits (e.g., grip strength, righting reflex and touch escape) in bf mutants, worsening with age. Histological examination of brain revealed significant Purkinje cell loss that was confirmed with staining for calbindin, a calcium binding protein enriched in Purkinje cells. This pathologic finding was progressive, as older bf mutants (13–14 months) showed a greater attrition of neurons, with their cerebella appearing to be particularly reduced (~30%) in size relative to those of age-matched-control cohorts. These studies suggest that loss of Purkinje neurons is the most obvious neurological atrophy in the bf mutant, a structural change that generates motor coordination deficits and impaired postural phenotypes. It is conceivable therefore that death of cerebellar cells may alsobea clinical feature of HPS patients, a pathological event which has not been reported in the literature. In general, the bf mutant may be a potentially new and useful model for understanding Purkinje cell development and function. PMID:19254700

Distribution of zebrin-immunoreactive Purkinje cell terminals in the cerebellar and vestibular nuclei of birds.

PubMed

Wylie, Douglas R; Pakan, Janelle M P; Huynh, Hang; Graham, David J; Iwaniuk, Andrew N

2012-05-01

Zebrin II (aldolase C) is expressed in a subset of Purkinje cells in the mammalian and avian cerebella such that there is a characteristic parasagittal organization of zebrin-immunopositive stripes alternating with zebrin-immunonegative stripes. Zebrin is expressed not only in the soma and dendrites of Purkinje cells but also in their axonal terminals. Here we describe the distribution of zebrin immunoreactivity in both the vestibular and the cerebellar nuclei of pigeons (Columba livia) and hummingbirds (Calypte anna, Selasphorus rufus). In the medial cerebellar nucleus, zebrin-positive labeling was particularly heavy in the “shell,” whereas the “core” was zebrin negative. In the lateral cerebellar nucleus, labeling was not as heavy, but a positive shell and negative core were also observed. In the vestibular nuclear complex, zebrin-positive terminal labeling was heavy in the dorsolateral vestibular nucleus and the lateral margin of the superior vestibular nucleus. The central and medial regions of the superior nucleus were generally zebrin negative. Labeling was moderate to heavy in the medial vestibular nucleus, particulary the rostral half of the parvocellular subnucleus. A moderate amount of zebrin-positive labeling was present in the descending vestibular nucleus: this was heaviest laterally, and the central region was generally zebrin negative. Zebrin-positive terminals were also observed in the the cerebellovestibular process, prepositus hypoglossi, and lateral tangential nucleus. We discuss our findings in light of similar studies in rats and with respect to the corticonuclear projections to the cerebellar nuclei and the functional connections of the vestibulocerebellum with the vestibular nuclei. Copyright © 2011 Wiley Periodicals, Inc.

Excess influx of Zn(2+) into dentate granule cells affects object recognition memory via attenuated LTP.

PubMed

Suzuki, Miki; Fujise, Yuki; Tsuchiya, Yuka; Tamano, Haruna; Takeda, Atsushi

2015-08-01

The influx of extracellular Zn(2+) into dentate granule cells is nonessential for dentate gyrus long-term potentiation (LTP) and the physiological significance of extracellular Zn(2+) dynamics is unknown in the dentate gyrus. Excess increase in extracellular Zn(2+) in the hippocampal CA1, which is induced with excitation of zincergic neurons, induces memory deficit via excess influx of Zn(2+) into CA1 pyramidal cells. In the present study, it was examined whether extracellular Zn(2+) induces object recognition memory deficit via excess influx of Zn(2+) into dentate granule cells. KCl (100 mM, 2 µl) was locally injected into the dentate gyrus. The increase in intracellular Zn(2+) in dentate granule cells induced with high K(+) was blocked by co-injection of CaEDTA and CNQX, an extracellular Zn(2+) chelator and an AMPA receptor antagonist, respectively, suggesting that high K(+) increases the influx of Zn(2+) into dentate granule cells via AMPA receptor activation. Dentate gyrus LTP induction was attenuated 1 h after KCl injection into the dentate gyrus and also attenuated when KCl was injected 5 min after the induction. Memory deficit was induced when training of object recognition test was performed 1 h after KCl injection into the dentate gyrus and also induced when KCl was injected 5 min after the training. High K(+)-induced impairments of LTP and memory were rescued by co-injection of CaEDTA. These results indicate that excess influx of Zn(2+) into dentate granule cells via AMPA receptor activation affects object recognition memory via attenuated LTP induction. Even in the dentate gyrus where is scarcely innervated by zincergic neurons, it is likely that extracellular Zn(2+) homeostasis is strictly regulated for cognition. Copyright © 2015 Elsevier Ltd. All rights reserved.

Updates to a 13C metabolic flux analysis model for evaluating energy metabolism in cultured cerebellar granule neurons from neonatal rats.

PubMed

Jekabsons, Mika B; Gebril, Hoda M; Wang, Yan-Hong; Avula, Bharathi; Khan, Ikhlas A

2017-10-01

A hexose phosphate recycling model previously developed to infer fluxes through the major glucose consuming pathways in cultured cerebellar granule neurons (CGNs) from neonatal rats metabolizing [1,2- 13 C 2 ]glucose was revised by considering reverse flux through the non-oxidative pentose phosphate pathway (PPP) and symmetrical succinate oxidation within the tricarboxylic acid (TCA) cycle. The model adjusts three flux ratios to effect 13 C distribution in the hexose, pentose, and triose phosphate pools, and in TCA cycle malate to minimize the error between predicted and measured 13 C labeling in exported lactate (i.e., unlabeled, single-, double-, and triple-labeled; M, M1, M2, and M3, respectively). Inclusion of reverse non-oxidative PPP flux substantially increased the number of calculations but ultimately had relatively minor effects on the labeling of glycolytic metabolites. From the error-minimized solution in which the predicted M-M3 lactate differed by 0.49% from that measured by liquid chromatography-triple quadrupole mass spectrometry, the neurons exhibited negligible forward non-oxidative PPP flux. Thus, no glucose was used by the pentose cycle despite explicit consideration of hexose phosphate recycling. Mitochondria consumed only 16% of glucose while 45% was exported as lactate by aerobic glycolysis. The remaining 39% of glucose was shunted to pentose phosphates presumably for de novo nucleotide synthesis, but the proportion metabolized through the oxidative PPP vs. the reverse non-oxidative PPP could not be determined. The lactate exported as M1 (2.5%) and M3 (1.2%) was attributed to malic enzyme, which was responsible for 7.8% of pyruvate production (vs. 92.2% by glycolysis). The updated model is more broadly applicable to different cell types by considering bi-directional flux through the non-oxidative PPP. Its application to cultured neurons utilizing glucose as the sole exogenous substrate has demonstrated substantial oxygen-independent glucose

HCN1 channels in cerebellar Purkinje cells promote late stages of learning and constrain synaptic inhibition

PubMed Central

Rinaldi, Arianna; Defterali, Cagla; Mialot, Antoine; Garden, Derek L F; Beraneck, Mathieu; Nolan, Matthew F

2013-01-01

Neural computations rely on ion channels that modify neuronal responses to synaptic inputs. While single cell recordings suggest diverse and neurone type-specific computational functions for HCN1 channels, their behavioural roles in any single neurone type are not clear. Using a battery of behavioural assays, including analysis of motor learning in vestibulo-ocular reflex and rotarod tests, we find that deletion of HCN1 channels from cerebellar Purkinje cells selectively impairs late stages of motor learning. Because deletion of HCN1 modifies only a subset of behaviours involving Purkinje cells, we asked whether the channel also has functional specificity at a cellular level. We find that HCN1 channels in cerebellar Purkinje cells reduce the duration of inhibitory synaptic responses but, in the absence of membrane hyperpolarization, do not affect responses to excitatory inputs. Our results indicate that manipulation of subthreshold computation in a single neurone type causes specific modifications to behaviour. PMID:24000178

Self-Organized Cerebellar Tissue from Human Pluripotent Stem Cells and Disease Modeling with Patient-Derived iPSCs.

PubMed

Muguruma, Keiko

2018-02-01

Recent advances in the techniques that differentiate induced pluripotent stem cells (iPSCs) into specific types of cells enabled us to establish in vitro cell-based models as a platform for drug discovery. iPSC-derived disease models are advantageous to generation of a large number of cells required for high-throughput screening. Furthermore, disease-relevant cells differentiated from patient-derived iPSCs are expected to recapitulate the disorder-specific pathogenesis and physiology in vitro. Such disease-relevant cells will be useful for developing effective therapies. We demonstrated that cerebellar tissues are generated from human PSCs (hPSCs) in 3D culture systems that recapitulate the in vivo microenvironments associated with the isthmic organizer. Recently, we have succeeded in generation of spinocerebellar ataxia (SCA) patient-derived Purkinje cells by combining the iPSC technology and the self-organizing stem cell 3D culture technology. We demonstrated that SCA6-derived Purkinje cells exhibit vulnerability to triiodothyronine depletion, which is suppressed by treatment with thyrotropin-releasing hormone and Riluzole. We further discuss applications of patient-specific iPSCs to intractable cerebellar disease.

The Long Adventurous Journey of Rhombic Lip Cells in Jawed Vertebrates: A Comparative Developmental Analysis

PubMed Central

Wullimann, Mario F.; Mueller, Thomas; Distel, Martin; Babaryka, Andreas; Grothe, Benedikt; Köster, Reinhard W.

2011-01-01

This review summarizes vertebrate rhombic lip and early cerebellar development covering classic approaches up to modern developmental genetics which identifies the relevant differential gene expression domains and their progeny. Most of this information is derived from amniotes. However, progress in anamniotes, particularly in the zebrafish, has recently been made. The current picture suggests that rhombic lip and cerebellar development in jawed vertebrates (gnathostomes) share many characteristics. Regarding cerebellar development, these include a ptf1a expressing ventral cerebellar proliferation (VCP) giving rise to Purkinje cells and other inhibitory cerebellar cell types, and an atoh1 expressing upper rhombic lip giving rise to an external granular layer (EGL, i.e., excitatory granule cells) and an early ventral migration into the anterior rhombencephalon (cholinergic nuclei). As for the lower rhombic lip (LRL), gnathostome commonalities likely include the formation of precerebellar nuclei (mossy fiber origins) and partially primary auditory nuclei (likely convergently evolved) from the atoh1 expressing dorsal zone. The fate of the ptf1a expressing ventral LRL zone which gives rise to (excitatory cells of) the inferior olive (climbing fiber origin) and (inhibitory cells of ) cochlear nuclei in amniotes, has not been determined in anamniotes. Special for the zebrafish in comparison to amniotes is the predominant origin of anamniote excitatory deep cerebellar nuclei homologs (i.e., eurydendroid cells) from ptf1a expressing VCP cells, the sequential activity of various atoh1 paralogs and the incomplete coverage of the subpial cerebellar plate with proliferative EGL cells. Nevertheless, the conclusion that a rhombic lip and its major derivatives evolved with gnathostome vertebrates only and are thus not an ancestral craniate character complex is supported by the absence of a cerebellum (and likely absence of its afferent and efferent nuclei) in jawless fishes PMID

Entorhinal Denervation Induces Homeostatic Synaptic Scaling of Excitatory Postsynapses of Dentate Granule Cells in Mouse Organotypic Slice Cultures

PubMed Central

Vlachos, Andreas; Becker, Denise; Jedlicka, Peter; Winkels, Raphael; Roeper, Jochen; Deller, Thomas

2012-01-01

Denervation-induced changes in excitatory synaptic strength were studied following entorhinal deafferentation of hippocampal granule cells in mature (≥3 weeks old) mouse organotypic entorhino-hippocampal slice cultures. Whole-cell patch-clamp recordings revealed an increase in excitatory synaptic strength in response to denervation during the first week after denervation. By the end of the second week synaptic strength had returned to baseline. Because these adaptations occurred in response to the loss of excitatory afferents, they appeared to be in line with a homeostatic adjustment of excitatory synaptic strength. To test whether denervation-induced changes in synaptic strength exploit similar mechanisms as homeostatic synaptic scaling following pharmacological activity blockade, we treated denervated cultures at 2 days post lesion for 2 days with tetrodotoxin. In these cultures, the effects of denervation and activity blockade were not additive, suggesting that similar mechanisms are involved. Finally, we investigated whether entorhinal denervation, which removes afferents from the distal dendrites of granule cells while leaving the associational afferents to the proximal dendrites of granule cells intact, results in a global or a local up-scaling of granule cell synapses. By using computational modeling and local electrical stimulations in Strontium (Sr2+)-containing bath solution, we found evidence for a lamina-specific increase in excitatory synaptic strength in the denervated outer molecular layer at 3–4 days post lesion. Taken together, our data show that entorhinal denervation results in homeostatic functional changes of excitatory postsynapses of denervated dentate granule cells in vitro. PMID:22403720

Glucokinase is an integral component of the insulin granules in glucose-responsive insulin secretory cells and does not translocate during glucose stimulation.

PubMed

Arden, Catherine; Harbottle, Andrew; Baltrusch, Simone; Tiedge, Markus; Agius, Loranne

2004-09-01

The association of glucokinase with insulin secretory granules has been shown by cell microscopy techniques. We used MIN6 insulin-secretory cells and organelle fractionation to determine the effects of glucose on the subcellular distribution of glucokinase. After permeabilization with digitonin, 50% of total glucokinase remained bound intracellularly, while 30% was associated with the 13,000g particulate fraction. After density gradient fractionation of the organelles, immunoreactive glucokinase was distributed approximately equally between dense insulin granules and low-density organelles that cofractionate with mitochondria. Although MIN6 cells show glucose-responsive insulin secretion, glucokinase association with the granules and low-density organelles was not affected by glucose. Subfractionation of the insulin granule components by hypotonic lysis followed by sucrose gradient centrifugation showed that glucokinase colocalized with the granule membrane marker phogrin and not with insulin. PFK2 (6-phosphofructo-2-kinase-2/fructose-2,6-bisphosphatase)/FDPase-2, a glucokinase-binding protein, and glyceraldehyde phosphate dehydrogenase, which has been implicated in granule fusion, also colocalized with glucokinase after hypotonic lysis or detergent extaction of the granules. The results suggest that glucokinase is an integral component of the granule and does not translocate during glucose stimulation.

Inhibitors of the alpha-ketoglutarate dehydrogenase complex alter [1-13C]glucose and [U-13C]glutamate metabolism in cerebellar granule neurons.

PubMed

Santos, Sónia Sá; Gibson, Gary E; Cooper, Arthur J L; Denton, Travis T; Thompson, Charles M; Bunik, Victoria I; Alves, Paula M; Sonnewald, Ursula

2006-02-15

Diminished activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), an important component of the tricarboxylic acid (TCA) cycle, occurs in several neurological diseases. The effect of specific KGDHC inhibitors [phosphonoethyl ester of succinyl phosphonate (PESP) and the carboxy ethyl ester of succinyl phosphonate (CESP)] on [1-13C]glucose and [U-13C]glutamate metabolism in intact cerebellar granule neurons was investigated. Both inhibitors decreased formation of [4-13C]glutamate from [1-13C]glucose, a reduction in label in glutamate derived from [1-13C]glucose/[U-13C]glutamate through a second turn of the TCA cycle and a decline in the amounts of gamma-aminobutyric acid (GABA), aspartate, and alanine. PESP decreased formation of [U-13C]aspartate and total glutathione, whereas CESP decreased concentrations of valine and leucine. The findings are consistent with decreased KGDHC activity; increased alpha-ketoglutarate formation; increased transamination of alpha-ketoglutarate with valine, leucine, and GABA; and new equilibrium position of the aspartate aminotransferase reaction. Overall, the findings also suggest that some carbon derived from alpha-ketoglutarate may bypass the block in the TCA cycle at KGDHC by means of the GABA shunt and/or conversion of valine to succinate. The results suggest the potential of succinyl phosphonate esters for modeling the biochemical and pathophysiological consequences of reduced KGDHC activity in brain diseases.

Mutant ataxin1 disrupts cerebellar development in spinocerebellar ataxia type 1.

PubMed

Edamakanti, Chandrakanth Reddy; Do, Jeehaeh; Didonna, Alessandro; Martina, Marco; Opal, Puneet

2018-06-01

Spinocerebellar ataxia type 1 (SCA1) is an adult-onset neurodegenerative disease caused by a polyglutamine expansion in the protein ATXN1, which is involved in transcriptional regulation. Although symptoms appear relatively late in life, primarily from cerebellar dysfunction, pathogenesis begins early, with transcriptional changes detectable as early as a week after birth in SCA1-knockin mice. Given the importance of this postnatal period for cerebellar development, we asked whether this region might be developmentally altered by mutant ATXN1. We found that expanded ATXN1 stimulates the proliferation of postnatal cerebellar stem cells in SCA1 mice. These hyperproliferating stem cells tended to differentiate into GABAergic inhibitory interneurons rather than astrocytes; this significantly increased the GABAergic inhibitory interneuron synaptic connections, disrupting cerebellar Purkinje cell function in a non-cell autonomous manner. We confirmed the increased basket cell-Purkinje cell connectivity in human SCA1 patients. Mutant ATXN1 thus alters the neural circuitry of the developing cerebellum, setting the stage for the later vulnerability of Purkinje cells to SCA1. We propose that other late-onset degenerative diseases may also be rooted in subtle developmental derailments.

Loss of Melanin by Eye Retinal Pigment Epithelium Cells Is Associated with Its Oxidative Destruction in Melanolipofuscin Granules.

PubMed

Dontsov, A E; Sakina, N L; Ostrovsky, M A

2017-08-01

The effect of superoxide radicals on melanin destruction and degradation of melanosomes isolated from cells of retinal pigment epithelium (RPE) of the human eye was studied. We found that potassium superoxide causes destruction of melanin in melanosomes of human and bovine RPE, as well as destruction of melanin from the ink bag of squid, with the formation of fluorescent decay products having an emission maximum at 520-525 nm. The initial kinetics of the accumulation of the fluorescent decay products is linear. Superoxide radicals lead simultaneously to a decrease in the number of melanosomes and to a decrease in concentration of paramagnetic centers in them. Complete degradation of melanosomes leads to the formation of a transparent solution containing dissolved proteins and melanin degradation products that do not exhibit paramagnetic properties. To completely degrade one melanosome of human RPE, 650 ± 100 fmol of superoxide are sufficient. The concentration of paramagnetic centers in a melanolipofuscin granule of human RPE is on average 32.5 ± 10.4% (p < 0.05, 150 eyes) lower than in a melanosome, which indicates melanin undergoing a destruction process in these granules. RPE cells also contain intermediate granules that have an EPR signal with a lower intensity than that of melanolipofuscin granules, but higher than that of lipofuscin granules. This signal is due to the presence of residual melanin in these granules. Irradiation of a mixture of melanosomes with lipofuscin granules with blue light (450 nm), in contrast to irradiation of only melanosomes, results in the appearance of fluorescent melanin degradation products. We suggest that one of the main mechanisms of age-related decrease in melanin concentration in human RPE cells is its destruction in melanolipofuscin granules under the action of superoxide radicals formed during photoinduced oxygen reduction by lipofuscin fluorophores.

Imaging of zymogen granules in fully wet cells: evidence for restricted mechanism of granule growth.

PubMed

Hammel, Ilan; Anaby, Debbie

2007-09-01

The introduction of wet SEM imaging technology permits electron microscopy of wet samples. Samples are placed in sealed specimen capsules and are insulated from the vacuum in the SEM chamber by an impermeable, electron-transparent membrane. The complete insulation of the sample from the vacuum allows direct imaging of fully hydrated, whole-mount tissue. In the current work, we demonstrate direct inspection of thick pancreatic tissue slices (above 400 mum). In the case of scanning of the pancreatic surface, the boundaries of intracellular features are seen directly. Thus no unfolding is required to ascertain the actual particle size distribution based on the sizes of the sections. This method enabled us to investigate the true granule size distribution and confirm early studies of improved conformity to a Poisson-like distribution, suggesting that the homotypic granule growth results from a mechanism, which favors the addition of a single unit granule to mature granules.

The life cycle of platelet granules.

PubMed

Sharda, Anish; Flaumenhaft, Robert

2018-01-01

Platelet granules are unique among secretory vesicles in both their content and their life cycle. Platelets contain three major granule types-dense granules, α-granules, and lysosomes-although other granule types have been reported. Dense granules and α-granules are the most well-studied and the most physiologically important. Platelet granules are formed in large, multilobulated cells, termed megakaryocytes, prior to transport into platelets. The biogenesis of dense granules and α-granules involves common but also distinct pathways. Both are formed from the trans -Golgi network and early endosomes and mature in multivesicular bodies, but the formation of dense granules requires trafficking machinery different from that of α-granules. Following formation in the megakaryocyte body, both granule types are transported through and mature in long proplatelet extensions prior to the release of nascent platelets into the bloodstream. Granules remain stored in circulating platelets until platelet activation triggers the exocytosis of their contents. Soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, located on both the granules and target membranes, provide the mechanical energy that enables membrane fusion during both granulogenesis and exocytosis. The function of these core fusion engines is controlled by SNARE regulators, which direct the site, timing, and extent to which these SNAREs interact and consequently the resulting membrane fusion. In this review, we assess new developments in the study of platelet granules, from their generation to their exocytosis.

Expression of the alpha and beta subunits of Ca2+/calmodulin kinase II in the cerebellum of jaundiced Gunn rats during development: a quantitative light microscopic analysis.

PubMed

Conlee, J W; Shapiro, S M; Churn, S B

2000-04-01

The homozygous (jj) jaundiced Gunn rat model for hyperbilirubinemia displays pronounced cerebellar hypoplasia. To examine the cellular mechanisms involved in bilirubin toxicity, this study focused on the effect of hyperbilirubinemia on calcium/calmodulin-dependent kinase II (CaM kinase II). CaM kinase II is a neuronally enriched enzyme which performs several important functions. Immunohistochemical analysis of alternating serial sections were performed using monoclonal antibodies for the alpha and beta subunits of CaM kinase II. Measurements were made of the total numbers of stained cells in each of the deep cerebellar nuclei and of Purkinje and granule cell densities in cerebellar lobules II, VI, and IX. The beta subunit was present in Purkinje cells and deep cerebellar nuclei of both groups at all ages, but only granule cells which had migrated through the Purkinje cell layer showed staining for beta subunit; external granule cells were completely negative. Many Purkinje cells had degenerated in the older animals, and the percent of granule cells stained for beta subunit was significantly reduced. The alpha subunit was found exclusively in Purkinje cells, although its appearance was delayed in the jaundiced animals. Sulfadimethoxine was administered to some jj rats 24 h or 15 days prior to sacrifice to increase brain bilirubin concentration. Results showed that bilirubin exposure modulated both alpha and beta CaM kinase II subunit expression in selective neuronal populations, but sulfadimethoxine had no acute effect on enzyme immunoreactivity. Thus, developmental expression of the alpha and beta subunits of CaM kinase II was affected by chronic bilirubin exposure during early postnatal development of jaundiced Gunn rats.

Cellular stress induces cytoplasmic RNA granules in fission yeast.

PubMed

Nilsson, Daniel; Sunnerhagen, Per

2011-01-01

Severe stress causes plant and animal cells to form large cytoplasmic granules containing RNA and proteins. Here, we demonstrate the existence of stress-induced cytoplasmic RNA granules in Schizosaccharomyces pombe. Homologs to several known protein components of mammalian processing bodies and stress granules are found in fission yeast RNA granules. In contrast to mammalian cells, poly(A)-binding protein (Pabp) colocalizes in stress-induced granules with decapping protein. After glucose deprivation, protein kinase A (PKA) is required for accumulation of Pabp-positive granules and translational down-regulation. This is the first demonstration of a role for PKA in RNA granule formation. In mammals, the translation initiation protein eIF2α is a key regulator of formation of granules containing poly(A)-binding protein. In S. pombe, nonphosphorylatable eIF2α does not block but delays granule formation and subsequent clearance after exposure to hyperosmosis. At least two separate pathways in S. pombe appear to regulate stress-induced granules: pka1 mutants are fully proficient to form granules after hyperosmotic shock; conversely, eIF2α does not affect granule formation in glucose starvation. Further, we demonstrate a Pka1-dependent link between calcium perturbation and RNA granules, which has not been described earlier in any organism.

Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell-mediated cytotoxicity.

PubMed

Tuli, Amit; Thiery, Jerome; James, Ashley M; Michelet, Xavier; Sharma, Mahak; Garg, Salil; Sanborn, Keri B; Orange, Jordan S; Lieberman, Judy; Brenner, Michael B

2013-12-01

Natural killer (NK) lymphocytes contain lysosome-related organelles (LROs), known as lytic granules, which upon formation of immune synapse with the target cell, polarize toward the immune synapse to deliver their contents to the target cell membrane. Here, we identify a small GTP-binding protein, ADP-ribosylation factor-like 8b (Arl8b), as a critical factor required for NK cell-mediated cytotoxicity. Our findings indicate that Arl8b drives the polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between effector NK lymphocytes and target cells. Using a glutathione S-transferase pull-down approach, we identify kinesin family member 5B (KIF5B; the heavy chain of kinesin-1) as an interaction partner of Arl8b from NK cell lysates. Previous studies showed that interaction between kinesin-1 and Arl8b is mediated by SifA and kinesin-interacting protein (SKIP) and the tripartite complex drives the anterograde movement of lysosomes. Silencing of both KIF5B and SKIP in NK cells, similar to Arl8b, led to failure of MTOC-lytic granule polarization to the immune synapse, suggesting that Arl8b and kinesin-1 together control this critical step in NK cell cytotoxicity.

Protease nexin-1 promotes secretory granule biogenesis by preventing granule protein degradation.

PubMed

Kim, Taeyoon; Loh, Y Peng

2006-02-01

Dense-core secretory granule (DCG) biogenesis is a prerequisite step for the sorting, processing, and secretion of neuropeptides and hormones in (neuro)endocrine cells. Previously, chromogranin A (CgA) has been shown to play a key role in the regulation of DCG biogenesis in vitro and in vivo. However, the underlying mechanism of CgA-mediated DCG biogenesis has not been explored. In this study, we have uncovered a novel mechanism for the regulation of CgA-mediated DCG biogenesis. Transfection of CgA into endocrine 6T3 cells lacking CgA and DCGs not only recovered DCG formation and regulated secretion but also prevented granule protein degradation. Genetic profiling of CgA-expressing 6T3 versus CgA- and DCG-deficient 6T3 cells, followed by real-time reverse transcription-polymerase chain reaction and Western blotting analyses, revealed that a serine protease inhibitor, protease nexin-1 (PN-1), was significantly up-regulated in CgA-expressing 6T3 cells. Overexpression of PN-1 in CgA-deficient 6T3 cells prevented degradation of DCG proteins at the Golgi apparatus, enhanced DCG biogenesis, and recovered regulated secretion. Moreover, depletion of PN-1 by antisense RNAs in CgA-expressing 6T3 cells resulted in the specific degradation of DCG proteins. We conclude that CgA increases DCG biogenesis in endocrine cells by up-regulating PN-1 expression to stabilize granule proteins against degradation.

Protease Nexin-1 Promotes Secretory Granule Biogenesis by Preventing Granule Protein Degradation

PubMed Central

Kim, Taeyoon; Loh, Y. Peng

2006-01-01

Dense-core secretory granule (DCG) biogenesis is a prerequisite step for the sorting, processing, and secretion of neuropeptides and hormones in (neuro)endocrine cells. Previously, chromogranin A (CgA) has been shown to play a key role in the regulation of DCG biogenesis in vitro and in vivo. However, the underlying mechanism of CgA-mediated DCG biogenesis has not been explored. In this study, we have uncovered a novel mechanism for the regulation of CgA-mediated DCG biogenesis. Transfection of CgA into endocrine 6T3 cells lacking CgA and DCGs not only recovered DCG formation and regulated secretion but also prevented granule protein degradation. Genetic profiling of CgA-expressing 6T3 versus CgA- and DCG-deficient 6T3 cells, followed by real-time reverse transcription-polymerase chain reaction and Western blotting analyses, revealed that a serine protease inhibitor, protease nexin-1 (PN-1), was significantly up-regulated in CgA-expressing 6T3 cells. Overexpression of PN-1 in CgA-deficient 6T3 cells prevented degradation of DCG proteins at the Golgi apparatus, enhanced DCG biogenesis, and recovered regulated secretion. Moreover, depletion of PN-1 by antisense RNAs in CgA-expressing 6T3 cells resulted in the specific degradation of DCG proteins. We conclude that CgA increases DCG biogenesis in endocrine cells by up-regulating PN-1 expression to stabilize granule proteins against degradation. PMID:16319172

Non-synaptic signaling from cerebellar climbing fibers modulates Golgi cell activity.

PubMed

Nietz, Angela K; Vaden, Jada H; Coddington, Luke T; Overstreet-Wadiche, Linda; Wadiche, Jacques I

2017-10-13

Golgi cells are the principal inhibitory neurons at the input stage of the cerebellum, providing feedforward and feedback inhibition through mossy fiber and parallel fiber synapses. In vivo studies have shown that Golgi cell activity is regulated by climbing fiber stimulation, yet there is little functional or anatomical evidence for synapses between climbing fibers and Golgi cells. Here, we show that glutamate released from climbing fibers activates ionotropic and metabotropic receptors on Golgi cells through spillover-mediated transmission. The interplay of excitatory and inhibitory conductances provides flexible control over Golgi cell spiking, allowing either excitation or a biphasic sequence of excitation and inhibition following single climbing fiber stimulation. Together with prior studies of spillover transmission to molecular layer interneurons, these results reveal that climbing fibers exert control over inhibition at both the input and output layers of the cerebellar cortex.

Cerebellar cortex development in the weaver condition presents regional and age-dependent abnormalities without differences in Purkinje cells neurogenesis.

PubMed

Martí, Joaquín; Santa-Cruz, María C; Hervás, José P; Bayer, Shirley A; Villegas, Sandra

2016-01-01

Ataxias are neurological disorders associated with the degeneration of Purkinje cells (PCs). Homozygous weaver mice (wv/wv) have been proposed as a model for hereditary cerebellar ataxia because they present motor abnormalities and PC loss. To ascertain the physiopathology of the weaver condition, the development of the cerebellar cortex lobes was examined at postnatal day (P): P8, P20 and P90. Three approaches were used: 1) quantitative determination of several cerebellar features; 2) qualitative evaluation of the developmental changes occurring in the cortical lobes; and 3) autoradiographic analyses of PC generation and placement. Our results revealed a reduction in the size of the wv/wv cerebellum as a whole, confirming previous results. However, as distinguished from these reports, we observed that quantified parameters contribute differently to the abnormal growth of the wv/wv cerebellar lobes. Qualitative analysis showed anomalies in wv/wv cerebellar cytoarchitecture, depending on the age and lobe analyzed. Such abnormalities included the presence of the external granular layer after P20 and, at P90, ectopic cells located in the molecular layer following several placement patterns. Finally, we obtained autoradiographic evidence that wild-type and wv/wv PCs presented similar neurogenetic timetables, as reported. However, the innovative character of this current work lies in the fact that the neurogenetic gradients of wv/wv PCs were not modified from P8 to P90. A tendency for the accumulation of late-formed PCs in the anterior and posterior lobes was found, whereas early-generated PCs were concentrated in the central and inferior lobes. These data suggested that wv/wv PCs may migrate properly to their final destinations. The extrapolation of our results to patients affected with cerebellar ataxias suggests that all cerebellar cortex lobes are affected with several age-dependent alterations in cytoarchitectonics. We also propose that PC loss may be regionally

Co-localization of glycine and gaba immunoreactivity in interneurons in Macaca monkey cerebellar cortex.

PubMed

Crook, J; Hendrickson, A; Robinson, F R

2006-09-15

Previous work demonstrates that the cerebellum uses glycine as a fast inhibitory neurotransmitter [Ottersen OP, Davanger S, Storm-Mathisen J (1987) Glycine-like immunoreactivity in the cerebellum of rat and Senegalese baboon, Papio papio: a comparison with the distribution of GABA-like immunoreactivity and with [3H]glycine and [3H]GABA uptake. Exp Brain Res 66(1):211-221; Ottersen OP, Storm-Mathisen J, Somogyi P (1988) Colocalization of glycine-like and GABA-like immunoreactivities in Golgi cell terminals in the rat cerebellum: a postembedding light and electron microscopic study. Brain Res 450(1-2):342-353; Dieudonne S (1995) Glycinergic synaptic currents in Golgi cells of the rat cerebellum. Proc Natl Acad Sci U S A 92:1441-1445; Dumoulin A, Triller A, Dieudonne S (2001) IPSC kinetics at identified GABAergic and mixed GABAergic and glycinergic synapses onto cerebellar Golgi cells. J Neurosci 21(16):6045-6057; Dugue GP, Dumoulin A, Triller A, Dieudonne S (2005) Target-dependent use of coreleased inhibitory transmitters at central synapses. J Neurosci 25(28):6490-6498; Zeilhofer HU, Studler B, Arabadzisz D, Schweizer C, Ahmadi S, Layh B, Bosl MR, Fritschy JM (2005) Glycinergic neurons expressing enhanced green fluorescent protein in bacterial artificial chromosome transgenic mice. J Comp Neurol 482(2):123-141]. In the rat cerebellum glycine is not released by itself but is released together with GABA by Lugaro cells onto Golgi cells [Dumoulin A, Triller A, Dieudonne S (2001) IPSC kinetics at identified GABAergic and mixed GABAergic and glycinergic synapses onto cerebellar Golgi cells. J Neurosci 21(16):6045-6057] and by Golgi cells onto unipolar brush and granule cells [Dugue GP, Dumoulin A, Triller A, Dieudonne S (2005) Target-dependent use of coreleased inhibitory transmitters at central synapses. J Neurosci 25(28):6490-6498]. Here we report, from immunolabeling evidence in Macaca cerebellum, that interneurons in the granular cell layer are glycine+ at a density

Large granulation cells on the surface of the giant star π1 Gruis

NASA Astrophysics Data System (ADS)

Paladini, C.; Baron, F.; Jorissen, A.; Le Bouquin, J.-B.; Freytag, B.; van Eck, S.; Wittkowski, M.; Hron, J.; Chiavassa, A.; Berger, J.-P.; Siopis, C.; Mayer, A.; Sadowski, G.; Kravchenko, K.; Shetye, S.; Kerschbaum, F.; Kluska, J.; Ramstedt, S.

2018-01-01

Convection plays a major part in many astrophysical processes, including energy transport, pulsation, dynamos and winds on evolved stars, in dust clouds and on brown dwarfs. Most of our knowledge about stellar convection has come from studying the Sun: about two million convective cells with typical sizes of around 2,000 kilometres across are present on the surface of the Sun—a phenomenon known as granulation. But on the surfaces of giant and supergiant stars there should be only a few large (several tens of thousands of times larger than those on the Sun) convective cells, owing to low surface gravity. Deriving the characteristic properties of convection (such as granule size and contrast) for the most evolved giant and supergiant stars is challenging because their photospheres are obscured by dust, which partially masks the convective patterns. These properties can be inferred from geometric model fitting, but this indirect method does not provide information about the physical origin of the convective cells. Here we report interferometric images of the surface of the evolved giant star π1 Gruis, of spectral type S5,7. Our images show a nearly circular, dust-free atmosphere, which is very compact and only weakly affected by molecular opacity. We find that the stellar surface has a complex convective pattern with an average intensity contrast of 12 per cent, which increases towards shorter wavelengths. We derive a characteristic horizontal granule size of about 1.2 × 1011 metres, which corresponds to 27 per cent of the diameter of the star. Our measurements fall along the scaling relations between granule size, effective temperature and surface gravity that are predicted by simulations of stellar surface convection.

Large granulation cells on the surface of the giant star π1 Gruis.

PubMed

Paladini, C; Baron, F; Jorissen, A; Le Bouquin, J-B; Freytag, B; Van Eck, S; Wittkowski, M; Hron, J; Chiavassa, A; Berger, J-P; Siopis, C; Mayer, A; Sadowski, G; Kravchenko, K; Shetye, S; Kerschbaum, F; Kluska, J; Ramstedt, S

2018-01-18

Convection plays a major part in many astrophysical processes, including energy transport, pulsation, dynamos and winds on evolved stars, in dust clouds and on brown dwarfs. Most of our knowledge about stellar convection has come from studying the Sun: about two million convective cells with typical sizes of around 2,000 kilometres across are present on the surface of the Sun-a phenomenon known as granulation. But on the surfaces of giant and supergiant stars there should be only a few large (several tens of thousands of times larger than those on the Sun) convective cells, owing to low surface gravity. Deriving the characteristic properties of convection (such as granule size and contrast) for the most evolved giant and supergiant stars is challenging because their photospheres are obscured by dust, which partially masks the convective patterns. These properties can be inferred from geometric model fitting, but this indirect method does not provide information about the physical origin of the convective cells. Here we report interferometric images of the surface of the evolved giant star π 1 Gruis, of spectral type S5,7. Our images show a nearly circular, dust-free atmosphere, which is very compact and only weakly affected by molecular opacity. We find that the stellar surface has a complex convective pattern with an average intensity contrast of 12 per cent, which increases towards shorter wavelengths. We derive a characteristic horizontal granule size of about 1.2 × 10 11 metres, which corresponds to 27 per cent of the diameter of the star. Our measurements fall along the scaling relations between granule size, effective temperature and surface gravity that are predicted by simulations of stellar surface convection.

[Cerebellar cognitive affective syndrome secondary to a cerebellar tumour].

PubMed

Domínguez-Carral, J; Carreras-Sáez, I; García-Peñas, J J; Fournier-Del Castillo, C; Villalobos-Reales, J

2015-01-01

Cerebellar cognitive affective syndrome is characterized by disturbances of executive function, impaired spatial cognition, linguistic difficulties, and personality change. The case of an 11 year old boy is presented, with behavior problems, learning difficulties and social interaction problems. In the physical examination he had poor visual contact, immature behavior, reduced expressive language and global motor disability with gait dyspraxia, with no defined cerebellar motor signs. In the neuropsychological evaluation he has a full scale overall intellectual quotient of 84, with signs of cerebellar cognitive affective syndrome. A tumour affecting inferior cerebellar vermis was observed in the magnetic resonance imaging, which had not significantly grown during 5 years of follow up. The cerebellum participates in controlling cognitive and affective functions. Cerebellar pathology must be considered in the differential diagnosis of children with cognitive or learning disorder with associated behavioral and emotional components. Copyright © 2013 Asociación Española de Pediatría. Published by Elsevier Espana. All rights reserved.

Facilitation of granule cell epileptiform activity by mossy fiber-released zinc in the pilocarpine model of temporal lobe epilepsy.

PubMed

Timofeeva, Olga; Nadler, J Victor

2006-03-17

Recurrent mossy fiber synapses in the dentate gyrus of epileptic brain facilitate the synchronous firing of granule cells and may promote seizure propagation. Mossy fiber terminals contain and release zinc. Released zinc inhibits the activation of NMDA receptors and may therefore oppose the development of granule cell epileptiform activity. Hippocampal slices from rats that had experienced pilocarpine-induced status epilepticus and developed a recurrent mossy fiber pathway were used to investigate this possibility. Actions of released zinc were inferred from the effects of chelation with 1 mM calcium disodium EDTA (CaEDTA). When granule cell population bursts were evoked by mossy fiber stimulation in the presence of 6 mM K(+) and 30 microM bicuculline, CaEDTA slowed the rate at which evoked bursting developed, but did not change the magnitude of the bursts once they had developed fully. The effects of CaEDTA were then studied on the pharmacologically isolated NMDA receptor- and AMPA/kainate receptor-mediated components of the fully developed bursts. CaEDTA increased the magnitude of NMDA receptor-mediated bursts and reduced the magnitude of AMPA/kainate receptor-mediated bursts. CaEDTA did not affect the granule cell bursts evoked in slices from untreated rats by stimulating the perforant path in the presence of bicuculline and 6 mM K(+). These results suggest that zinc released from the recurrent mossy fibers serves mainly to facilitate the recruitment of dentate granule cells into population bursts.

Stimulation of microtubule-based transport by nucleation of microtubules on pigment granules

PubMed Central

Semenova, Irina; Gupta, Dipika; Usui, Takeo; Hayakawa, Ichiro; Cowan, Ann; Rodionov, Vladimir

2017-01-01

Microtubule (MT)-based transport can be regulated through changes in organization of MT transport tracks, but the mechanisms that regulate these changes are poorly understood. In Xenopus melanophores, aggregation of pigment granules in the cell center involves their capture by the tips of MTs growing toward the cell periphery, and granule aggregation signals facilitate capture by increasing the number of growing MT tips. This increase could be explained by stimulation of MT nucleation either on the centrosome or on the aggregate of pigment granules that gradually forms in the cell center. We blocked movement of pigment granules to the cell center and compared the MT-nucleation activity of the centrosome in the same cells in two signaling states. We found that granule aggregation signals did not stimulate MT nucleation on the centrosome but did increase MT nucleation activity of pigment granules. Elevation of MT-nucleation activity correlated with the recruitment to pigment granules of a major component of MT-nucleation templates, γ-tubulin, and was suppressed by γ-tubulin inhibitors. We conclude that generation of new MT transport tracks by concentration of the leading pigment granules provides a positive feedback loop that enhances delivery of trailing granules to the cell center. PMID:28381426

Tubulin-related cerebellar dysplasia: definition of a distinct pattern of cerebellar malformation.

PubMed

Romaniello, Romina; Arrigoni, Filippo; Panzeri, Elena; Poretti, Andrea; Micalizzi, Alessia; Citterio, Andrea; Bedeschi, Maria Francesca; Berardinelli, Angela; Cusmai, Raffaella; D'Arrigo, Stefano; Ferraris, Alessandro; Hackenberg, Annette; Kuechler, Alma; Mancardi, Margherita; Nuovo, Sara; Oehl-Jaschkowitz, Barbara; Rossi, Andrea; Signorini, Sabrina; Tüttelmann, Frank; Wahl, Dagmar; Hehr, Ute; Boltshauser, Eugen; Bassi, Maria Teresa; Valente, Enza Maria; Borgatti, Renato

2017-12-01

To determine the neuroimaging pattern of cerebellar dysplasia (CD) and other posterior fossa morphological anomalies associated with mutations in tubulin genes and to perform clinical and genetic correlations. Twenty-eight patients harbouring 23 heterozygous pathogenic variants (ten novel) in tubulin genes TUBA1A (n = 10), TUBB2B (n = 8) or TUBB3 (n = 5) were studied by a brain MRI scan performed either on a 1.5 T (n = 10) or 3 T (n = 18) MR scanner with focus on the posterior fossa. Cerebellar anomalies were detected in 24/28 patients (86%). CD was recognised in 19/28 (68%) including cortical cerebellar dysplasia (CCD) in 18/28, either involving only the cerebellar hemispheres (12/28) or associated with vermis dysplasia (6/28). CCD was located only in the right hemisphere in 13/18 (72%), including four TUBB2B-, four TUBB3- and five TUBA1A-mutated patients, while in the other five TUBA1A cases it was located only in the left hemisphere or in both hemispheres. The postero-superior region of the cerebellar hemispheres was most frequently affected. The cerebellar involvement in tubulinopathies shows specific features that may be labelled as 'tubulin-related CD'. This pattern is unique and differs from other genetic causes of cerebellar dysplasia. • Cortical cerebellar dysplasia without cysts is suggestive of tubulin-related disorder. • Cerebellar dysplasia in tubulinopathies shows specific features labelled as 'tubulin-related CD'. • Focal and unilateral involvement of cerebellar hemispheres has important implications for counselling.

The presence of PHB granules in cytoplasm protects non-halophilic bacterial cells against the harmful impact of hypertonic environments.

PubMed

Obruca, Stanislav; Sedlacek, Petr; Mravec, Filip; Krzyzanek, Vladislav; Nebesarova, Jana; Samek, Ota; Kucera, Dan; Benesova, Pavla; Hrubanova, Kamila; Milerova, Miluse; Marova, Ivana

2017-10-25

Numerous prokaryotes accumulate polyhydroxybutyrate (PHB) intracellularly as a storage material. It has also been proposed that PHB accumulation improves bacterial stress resistance. Cupriavidus necator and its PHB non-accumulating mutant were employed to investigate the protective role of PHB under hypertonic conditions. The presence of PHB granules enhanced survival of the bacteria after exposure to hypertonic conditions. Surprisingly, when coping with such conditions, the bacteria did not utilize PHB to harvest carbon or energy, suggesting that, in the osmotic upshock of C. necator, the protective mechanism of PHB granules is not associated with their hydrolysis. The presence of PHB granules influenced the overall properties of the cells, since challenged PHB-free cells underwent massive plasmolysis accompanied by damage to the cell membrane and the leakage of cytoplasm content, while no such effects were observed in PHB containing bacteria. Moreover, PHB granules demonstrated "liquid-like" properties indicating that they can partially repair and stabilize cell membranes by plugging small gaps formed during plasmolysis. In addition, the level of dehydration and changes in intracellular pH in osmotically challenged cells were less pronounced for PHB-containing cultures, demonstrating the important role of PHB for bacterial survival under hyperosmotic conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

Isolation of zymogen granules from rat pancreas.

PubMed

Rindler, Michael J

2006-01-01

This unit describes methods for preparing zymogen granules from rat pancreas. Zymogen granules are storage organelles in pancreatic acinar cells containing digestive enzymes that are released into the pancreatic duct. The protocols in this unit take advantage of the large size (up to 1 microm diameter) and high density (>1.20 g/cm(3) on sucrose gradients) of the granules as compared to other cellular organelles. They use a combination of differential sedimentation and density gradient separation to accomplish the purification. Similar procedures can be used to isolate zymogen granules from mouse pancreas and canine pancreas. A protocol for preparing zymogen granules from dog pancreas is also included.

Time-dependent effects of perfluorinated compounds on viability in cerebellar granule neurons: Dependence on carbon chain length and functional group attached.

PubMed

Berntsen, Hanne Friis; Bjørklund, Cesilie Granum; Audinot, Jean-Nicolas; Hofer, Tim; Verhaegen, Steven; Lentzen, Esther; Gutleb, Arno Christian; Ropstad, Erik

2017-12-01

The toxicity of long chained perfluoroalkyl acids (PFAAs) has previously been reported to be related to the length of the perfluorinated carbon chain and functional group attached. In the present study, we compared the cytotoxicity of six PFAAs, using primary cultures of rat cerebellar granule neurons (CGNs). Two perfluoroalkyl sulfonic acids (PFSAs, chain length C 6 and C 8 ) and four perfluoroalkyl carboxylic acids (PFCAs, chain length C 8 -C 11 ) were studied. These PFAAs have been detected in human blood and the brain tissue of mammals. The cell viability trypan blue and MTT assays were used to determine toxicity potencies (based on LC 50 values) after 24h exposure (in descending order): perfluoroundecanoic acid (PFUnDA)≥perfluorodecanoic acid (PFDA)>perfluorooctanesulfonic acid potassium salt (PFOS)>perfluorononanoic acid (PFNA)>perfluorooctanoic acid (PFOA)>perfluorohexanesulfonic acid potassium salt (PFHxS). Concentrations of the six PFAAs that produced equipotent effects after 24h exposure were used to further explore the dynamics of viability changes during this period. Therefore viability was assessed at 10, 30, 60, 90, 120 and 180min as well as 6, 12, 18 and 24h. A difference in the onset of reduction in viability was observed, occurring relatively quickly (30-60min) for PFOS, PFDA and PFUnDA, and much slower (12-24h) for PFHxS, PFOA and PFNA. A slight protective effect of vitamin E against PFOA, PFNA and PFOS-induced reduction in viability indicated a possible involvement of oxidative stress. PFOA and PFOS did not induce lipid peroxidation on their own, but significantly accelerated cumene hydroperoxide-induced lipid peroxidation. When distribution of the six PFAAs in the CGN-membrane was investigated using NanoSIMS50 imaging, two distinct patterns appeared. Whereas PFHxS, PFOS and PFUnDA aggregated in large hotspots, PFOA, PFNA and PFDA showed a more dispersed distribution pattern. In conclusion, the toxicity of the investigated PFAAs increased with

Glucocorticoid Effects on Cerebellar Development in a Chicken Embryo Model: Exploring Changes in PAX6 and Metalloproteinase-9 After Exposure to Dexamethasone.

PubMed

Austdal, L P E; Bjørnstad, S; Mathisen, G H; Aden, P K; Mikkola, I; Paulsen, R E; Rakkestad, K E

2016-12-01

The developing cerebellum is vulnerable to effects of glucocorticoids and cerebellar dysfunction is associated with neurodevelopmental disorders (e.g. autism). Transcription factor PAX6 and matrix metalloproteinase-9 (MMP-9) are critical for normal cerebellar development and are highly expressed in migrating neurones. Alterations in MMP-9 and PAX6 are associated with altered cerebellar development. In the present study, we characterised the growth rate and development of the cortical layers, and further investigated how the levels of PAX6 and MMP-9, as well as glucocorticoid receptor (GR) and proliferating cell nuclear antigen (PCNA), change in the cerebellum during the foetal period [embryonic day (E)12-21] in chicken, which corresponds to the human perinatal period. Dexamethasone (DEX) was administered in ovo at E13 and E16, aiming to investigate how prenatal exposure to glucocorticoids interferes with normal development. DEX reduced foetal and cerebellar weight at E17 in a dose-dependent manner linked to a reduced level of PCNA and, over time, down-regulation of GR. We report that promoter activity of PAX6 and MMP-9 increased as a result of GR-stimulation in vitro. Prenatal DEX increased the protein level of PAX6 in a transient manner. PAX6 is reduced in mature granule neurones, and this occurred earlier in embryos exposed to DEX than in non-exposed controls. DEX exposure also led to a slow-onset down-regulation of MMP-9. Taken together, these findings indicate that excess prenatal glucocorticoid stimulation disturbs normal development of the cerebellum through mechanisms associated with reduced proliferation and accelerated maturation where PAX6 and MMP-9 play important roles. © 2016 British Society for Neuroendocrinology.

Chromaffin granules in the rat adrenal medulla release their secretory content in a particulate fashion.

PubMed

Crivellato, Enrico; Belloni, Anna; Nico, Beatrice; Nussdorfer, Gastone G; Ribatti, Domenico

2004-03-01

Exocytosis is considered the main route of granule discharge in chromaffin cells. We recently provided ultrastructural evidence suggesting that piecemeal degranulation (PMD) occurs in mouse adrenal chromaffin cells. In the present study, we processed rat adrenal glands for transmission electron microscopy (TEM), and examined chromaffin cells for changes characteristic of PMD. Both adrenaline (A)- and noradrenaline (NA)-storing cells express ultrastructural features suggestive of a slow and particulate mode of granule discharge. In adrenaline-containing cells, some granules present enlarged dimensions accompanied by eroded or dissolved matrices. Likewise, a number of granules in NA-releasing cells show content reduction with variably expanded granule chambers. Dilated, empty granule containers are recognizable in the cytoplasm of both cell types. Characteristically, altered granules and empty containers are seen intermingled with normal, resting granules. In addition, chromaffin granules often show irregular profiles, with budding or tail-like projections of their limiting membranes. Thirty 150-nm-diameter membrane-bound vesicles with a moderately electron-dense or -lucent internal structure are observable in the cytoplasm of both cell types. These vesicles are seen among the granules and some of them are fused with the perigranule membranes in the process of attachment to or budding from the granules. These data add further support to the concept that PMD may be an alternative secretory pathway in adrenal chromaffin cells. Copyright 2004 Wiley-Liss, Inc.

BK Channels Localize to the Paranodal Junction and Regulate Action Potentials in Myelinated Axons of Cerebellar Purkinje Cells.

PubMed

Hirono, Moritoshi; Ogawa, Yasuhiro; Misono, Kaori; Zollinger, Daniel R; Trimmer, James S; Rasband, Matthew N; Misonou, Hiroaki

2015-05-06

In myelinated axons, K(+) channels are clustered in distinct membrane domains to regulate action potentials (APs). At nodes of Ranvier, Kv7 channels are expressed with Na(+) channels, whereas Kv1 channels flank nodes at juxtaparanodes. Regulation of axonal APs by K(+) channels would be particularly important in fast-spiking projection neurons such as cerebellar Purkinje cells. Here, we show that BK/Slo1 channels are clustered at the paranodal junctions of myelinated Purkinje cell axons of rat and mouse. The paranodal junction is formed by a set of cell-adhesion molecules, including Caspr, between the node and juxtaparanodes in which it separates nodal from internodal membrane domains. Remarkably, only Purkinje cell axons have detectable paranodal BK channels, whose clustering requires the formation of the paranodal junction via Caspr. Thus, BK channels occupy this unique domain in Purkinje cell axons along with the other K(+) channel complexes at nodes and juxtaparanodes. To investigate the physiological role of novel paranodal BK channels, we examined the effect of BK channel blockers on antidromic AP conduction. We found that local application of blockers to the axon resulted in a significant increase in antidromic AP failure at frequencies above 100 Hz. We also found that Ni(2+) elicited a similar effect on APs, indicating the involvement of Ni(2+)-sensitive Ca(2+) channels. Furthermore, axonal application of BK channel blockers decreased the inhibitory synaptic response in the deep cerebellar nuclei. Thus, paranodal BK channels uniquely support high-fidelity firing of APs in myelinated Purkinje cell axons, thereby underpinning the output of the cerebellar cortex. Copyright © 2015 the authors 0270-6474/15/357082-13$15.00/0.

Treadmill performance of mice with cerebellar lesions: 1. Purkinje cell degeneration mutant mice.

PubMed

Le Marec, N; Lalonde, R

1998-02-01

The purpose of this study was to evaluate the sensorimotor skills of a spontaneous mouse mutant, Purkinje cell degeneration (PCD), marked by selective cerebellar cortical atrophy on a treadmill activated at 1 of 2 speeds and at 1 of 3 slopes, requiring forward movements to avoid footshocks. There was no difference in latencies before falling from the belt between PCD mutants and controls during acquisition. However, PCD mutants were impaired on the fast treadmill during retention, implicating the cerebellum in the memory of a motor skill. During acquisition of the slow treadmill task at the 2 lowest slopes of inclination, PCD mutants spent more time walking than controls, an indication of a decreased ability of coordinating whole body movements. The same pattern of higher walking time on the slow treadmill in PCD mutants was evident during retention. These results indicate that the cerebellar cortex is involved in the acquisition and the retention of a task requiring equilibrium.

Gene expression signature of cerebellar hypoplasia in a mouse model of Down syndrome during postnatal development

PubMed Central

Laffaire, Julien; Rivals, Isabelle; Dauphinot, Luce; Pasteau, Fabien; Wehrle, Rosine; Larrat, Benoit; Vitalis, Tania; Moldrich, Randal X; Rossier, Jean; Sinkus, Ralph; Herault, Yann; Dusart, Isabelle; Potier, Marie-Claude

2009-01-01

Background Down syndrome is a chromosomal disorder caused by the presence of three copies of chromosome 21. The mechanisms by which this aneuploidy produces the complex and variable phenotype observed in people with Down syndrome are still under discussion. Recent studies have demonstrated an increased transcript level of the three-copy genes with some dosage compensation or amplification for a subset of them. The impact of this gene dosage effect on the whole transcriptome is still debated and longitudinal studies assessing the variability among samples, tissues and developmental stages are needed. Results We thus designed a large scale gene expression study in mice (the Ts1Cje Down syndrome mouse model) in which we could measure the effects of trisomy 21 on a large number of samples (74 in total) in a tissue that is affected in Down syndrome (the cerebellum) and where we could quantify the defect during postnatal development in order to correlate gene expression changes to the phenotype observed. Statistical analysis of microarray data revealed a major gene dosage effect: for the three-copy genes as well as for a 2 Mb segment from mouse chromosome 12 that we show for the first time as being deleted in the Ts1Cje mice. This gene dosage effect impacts moderately on the expression of euploid genes (2.4 to 7.5% differentially expressed). Only 13 genes were significantly dysregulated in Ts1Cje mice at all four postnatal development stages studied from birth to 10 days after birth, and among them are 6 three-copy genes. The decrease in granule cell proliferation demonstrated in newborn Ts1Cje cerebellum was correlated with a major gene dosage effect on the transcriptome in dissected cerebellar external granule cell layer. Conclusion High throughput gene expression analysis in the cerebellum of a large number of samples of Ts1Cje and euploid mice has revealed a prevailing gene dosage effect on triplicated genes. Moreover using an enriched cell population that is thought

Formation of artificial granules for proving gelation as the main mechanism of aerobic granulation in biological wastewater treatment.

PubMed

Li, Yun; Yang, Shu-Fang; Zhang, Jian-Jun; Li, Xiao-Yan

2014-01-01

In this study, gelation-facilitated biofilm formation as a new mechanism is proposed for the phenomenon of aerobic granulation in biological wastewater treatment. To obtain an experimental proof for the gelation-based theory, the granulation process was simulated in a chemical system using latex particles for bacterial cells and organic polymers (alginate and peptone) for extracellular polymeric substances (EPS) in a solution with the addition of cations (Ca²⁺, Mg²⁺ and Fe³⁺). The results showed that at a low alginate content (70 mg g⁻¹ mixed liquid suspended solids (MLSS)) flocculation was observed in the suspension with loose flocs. At a higher alginate content (180 mg g⁻¹ MLSS), together with discharge of small flocs, formation of artificial gel granules was successfully achieved leading to granulation. The artificial granules show a morphological property similar to that of actual microbial granules. However, if the protein content increased, granulation became difficult with little gel formation. The experimental work demonstrates the importance of the bonding interactions between EPS functional groups and cations in gel formation and granulation. The laboratory results on the formation of artificial granules provide a sound proof for the theory of gelation-facilitated biofilm formation as the main mechanism for aerobic granulation in sludge suspensions.

Roll compaction/dry granulation: comparison between roll mill and oscillating granulator in dry granulation.

PubMed

Sakwanichol, Jarunee; Puttipipatkhachorn, Satit; Ingenerf, Gernot; Kleinebudde, Peter

2012-01-01

Different experimental factorial designs were employed to evaluate granule properties obtained from oscillating granulator and roll mill. Four oscillating-granulator parameters were varied, i.e. rotor speed, oscillating angle, aperture of mesh screen and rotor type. Six roll-mill parameters that were throughput, speed ratio in both first and second stages, gap between roll pair in both stages and roll-surface texture were also investigated. Afterwards, the granule properties obtained from two milling types with similar median particle size were compared. All milling parameters in both milling types affected significantly the median particle size, size distribution and amount of fine particles (P < 0.05), except the rotor types of oscillating granulator on fines. Only three milling parameters influenced significantly the flowability (P < 0.05). These were the throughput and the gap size in the first stage of roll mill and the sieve size of oscillating granulator. In comparison between milling types, the differences of granule properties were not practically relevant. However, the roll mill had much higher capacity than the oscillating granulator about seven times, resulting in improving energy savings per unit of product. Consequently, the roll mill can be applied instead of oscillating granulator for roll compaction/dry granulation technique.

Visualization of Cytolytic T Cell Differentiation and Granule Exocytosis with T Cells from Mice Expressing Active Fluorescent Granzyme B

PubMed Central

Mouchacca, Pierre; Schmitt-Verhulst, Anne-Marie; Boyer, Claude

2013-01-01

To evaluate acquisition and activation of cytolytic functions during immune responses we generated knock in (KI) mice expressing Granzyme B (GZMB) as a fusion protein with red fluorescent tdTomato (GZMB-Tom). As for GZMB in wild type (WT) lymphocytes, GZMB-Tom was absent from naïve CD8 and CD4 T cells in GZMB-Tom-KI mice. It was rapidly induced in most CD8 T cells and in a subpopulation of CD4 T cells in response to stimulation with antibodies to CD3/CD28. A fraction of splenic NK cells expressed GZMB-Tom ex vivo with most becoming positive upon culture in IL-2. GZMB-Tom was present in CTL granules and active as a protease when these degranulated into cognate target cells, as shown with target cells expressing a specific FRET reporter construct. Using T cells from mice expressing GZMB-Tom but lacking perforin, we show that the transfer of fluorescent GZMB-Tom into target cells was dependent on perforin, favoring a role for perforin in delivery of GZMB at the target cells’ plasma membranes. Time-lapse video microscopy showed Ca++ signaling in CTL upon interaction with cognate targets, followed by relocalization of GZMB-Tom-containing granules to the synaptic contact zone. A perforin-dependent step was next visualized by the fluorescence signal from the non-permeant dye TO-PRO-3 at the synaptic cleft, minutes before the labeling of the target cell nucleus, characterizing a previously undescribed synaptic event in CTL cytolysis. Transferred OVA-specific GZMB-Tom-expressing CD8 T cells acquired GZMB-Tom expression in Listeria monocytogenes-OVA infected mice as soon as 48h after infection. These GZMB-Tom positive CD8 T cells localized in the splenic T-zone where they interacted with CD11c positive dendritic cells (DC), as shown by GZMB-Tom granule redistribution to the T/DC contact zone. GZMB-Tom-KI mice thus also provide tools to visualize acquisition and activation of cytolytic function in vivo. PMID:23840635

Cerebellar infarction in the territory of the medial branch of the superior cerebellar artery.

PubMed

Sohn, Sung-Il; Lee, Hyung; Lee, Seong-Ryong; Baloh, Robert W

2006-01-10

The authors studied 14 patients with an isolated cerebellar infarct in the territory of the medial branch of the superior cerebellar artery (MSCA). The most common clinical finding was severe gait ataxia with sudden falling (n = 9) or severe veering (n = 2). Cerebellar dysarthria was found in 8 patients. Eight patients had a mild unilateral limb ataxia. These findings emphasize that MSCA territory cerebellar infarction presented with the prominent gait ataxia and cerebellar dysarthria.

Imaging Ca2+-triggered exocytosis of single secretory granules on plasma membrane lawns from neuroendocrine cells.

PubMed

Lang, Thorsten

2008-01-01

This cell-free assay for exocytosis is particularly useful when spatial information about exocytotic sites and biochemical access to the plasma membrane within less than a minute is required. It is based on the study of plasma membrane lawns from secretory cells exhibiting secretory granules filled with neuropeptide Y-green fluorescent protein (NPY-GFP). The sample is prepared by subjecting NPY-GFP-expressing cells to a brief ultrasound pulse, leaving behind a basal, flat plasma membrane with fluorescent attached secretory organelles. These sheets can then be incubated in defined solutions with the benefit that complete solution changes can be achieved in less than 1 min. Individual secretory granules are monitored in the docked state and during exocytosis by video microscopy.

Secretory granule formation and membrane recycling by the trans-Golgi network in adipokinetic cells of Locusta migratoria in relation to flight and rest.

PubMed

Diederen, J H; Vullings, H G

1995-03-01

The influence of flight activity on the formation of secretory granules and the concomitant membrane recycling by the trans-Golgi network in the peptidergic neurosecretory adipokinetic cells of Locusta migratoria was investigated by means of ultrastructural morphometric methods. The patterns of labelling of the trans-Golgi network by the exogenous adsorptive endocytotic tracer wheat-germ agglutinin-conjugated horse-radish peroxidase and by the endogenous marker enzyme acid phosphatase were used as parameters and were measured by an automatic image analysis system. The results show that endocytosed fragments of plasma membrane with bound peroxidase label were transported to the trans-Golgi network and used to build new secretory granules. The amounts of peroxidase and especially of acid phosphatase within the trans-Golgi network showed a strong tendency to be smaller in flight-stimulated cells than in non-stimulated cells. The amounts of acid phosphatase in the immature secretory granules originating from the trans-Golgi network were significantly smaller in stimulated cells. The number of immature secretory granules positive for acid phosphatase tended to be higher in stimulated cells. Thus, flight stimulation of adipokinetic cells for 1 h influences the functioning of the trans-Golgi network; this most probably results in a slight enhancement of the production of secretory granules by the trans-Golgi network.

Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex

PubMed Central

Parthier, Daniel; Frings, Stephan; Möhrlen, Frank

2015-01-01

Calcium-activated chloride channels of the anoctamin (alias TMEM16) protein family fulfill critical functions in epithelial fluid transport, smooth muscle contraction and sensory signal processing. Little is known, however, about their contribution to information processing in the central nervous system. Here we examined the recent finding that a calcium-dependent chloride conductance impacts on GABAergic synaptic inhibition in Purkinje cells of the cerebellum. We asked whether anoctamin channels may underlie this chloride conductance. We identified two anoctamin channel proteins, ANO1 and ANO2, in the cerebellar cortex. ANO1 was expressed in inhibitory interneurons of the molecular layer and the granule cell layer. Both channels were expressed in Purkinje cells but, while ANO1 appeared to be retained in the cell body, ANO2 was targeted to the dendritic tree. Functional studies confirmed that ANO2 was involved in a calcium-dependent mode of ionic plasticity that reduces the efficacy of GABAergic synapses. ANO2 channels attenuated GABAergic transmission by increasing the postsynaptic chloride concentration, hence reducing the driving force for chloride influx. Our data suggest that ANO2 channels are involved in a Ca2+-dependent regulation of synaptic weight in GABAergic inhibition. Thus, in balance with the chloride extrusion mechanism via the co-transporter KCC2, ANO2 appears to regulate ionic plasticity in the cerebellum. PMID:26558388

Inositol Hexakisphosphate Kinase-3 Regulates the Morphology and Synapse Formation of Cerebellar Purkinje Cells via Spectrin/Adducin

PubMed Central

Fu, Chenglai; Xu, Jing; Li, Ruo-Jing; Crawford, Joshua A.; Khan, A. Basit; Ma, Ting Martin; Cha, Jiyoung Y.; Snowman, Adele M.; Pletnikov, Mikhail V.

2015-01-01

The inositol hexakisphosphate kinases (IP6Ks) are the principal enzymes that generate inositol pyrophosphates. There are three IP6Ks (IP6K1, 2, and 3). Functions of IP6K1 and IP6K2 have been substantially delineated, but little is known of IP6K3's role in normal physiology, especially in the brain. To elucidate functions of IP6K3, we generated mice with targeted deletion of IP6K3. We demonstrate that IP6K3 is highly concentrated in the brain in cerebellar Purkinje cells. IP6K3 physiologically binds to the cytoskeletal proteins adducin and spectrin, whose mutual interactions are perturbed in IP6K3-null mutants. Consequently, IP6K3 knock-out cerebella manifest abnormalities in Purkinje cell structure and synapse number, and the mutant mice display deficits in motor learning and coordination. Thus, IP6K3 is a major determinant of cytoskeletal disposition and function of cerebellar Purkinje cells. SIGNIFICANCE STATEMENT We identified and cloned a family of three inositol hexakisphosphate kinases (IP6Ks) that generate the inositol pyrophosphates, most notably 5-diphosphoinositol pentakisphosphate (IP7). Of these, IP6K3 has been least characterized. In the present study we generated IP6K3 knock-out mice and show that IP6K3 is highly expressed in cerebellar Purkinje cells. IP6K3-deleted mice display defects of motor learning and coordination. IP6K3-null mice manifest aberrations of Purkinje cells with a diminished number of synapses. IP6K3 interacts with the cytoskeletal proteins spectrin and adducin whose altered disposition in IP6K3 knock-out mice may mediate phenotypic features of the mutant mice. These findings afford molecular/cytoskeletal mechanisms by which the inositol polyphosphate system impacts brain function. PMID:26245967

Structural Basis of Cerebellar Microcircuits in the Rat

PubMed Central

Cerminara, Nadia L.; Aoki, Hanako; Loft, Michaela; Apps, Richard

2013-01-01

The topography of the cerebellar cortex is described by at least three different maps, with the basic units of each map termed “microzones,” “patches,” and “bands.” These are defined, respectively, by different patterns of climbing fiber input, mossy fiber input, and Purkinje cell (PC) phenotype. Based on embryological development, the “one-map” hypothesis proposes that the basic units of each map align in the adult animal and the aim of the present study was to test this possibility. In barbiturate anesthetized adult rats, nanoinjections of bidirectional tracer (Retrobeads and biotinylated dextran amine) were made into somatotopically identified regions within the hindlimb C1 zone in copula pyramidis. Injection sites were mapped relative to PC bands defined by the molecular marker zebrin II and were correlated with the pattern of retrograde cell labeling within the inferior olive and in the basilar pontine nuclei to determine connectivity of microzones and patches, respectively, and also with the distributions of biotinylated dextran amine-labeled PC terminals in the cerebellar nuclei. Zebrin bands were found to be related to both climbing fiber and mossy fiber inputs and also to cortical representation of different parts of the ipsilateral hindpaw, indicating a precise spatial organization within cerebellar microcircuitry. This precise connectivity extends to PC terminal fields in the cerebellar nuclei and olivonuclear projections. These findings strongly support the one-map hypothesis and suggest that, at the microcircuit level of resolution, the cerebellar cortex has a common plan of spatial organization for major inputs, outputs, and PC phenotype. PMID:24133249

Anti-Yo antibody uptake and interaction with its intracellular target antigen causes Purkinje cell death in rat cerebellar slice cultures: a possible mechanism for paraneoplastic cerebellar degeneration in humans with gynecological or breast cancers.

PubMed

Greenlee, John E; Clawson, Susan A; Hill, Kenneth E; Wood, Blair; Clardy, Stacey L; Tsunoda, Ikuo; Carlson, Noel G

2015-01-01

Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not

Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological or Breast Cancers

PubMed Central

Greenlee, John E.; Clawson, Susan A.; Hill, Kenneth E.; Wood, Blair; Clardy, Stacey L.; Tsunoda, Ikuo; Carlson, Noel G.

2015-01-01

Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not

Geminin deficiency enhances survival in a murine medulloblastoma model by inducing apoptosis of preneoplastic granule neuron precursors

PubMed Central

Sankar, Savita; Patterson, Ethan; Lewis, Emily M.; Waller, Laura E.; Tong, Caili; Dearborn, Joshua; Wozniak, David; Rubin, Joshua B.; Kroll, Kristen L.

2017-01-01

Medulloblastoma is the most common malignant brain cancer of childhood. Further understanding of tumorigenic mechanisms may define new therapeutic targets. Geminin maintains genome fidelity by controlling re-initiation of DNA replication within a cell cycle. In some contexts, Geminin inhibition induces cancer-selective cell cycle arrest and apoptosis and/or sensitizes cancer cells to Topoisomerase IIα inhibitors such as etoposide, which is used in combination chemotherapies for medulloblastoma. However, Geminin's potential role in medulloblastoma tumorigenesis remained undefined. Here, we found that Geminin is highly expressed in human and mouse medulloblastomas and in murine granule neuron precursor (GNP) cells during cerebellar development. Conditional Geminin loss significantly enhanced survival in the SmoA1 mouse medulloblastoma model. Geminin loss in this model also reduced numbers of preneoplastic GNPs persisting at one postnatal month, while at two postnatal weeks these cells exhibited an elevated DNA damage response and apoptosis. Geminin knockdown likewise impaired human medulloblastoma cell growth, activating G2 checkpoint and DNA damage response pathways, triggering spontaneous apoptosis, and enhancing G2 accumulation of cells in response to etoposide treatment. Together, these data suggest preneoplastic and cancer cell-selective roles for Geminin in medulloblastoma, and suggest that targeting Geminin may impair tumor growth and enhance responsiveness to Topoisomerase IIα-directed chemotherapies. PMID:29234490

Sensorimotor-correlated discharge recorded from ensembles of cerebellar Purkinje cells varies across the estrous cycle of the rat.

PubMed

Smith, S S

1995-09-01

1. In the present study, locomotor-correlated activity of cerebellar Purkinje cells, recorded using arrays of microwires chronically implanted in adult female rats, was examined across estrous-cycle-associated fluctuations in endogenous sex steroids. Ongoing studies from this laboratory have shown that systemic and local administration of the sex steroid 17 beta-estradiol (E2) augments excitatory responses of cerebellar Purkinje cells to iontophoretically applied glutamate, recorded in vivo from anesthetized female rats. In addition, this steroid potentiated discharge correlated with limb movement. For the present study, extracellular single-unit activity was recorded from as many as 5-11 Purkinje cells simultaneously during treadmill locomotion paradigms. Motor modulation of activity was recorded across three to five consecutive estrous cycles from behaviorally identified cohorts of neurons to test the hypothesis that fluctuations in endogenous sex steroids alter motor modulation of Purkinje cell discharge. 2. Locomotor-associated discharge correlated with treadmill locomotion was increased by a mean of 47% on proestrus, when E2 levels are elevated, relative to diestrus 1. These changes in discharge rate during treadmill locomotion were of significantly greater magnitude than corresponding cyclic alterations in discharge during stationary periods. 3. Correlations with the circadian cycle were also significant, because peak levels of locomotor-associated discharge on the night of behavioral estrus, following elevations in circulating E2, were on average 67% greater than corresponding discharge recorded during the light (proestrus). 4. Alterations in the step cycle were also observed across the estrous cycle: significant decreases in the duration of the flexion phase (by 265 ms, P < 0.05) were noted on estrus compared with diestrus. 5. When recorded on estrus, Purkinje cell discharge correlated with the stance or flexion phase of the step cycle was greater in

Interneuron- and GABAA receptor-specific inhibitory synaptic plasticity in cerebellar Purkinje cells

NASA Astrophysics Data System (ADS)

He, Qionger; Duguid, Ian; Clark, Beverley; Panzanelli, Patrizia; Patel, Bijal; Thomas, Philip; Fritschy, Jean-Marc; Smart, Trevor G.

2015-07-01

Inhibitory synaptic plasticity is important for shaping both neuronal excitability and network activity. Here we investigate the input and GABAA receptor subunit specificity of inhibitory synaptic plasticity by studying cerebellar interneuron-Purkinje cell (PC) synapses. Depolarizing PCs initiated a long-lasting increase in GABA-mediated synaptic currents. By stimulating individual interneurons, this plasticity was observed at somatodendritic basket cell synapses, but not at distal dendritic stellate cell synapses. Basket cell synapses predominantly express β2-subunit-containing GABAA receptors; deletion of the β2-subunit ablates this plasticity, demonstrating its reliance on GABAA receptor subunit composition. The increase in synaptic currents is dependent upon an increase in newly synthesized cell surface synaptic GABAA receptors and is abolished by preventing CaMKII phosphorylation of GABAA receptors. Our results reveal a novel GABAA receptor subunit- and input-specific form of inhibitory synaptic plasticity that regulates the temporal firing pattern of the principal output cells of the cerebellum.

Beta cell chromogranin B is partially segregated in distinct granules and can be released separately from insulin in response to stimulation.

PubMed

Giordano, T; Brigatti, C; Podini, P; Bonifacio, E; Meldolesi, J; Malosio, M L

2008-06-01

We investigated, in three beta cell lines (INS-1E, RIN-5AH, betaTC3) and in human and rodent primary beta cells, the storage and release of chromogranin B, a secretory protein expressed in beta cells and postulated to play an autocrine role. We asked whether chromogranin B is stored together with and discharged in constant ratio to insulin upon various stimuli. The intracellular distribution of insulin and chromogranin B was revealed by immunofluorescence followed by three-dimensional image reconstruction and by immunoelectron microscopy; their stimulated discharge was measured by ELISA and immunoblot analysis of homogenates and incubation media. Insulin and chromogranin B, co-localised in the Golgi complex/trans-Golgi network, appeared largely segregated from each other in the secretory granule compartment. In INS-1E cells, the percentage of granules positive only for insulin or chromogranin B and of those positive for both was 66, 7 and 27%, respectively. In resting cells, both insulin and chromogranin B were concentrated in the granule cores; upon stimulation, chromogranin B (but not insulin) was largely redistributed to the core periphery and the surrounding halo. Strong stimulation with a secretagogue mixture induced parallel release of insulin and chromogranin B, whereas with 3-isobutyl-1-methylxantine and forskolin +/- high glucose release of chromogranin B predominated. Weak, Ca(2+)-dependent stimulation with ionomycin or carbachol induced exclusive release of chromogranin B, suggesting a higher Ca(2+) sensitivity of the specific granules. The unexpected complexity of the beta cell granule population in terms of heterogeneity, molecular plasticity and the differential discharge, could play an important role in physiological control of insulin release and possibly also in beta cell pathology.

Gating of Long-Term Potentiation by Nicotinic Acetylcholine Receptors at the Cerebellum Input Stage

PubMed Central

Prestori, Francesca; Bonardi, Claudia; Mapelli, Lisa; Lombardo, Paola; Goselink, Rianne; De Stefano, Maria Egle; Gandolfi, Daniela; Mapelli, Jonathan; Bertrand, Daniel; Schonewille, Martijn; De Zeeuw, Chris; D’Angelo, Egidio

2013-01-01

The brain needs mechanisms able to correlate plastic changes with local circuit activity and internal functional states. At the cerebellum input stage, uncontrolled induction of long-term potentiation or depression (LTP or LTD) between mossy fibres and granule cells can saturate synaptic capacity and impair cerebellar functioning, which suggests that neuromodulators are required to gate plasticity processes. Cholinergic systems innervating the cerebellum are thought to enhance procedural learning and memory. Here we show that a specific subtype of acetylcholine receptors, the α7-nAChRs, are distributed both in cerebellar mossy fibre terminals and granule cell dendrites and contribute substantially to synaptic regulation. Selective α7-nAChR activation enhances the postsynaptic calcium increase, allowing weak mossy fibre bursts, which would otherwise cause LTD, to generate robust LTP. The local microperfusion of α7-nAChR agonists could also lead to in vivo switching of LTD to LTP following sensory stimulation of the whisker pad. In the cerebellar flocculus, α7-nAChR pharmacological activation impaired vestibulo-ocular-reflex adaptation, probably because LTP was saturated, preventing the fine adjustment of synaptic weights. These results show that gating mechanisms mediated by specific subtypes of nicotinic receptors are required to control the LTD/LTP balance at the mossy fibre-granule cell relay in order to regulate cerebellar plasticity and behavioural adaptation. PMID:23741401

AMP Kinase Activation Alters Oxidant-Induced Stress Granule Assembly by Modulating Cell Signaling and Microtubule Organization.

PubMed

Mahboubi, Hicham; Koromilas, Antonis E; Stochaj, Ursula

2016-10-01

Eukaryotic cells assemble stress granules (SGs) when translation initiation is inhibited. Different cell signaling pathways regulate SG production. Particularly relevant to this process is 5'-AMP-activated protein kinase (AMPK), which functions as a stress sensor and is transiently activated by adverse physiologic conditions. Here, we dissected the role of AMPK for oxidant-induced SG formation. Our studies identified multiple steps of de novo SG assembly that are controlled by the kinase. Single-cell analyses demonstrated that pharmacological AMPK activation prior to stress exposure changed SG properties, because the granules became more abundant and smaller in size. These altered SG characteristics correlated with specific changes in cell survival, cell signaling, cytoskeletal organization, and the abundance of translation initiation factors. Specifically, AMPK activation increased stress-induced eukaryotic initiation factor (eIF) 2α phosphorylation and reduced the concentration of eIF4F complex subunits eIF4G and eIF4E. At the same time, the abundance of histone deacetylase 6 (HDAC6) was diminished. This loss of HDAC6 was accompanied by increased acetylation of α-tubulin on Lys40. Pharmacological studies further confirmed this novel AMPK-HDAC6 interplay and its importance for SG biology. Taken together, we provide mechanistic insights into the regulation of SG formation. We propose that AMPK activation stimulates oxidant-induced SG formation but limits their fusion into larger granules. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Evolution of the cerebellar cortex: the selective expansion of prefrontal-projecting cerebellar lobules.

PubMed

Balsters, J H; Cussans, E; Diedrichsen, J; Phillips, K A; Preuss, T M; Rilling, J K; Ramnani, N

2010-02-01

It has been suggested that interconnected brain areas evolve in tandem because evolutionary pressures act on complete functional systems rather than on individual brain areas. The cerebellar cortex has reciprocal connections with both the prefrontal cortex and motor cortex, forming independent loops with each. Specifically, in capuchin monkeys cerebellar cortical lobules Crus I and Crus II connect with prefrontal cortex, whereas the primary motor cortex connects with cerebellar lobules V, VI, VIIb, and VIIIa. Comparisons of extant primate species suggest that the prefrontal cortex has expanded more than cortical motor areas in human evolution. Given the enlargement of the prefrontal cortex relative to motor cortex in humans, our hypothesis would predict corresponding volumetric increases in the parts of the cerebellum connected to the prefrontal cortex, relative to cerebellar lobules connected to the motor cortex. We tested the hypothesis by comparing the volumes of cerebellar lobules in structural MRI scans in capuchins, chimpanzees and humans. The fractions of cerebellar volume occupied by Crus I and Crus II were significantly larger in humans compared to chimpanzees and capuchins. Our results therefore support the hypothesis that in the cortico-cerebellar system, functionally related structures evolve in concert with each other. The evolutionary expansion of these prefrontal-projecting cerebellar territories might contribute to the evolution of the higher cognitive functions of humans. Copyright (c) 2009 Elsevier Inc. All rights reserved.

PHB granules are attached to the nucleoid via PhaM in Ralstonia eutropha.

PubMed

Wahl, Andreas; Schuth, Nora; Pfeiffer, Daniel; Nussberger, Stephan; Jendrossek, Dieter

2012-11-16

Poly(3-hydroxybutyrate) (PHB) granules are important storage compounds of carbon and energy in many prokaryotes which allow survival of the cells in the absence of suitable carbon sources. Formation and subcellular localization of PHB granules was previously assumed to occur randomly in the cytoplasm of PHB accumulating bacteria. However, contradictionary results on subcellular localization of PHB granules in Ralstonia eutropha were published, recently. Here, we provide evidence by transmission electron microscopy that PHB granules are localized in close contact to the nucleoid region in R. eutropha during growth on nutrient broth. Binding of PHB granules to the nucleoid is mediated by PhaM, a PHB granule associated protein with phasin-like properties that is also able to bind to DNA and to phasin PhaP5. Over-expression of PhaM resulted in formation of many small PHB granules that were always attached to the nucleoid region. In contrast, PHB granules of ∆phaM strains became very large and distribution of granules to daughter cells was impaired. Association of PHB granules to the nucleoid region was prevented by over-expression of PhaP5 and clusters of several PHB granules were mainly localized near the cell poles. Subcellular localization of PHB granules is controlled in R. eutropha and depends on the presence and concentrations of at least two PHB granule associated proteins, PhaM and PhaP5.

Changes in the cerebellar and cerebro-cerebellar circuit in type 2 diabetes.

PubMed

Fang, Peng; An, Jie; Tan, Xin; Zeng, Ling-Li; Shen, Hui; Qiu, Shijun; Hu, Dewen

2017-04-01

Currently, 422 million adults suffer from diabetes worldwide, leading to tremendous disabilities and a great burden to families and society. Functional and structural MRIs have demonstrated that patients with type 2 diabetes mellitus (T2DM) exhibit abnormalities in brain regions in the cerebral cortex. However, the changes of cerebellar anatomical connections in diabetic patients remains unclear. In the current study, diffusion tensor imaging deterministic tractography and statistical analysis were employed to investigate abnormal cerebellar anatomical connections in diabetic patients. This is the first study to investigate the altered cerebellar anatomical connectivity in T2DM patients. Decreased anatomical connections were found in the cerebellar and cerebro-cerebellar circuits of T2DM patients, providing valuable new insights into the potential neuro-pathophysiology of diabetes-related motor and cognitive deficits. Copyright © 2017. Published by Elsevier Inc.

Exocyst sec5 regulates exocytosis of newcomer insulin granules underlying biphasic insulin secretion.

PubMed

Xie, Li; Zhu, Dan; Kang, Youhou; Liang, Tao; He, Yu; Gaisano, Herbert Y

2013-01-01

The exocyst complex subunit Sec5 is a downstream effector of RalA-GTPase which promotes RalA-exocyst interactions and exocyst assembly, serving to tether secretory granules to docking sites on the plasma membrane. We recently reported that RalA regulates biphasic insulin secretion in pancreatic islet β cells in part by tethering insulin secretory granules to Ca(2+) channels to assist excitosome assembly. Here, we assessed β cell exocytosis by patch clamp membrane capacitance measurement and total internal reflection fluorescence microscopy to investigate the role of Sec5 in regulating insulin secretion. Sec5 is present in human and rodent islet β cells, localized to insulin granules. Sec5 protein depletion in rat INS-1 cells inhibited depolarization-induced release of primed insulin granules from both readily-releasable pool and mobilization from the reserve pool. This reduction in insulin exocytosis was attributed mainly to reduction in recruitment and exocytosis of newcomer insulin granules that undergo minimal docking time at the plasma membrane, but which encompassed a larger portion of biphasic glucose stimulated insulin secretion. Sec5 protein knockdown had little effect on predocked granules, unless vigorously stimulated by KCl depolarization. Taken together, newcomer insulin granules in β cells are more sensitive than predocked granules to Sec5 regulation.

Interactions between Inhibitory Interneurons and Excitatory Associational Circuitry in Determining Spatio-Temporal Dynamics of Hippocampal Dentate Granule Cells: A Large-Scale Computational Study

PubMed Central

Hendrickson, Phillip J.; Yu, Gene J.; Song, Dong; Berger, Theodore W.

2015-01-01

This paper reports on findings from a million-cell granule cell model of the rat dentate gyrus that was used to explore the contributions of local interneuronal and associational circuits to network-level activity. The model contains experimentally derived morphological parameters for granule cells, which each contain approximately 200 compartments, and biophysical parameters for granule cells, basket cells, and mossy cells that were based both on electrophysiological data and previously published models. Synaptic input to cells in the model consisted of glutamatergic AMPA-like EPSPs and GABAergic-like IPSPs from excitatory and inhibitory neurons, respectively. The main source of input to the model was from layer II entorhinal cortical neurons. Network connectivity was constrained by the topography of the system, and was derived from axonal transport studies, which provided details about the spatial spread of axonal terminal fields, as well as how subregions of the medial and lateral entorhinal cortices project to subregions of the dentate gyrus. Results of this study show that strong feedback inhibition from the basket cell population can cause high-frequency rhythmicity in granule cells, while the strength of feedforward inhibition serves to scale the total amount of granule cell activity. Results furthermore show that the topography of local interneuronal circuits can have just as strong an impact on the development of spatio-temporal clusters in the granule cell population as the perforant path topography does, both sharpening existing clusters and introducing new ones with a greater spatial extent. Finally, results show that the interactions between the inhibitory and associational loops can cause high frequency oscillations that are modulated by a low-frequency oscillatory signal. These results serve to further illustrate the importance of topographical constraints on a global signal processing feature of a neural network, while also illustrating how rich

Docking is not a prerequisite but a temporal constraint for fusion of secretory granules.

PubMed

Kasai, Kazuo; Fujita, Takuji; Gomi, Hiroshi; Izumi, Tetsuro

2008-07-01

We examined secretory granule dynamics using total internal reflection fluorescence microscopy in normal pancreatic beta cells and their mutants devoid of Rab27a and/or its effector, granuphilin, which play critical roles in the docking and recruitment of insulin granules to the plasma membrane. In the early phase of glucose stimulation in wild-type cells, we observed marked fusion of granules recruited from a relatively distant area, in parallel with that from granules located underneath the plasma membrane. Furthermore, despite a lack of granules directly attached to the plasma membrane, both spontaneous and evoked fusion was increased in granuphilin-null cells. In addition to these granuphilin-null phenotypes, Rab27a/granuphilin doubly deficient cells showed the decreases in granules located next to the docked area and in fusion from granules near the plasma membrane in the early phase of glucose-stimulated secretion, similar to Rab27a-mutated cells. Thus, the two proteins play nonoverlapping roles in insulin exocytosis: granuphilin acts on the granules underneath the plasma membrane, whereas Rab27a acts on those in a more distal area. These findings demonstrate that, in contrast to our conventional understanding, stable attachment of secretory granules to the plasma membrane is not prerequisite but temporally inhibitory for both spontaneous and evoked fusion.

Shp2-Dependent ERK Signaling Is Essential for Induction of Bergmann Glia and Foliation of the Cerebellum

PubMed Central

Li, Kairong; Leung, Alan W.; Guo, Qiuxia; Yang, Wentian

2014-01-01

Folding of the cortex and the persistence of radial glia (RG)-like cells called Bergmann glia (BG) are hallmarks of the mammalian cerebellum. Similar to basal RG in the embryonic neocortex, BG maintain only basal processes and continuously express neural stem cell markers. Past studies had focused on the function of BG in granule cell migration and how granule cell progenitors (GCP) regulate cerebellar foliation. The molecular control of BG generation and its role in cerebellar foliation are less understood. Here, we have analyzed the function of the protein tyrosine phosphatase Shp2 in mice by deleting its gene Ptpn11 in the entire cerebellum or selectively in the GCP lineage. Deleting Ptpn11 in the entire cerebellum by En1-cre blocks transformation of RG into BG but preserves other major cerebellar cell types. In the absence of BG, inward invagination of GCP persists but is uncoupled from the folding of the Purkinje cell layer and the basement membrane, leading to disorganized lamination and an absence of cerebellar folia. In contrast, removing Ptpn11 in the GCP lineage by Atoh1-cre has no effect on cerebellar development, indicating that Shp2 is not cell autonomously required in GCP. Furthermore, we demonstrate that Ptpn11 interacts with Fgf8 and is essential for ERK activation in RG and nascent BG. Finally, expressing constitutively active MEK1 rescues BG formation and cerebellar foliation in Shp2-deficient cerebella. Our results demonstrate an essential role of Shp2 in BG specification via fibroblast growth factor/extracellular signal-regulated protein kinase signaling, and reveal a crucial function of BG in organizing cerebellar foliation. PMID:24431450

Correlation between loose density and compactibility of granules prepared by various granulation methods.

PubMed

Murakami, H; Yoneyama, T; Nakajima, K; Kobayashi, M

2001-03-23

The objectives of this study were to prepare the lactose granules by various granulation methods using polyethylene glycol 6000 (PEG 6000) as a binder and to evaluate the effects of granulation methods on the compressibility and compactibility of granules in tabletting. Lactose was granulated by seven granulation methods -- four wet granulations including wet massing granulation, wet high-speed mixer granulation, wet fluidized bed granulation and wet tumbling fluidized bed granulation; and three melt granulations including melt high-speed mixer granulation, melt fluidized bed granulation and melt tumbling fluidized bed granulation. The loose density, angle of repose, granule size distribution, mean diameter of granules, and the tensile strength and porosity of tablets were evaluated. The compactibilities of granules were varied by the granulation methods. However, the difference in compactibility of granules could not be explained due to the difference in compressibility, since there was no difference in Heckel plots due to granulation methods. Among their granule properties, the loose density of granules seemed to have a correlation with the tablet strength regardless of the granulation methods.

Stimulation of insulin release by glucose is associated with an increase in the number of docked granules in the beta-cells of rat pancreatic islets.

PubMed

Straub, Susanne G; Shanmugam, Geetha; Sharp, Geoffrey W G

2004-12-01

Electron microscopy and quantitative stereological techniques were used to study the dynamics of the docked granule pool in the rat pancreatic beta-cell. The mean number of granules per beta-cell was 11,136. After equilibration in RPMI containing 5.6 mmol/l glucose, 6.4% of the granules (approximately 700) were docked at the plasma membrane (also measured as [means +/- SE] 4.3 +/- 0.6 docked granules per 10 microm of plasma membrane at the perimeter of the cell sections). After a 40-min exposure to 16.7 mmol/l glucose, 10.2% of the granules (approximately 1,060) were docked (6.4 +/- 0.8 granules per 10 microm of plasma membrane). Thus, the docked pool increased by 50% during stimulation with glucose. Islets were also exposed to 16.7 mmol/l glucose in the absence or presence of 10 micromol/l nitrendipine. In the absence and presence of nitrendipine, there were 6.1 +/- 0.7 and 6.3 +/- 0.6 granules per 10 microm of membrane, respectively. Thus, glucose increased granule docking independently of increased [Ca2+]i and exocytosis. The data suggest a limit to the number of docking sites. As the rate of docking exceeded the rate of exocytosis, docking is not rate limiting for insulin release. Only with extremely high release rates, glucose stimulation after a 4-h incubation with a high concentration of fatty acid-free BSA, was the docked granule pool reduced in size.

MICROLESIONS OF THE INFERIOR OLIVE REDUCE VESTIBULAR MODULATION OF PURKINJE CELL COMPLEX AND SIMPLE SPIKES IN MOUSE CEREBELLUM

PubMed Central

Barmack, N.H.; Yakhnitsa, V.

2011-01-01

Cerebellar Purkinje cells have two distinct action potentials: Complex spikes (CSs) are evoked by single climbing fibers that originate from the contralateral inferior olive. Simple spikes (SSs) are often ascribed to mossy fiber---granule cell---parallel fiber inputs to Purkinje cells. Although generally accepted, this view lacks experimental support. Vestibular stimulation independently activates primary afferent mossy fibers and tertiary afferent climbing fibers that project to theuvula-nodulus (folia 8-10). CSs and SSs normally discharge antiphasically during sinusoidal roll-tilt. When CSs increase, SSs decrease. We tested the relative independence of these pathways in mice by making electrolytic microlesions of the two inferior olivary nuclei from which vestibular climbing fibers originate; the β-nucleus and dorsomedial cell column (DMCC). This reduced vestibular climbing fiber signaling to the contralateral folia 8-10, while leaving intact vestibular primary and secondary afferent mossy fibers. We recorded from Purkinje cells and interneurons in folia 8-10, identified by juxtacellular labeling with neurobiotin. Microlesions of the inferior olive increased the spontaneous discharge of SSs in contralateral folia 8-10, but blocked their modulation during vestibular stimulation. The vestibularly-evoked discharge of excitatory cerebellar interneurons (granule cells and unipolar brush cells) was not modified by olivary microlesions. The modulated discharge of stellate cells, but not Golgi cells was reduced by olivary microlesions. We conclude that vestibular modulation of CSs and SSs depends on intact climbing fibers. The absence of vestibularly-modulated SSs following olivary microlesions reflects the loss of climbing fiber-evoked stellate cell discharge. PMID:21734274

Biogenesis of zinc storage granules in Drosophila melanogaster.

PubMed

Tejeda-Guzmán, Carlos; Rosas-Arellano, Abraham; Kroll, Thomas; Webb, Samuel M; Barajas-Aceves, Martha; Osorio, Beatriz; Missirlis, Fanis

2018-03-19

Membrane transporters and sequestration mechanisms concentrate metal ions differentially into discrete subcellular microenvironments for use in protein cofactors, signalling, storage or excretion. Here we identify zinc storage granules as the insect's major zinc reservoir in principal Malpighian tubule epithelial cells of Drosophila melanogaster The concerted action of Adaptor Protein-3, Rab32, HOPS and BLOC complexes as well as of the white-scarlet (ABCG2-like) and ZnT35C (ZnT2/ZnT3/ZnT8-like) transporters is required for zinc storage granule biogenesis. Due to lysosome-related organelle defects caused by mutations in the homologous human genes, patients with Hermansky-Pudlak syndrome may lack zinc granules in beta pancreatic cells, intestinal paneth cells and presynaptic vesicles of hippocampal mossy fibers. © 2018. Published by The Company of Biologists Ltd.

PHB granules are attached to the nucleoid via PhaM in Ralstonia eutropha

PubMed Central

2012-01-01

Background Poly(3-hydroxybutyrate) (PHB) granules are important storage compounds of carbon and energy in many prokaryotes which allow survival of the cells in the absence of suitable carbon sources. Formation and subcellular localization of PHB granules was previously assumed to occur randomly in the cytoplasm of PHB accumulating bacteria. However, contradictionary results on subcellular localization of PHB granules in Ralstonia eutropha were published, recently. Results Here, we provide evidence by transmission electron microscopy that PHB granules are localized in close contact to the nucleoid region in R. eutropha during growth on nutrient broth. Binding of PHB granules to the nucleoid is mediated by PhaM, a PHB granule associated protein with phasin-like properties that is also able to bind to DNA and to phasin PhaP5. Over-expression of PhaM resulted in formation of many small PHB granules that were always attached to the nucleoid region. In contrast, PHB granules of ∆phaM strains became very large and distribution of granules to daughter cells was impaired. Association of PHB granules to the nucleoid region was prevented by over-expression of PhaP5 and clusters of several PHB granules were mainly localized near the cell poles. Conclusion Subcellular localization of PHB granules is controlled in R. eutropha and depends on the presence and concentrations of at least two PHB granule associated proteins, PhaM and PhaP5. PMID:23157596

Autophagy meets fused in sarcoma-positive stress granules.

PubMed

Matus, Soledad; Bosco, Daryl A; Hetz, Claudio

2014-12-01

Mutations in fused in sarcoma and/or translocated in liposarcoma (FUS, TLS or FUS) are linked to familial cases of amyotrophic lateral sclerosis (ALS). Mutant FUS selectively accumulates into discrete cytosolic structures known as stress granules under various stress conditions. In addition, mutant FUS expression can alter the dynamics and morphology of stress granules. Although the link between mutant FUS and stress granules is well established, the mechanisms modulating stress granule formation and disassembly in the context of ALS are poorly understood. In this issue of Neurobiology of Aging, Ryu et al. uncover the impact of autophagy on the potential toxicity of mutant FUS-positive stress granules. The authors provide evidence indicating that enhanced autophagy activity reduces the number of stress granules, which in the case of cells containing mutant FUS-positive stress granules, is neuroprotective. Overall, this study identifies an intersection between the proteostasis network and alterations in RNA metabolism in ALS through the dynamic assembly and disassembly of stress granules. Copyright © 2014 Elsevier Inc. All rights reserved.

Neuroprotective Effect of Carnosine on Primary Culture of Rat Cerebellar Cells under Oxidative Stress.

PubMed

Lopachev, A V; Lopacheva, O M; Abaimov, D A; Koroleva, O V; Vladychenskaya, E A; Erukhimovich, A A; Fedorova, T N

2016-05-01

Dipeptide carnosine (β-alanyl-L-histidine) is a natural antioxidant, but its protective effect under oxidative stress induced by neurotoxins is studied insufficiently. In this work, we show the neuroprotective effect of carnosine in primary cultures of rat cerebellar cells under oxidative stress induced by 1 mM 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), which directly generates free radicals both in the medium and in the cells, and 20 nM rotenone, which increases the amount of intracellular reactive oxygen species (ROS). In both models, adding 2 mM carnosine to the incubation medium decreased cell death calculated using fluorescence microscopy and enhanced cell viability estimated by the MTT assay. The antioxidant effect of carnosine inside cultured cells was demonstrated using the fluorescence probe dichlorofluorescein. Carnosine reduced by half the increase in the number of ROS in neurons induced by 20 nM rotenone. Using iron-induced chemiluminescence, we showed that preincubation of primary neuronal cultures with 2 mM carnosine prevents the decrease in endogenous antioxidant potential of cells induced by 1 mM AAPH and 20 nM rotenone. Using liquid chromatography-mass spectrometry, we showed that a 10-min incubation of neuronal cultures with 2 mM carnosine leads to a 14.5-fold increase in carnosine content in cell lysates. Thus, carnosine is able to penetrate neurons and exerts an antioxidant effect. Western blot analysis revealed the presence of the peptide transporter PEPT2 in rat cerebellar cells, which suggests the possibility of carnosine transport into the cells. At the same time, Western blot analysis showed no carnosine-induced changes in the level of apoptosis regulating proteins of the Bcl-2 family and in the phosphorylation of MAP kinases, which suggests that carnosine could have minimal or no side effects on proliferation and apoptosis control systems in normal cells.

Loss of protohaem IX farnesyltransferase in mature dentate granule cells impairs short-term facilitation at mossy fibre to CA3 pyramidal cell synapses.

PubMed

Booker, Sam A; Campbell, Graham R; Mysiak, Karolina S; Brophy, Peter J; Kind, Peter C; Mahad, Don J; Wyllie, David J A

2017-03-15

Neurodegenerative disorders can exhibit dysfunctional mitochondrial respiratory chain complex IV activity. Conditional deletion of cytochrome c oxidase, the terminal enzyme in the respiratory electron transport chain of mitochondria, from hippocampal dentate granule cells in mice does not affect low-frequency dentate to CA3 glutamatergic synaptic transmission. High-frequency dentate to CA3 glutamatergic synaptic transmission and feedforward inhibition are significantly attenuated in cytochrome c oxidase-deficient mice. Intact presynaptic mitochondrial function is critical for the short-term dynamics of mossy fibre to CA3 synaptic function. Neurodegenerative disorders are characterized by peripheral and central symptoms including cognitive impairments which have been associated with reduced mitochondrial function, in particular mitochondrial respiratory chain complex IV or cytochrome c oxidase activity. In the present study we conditionally removed a key component of complex IV, protohaem IX farnesyltransferase encoded by the COX10 gene, in granule cells of the adult dentate gyrus. Utilizing whole-cell patch-clamp recordings from morphologically identified CA3 pyramidal cells from control and complex IV-deficient mice, we found that reduced mitochondrial function did not result in overt deficits in basal glutamatergic synaptic transmission at the mossy-fibre synapse because the amplitude, input-output relationship and 50 ms paired-pulse facilitation were unchanged following COX10 removal from dentate granule cells. However, trains of stimuli given at high frequency (> 20 Hz) resulted in dramatic reductions in short-term facilitation and, at the highest frequencies (> 50 Hz), also reduced paired-pulse facilitation, suggesting a requirement for adequate mitochondrial function to maintain glutamate release during physiologically relevant activity patterns. Interestingly, local inhibition was reduced, suggesting the effect observed was not restricted to synapses

Purification of a factor from the granules of a rat natural killer cell line (RNK) that reduces tumor cell growth and changes tumor morphology. Molecular identity with a granule serine protease (RNKP-1).

PubMed

Sayers, T J; Wiltrout, T A; Sowder, R; Munger, W L; Smyth, M J; Henderson, L E

1992-01-01

We have purified a protein from the granules of the rat NK leukemia cell line (RNK) that is cytostatic to a variety of tumor cells. This protein shows no species specificity because certain tumor cell lines of mouse, rat, and human origin were equally sensitive to its growth inhibitory effects. Treatment of sensitive cells resulted in a rounding of the cells followed by homotypic aggregation into large aggregates. The granule protein was distinct from cytolysin, Na-Cbz-Lys-thiobenzylester-esterase, or leukolexin. It had a molecular mass of 29 to 31 kDa, bound strongly to heparin, was inactivated by heating at 70 degrees C for 5 min or reduction, but was stable to trypsin treatment. By using molecular sieve chromatography, heparin agarose chromatography, and reverse phase HPLC, this protein was purified to homogeneity. The first 33 amino acids of the N-terminal amino acid sequence showed complete identity to the sequence predicted from a rat serine protease gene recently cloned and designated RNKP-1. Therefore we have purified a novel serine protease and demonstrated that it has effects on the growth and morphology of certain tumor cells. Other serine proteases that were structurally related and have substantial homology with RNKP-1 at the amino acid level showed neither growth inhibitory properties nor affected the morphology of the tumor target cells we used.

Cerebellar modulation of frontal cortex dopamine efflux in mice: relevance to autism and schizophrenia.

PubMed

Mittleman, Guy; Goldowitz, Daniel; Heck, Detlef H; Blaha, Charles D

2008-07-01

Cerebellar and frontal cortical pathologies have been commonly reported in schizophrenia, autism, and other developmental disorders. Whether there is a relationship between prefrontal and cerebellar pathologies is unknown. Using fixed potential amperometry, dopamine (DA) efflux evoked by cerebellar or, dentate nucleus electrical stimulation (50 Hz, 200 muA) was recorded in prefrontal cortex of urethane anesthetized lurcher (Lc/+) mice with 100% loss of cerebellar Purkinje cells and wildtype (+/+) control mice. Cerebellar stimulation with 25 and 100 pulses evoked prefrontal cortex DA efflux in +/+ mice that persisted for 12 and 25 s poststimulation, respectively. In contrast, 25 pulse cerebellar stimulation failed to evoke prefrontal cortex DA efflux in Lc/+ mice indicating a dependency on cerebellar Purkinje cell outputs. Dentate nucleus stimulation (25 pulses) evoked a comparable but briefer (baseline recovery within 7 s) increase in prefrontal cortex DA efflux compared to similar cerebellar stimulation in +/+ mice. However, in Lc/+ mice 25 pulse dentate nucleus evoked prefrontal cortex DA efflux was attenuated by 60% with baseline recovery within 4 s suggesting that dentate nucleus outputs to prefrontal cortex remain partially functional. DA reuptake blockade enhanced 100 pulse stimulation evoked prefrontal cortex responses, while serotonin or norepinephrine reuptake blockade were without effect indicating the specificity of the amperometric recordings to DA. Results provide neurochemical evidence that the cerebellum can modulate DA efflux in the prefrontal cortex. Together, these findings may explain why cerebellar and frontal cortical pathologies co-occur, and may provide a mechanism that accounts for the diversity of symptoms common to multiple developmental disorders.

Cerebellar Modulation of Frontal Cortex Dopamine Efflux in Mice: Relevance to Autism and Schizophrenia

PubMed Central

MITTLEMAN, GUY; GOLDOWITZ, DANIEL; HECK, DETLEF H.; BLAHA, CHARLES D.

2013-01-01

Cerebellar and frontal cortical pathologies have been commonly reported in schizophrenia, autism, and other developmental disorders. Whether there is a relationship between prefrontal and cerebellar pathologies is unknown. Using fixed potential amperometry, dopamine (DA) efflux evoked by cerebellar or, dentate nucleus electrical stimulation (50 Hz, 200 μA) was recorded in prefrontal cortex of urethane anesthetized lurcher (Lc/+) mice with 100% loss of cerebellar Purkinje cells and wildtype (+/+) control mice. Cerebellar stimulation with 25 and 100 pulses evoked prefrontal cortex DA efflux in +/+ mice that persisted for 12 and 25 s poststimulation, respectively. In contrast, 25 pulse cerebellar stimulation failed to evoke prefrontal cortex DA efflux in Lc/+ mice indicating a dependency on cerebellar Purkinje cell outputs. Dentate nucleus stimulation (25 pulses) evoked a comparable but briefer (baseline recovery within 7 s) increase in prefrontal cortex DA efflux compared to similar cerebellar stimulation in +/+ mice. However, in Lc/+ mice 25 pulse dentate nucleus evoked prefrontal cortex DA efflux was attenuated by 60% with baseline recovery within 4 s suggesting that dentate nucleus outputs to prefrontal cortex remain partially functional. DA reuptake blockade enhanced 100 pulse stimulation evoked pre-frontal cortex responses, while serotonin or norepinephrine reuptake blockade were without effect indicating the specificity of the amperometric recordings to DA. Results provide neurochemical evidence that the cerebellum can modulate DA efflux in the prefrontal cortex. Together, these findings may explain why cerebellar and frontal cortical pathologies co-occur, and may provide a mechanism that accounts for the diversity of symptoms common to multiple developmental disorders. PMID:18435424

Phospholipase B activity and organophosphorus compound toxicity in cultured neural cells

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

Read, David J.; Langford, Lynda; Barbour, Helen R.

2007-03-15

Organophosphorus compounds (OP) such as phenyl saligenin phosphate (PSP) and mipafox (MPX) which cause delayed neuropathy, inhibit neuropathy target esterase (NTE), while OPs such as paraoxon (PXN) react more readily with acetylcholinesterase. In yeast and mammalian cell lines, NTE has been shown to have phospholipase B (PLB) activity which deacylates intracellular phosphatidylcholine to glycerophosphocholine (GroPCho) and can be detected by metabolic labeling with [{sup 14}C]choline. Here we investigated PLB activity in primary cultures of mouse neural cells. In cortical and cerebellar granule neurons and astrocytes, [{sup 14}C]GroPCho labeling was inhibited by PSP and MPX: phenyl dipentylphosphinate (PDPP), a non-neuropathic NTEmore » inhibitor, was more potent, while PXN, was substantially less so. In all three cell types, conversion of [{sup 14}C]phosphatidylcholine to [{sup 14}C]GroPCho over 24 h was relatively small (2.3-14%). Consequently, even with > 80% inhibition of [{sup 14}C]GroPCho production, increased [{sup 14}C]phosphatidylcholine was not detected. At concentrations of 1-10 {mu}M, only PSP was cytotoxic to cortical and cerebellar granule neurons after 24-h exposure. Moreover, dramatic changes in glial cell morphology were induced by PSP, but not PDPP or MPX, with rapid (2-3 h) rounding up of astrocytes and of Schwann cells in cultures of dissociated mouse dorsal root ganglia. We conclude that PLB activity is present in a variety of cultured mouse neural cell types but that acute loss of this activity is not cytotoxic. Conversely, the rapid toxic effects of PSP in vitro suggest that a serine hydrolase distinct from NTE is required continuously by neurons and glia.« less

Detection of component segregation in granules manufactured by high shear granulation with over-granulation conditions using near-infrared chemical imaging.

PubMed

Koide, Tatsuo; Nagato, Takuya; Kanou, Yoshiyuki; Matsui, Kou; Natsuyama, Susumu; Kawanishi, Toru; Hiyama, Yukio

2013-01-30

The objective of this study was to evaluate the high shear granulation process using near-infrared (NIR) chemical imaging technique and to make the findings available for pharmaceutical development. We prepared granules and tablets made under appropriate- and over-granulation conditions with high shear granulation and observed these granules and tablets using NIR chemical imaging system. We found an interesting phenomenon: lactose agglomeration and segregation of ingredients occurred in experimental tablets when over-granulation conditions, including greater impeller rotation speeds and longer granulation times, were employed. Granules prepared using over-granulation conditions were larger and had progressed to the consolidation stage; segregation between ethenzamide and lactose occurred within larger granules. The segregation observed here is not detectable using conventional analytical technologies such as high pressure liquid chromatography (HPLC) because the content of the granules remained uniform despite the segregation. Therefore, granule visualization using NIR chemical imaging is an effective method for investigating and evaluating the granulation process. Copyright © 2012 Elsevier B.V. All rights reserved.

Iron and cell death in Parkinson's disease: a nuclear microscopic study into iron-rich granules in the parkinsonian substantia nigra of primate models

NASA Astrophysics Data System (ADS)

Thong, P. S. P.; Watt, F.; Ponraj, D.; Leong, S. K.; He, Y.; Lee, T. K. Y.

1999-10-01

Parkinson's disease is a degenerative brain disease characterised by a loss of cells in the substantia nigra (SN) region of the brain and accompanying biochemical changes such as inhibition of mitochondrial function, increased iron concentrations and decreased glutathione levels in the parkinsonian SN. Though the aetiology of the disease is still unknown, the observed biochemical changes point to the involvement of oxidative stress. In particular, iron is suspected to play a role by promoting free radical production, leading to oxidative stress and cell death. The increase in iron in the parkinsonian SN has been confirmed by several research groups, both in human post-mortem brains and in brain tissue from parkinsonian animal models. However, the question remains as to whether the observed increase in iron is a cause or a consequence of the SN cell death process. Our previous study using unilaterally 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP)-lesioned monkeys in a time sequence experiment has shown that the increase in bulk iron concentrations follow rather than precede dopaminergic cell death. However, changes in the localised iron concentrations, which may play a more direct role in SN cell death, may not be reflected at the bulk level. Indeed, we have observed iron-rich granules in parkinsonian SNs. From this time sequence study into the iron content of iron-rich granules in the SNs of an untreated control and unilaterally MPTP-lesioned parkinsonian models, we present the following observations: (1) Iron-rich granules are found in both control and parkinsonian SNs and are variable in size and iron content in any one model. (2) These iron-rich granules may be associated with neuromelanin granules found in the SN and are known to accumulate transition metal ions such as iron. (3) The early onset of bulk SN cell loss (35%) was accompanied by a significant elevation of iron in granules found in the MPTP-injected SN compared to the contra-lateral SN. This

Anticoagulation therapy is harmful to large-sized cerebellar infarction.

PubMed

Zhang, She-Qing; Wang, Wei; Ma, Xiao-Long; Xia, Yu-Ye; Liu, Ai-Jun

2014-09-01

Anticoagulants are commonly used to treat ischemic stroke. Its impact on cerebellar infarction has not been fully understood. In the clinical study, we reviewed a consecutive series of patients with large-sized cerebellar infarction (diameter > 3 cm, n = 30) treated with or without anticoagulation. In animal study, cerebellar infarction operation was performed in 12 Cynomolgus monkeys. Then the animals were administrated with low molecular weight heparin (LMWH) or vehicle for 14 days. Six patients died during the following treatment. All the subjects that died received anticoagulation therapy, while nobody in the survival group received such a therapy. Compared with sham-operated animals, all monkeys with cerebellar infarction have obvious neurological deficits. The number and size of the Purkinje cells in the cerebellar area were also reduced. Two animals in the LMWH group (33%) died, while all animals in the vehicle control group survived. Compared with the vehicle group, the neurological score in the LMWH group was significantly increased (P < 0.05). The water content in the cerebella was also significantly higher (P < 0.05). Edema, hemorrhage, and subarachnoid hemorrhage occurred in the cerebella as well as brainstem of all the LMWH treated animals. These results indicated the harmful effects of anticoagulation therapy on large-sized cerebellar infarction. © 2014 John Wiley & Sons Ltd.

Solar granulation and statistical crystallography: A modeling approach using size-shape relations

NASA Technical Reports Server (NTRS)

Noever, D. A.

1994-01-01

The irregular polygonal pattern of solar granulation is analyzed for size-shape relations using statistical crystallography. In contrast to previous work which has assumed perfectly hexagonal patterns for granulation, more realistic accounting of cell (granule) shapes reveals a broader basis for quantitative analysis. Several features emerge as noteworthy: (1) a linear correlation between number of cell-sides and neighboring shapes (called Aboav-Weaire's law); (2) a linear correlation between both average cell area and perimeter and the number of cell-sides (called Lewis's law and a perimeter law, respectively) and (3) a linear correlation between cell area and squared perimeter (called convolution index). This statistical picture of granulation is consistent with a finding of no correlation in cell shapes beyond nearest neighbors. A comparative calculation between existing model predictions taken from luminosity data and the present analysis shows substantial agreements for cell-size distributions. A model for understanding grain lifetimes is proposed which links convective times to cell shape using crystallographic results.

Polyhydroxyalkanoate (PHA) Granules Have no Phospholipids.

PubMed

Bresan, Stephanie; Sznajder, Anna; Hauf, Waldemar; Forchhammer, Karl; Pfeiffer, Daniel; Jendrossek, Dieter

2016-05-25

Polyhydroxybutyrate (PHB) granules, also designated as carbonosomes, are supra-molecular complexes in prokaryotes consisting of a PHB polymer core and a surface layer of structural and functional proteins. The presence of suspected phospholipids in the surface layer is based on in vitro data of isolated PHB granules and is often shown in cartoons of the PHB granule structure in reviews on PHB metabolism. However, the in vivo presence of a phospholipid layer has never been demonstrated. We addressed this topic by the expression of fusion proteins of DsRed2EC and other fluorescent proteins with the phospholipid-binding domain (LactC2) of lactadherin in three model organisms. The fusion proteins specifically localized at the cell membrane of Ralstonia eutropha but did not co-localize with PHB granules. The same result was obtained for Pseudomonas putida, a species that accumulates another type of polyhydroxyalkanoate (PHA) granules related to PHB. Notably, DsRed2EC-LactC2 expressed in Magnetospirillum gryphiswaldense was detected at the position of membrane-enclosed magnetosome chains and at the cytoplasmic membrane but not at PHB granules. In conclusion, the carbonosomes of representatives of α-proteobacteria, β-proteobacteria and γ-proteobacteria have no phospholipids in vivo and we postulate that the PHB/PHA granule surface layers in natural producers generally are free of phospholipids and consist of proteins only.

Polyhydroxyalkanoate (PHA) Granules Have no Phospholipids

PubMed Central

Bresan, Stephanie; Sznajder, Anna; Hauf, Waldemar; Forchhammer, Karl; Pfeiffer, Daniel; Jendrossek, Dieter

2016-01-01

Polyhydroxybutyrate (PHB) granules, also designated as carbonosomes, are supra-molecular complexes in prokaryotes consisting of a PHB polymer core and a surface layer of structural and functional proteins. The presence of suspected phospholipids in the surface layer is based on in vitro data of isolated PHB granules and is often shown in cartoons of the PHB granule structure in reviews on PHB metabolism. However, the in vivo presence of a phospholipid layer has never been demonstrated. We addressed this topic by the expression of fusion proteins of DsRed2EC and other fluorescent proteins with the phospholipid-binding domain (LactC2) of lactadherin in three model organisms. The fusion proteins specifically localized at the cell membrane of Ralstonia eutropha but did not co-localize with PHB granules. The same result was obtained for Pseudomonas putida, a species that accumulates another type of polyhydroxyalkanoate (PHA) granules related to PHB. Notably, DsRed2EC-LactC2 expressed in Magnetospirillum gryphiswaldense was detected at the position of membrane-enclosed magnetosome chains and at the cytoplasmic membrane but not at PHB granules. In conclusion, the carbonosomes of representatives of α-proteobacteria, β-proteobacteria and γ-proteobacteria have no phospholipids in vivo and we postulate that the PHB/PHA granule surface layers in natural producers generally are free of phospholipids and consist of proteins only. PMID:27222167

A New Mouse Allele of Glutamate Receptor Delta 2 with Cerebellar Atrophy and Progressive Ataxia

PubMed Central

Miyoshi, Yuka; Yoshioka, Yoshichika; Suzuki, Kinuko; Miyazaki, Taisuke; Koura, Minako; Saigoh, Kazumasa; Kajimura, Naoko; Monobe, Yoko; Kusunoki, Susumu; Matsuda, Junichiro; Watanabe, Masahiko; Hayasaka, Naoto

2014-01-01

Spinocerebellar degenerations (SCDs) are a large class of sporadic or hereditary neurodegenerative disorders characterized by progressive motion defects and degenerative changes in the cerebellum and other parts of the CNS. Here we report the identification and establishment from a C57BL/6J mouse colony of a novel mouse line developing spontaneous progressive ataxia, which we refer to as ts3. Frequency of the phenotypic expression was consistent with an autosomal recessive Mendelian trait of inheritance, suggesting that a single gene mutation is responsible for the ataxic phenotype of this line. The onset of ataxia was observed at about three weeks of age, which slowly progressed until the hind limbs became entirely paralyzed in many cases. Micro-MRI study revealed significant cerebellar atrophy in all the ataxic mice, although individual variations were observed. Detailed histological analyses demonstrated significant atrophy of the anterior folia with reduced granule cells (GC) and abnormal morphology of cerebellar Purkinje cells (PC). Study by ultra-high voltage electron microscopy (UHVEM) further indicated aberrant morphology of PC dendrites and their spines, suggesting both morphological and functional abnormalities of the PC in the mutants. Immunohistochemical studies also revealed defects in parallel fiber (PF)–PC synapse formation and abnormal distal extension of climbing fibers (CF). Based on the phenotypic similarities of the ts3 mutant with other known ataxic mutants, we performed immunohistological analyses and found that expression levels of two genes and their products, glutamate receptor delta2 (grid2) and its ligand, cerebellin1 (Cbln1), are significantly reduced or undetectable. Finally, we sequenced the candidate genes and detected a large deletion in the coding region of the grid2 gene. Our present study suggests that ts3 is a new allele of the grid2 gene, which causes similar but different phenotypes as compared to other grid2 mutants. PMID

Cerebro-Cerebellar Functional Connectivity is Associated with Cerebellar Excitation-Inhibition Balance in Autism Spectrum Disorder.

PubMed

Hegarty, John P; Weber, Dylan J; Cirstea, Carmen M; Beversdorf, David Q

2018-05-23

Atypical functional connectivity (FC) and an imbalance of excitation-to-inhibition (E/I) have been previously reported in cerebro-cerebellar circuits in autism spectrum disorder (ASD). The current investigation used resting state fMRI and proton magnetic resonance spectroscopy ( 1 H-MRS) to examine the relationships between E/I (glutamate + glutamine/GABA) and FC of the dorsolateral prefrontal cortex and posterolateral cerebellar hemisphere from 14 adolescents/adults with ASD and 12 age/sex/IQ-matched controls. In this pilot sample, cerebro-cerebellar FC was positively associated with cerebellar E/I and listening comprehension abilities in individuals with ASD but not controls. Additionally, a subgroup of individuals with ASD and low FC (n = 5) exhibited reduced E/I and impaired listening comprehension. Thus, altered functional coherence of cerebro-cerebellar circuits in ASD may be related with a cerebellar E/I imbalance.

Identification of SNAREs that mediate zymogen granule exocytosis

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

Pickett, James A.; Campos-Toimil, Manuel; Thomas, Paul

2007-08-03

A secretagogue-stimulated pancreatic acinar cell releases digestive enzymes from its apical pole. We attempted to identify the SNAREs involved in zymogen granule exocytosis. Antibodies against syntaxins 2 and 3, SNAP-23 and VAMP 8, and the corresponding recombinant SNAREs, inhibited amylase secretion from streptolysin O-permeabilised acini; other anti-SNARE antibodies and SNAREs had no effect. Botulinum neurotoxin C, which cleaved syntaxin 2 and (to a lesser extent) syntaxin 3, but not syntaxins 4, 7 or 8, also inhibited exocytosis. We propose that syntaxin 2, SNAP-23 and VAMP 8 mediate primary granule-plasma membrane fusion. Syntaxin 3 may be involved in secondary granule-granule fusion.

Contribution of cerebellar intracortical inhibition to Purkinje cell response during vestibulo-ocular reflex of alert rabbits.

PubMed Central

Miyashita, Y; Nagao, S

1984-01-01

Ionophoretic application of bicuculline, an antagonist of gamma-aminobutyric acid (GABA), was used to examine the contribution of intracortical inhibition to vestibular responses of Purkinje cells in the cerebellar flocculus of alert rabbits. Purkinje cells were sampled extracellularly (with triple-barrelled micropipettes) from the floccular area where electrical stimulation through the micro-electrode evoked abduction of the ipsilateral eye, indicating its close functional relationship to the horizontal vestibulo-ocular reflex. These cells exhibited frequency modulation of simple spike discharges in-phase or out-phase with sinusoidal head rotation (0.5 cycles/s, 5 degrees peak-to-peak) in the horizontal plane. Bicuculline was ejected ionophoretically through one barrel with a 20-60 nA current. The pharmacological effectiveness of the ejected bicuculline was confirmed for each Purkinje cell by its blocking action upon the depressant action of GABA applied ionophoretically through another barrel. Bicuculline usually shifted the simple spike modulation in the in-phase direction: it reduced the amplitude of out-phase modulation in three cells, converted out-phase modulation to the in-phase type in four cells, and increased in-phase modulation in five cells. In three other cells, however, bicuculline shifted the modulation in the out-phase direction. Because bicuculline application usually increased the resting discharge level of a Purkinje cell, ionophoretic application of DL-homocysteate was used in ten Purkinje cells to control for the effect of a generalized increase in excitability. In contrast to bicuculline, DL-homocysteate generally induced a slight increase of the simple spike modulation regardless of the phase relationship. Since frequency modulation of the simple spike discharges of flocculus Purkinje cells is presumed to contribute to the control of vestibulo-ocular reflexes, these results point to an important functional role of intracortical post

Physicochemical characteristics of insulin secretion granules

PubMed Central

Coore, H. G.; Hellman, B.; Pihl, E.; Täljedal, I.-B.

1969-01-01

β-Granules were prepared from micro-dissected pancreatic islets of obese–hyperglycaemic mice. This fraction contained 60% of the insulin, 30% of the cytochrome oxidase, 16% of the acid phosphatase activity and 20% of the protein present in whole islets. The isolated granules retained a heavy metal during fractionation. Optimum conditions for granule stability were low ionic strength and pH6, the granules being unexpectedly fragile at pH7·4. The stability of the granules was unaffected by sucrose in the concentration range 50–320mm, but 1% (w/v) sodium deoxycholate released all insulin. A solubilizing effect was also noted with ATP and citrate. Spinning through 1·6m-sucrose yielded a further purification in relation to mitochondria and acid-phosphatase-carrying particles but virtually no purification in relation to protein. Electron microscopy revealed that the major contaminants were rough-surfaced vesicles and membranes. A separation of granules from acid phosphatase was achieved by phase distribution in polyethylene glycol and dextran. The location of the enzyme to the interphase was so pronounced in systems buffered with lithium phosphate that the technique may be used for future purification of acid-phosphatase-carrying particles from the β-cells. ImagesPLATE 1 PMID:4887194

Convergent evolution of germ granule nucleators: A hypothesis.

PubMed

Kulkarni, Arpita; Extavour, Cassandra G

2017-10-01

Germ cells have been considered "the ultimate stem cell" because they alone, during normal development of sexually reproducing organisms, are able to give rise to all organismal cell types. Morphological descriptions of a specialized cytoplasm termed 'germ plasm' and associated electron dense ribonucleoprotein (RNP) structures called 'germ granules' within germ cells date back as early as the 1800s. Both germ plasm and germ granules are implicated in germ line specification across metazoans. However, at a molecular level, little is currently understood about the molecular mechanisms that assemble these entities in germ cells. The discovery that in some animals, the gene products of a small number of lineage-specific genes initiate the assembly (also termed nucleation) of germ granules and/or germ plasm is the first step towards facilitating a better understanding of these complex biological processes. Here, we draw on research spanning over 100years that supports the hypothesis that these nucleator genes may have evolved convergently, allowing them to perform analogous roles across animal lineages. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Zinc sulfide in intestinal cell granules of Ancylostoma caninum adults

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

Gianotti, A.J.; Clark, D.T.; Dash, J.

1991-04-01

A source of confusion has existed since the turn of the century about the reddish brown, weakly birefringent 'sphaerocrystals' located in the intestines of strongyle nematodes, Strongylus and Ancylostoma. X-ray diffraction and energy dispersive spectrometric analyses were used for accurate determination of the crystalline order and elemental composition of the granules in the canine hookworm Ancylostoma caninum. The composition of the intestinal pigmented granules was identified unequivocally as zinc sulfide. It seems most probable that the granules serve to detoxify high levels of metallic ions (specifically zinc) present due to the large intake of host blood.

Frequency-dependent reliability of spike propagation is function of axonal voltage-gated sodium channels in cerebellar Purkinje cells.