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Sample records for acute hippocampal slices

  1. Neuroprotection against diisopropylfluorophosphate in acute hippocampal slices

    PubMed Central

    Ferchmin, P. A.; Pérez, Dinely; Cuadrado, Brenda L.; Carrasco, Marimée; Martins, Antonio H.; Eterović, Vesna A.

    2015-01-01

    Diisopropylfluorophosphate (DFP) is an irreversible inhibitor of acetylcholine esterase (AChE) and a surrogate of the organophosphorus (OP) nerve agent sarin. The neurotoxicity of DFP was assessed as a reduction of population spike (PS) area elicited by synaptic stimulation in acute hippocampal slices. Two classical antidotes, atropine, and pralidoxime, and two novel antidotes, 4R-cembranotriene-diol (4R) and a caspase 9 inhibitor, were tested. Atropine, pralidoxime, and 4R significantly protected when applied 30 min after DFP. The caspase inhibitor was neuroprotective when applied 5–10 min before or after DFP, suggesting that early synaptic apoptosis is responsible for the loss of PSs. It is likely that apoptosis starts at the synapses and, if antidotes are not applied, descends to the cell bodies, causing death. The acute slice is a reliable tool for mechanistic studies, and the assessment of neurotoxicity and neuroprotection with PS areas is, in general, pharmacologically congruent with in vivo results and predicts the effect of drugs in vivo. 4R was first found to be neuroprotective in slices and later we demonstrated that 4R is neuroprotective in vivo. The mechanism of neurotoxicity of OPs is not well understood, and there is a need for novel antidotes that could be discovered using acute slices. PMID:26438150

  2. A Cembranoid Protects Acute Hippocampal Slices Against Paraoxon Neurotoxicity

    PubMed Central

    Eterović, Vesna A.; Pérez, Dinely; Martins, Antonio H.; Cuadrado, Brenda L.; Carrasco, Marimée

    2011-01-01

    Many neurotoxic organophosphates (OPs) inhibit acetylcholinesterase (AChE) and as a result can cause a life threatening cholinergic crisis. Current medical countermeasures, which typically include atropine and oximes target the cholinergic crisis and are effective in decreasing mortality but do not sufficiently protect against delayed neurological deficits. There is, therefore, a need to develop neuroprotective drugs to prevent long-term neurological deficits. We used acute hippocampal slices to test the hypothesis that 4R,6R-cembratrienediol (4R) protects against functional damage caused by the OP paraoxon (POX). To assess hippocampal function, we measured synaptically evoked population spikes (PSs). Application of 4R reversed POX inhibition of PSs and the EC50 of this effect was 0.8 µM. Atropine alone did not protect against POX neurotoxicity, but it did enhance protection by 4R. Pralidoxime partially regenerated AChE activity and protected against POX inhibition of PSs. 4R did not regenerate AChE suggesting that under our experimental conditions, the deleterious effect of POX on hippocampal function is not directly related to AChE inhibition. In conclusion, 4R is a promising neuroprotective compound against OP neurotoxins. PMID:21569834

  3. Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

    PubMed Central

    Shetty, Mahesh Shivarama; Sharma, Mahima; Hui, Neo Sin; Dasgupta, Ananya; Gopinadhan, Suma; Sajikumar, Sreedharan

    2015-01-01

    Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats. PMID:26381286

  4. Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents.

    PubMed

    Shetty, Mahesh Shivarama; Sharma, Mahima; Hui, Neo Sin; Dasgupta, Ananya; Gopinadhan, Suma; Sajikumar, Sreedharan

    2015-09-04

    Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.

  5. Long-Term Potentiation by Theta-Burst Stimulation Using Extracellular Field Potential Recordings in Acute Hippocampal Slices.

    PubMed

    Abrahamsson, Therese; Lalanne, Txomin; Watt, Alanna J; Sjöström, P Jesper

    2016-06-01

    This protocol describes how to carry out theta-burst long-term potentiation (LTP) with extracellular field recordings in acute rodent hippocampal slices. This method is relatively simple and noninvasive and provides a way to sample many neurons simultaneously, making it suitable for applications requiring higher throughput than whole-cell recording.

  6. Long-Term Potentiation by Theta-Burst Stimulation using Extracellular Field Potential Recordings in Acute Hippocampal Slices

    PubMed Central

    Abrahamsson, Therese; Lalanne, Txomin; Watt, Alanna J.; Sjöström, P. Jesper

    2017-01-01

    This protocol describes how to carry out theta-burst long-term potentiation (LTP) with extracellular field recordings in acute rodent hippocampal slices. This method is relatively simple and noninvasive and provides a way to sample many neurons simultaneously, making it suitable for applications requiring higher throughput than whole-cell recording. PMID:27250947

  7. Acute death of astrocytes in blast-exposed rat organotypic hippocampal slice cultures

    PubMed Central

    Miller, Anna P.; Shah, Alok S.; Aperi, Brandy V.; Kurpad, Shekar N.; Stemper, Brian D.; Glavaski-Joksimovic, Aleksandra

    2017-01-01

    Blast traumatic brain injury (bTBI) affects civilians, soldiers, and veterans worldwide and presents significant health concerns. The mechanisms of neurodegeneration following bTBI remain elusive and current therapies are largely ineffective. It is important to better characterize blast-evoked cellular changes and underlying mechanisms in order to develop more effective therapies. In the present study, our group utilized rat organotypic hippocampal slice cultures (OHCs) as an in vitro system to model bTBI. OHCs were exposed to either 138 ± 22 kPa (low) or 273 ± 23 kPa (high) overpressures using an open-ended helium-driven shock tube, or were assigned to sham control group. At 2 hours (h) following injury, we have characterized the astrocytic response to a blast overpressure. Immunostaining against the astrocytic marker glial fibrillary acidic protein (GFAP) revealed acute shearing and morphological changes in astrocytes, including clasmatodendrosis. Moreover, overlap of GFAP immunostaining and propidium iodide (PI) indicated astrocytic death. Quantification of the number of dead astrocytes per counting area in the hippocampal cornu Ammonis 1 region (CA1), demonstrated a significant increase in dead astrocytes in the low- and high-blast, compared to sham control OHCs. However only a small number of GFAP-expressing astrocytes were co-labeled with the apoptotic marker Annexin V, suggesting necrosis as the primary type of cell death in the acute phase following blast exposure. Moreover, western blot analyses revealed calpain mediated breakdown of GFAP. The dextran exclusion additionally indicated membrane disruption as a potential mechanism of acute astrocytic death. Furthermore, although blast exposure did not evoke significant changes in glutamate transporter 1 (GLT-1) expression, loss of GLT-1-expressing astrocytes suggests dysregulation of glutamate uptake following injury. Our data illustrate the profound effect of blast overpressure on astrocytes in OHCs at 2 h

  8. Acute death of astrocytes in blast-exposed rat organotypic hippocampal slice cultures.

    PubMed

    Miller, Anna P; Shah, Alok S; Aperi, Brandy V; Kurpad, Shekar N; Stemper, Brian D; Glavaski-Joksimovic, Aleksandra

    2017-01-01

    Blast traumatic brain injury (bTBI) affects civilians, soldiers, and veterans worldwide and presents significant health concerns. The mechanisms of neurodegeneration following bTBI remain elusive and current therapies are largely ineffective. It is important to better characterize blast-evoked cellular changes and underlying mechanisms in order to develop more effective therapies. In the present study, our group utilized rat organotypic hippocampal slice cultures (OHCs) as an in vitro system to model bTBI. OHCs were exposed to either 138 ± 22 kPa (low) or 273 ± 23 kPa (high) overpressures using an open-ended helium-driven shock tube, or were assigned to sham control group. At 2 hours (h) following injury, we have characterized the astrocytic response to a blast overpressure. Immunostaining against the astrocytic marker glial fibrillary acidic protein (GFAP) revealed acute shearing and morphological changes in astrocytes, including clasmatodendrosis. Moreover, overlap of GFAP immunostaining and propidium iodide (PI) indicated astrocytic death. Quantification of the number of dead astrocytes per counting area in the hippocampal cornu Ammonis 1 region (CA1), demonstrated a significant increase in dead astrocytes in the low- and high-blast, compared to sham control OHCs. However only a small number of GFAP-expressing astrocytes were co-labeled with the apoptotic marker Annexin V, suggesting necrosis as the primary type of cell death in the acute phase following blast exposure. Moreover, western blot analyses revealed calpain mediated breakdown of GFAP. The dextran exclusion additionally indicated membrane disruption as a potential mechanism of acute astrocytic death. Furthermore, although blast exposure did not evoke significant changes in glutamate transporter 1 (GLT-1) expression, loss of GLT-1-expressing astrocytes suggests dysregulation of glutamate uptake following injury. Our data illustrate the profound effect of blast overpressure on astrocytes in OHCs at 2 h

  9. Effect of acute and repeated restraint stress on glucose oxidation to CO2 in hippocampal and cerebral cortex slices.

    PubMed

    Torres, I L; Gamaro, G D; Silveira-Cucco, S N; Michalowski, M B; Corrêa, J B; Perry, M L; Dalmaz, C

    2001-01-01

    It has been suggested that glucocorticoids released during stress might impair neuronal function by decreasing glucose uptake by hippocampal neurons. Previous work has demonstrated that glucose uptake is reduced in hippocampal and cerebral cortex slices 24 h after exposure to acute stress, while no effect was observed after repeated stress. Here, we report the effect of acute and repeated restraint stress on glucose oxidation to CO2 in hippocampal and cerebral cortex slices and on plasma glucose and corticosterone levels. Male adult Wistar rats were exposed to restraint 1 h/day for 50 days in the chronic model. In the acute model there was a single exposure. Immediately or 24 h after stress, the animals were sacrificed and the hippocampus and cerebral cortex were dissected, sliced, and incubated with Krebs buffer, pH 7.4, containing 5 mM glucose and 0.2 microCi D-[U-14C] glucose. CO2 production from glucose was estimated. Trunk blood was also collected, and both corticosterone and glucose were measured. The results showed that corticosterone levels after exposure to acute restraint were increased, but the increase was smaller when the animals were submitted to repeated stress. Blood glucose levels increased after both acute and repeated stress. However, glucose utilization, measured as CO2 production in hippocampal and cerebral cortex slices, was the same in stressed and control groups under conditions of both acute and chronic stress. We conclude that, although stress may induce a decrease in glucose uptake, this effect is not sufficient to affect the energy metabolism of these cells.

  10. Endogenous waves in hippocampal slices.

    PubMed

    Kubota, Don; Colgin, Laura Lee; Casale, Malcolm; Brucher, Fernando A; Lynch, Gary

    2003-01-01

    Sharp waves (SPWs) are thought to play a major role in intrinsic hippocampal operations during states in which subcortical and cortical inputs to hippocampus are reduced. This study describes evidence that such activity occurs spontaneously in appropriately prepared rat hippocampal slices. Irregular waves, with an average frequency of approximately 4 Hz, were recorded from field CA3 in slices prepared from the temporal region of hippocampus. The waves persisted for hours and were not accompanied by aberrant discharges. Multi-electrode analyses established that they were locally generated within each of the subfields of CA3 and yet were coherent between subfields. The sharp waves were reversibly blocked by either cholinergic or serotonergic stimulation. Various lines of evidence indicate that they are propagated by the CA3 associational system.

  11. A novel method for monitoring the cell surface expression of heteromeric protein complexes in dispersed neurons and acute hippocampal slices

    PubMed Central

    Holman, David; Henley, Jeremy M.

    2012-01-01

    The subunit composition of multimeric protein complexes is critical in determining their trafficking and functional properties. Despite there being multiple techniques to investigate the trafficking events of individual subunits there are currently limited means to monitor the trafficking properties of heteromeric protein complexes. Here, we combine surface biotinylation with co-immunoprecipitation to monitor the cell surface expression of native, heteromeric AMPA receptor complexes. Using this method, we demonstrate that the surface levels of GluR1/2 and GluR2/3 complexes are reduced following NMDA-evoked long-term depression (NMDA-LTD) in acute hippocampal slices. Finally, we discuss how this method can be adapted to monitor the cell surface expression of other heteromeric protein complexes. PMID:17083981

  12. Dopamine Modulates Spike Timing-Dependent Plasticity and Action Potential Properties in CA1 Pyramidal Neurons of Acute Rat Hippocampal Slices

    PubMed Central

    Edelmann, Elke; Lessmann, Volkmar

    2011-01-01

    Spike timing-dependent plasticity (STDP) is a cellular model of Hebbian synaptic plasticity which is believed to underlie memory formation. In an attempt to establish a STDP paradigm in CA1 of acute hippocampal slices from juvenile rats (P15–20), we found that changes in excitability resulting from different slice preparation protocols correlate with the success of STDP induction. Slice preparation with sucrose containing ACSF prolonged rise time, reduced frequency adaptation, and decreased latency of action potentials in CA1 pyramidal neurons compared to preparation in conventional ASCF, while other basal electrophysiological parameters remained unaffected. Whereas we observed prominent timing-dependent long-term potentiation (t-LTP) to 171 ± 10% of controls in conventional ACSF, STDP was absent in sucrose prepared slices. This sucrose-induced STDP deficit could not be rescued by stronger STDP paradigms, applying either more pre- and/or postsynaptic stimuli, or by a higher stimulation frequency. Importantly, slice preparation with sucrose containing ACSF did not eliminate theta-burst stimulation induced LTP in CA1 in field potential recordings in our rat hippocampal slices. Application of dopamine (for 10–20 min) to sucrose prepared slices completely rescued t-LTP and recovered action potential properties back to levels observed in ACSF prepared slices. Conversely, acute inhibition of D1 receptor signaling impaired t-LTP in ACSF prepared slices. No similar restoring effect for STDP as seen with dopamine was observed in response to the β-adrenergic agonist isoproterenol. ELISA measurements demonstrated a significant reduction of endogenous dopamine levels (to 61.9 ± 6.9% of ACSF values) in sucrose prepared slices. These results suggest that dopamine signaling is involved in regulating the efficiency to elicit STDP in CA1 pyramidal neurons. PMID:22065958

  13. Resveratrol attenuates early pyramidal neuron excitability impairment and death in acute rat hippocampal slices caused by oxygen-glucose deprivation

    PubMed Central

    Zhang, Huaqiu; Schools, Gary P.; Lei, Ting; Wang, Wei; Kimelberg, Harold; Zhou, Min

    2008-01-01

    Accumulating evidence indicates that the polyphenol resveratrol (trans-3, 5, 4′-trihydroxystibene, RVT) potently protects against cerebral ischemia neuronal damage due to its oxygen free radicals scavenging and antioxidant properties. However, it is unknown whether RVT can attenuate ischemia-induced early impairment of neuronal excitability. To address this question, we simulated ischemic conditions by applying oxygen-glucose deprivation (OGD) to acute rat hippocampal slices and examined the effect of RVT on OGD-induced pyramidal neuron excitability impairment using whole-cell patch clamp recording. 100 μM RVT largely inhibited the 15 min OGD-induced progressive membrane potential (Vm) depolarization and the reduction in evoked action potential frequency and amplitude in pyramidal neurons. In the parallel neuronal viability study using TO-PRO-3 iodide staining, 20 min OGD induced irreversible CA1 pyramidal neuronal death which was significantly reduced by 100 μM RVT. No similar effects were found with PQQ treatment, an antioxidant also showing potent neuroprotection in the rat rMCAO ischemia model. This suggests that antioxidant action per se, is unlikely accounting for the observed early effects of RVT. RVT also markedly reduced the frequency and amplitude of AMPA mediated spontaneous excitatory postsynaptic currents (sEPSCs) in pyramidal neurons, which is also an early consequence of OGD. RVT effects on neuronal excitability were inhibited by the large conductance potassium channel (BK channel) inhibitor paxilline. Together, these studies demonstrate that RVT attenuates OGD induced neuronal impairment occurring early in the simulated ischemia slice model by enhancing the activation of BK channel and reducing the OGD-enhanced AMPA/NMDA receptor mediated neuronal EPSCs. PMID:18495119

  14. Nuclear shrinkage in live mouse hippocampal slices.

    PubMed

    Kasischke, K; Büchner, M; Ludolph, A C; Riepe, M W

    2001-05-01

    Brain slices are used extensively for biochemical, electrophysiological and molecular investigations. However, only the time frame for electrophysiological and biochemical investigations has as yet been defined. The goal of the present study was to investigate the time course of nuclear structure in live brain slices. Hippocampal slices (300 microm) were prepared from male CD1 mice (25-30 g), stained with Hoechst 33342 (10 microM), calcein-AM (2 microM) and ethidium homodimer (4 microM), and imaged with single- and dual-photon microscopy. The volume of CA1 pyramidal cell nuclei decreased from 759+/-229 microm3 in 40-50 microm depth 25 min after preparation to 453+/-169 microm3 (P<0.001) after 60 min, 315+/-112 microm3 (P<0.001) after 120 min and 128+/-71 microm3 (P<0.001) after 8 h. Similar results were obtained on a prolonged time scale in 70-80 microm depth and with an accelerated time scale in 20-30 microm depth. Live-dead staining showed that cell damage is progressing from the surface to deeper layers of the slices in a time-dependent fashion. We conclude that nuclei of CA1 hippocampal pyramidal cells show a time- and depth-dependent shrinkage converging 8 h after slice preparation to a volume of 90-130 microm; in any depth between 20 and 80 microm. The nucleus in the superficial 80 microm of each side appears dysfunctional even at times suitable for electrophysiological and biochemical experimentation in hippocampal slices. Molecular analysis of cell regulation in brain slices may, therefore, be time-dependently distorted by progressing cell death in at least half of the tissue under investigation.

  15. Trafficking of astrocytic vesicles in hippocampal slices

    SciTech Connect

    Potokar, Maja; Kreft, Marko; Lee, So-Young; Takano, Hajime; Haydon, Philip G.; Zorec, Robert

    2009-12-25

    The increasingly appreciated role of astrocytes in neurophysiology dictates a thorough understanding of the mechanisms underlying the communication between astrocytes and neurons. In particular, the uptake and release of signaling substances into/from astrocytes is considered as crucial. The release of different gliotransmitters involves regulated exocytosis, consisting of the fusion between the vesicle and the plasma membranes. After fusion with the plasma membrane vesicles may be retrieved into the cytoplasm and may continue to recycle. To study the mobility implicated in the retrieval of secretory vesicles, these structures have been previously efficiently and specifically labeled in cultured astrocytes, by exposing live cells to primary and secondary antibodies. Since the vesicle labeling and the vesicle mobility properties may be an artifact of cell culture conditions, we here asked whether the retrieving exocytotic vesicles can be labeled in brain tissue slices and whether their mobility differs to that observed in cell cultures. We labeled astrocytic vesicles and recorded their mobility with two-photon microscopy in hippocampal slices from transgenic mice with fluorescently tagged astrocytes (GFP mice) and in wild-type mice with astrocytes labeled by Fluo4 fluorescence indicator. Glutamatergic vesicles and peptidergic granules were labeled by the anti-vesicular glutamate transporter 1 (vGlut1) and anti-atrial natriuretic peptide (ANP) antibodies, respectively. We report that the vesicle mobility parameters (velocity, maximal displacement and track length) recorded in astrocytes from tissue slices are similar to those reported previously in cultured astrocytes.

  16. Evaluation of Mitochondrial Function in the CNS of Rodent Models of Alzheimer's Disease - High Resolution Respirometry Applied to Acute Hippocampal Slices.

    PubMed

    Dias, Candida; Barbosa, Rui M; Laranjinha, Joao; Ledo, Ana

    2014-10-01

    Alzheimer's disease (AD) is a multifactorial disease characterized by extracellular deposits of amyloid plaques and intracellular neurofibrillary tangles. These hallmark alterations are preceded by synaptic deterioration, changes in neuromolecular plasticity phenomena, mitochondrial dysfunction, increase in oxidative damage to cellular constituents and decreased energy metabolism. The hippocampus is a structure of the temporal medial lobe implicated in specific forms of memory processes. It is also one of the first and most affected regions of the CNS in AD. Here we present a novel approach to the study if mitochondrial function/disfunction in 2 rodent models of AD: an acute rat model obtained by intracerebroventricular injection of the toxin streptozotocin (STZ) and a progressive triple transgenic mouse model (3TgAD) harboring PS1M146V, APPSwe, and tauP301L transgenes. Mitochondrial dysfunction has classically been assessed in such models by isolating mitochondria, synaptossoms or working with cell cultures. Anyone of these approaches destroys the intricate intercellular connectivity and cytoarchitecture of neuronal tissue. We used acute hippocampal slices obtained from the 2 models of AD and evaluated changes in mitochondrial function as a function of disease and/or age. Mitochondrial stress test were performed on the high resolution respirometry (Oroboros 2K Oxymeter). Upon analysis of oxygen consumption rates (OCR) we observed significant decreases in basal OCR, maximal respiratory capacity, ATP turnover and a tendency for decrease in sparing capacity in the STZ rat model compared to shame injected animals. Regarding the 3TgAD model we observed an age-dependent decrease in all parameters evaluated in the mitochondrial stress test, in both 3TgAD and NTg animals. However, although a tendency towards decreased OCR was observed when comparing 3TgAD and age-matched NTg animals, no statistically significant difference was observed. Copyright © 2014. Published by

  17. Spontaneous recurrent network activity in organotypic rat hippocampal slices.

    PubMed

    Mohajerani, Majid H; Cherubini, Enrico

    2005-07-01

    Organotypic hippocampal slices were prepared from postnatal day 4 rats and maintained in culture for >6 weeks. Cultured slices exhibited from 12 days in vitro spontaneous events which closely resembled giant depolarizing potentials (GDPs) recorded in neonatal hippocampal slices. GDP-like events occurred over the entire hippocampus with a delay of 30-60 ms between two adjacent regions as demonstrated by pair recordings from CA3-CA3, CA3-CA1 and interneurone-CA3 pyramidal cells. As in acute slices, spontaneous recurrent events were generated by the interplay of GABA and glutamate acting on AMPA receptors as they were reversibly blocked by bicuculline and 6,7-dinitroquinoxaline-2,3-dione but not by dl-2-amino-5-phosphonopentaoic acid. The equilibrium potentials for GABA measured in whole cell and gramicidin-perforated patch from interconnected interneurones-CA3 pyramidal cells were -70 and -56 mV, respectively. The resting membrane potential estimated from the reversal of N-methyl-D-aspartate-induced single-channel currents in cell-attach experiments was -75 mV. In spite of its depolarizing action, in the majority of cases GABA was still inhibitory as it blocked the firing of principal cells. The increased level of glutamatergic connectivity certainly contributed to network synchronization and to the development of interictal discharges after prolonged exposure to bicuculline. In spite of its inhibitory action, in a minority of cells GABA was still depolarizing and excitatory as it was able to bring principal cells to fire, suggesting that a certain degree of immaturity is still present in cultured slices. This was in line with the transient bicuculline-induced block of GDPs and with the isoguvacine-induced increase of GDP frequency.

  18. Unstable periodic orbits in human epileptic hippocampal slices.

    PubMed

    Pen-Ning Yu; Min-Chi Hsiao; Dong Song; Liu, Charles Y; Heck, Christi N; Millett, David; Berger, Theodore W

    2014-01-01

    Inter-ictal activity is studied in hippocampal slices resected from patients with epilepsy using local field potential recording. Inter-ictal activity in the dentate gyrus (DG) is induced by high-potassium (8 mM), low-magnesium (0.25 mM) aCSF with additional 100 μM 4-aminopyridine(4-AP). The dynamics of the inter-ictal activity is investigated by developing the first return map with inter-pulse intervals. Unstable periodic orbits (UPOs) are detected in the hippocampal slice at the DG area according to both the topological recurrence method and the periodic orbit transform method. Surrogate analysis suggests the presence of UPOs in hippocampal slices from patients with epilepsy. This finding also suggests that inter-ictal activity is a chaotic system and will allow us to apply chaos control techniques to manipulate inter-ictal activity.

  19. Cannabidiol inhibits synaptic transmission in rat hippocampal cultures and slices via multiple receptor pathways

    PubMed Central

    Ledgerwood, CJ; Greenwood, SM; Brett, RR; Pratt, JA; Bushell, TJ

    2011-01-01

    BACKGROUND AND PURPOSE Cannabidiol (CBD) has emerged as an interesting compound with therapeutic potential in several CNS disorders. However, whether it can modulate synaptic activity in the CNS remains unclear. Here, we have investigated whether CBD modulates synaptic transmission in rat hippocampal cultures and acute slices. EXPERIMENTAL APPROACH The effect of CBD on synaptic transmission was examined in rat hippocampal cultures and acute slices using whole cell patch clamp and standard extracellular recordings respectively. KEY RESULTS Cannabidiol decreased synaptic activity in hippocampal cultures in a concentration-dependent and Pertussis toxin-sensitive manner. The effects of CBD in culture were significantly reduced in the presence of the cannabinoid receptor (CB1) inverse agonist, LY320135 but were unaffected by the 5-HT1A receptor antagonist, WAY100135. In hippocampal slices, CBD inhibited basal synaptic transmission, an effect that was abolished by the proposed CB1 receptor antagonist, AM251, in addition to LY320135 and WAY100135. CONCLUSIONS AND IMPLICATIONS Cannabidiol reduces synaptic transmission in hippocampal in vitro preparations and we propose a role for both 5-HT1A and CB1 receptors in these CBD-mediated effects. These data offer some mechanistic insights into the effects of CBD and emphasize that further investigations into the actions of CBD in the CNS are required in order to elucidate the full therapeutic potential of CBD. PMID:20825410

  20. The Characteristics of LTP Induced in Hippocampal Slices Are Dependent on Slice-Recovery Conditions

    ERIC Educational Resources Information Center

    Godaux, Emile; Ris, Laurence; Capron, Brigitte; Sindic, Christian

    2006-01-01

    In area CA1 of hippocampal slices which are allowed to recover from slicing "in interface" and where recordings are carried out in interface, a single 1-sec train of 100-Hz stimulation triggers a short-lasting long-term potentiation (S-LTP), which lasts 1-2 h, whereas multiple 1-sec trains induce a long-lasting LTP (L-LTP), which lasts several…

  1. The Characteristics of LTP Induced in Hippocampal Slices Are Dependent on Slice-Recovery Conditions

    ERIC Educational Resources Information Center

    Godaux, Emile; Ris, Laurence; Capron, Brigitte; Sindic, Christian

    2006-01-01

    In area CA1 of hippocampal slices which are allowed to recover from slicing "in interface" and where recordings are carried out in interface, a single 1-sec train of 100-Hz stimulation triggers a short-lasting long-term potentiation (S-LTP), which lasts 1-2 h, whereas multiple 1-sec trains induce a long-lasting LTP (L-LTP), which lasts several…

  2. Adaptation of Microplate-based Respirometry for Hippocampal Slices and Analysis of Respiratory Capacity

    PubMed Central

    Schuh, Rosemary A.; Clerc, Pascaline; Hwang, Hyehyun; Mehrabian, Zara; Bittman, Kevin; Chen, Hegang; Polster, Brian M.

    2011-01-01

    Multiple neurodegenerative disorders are associated with altered mitochondrial bioenergetics. Although mitochondrial O2 consumption is frequently measured in isolated mitochondria, isolated synaptic nerve terminals (synaptosomes), or cultured cells, the absence of mature brain circuitry is a remaining limitation. Here we describe the development of a method that adapts the Seahorse Extracellular Flux Analyzer (XF24) for the microplate-based measurement of hippocampal slice O2 consumption. As a first evaluation of the technique, we compared whole slice bioenergetics to previous measurements made with synaptosomes or cultured neurons. We found that mitochondrial respiratory capacity and O2 consumption coupled to ATP synthesis could be estimated in cultured or acute hippocampal slices with preserved neural architecture. Mouse organotypic hippocampal slices oxidizing glucose displayed mitochondrial O2 consumption that was well-coupled, as determined by the sensitivity to the ATP synthase inhibitor oligomycin. However stimulation of respiration by uncoupler was modest (<120% of basal respiration) compared to previous measurements in cells or synaptosomes, although enhanced slightly (to ~150% of basal respiration) by the acute addition of the mitochondrial complex I-linked substrate pyruvate. These findings suggest a high basal utilization of respiratory capacity in slices and a limitation of glucose-derived substrate for maximal respiration. The improved throughput of microplate-based hippocampal respirometry over traditional O2 electrode-based methods is conducive to neuroprotective drug screening. When coupled with cell type-specific pharmacology or genetic manipulations, the ability to efficiently measure O2 consumption from whole slices should advance our understanding of mitochondrial roles in physiology and neuropathology. PMID:21520220

  3. Atorvastatin and Fluoxetine Prevent Oxidative Stress and Mitochondrial Dysfunction Evoked by Glutamate Toxicity in Hippocampal Slices.

    PubMed

    Ludka, Fabiana K; Dal-Cim, Tharine; Binder, Luisa Bandeira; Constantino, Leandra Celso; Massari, Caio; Tasca, Carla I

    2017-07-01

    Atorvastatin has been shown to exert a neuroprotective action by counteracting glutamatergic toxicity. Recently, we have shown atorvastatin also exerts an antidepressant-like effect that depends on both glutamatergic and serotonergic systems modulation. Excitotoxicity is involved in several brain disorders including depression; thus, it is suggested that antidepressants may target glutamatergic system as a final common pathway. In this study, a comparison of the mechanisms involved in the putative neuroprotective effect of a repetitive atorvastatin or fluoxetine treatment against glutamate toxicity in hippocampal slices was performed. Adult Swiss mice were treated with atorvastatin (10 mg/kg, p.o.) or fluoxetine (10 mg/kg, p.o.), once a day during seven consecutive days. On the eighth day, animals were killed and hippocampal slices were obtained and subjected to an in vitro protocol of glutamate toxicity. An acute treatment of atorvastatin or fluoxetine was not neuroprotective; however, the repeated atorvastatin or fluoxetine treatment prevented the decrease in cellular viability induced by glutamate in hippocampal slices. The loss of cellular viability induced by glutamate was accompanied by increased D-aspartate release, increased reactive oxygen species (ROS) and nitric oxide (NO) production, and impaired mitochondrial membrane potential. Atorvastatin or fluoxetine repeated treatment also presented an antidepressant-like effect in the tail suspension test. Atorvastatin or fluoxetine treatment was effective in protecting mice hippocampal slices from glutamate toxicity by preventing the oxidative stress and mitochondrial dysfunction.

  4. Microglial polarization and plasticity: evidence from organotypic hippocampal slice cultures.

    PubMed

    Ajmone-Cat, Maria Antonietta; Mancini, Melissa; De Simone, Roberta; Cilli, Piera; Minghetti, Luisa

    2013-10-01

    Increasing evidence indicates that "functional plasticity" is not solely a neuronal attribute but a hallmark of microglial cells, the main brain resident macrophage population. Far from being a univocal phenomenon, microglial activation can originate a plethora of functional phenotypes, encompassing the classic M1 proinflammatory and the alternative M2 anti-inflammatory phenotypes. This concept overturns the popular view of microglial activation as a synonym of neurotoxicity and neurogenesis failure in brain disorders. The characterization of the alternative programs is a matter of intense investigation, but still scarce information is available on the course of microglial activation, on the reversibility of the different commitments and on the capability of preserving molecular memory of previous priming stimuli. By using organotypic hippocampal slice cultures as a model, we developed paradigms of stimulation aimed at shedding light on some of these aspects. We show that persistent stimulation of TLR4 signaling promotes an anti-inflammatory response and microglial polarization toward M2-like phenotype. Moreover, acute and chronic preconditioning regimens permanently affect the capability to respond to a later challenge, suggesting the onset of mechanisms of molecular memory. Similar phenomena could occur in the intact brain and differently affect the vulnerability of mature and newborn neurons to noxious signals. Copyright © 2013 Wiley Periodicals, Inc.

  5. Stochastic neural network model for spontaneous bursting in hippocampal slices.

    PubMed

    Biswal, B; Dasgupta, C

    2002-11-01

    A biologically plausible, stochastic, neural network model that exhibits spontaneous transitions between a low-activity (normal) state and a high-activity (epileptic) state is studied by computer simulation. Brief excursions of the network to the high-activity state lead to spontaneous population bursting similar to the behavior observed in hippocampal slices bathed in a high-potassium medium. Although the variability of interburst intervals in this model is due to stochasticity, first return maps of successive interburst intervals show trajectories that resemble the behavior expected near unstable periodic orbits (UPOs) of systems exhibiting deterministic chaos. Simulations of the effects of the application of chaos control, periodic pacing, and anticontrol to the network model yield results that are qualitatively similar to those obtained in experiments on hippocampal slices. Estimation of the statistical significance of UPOs through surrogate data analysis also leads to results that resemble those of similar analysis of data obtained from slice experiments and human epileptic activity. These results suggest that spontaneous population bursting in hippocampal slices may be a manifestation of stochastic bistable dynamics, rather than of deterministic chaos. Our results also question the reliability of some of the recently proposed, UPO-based, statistical methods for detecting determinism and chaos in experimental time-series data.

  6. Differential Conditioning of Associative Synaptic Enhancement in Hippocampal Brain Slices

    NASA Astrophysics Data System (ADS)

    Kelso, Stephen R.; Brown, Thomas H.

    1986-04-01

    An electrophysiological stimulation paradigm similar to one that produces Pavlovian conditioning was applied to synaptic inputs to pyramidal neurons of hippocampal brain slices. Persistent synaptic enhancement was induced in one of two weak synaptic inputs by pairing high-frequency electrical stimulation of the weak input with stimulation of a third, stronger input to the same region. Forward (temporally overlapping) but not backward (temporally separate) pairings caused this enhancement. Thus hippocampal synapses in vitro can undergo the conditional and selective type of associative modification that could provide the substrate for some of the mnemonic functions in which the hippocampus is thought to participate.

  7. Brevetoxin Depresses Synaptic Transmission in Guinea Pig Hippocampal Slices

    DTIC Science & Technology

    1993-01-01

    literature publication 4. TITLE AND SUBTITLE IS. FUNDING NUMBERS Brevetoxin depresses synpatic transmission in guinea pig hippocampal slices 61102A...The toxin produced a concentration -dependent depression of the orlhodroiiiicallk esoked population spl, , i~h an EC50 of 37 5 nM. Brevetoxin...precise mechanism b) which PbTx-3 depresses evoked responses is not certain, depolarization of the presynaptic nerve terminals leading to failure of

  8. Holographic Photolysis for Multiple Cell Stimulation in Mouse Hippocampal Slices

    PubMed Central

    Papagiakoumou, Eirini; Ventalon, Cathie; Angulo, María Cecilia; Emiliani, Valentina

    2010-01-01

    Background Advanced light microscopy offers sensitive and non-invasive means to image neural activity and to control signaling with photolysable molecules and, recently, light-gated channels. These approaches require precise and yet flexible light excitation patterns. For synchronous stimulation of subsets of cells, they also require large excitation areas with millisecond and micrometric resolution. We have recently developed a new method for such optical control using a phase holographic modulation of optical wave-fronts, which minimizes power loss, enables rapid switching between excitation patterns, and allows a true 3D sculpting of the excitation volumes. In previous studies we have used holographic photololysis to control glutamate uncaging on single neuronal cells. Here, we extend the use of holographic photolysis for the excitation of multiple neurons and of glial cells. Methods/Principal Findings The system combines a liquid crystal device for holographic patterned photostimulation, high-resolution optical imaging, the HiLo microscopy, to define the stimulated regions and a conventional Ca2+ imaging system to detect neural activity. By means of electrophysiological recordings and calcium imaging in acute hippocampal slices, we show that the use of excitation patterns precisely tailored to the shape of multiple neuronal somata represents a very efficient way for the simultaneous excitation of a group of neurons. In addition, we demonstrate that fast shaped illumination patterns also induce reliable responses in single glial cells. Conclusions/Significance We show that the main advantage of holographic illumination is that it allows for an efficient excitation of multiple cells with a spatiotemporal resolution unachievable with other existing approaches. Although this paper focuses on the photoactivation of caged molecules, our approach will surely prove very efficient for other probes, such as light-gated channels, genetically encoded photoactivatable

  9. Measurement of Inositol Triphosphate Levels from Rat Hippocampal Slices

    PubMed Central

    Tabatadze, Nino; Woolley, Catherine

    2016-01-01

    Inositol triphosphate (IP3) is an important second messenger that participates in signal transduction pathways in diverse cell types including hippocampal neurons. Stimulation of phospholipase C in response to various stimuli (hormones, growth factors, neurotransmitters, neurotrophins, neuromodulators, odorants, light, etc) results in hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2), a phospholipid that is located in the plasma membrane, and leads to the production of IP3 and diacylglycerol. Binding of IP3 to the IP3 receptor (IP3R) induces Ca2+ release from intracellular stores and enables the initiation of intracellular Ca2+-dependent signaling. Here we describe a procedure for the measurement of cellular IP3 levels in tissue homogenates prepared from rat hippocampal slices. PMID:27468425

  10. 5-HT4-Receptors Modulate Induction of Long-Term Depression but Not Potentiation at Hippocampal Output Synapses in Acute Rat Brain Slices

    PubMed Central

    Wawra, Matthias; Fidzinski, Pawel; Heinemann, Uwe; Mody, Istvan; Behr, Joachim

    2014-01-01

    The subiculum is the principal target of CA1 pyramidal cells and mediates hippocampal output to various cortical and subcortical regions of the brain. The majority of subicular pyramidal cells are burst-spiking neurons. Previous studies indicated that high frequency stimulation in subicular burst-spiking cells causes presynaptic NMDA-receptor dependent long-term potentiation (LTP) whereas low frequency stimulation induces postsynaptic NMDA-receptor-dependent long-term depression (LTD). In the present study, we investigate the effect of 5-hydroxytryptamine type 4 (5-HT4) receptor activation and blockade on both forms of synaptic plasticity in burst-spiking cells. We demonstrate that neither activation nor block of 5-HT4 receptors modulate the induction or expression of LTP. In contrast, activation of 5-HT4 receptors facilitates expression of LTD, and block of the 5-HT4 receptor prevents induction of short-term depression and LTD. As 5-HT4 receptors are positively coupled to adenylate cyclase 1 (AC1), 5-HT4 receptors might modulate PKA activity through AC1. Since LTD is blocked in the presence of 5-HT4 receptor antagonists, our data are consistent with 5-HT4 receptor activation by ambient serotonin or intrinsically active 5-HT4 receptors. Our findings provide new insight into aminergic modulation of hippocampal output. PMID:24505387

  11. Astrocyte calcium signalling orchestrates neuronal synchronization in organotypic hippocampal slices

    PubMed Central

    Sasaki, Takuya; Ishikawa, Tomoe; Abe, Reimi; Nakayama, Ryota; Asada, Akiko; Matsuki, Norio; Ikegaya, Yuji

    2014-01-01

    Astrocytes are thought to detect neuronal activity in the form of intracellular calcium elevations; thereby, astrocytes can regulate neuronal excitability and synaptic transmission. Little is known, however, about how the astrocyte calcium signal regulates the activity of neuronal populations. In this study, we addressed this issue using functional multineuron calcium imaging in hippocampal slice cultures. Under normal conditions, CA3 neuronal networks exhibited temporally correlated activity patterns, occasionally generating large synchronization among a subset of cells. The synchronized neuronal activity was correlated with astrocyte calcium events. Calcium buffering by an intracellular injection of a calcium chelator into multiple astrocytes reduced the synaptic strength of unitary transmission between pairs of surrounding pyramidal cells and caused desynchronization of the neuronal networks. Uncaging the calcium in the astrocytes increased the frequency of neuronal synchronization. These data suggest an essential role of the astrocyte calcium signal in the maintenance of basal neuronal function at the circuit level. PMID:24710057

  12. Electrical coupling between hippocampal astrocytes in rat brain slices.

    PubMed

    Meme, William; Vandecasteele, Marie; Giaume, Christian; Venance, Laurent

    2009-04-01

    Gap junctions in astrocytes play a crucial role in intercellular communication by supporting both biochemical and electrical coupling between adjacent cells. Despite the critical role of electrical coupling in the network organization of these glial cells, the electrophysiological properties of gap junctions have been characterized in cultures while no direct evidence has been sought in situ. In the present study, gap-junctional currents were investigated using simultaneous dual whole-cell patch-clamp recordings between astrocytes from rat hippocampal slices. Bidirectional electrotonic coupling was observed in 82% of the cell pairs with an average coupling coefficient of 5.1%. Double patch-clamp analysis indicated that junctional currents were independent of the transjunctional voltage over a range from -100 to +110 mV. Interestingly, astrocytic electrical coupling displayed weak low-pass filtering properties compared to neuronal electrical synapses. Finally, during uncoupling processes triggered by either the gap-junction inhibitor carbenoxolone or endothelin-1, an increase in the input resistance in the injected cell paralleled the decrease in the coupling coefficient. Altogether, these results demonstrate that hippocampal astrocytes are electrically coupled through gap-junction channels characterized by properties that are distinct from those of electrical synapses between neurons. In addition, gap-junctional communication is efficiently regulated by endogenous compounds. This is taken to represent a mode of communication that may have important implications for the functional role of astrocyte networks in situ.

  13. Delivery of recombinant alphavirus into hippocampal slice tissue culture.

    PubMed

    Lundstrom, Kenneth

    2012-08-01

    The alphaviruses Semliki Forest virus (SFV) and Sindbis virus (SIN) have been used frequently as expression vectors in vitro and in vivo. Usually, these systems consist of replication-deficient vectors that require a helper vector for packaging of recombinant particles. Replication-proficient vectors have also been engineered. Alphaviral vectors can be used as nucleic-acid-based vectors (DNA and RNA) or infectious particles. High-titer viral production is achieved in <2 d. The broad host range of alphaviruses facilitates studies in mammalian and nonmammalian cell lines, primary cells in culture, and in vivo. The strong preference for expression in neuronal cells has made alphaviruses particularly useful in neurobiological studies. Unfortunately, their strong cytotoxic effect on host cells, relatively short-term transient expression patterns, and the reasonably high cost of viral production remain drawbacks. However, novel mutant alphaviruses have shown reduced cytotoxicity and prolonged expression. This protocol describes gene delivery of recombinant alphavirus to hippocampal slice cultures. Organotypic slices are covered by a layer of glial cells that impedes the penetration of viral particles to the neurons. Thus, viral particles should be injected manually into the extracellular space of the tissue.

  14. Temperature effects on evoked potentials of hippocampal slices from euthermic chipmunks, hamsters and rats

    NASA Technical Reports Server (NTRS)

    Hooper, D. C.; Martin, S. M.; Horowitz, J. M.

    1985-01-01

    1. Neural activity was recorded in hippocampal slices from euthermic chipmunks, hamsters and rats. 2. While recording the evoked potentials, the temperature of the Ringer's solution bathing the slice was varied by controlling the temperature of an outer chamber jacketing the recording chamber. 3. The temperature just below that at which a population spike could be evoked, Tt, was 10.4 +/- 0.3 degrees C (mean +/- SEM) for chipmunk slices, 14.1 +/- 0.4 degrees C for rat slices and 14.8 +/- 0.4 degrees C for hamster slices. Tt was significantly lower in the chipmunk slices (P<0.01) than in the rat and hamster slices. 4. Data were interpreted as consistent with the hypothesis that chipmunk hippocampal neurons are intrinsically cold resistant.

  15. Temperature effects on evoked potentials of hippocampal slices from euthermic chipmunks, hamsters and rats

    NASA Technical Reports Server (NTRS)

    Hooper, D. C.; Martin, S. M.; Horowitz, J. M.

    1985-01-01

    1. Neural activity was recorded in hippocampal slices from euthermic chipmunks, hamsters and rats. 2. While recording the evoked potentials, the temperature of the Ringer's solution bathing the slice was varied by controlling the temperature of an outer chamber jacketing the recording chamber. 3. The temperature just below that at which a population spike could be evoked, Tt, was 10.4 +/- 0.3 degrees C (mean +/- SEM) for chipmunk slices, 14.1 +/- 0.4 degrees C for rat slices and 14.8 +/- 0.4 degrees C for hamster slices. Tt was significantly lower in the chipmunk slices (P<0.01) than in the rat and hamster slices. 4. Data were interpreted as consistent with the hypothesis that chipmunk hippocampal neurons are intrinsically cold resistant.

  16. Acetylcholinesterase inhibition reveals endogenous nicotinic modulation of glutamate inputs to CA1 stratum radiatum interneurons in hippocampal slices.

    PubMed

    Alkondon, Manickavasagom; Albuquerque, Edson X; Pereira, Edna F R

    2013-05-01

    The involvement of brain nicotinic acetylcholine receptors (nAChRs) in the neurotoxicological effects of soman, a potent acetylcholinesterase (AChE) inhibitor and a chemical warfare agent, is not clear. This is partly due to a poor understanding of the role of AChE in brain nAChR-mediated functions. To test the hypothesis that AChE inhibition builds sufficient acetylcholine (ACh) in the brain and facilitates nAChR-dependent glutamate transmission, we used whole-cell patch-clamp technique to record spontaneous glutamate excitatory postsynaptic currents (EPSCs) from CA1 stratum radiatum interneurons (SRI) in hippocampal slices. First, the frequency, amplitude and kinetics of EPSCs recorded from slices of control guinea pigs were compared to those recorded from slices of guinea pigs after a single injection of the irreversible AChE inhibitor soman (25.2μg/kg, s.c.). Second, EPSCs were recorded from rat hippocampal slices before and after their superfusion with the reversible AChE inhibitor donepezil (100nM). The frequency of EPSCs was significantly higher in slices taken from guinea pigs 24h but not 7 days after the soman injection than in slices from control animals. In 52% of the rat hippocampal slices tested, bath application of donepezil increased the frequency of EPSCs. Further, exposure to donepezil increased both burst-like and large-amplitude EPSCs, and increased the proportion of short (20-100ms) inter-event intervals. Donepezil's effects were suppressed significantly in presence of 10μM mecamylamine or 10nM methyllycaconitine. These results support the concept that AChE inhibition is able to recruit nAChR-dependent glutamate transmission in the hippocampus and such a mechanism can contribute to the acute neurotoxicological actions of soman.

  17. Effects of Relative Hypoglycemia on LTP and NADH Imaging in Rat Hippocampal Slices

    PubMed Central

    Sadgrove, Matthew P.; Beaver, Christopher J.; Turner, Dennis A.

    2007-01-01

    Cognitive and neuronal impairment in diabetes may be associated with iatrogenic hypoglycemia, particularly at low serum glucose levels (< 3 mM). To evaluate cellular impairment, we assessed acute hippocampal slice functioning during decreased ambient glucose, by monitoring evoked field excitatory post-synaptic potentials (fEPSP), and slice nicotinamide adenine dinucleotide (NADH) fluorescence. The effects of lowered glucose levels (60 min) were analyzed by examining the induction and maintenance of long-term potentiation (LTP), and NADH metabolic imaging in the CA1 region. The basal fEPSP response was reduced by lowered ambient glucose, an effect that was reversible in 2.5 mM glucose, partially reversible in 1.25 mM glucose and irreversible in 0 mM glucose, after 25 min recovery. LTP induction and maintenance declined during glucose restriction, demonstrating reversibly failed maintenance in 5 mM and 2.5 mM ambient glucose, and absent induction in 1.25 mM glucose. Peak NADH levels observed during train-induced biphasic transients were significantly reduced during 1.25 mM and 2.5 mM glucose. Significant functional compromise in our slice model occurred at 2.5 mM ambient glucose, equivalent to <1mM tissue glucose in the slice center, due to diffusion limitations and active glucose utilization. This tissue glucose level correlates with human observations of a serum threshold of <3mM for cognitive impairment, since brain tissue glucose is approximately one third of serum levels. The physiological effects of hypoglycemia in our slice model, assessed through fEPSP, LTP, and NADH responses, replicate closely the in vivo situation, confirming the usefulness of this model in assessing consequences of relative hypoglycemia. PMID:17651706

  18. Neuroprotective effects of mild hypoxia in organotypic hippocampal slice cultures

    PubMed Central

    Kim, Seh Hyun; Lee, Woo Soon; Lee, Na Mi; Yun, Sin Weon

    2015-01-01

    Purpose The aim of this study was to investigate the potential effects of mild hypoxia in the mature and immature brain. Methods We prepared organotypic slice cultures of the hippocampus and used hippocampal tissue cultures at 7 and 14 days in vitro (DIV) to represent the immature and mature brain, respectively. Tissue cultures were exposed to 10% oxygen for 60 minutes. Twenty-four hours after this hypoxic insult, propidium iodide fluorescence images were obtained, and the damaged areas in the cornu ammonis 1 (CA1), CA3, and dentate gyrus (DG) were measured using image analysis. Results In the 7-DIV group compared to control tissue, hypoxia-exposed tissue showed decreased damage in two regions (CA1: 5.59%±2.99% vs. 4.80%±1.37%, P=0.900; DG: 33.88%±12.53% vs. 15.98%±2.37%, P=0.166), but this decrease was not statistically significant. In the 14-DIV group, hypoxia-exposed tissue showed decreased damage compared to control tissues; this decrease was not significant in the CA3 (24.51%±6.05% vs. 18.31%±3.28%, P=0.373) or DG (15.72%±3.47% vs. 9.91%±2.11%, P=0.134), but was significant in the CA1 (50.91%±5.90% vs. 32.30%±3.34%, P=0.004). Conclusion Although only CA1 tissues cultured for 14 DIV showed significantly less damage after exposure to hypoxia, the other tissues examined in this study showed a tendency towards less damage after hypoxic exposure. Therefore, mild hypoxia might play a protective role in the brain. PMID:25932036

  19. SCH 58261 differentially influences quinolinic acid-induced effects in striatal and in hippocampal slices.

    PubMed

    Tebano, Maria Teresa; Domenici, Maria Rosaria; Popoli, Patrizia

    2002-08-30

    The influence of the adenosine A(2A) receptor antagonist SCH 58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-trizolo[1,5-c] pyrimidine) (50, 200 nM, 1 microM) on quinolinic acid effects has been studied in rat striatal and hippocampal slices. Quinolinic acid induced disappearance of field potentials at concentrations of 500 microM and 2 mM in hippocampal and corticostriatal slices, respectively. We found that 1 microM SCH 58261 prevented quinolinic acid-induced field potential disappearance in corticostriatal but not in hippocampal slices. This finding demonstrates that the peculiar binding profile of SCH 58261 and the predominance in the hippocampus of "atypical" adenosine A(2A) receptor population (not recognized by SCH 58261) could have a functional relevance in the occurrence of region-specific neuroprotective effects.

  20. Effects of Acetylcholinesterase Inhibition on Cholinergic Transmission in the Hippocampal Slice.

    DTIC Science & Technology

    1985-02-08

    examined using a completely different experimental paradigm involving the biochemical measurement of sodium fluxes in slices of hippocampus . All of the...Sum OR. Acetylcholinesterase, long-term effects, hippocampus IS. A AC 4rCO.hImaw so M if weem’y andid Wit Ufy ft 61W& awmwrr is research program is...physiological response has been identified in ,the in vitro hippocampal slice, (2) the response of the hippocampus to repeated applications of cholTnerg-g

  1. An organotypic hippocampal slice culture model of excitotoxic injury induced spontaneous recurrent epileptiform discharges

    PubMed Central

    Ziobro, Julie M.; Deshpande, Laxmikant S.; DeLorenzo, Robert J.

    2011-01-01

    Stroke is the major cause of acquired epilepsy in the adult population. The mechanisms of ischemia-induced epileptogenesis are not completely understood, but glutamate is associated with both ischemia-induced injury and epileptogenesis. The objective of this study was to develop an in vitro model of epileptogenesis induced by glutamate injury in organotypic hippocampal slice cultures (OHSCs), as observed in stroke-induced acquired epilepsy. OHSCs were prepared from 1-week old Sprague-Dawley rat pups. They were exposed to 3.5 mM glutamate for 35 minutes at 21 days in vitro. Field potential recordings and whole-cell current clamp electrophysiology were used to monitor the development of in vitro seizure events up to 19 days after injury. Propidium iodide uptake assays were used to examine acute cell death following injury. Glutamate exposure produced a subset of hippocampal neurons that died acutely and a larger population of injured but surviving neurons. These surviving neurons manifested spontaneous, recurrent epileptiform discharges in neural networks, characterized by paroxysmal depolarizing shifts and high frequency spiking in both field potential and intracellular recordings. This model also exhibited anticonvulsant sensitivity similar to in vivo models. Our study is the first demonstration of a chronic model of acquired epilepsy in OHSCs following a glutamate injury. This in vitro model of glutamate injury–induced epileptogenesis may help develop therapeutic strategies to prevent epileptogenesis after stroke and elucidate some of the mechanisms that underlie stroke-induced epilepsy in a more anatomically in-tact system. PMID:21111720

  2. Network hyperexcitability in hippocampal slices from Mecp2 mutant mice revealed by voltage-sensitive dye imaging

    PubMed Central

    Calfa, Gaston; Hablitz, John J.

    2011-01-01

    Dysfunctions of neuronal and network excitability have emerged as common features in disorders associated with intellectual disabilities, autism, and seizure activity, all common clinical manifestations of Rett syndrome (RTT), a neurodevelopmental disorder caused by loss-of-function mutations in the transcriptional regulator methyl-CpG-binding protein 2 (MeCP2). Here, we evaluated the consequences of Mecp2 mutation on hippocampal network excitability, as well as synapse structure and function using a combination of imaging and electrophysiological approaches in acute slices. Imaging the amplitude and spatiotemporal spread of neuronal depolarizations with voltage-sensitive dyes (VSD) revealed that the CA1 and CA3 regions of hippocampal slices from symptomatic male Mecp2 mutant mice are highly hyperexcitable. However, only the density of docked synaptic vesicles and the rate of release from the readily releasable pool are impaired in Mecp2 mutant mice, while synapse density and morphology are unaffected. The differences in network excitability were not observed in surgically isolated CA1 minislices, and blockade of GABAergic inhibition enhanced VSD signals to the same extent in Mecp2 mutant and wild-type mice, suggesting that network excitability originates in area CA3. Indeed, extracellular multiunit recordings revealed a higher level of spontaneous firing of CA3 pyramidal neurons in slices from symptomatic Mecp2 mutant mice. The neuromodulator adenosine reduced the amplitude and spatiotemporal spread of VSD signals evoked in CA1 of Mecp2 mutant slices to wild-type levels, suggesting its potential use as an anticonvulsant in RTT individuals. The present results suggest that hyperactive CA3 pyramidal neurons contribute to hippocampal dysfunction and possibly to limbic seizures observed in Mecp2 mutant mice and RTT individuals. PMID:21307327

  3. Chronic Mild Stress Modulates Activity-Dependent Transcription of BDNF in Rat Hippocampal Slices.

    PubMed

    Molteni, Raffaella; Rossetti, Andrea C; Savino, Elisa; Racagni, Giorgio; Calabrese, Francesca

    2016-01-01

    Although activity-dependent transcription represents a crucial mechanism for long-lasting experience-dependent changes in the hippocampus, limited data exist on its contribution to pathological conditions. We aim to investigate the influence of chronic stress on the activity-dependent transcription of brain-derived neurotrophic factor (BDNF). The ex vivo methodology of acute stimulation of hippocampal slices obtained from rats exposed to chronic mild stress (CMS) was used to evaluate whether the adverse experience may alter activity-dependent BDNF gene expression. CMS reduces BDNF expression and that acute depolarization significantly upregulates total BDNF mRNA levels only in control animals, showing that CMS exposure may alter BDNF transcription under basal conditions and during neuronal activation. Moreover, while the basal effect of CMS on total BDNF reflects parallel modulations of all the transcripts examined, isoform-specific changes were found after depolarization. This different effect was also observed in the activation of intracellular signaling pathways related to the neurotrophin. In conclusion, our study discloses a functional alteration of BDNF transcription as a consequence of stress. Being the activity-regulated transcription a critical process in synaptic and neuronal plasticity, the different regulation of individual BDNF promoters may contribute to long-lasting changes, which are fundamental for the vulnerability of the hippocampus to stress-related diseases.

  4. Thermal dependence of neural activity in the hamster hippocampal slice preparation

    NASA Technical Reports Server (NTRS)

    Horowitz, J. M.; Thomas, M. P.; Eckerman, P.

    1987-01-01

    1. Neural activity was recorded in an in vitro hamster hippocampal slice preparation while the temperature of the Ringer's solution bathing in the slice was controlled at selected levels. 2. The amplitude of the population spike (action potentials from a group of pyramidal cells) was measured as bath temperature was lowered from 35 degrees C to temperatures where a response could not be evoked. 3. Plots of population spike amplitude versus temperature have bell-shaped curves. The population spikes increased in amplitude as temperature was lowered from 35 degrees C, reached a peak amplitude between 25 and 20 degrees C, and then decreased until a response could not be evoked when temperature was further lowered. 4. These in vitro results obtained in the slice preparation are related to in vivo hippocampal studies. Results are interpreted as consistent with the proposal reviewed here that neural activity in the hippocampus plays a role at specific stages of entrance into and arousal from hibernation.

  5. Comparison of carbon monoxide and nitrogen induced effects on synaptic transmission in the rat hippocampal slice.

    PubMed

    Doolette, D J; Kerr, D I

    1992-04-13

    A comparison has been made of the effects of carbon monoxide (CO) or nitrogen (N2) exposure on synaptic transmission in the hippocampal slice. CA1 field potentials, evoked by Schaffer collateral stimulation, were unaffected by superfusion of slices with artificial cerebral spinal fluid (ACSF) equilibrated with either 15% CO or 15% N2 for 120 min. However, superfusion with hypoxic ACSF equilibrated with either 85% CO or 85% N2 caused a rapid depression of synaptic transmission. Reperfusion with control ACSF following 30 min hypoxia led to recovery of evoked responses and a slight hyperexcitability. In the hippocampal slice, synaptic transmission, as assessed by input/output curves, was not different during or following hypoxia induced by exposure to CO or N2. In the short term, CO is not toxic.

  6. Preparation of postsynaptic density fraction from hippocampal slices and proteomic analysis

    SciTech Connect

    Dosemeci, Ayse . E-mail: dosemeca@mail.nih.gov; Tao-Cheng, J.-H.; Vinade, Lucia; Jaffe, Howard

    2006-01-13

    Hippocampal slices offer an excellent experimental system for the study of activity-induced changes in the postsynaptic density (PSD). While studies have documented electrophysiological and structural changes at synapses in response to precise manipulations of hippocampal slices, parallel biochemical and proteomic analyses were hampered by the lack of subcellular fractionation techniques applicable to starting tissue about three orders of magnitude smaller than that used in conventional protocols. Here, we describe a simple and convenient method for the preparation of PSD fractions from hippocampal slices and the identification of its components by proteomic techniques. The 'micro PSD fraction' obtained following two consecutive extractions of a synaptosomal fraction with Triton X-100 shows a significant enrichment in the marker protein PSD-95. Thin section electron microscopy shows PSDs similar to those observed in situ. However, other particulate material, especially myelin, and membrane vesicles are also present. The composition of the PSD fraction from hippocampal slices was analyzed by 2D LC/MS/MS. The proteomic approach which utilizes as little as 10 {mu}g total protein allowed the identification of >100 proteins. Many of the proteins detected in the fraction are the same as those identified in conventional PSD preparations including specialized PSD-scaffolding proteins, signaling molecules, cytoskeletal elements as well as certain contaminants. The results show the feasibility of the preparation of a PSD fraction from hippocampal slices of reasonable purity and of sufficient yield for proteomic analyses. In addition, we show that further purification of PSDs is possible using magnetic beads coated with a PSD-95 antibody.

  7. Neuroprotective effects of arachidonic acid against oxidative stress on rat hippocampal slices.

    PubMed

    Wang, Ze-Jian; Liang, Cui-Ling; Li, Guang-Mei; Yu, Cai-Yi; Yin, Ming

    2006-11-07

    Arachidonic acid (AA), 5,8,11,14-eicosateraenoic acid is abundant, active and necessary in the human body. In the present study, we reported the neuroprotective effects and mechanism of arachidonic acid on hippocampal slices insulted by glutamate, NaN(3) or H(2)O(2)in vitro. Different types of models of brain injury in vitro were developed by 1mM glutamate, 10mM NaN(3) or 2mM H(2)O(2). After 30 min of preincubation with arachidonic acid or linoleic acid, hippocampal slices were subjected to glutamate, NaN(3) or H(2)O(2), then the tissue activities were evaluated by using the 2,3,5-triphenyltetrazolium chloride method. Endogenous antioxidant enzymes activities (SOD, GSH-PX and catalase) in hippocampal slices were evaluated during the course of incubation. MK886 (5 microM; a noncompetitive inhibitor of proliferator-activated receptor [PPAR]alpha), BADGE (bisphenol A diglycidyl ether; 100 microM; an antagonist of PPARgamma) and cycloheximide (CHX; 30 microM; an inhibitor of protein synthesis) were tested for their effects on the neuroprotection afforded by arachidonic acid. Population spikes were recorded in randomly selected hippocapal slices. Arachidonic acid (1-10 microM) dose dependently protected hippocampal slices from glutamate and H(2)O(2) injury (P<0.01), and arachidonic acid (10 microM) can significantly improve the activities of Cu/Zn-SOD in hippocampal slices after 1h incubation. In addition, 10 microM arachidonic acid significantly increased the activity of Mn-SOD and catalase, and decreased the activities of Cu/Zn-SOD to control value after 3h incubation. These secondary changes of SOD during incubation can be reversed by indomethacine (10 microM; a nonspecific cyclooxygenase inhibitor) or AA 861 (20 microM; a 5-lipoxygenase inhibitor). Its neuroprotective effect was completely abolished by BADGE and CHX. These observations reveal that arachidonic acid can defense against oxidative stress by boosting the internal antioxidant system of hippocampal slices

  8. Extrasynaptic and synaptic NMDA receptors form stable and uniform pools in rat hippocampal slices

    PubMed Central

    Harris, Alexander Z; Pettit, Diana L

    2007-01-01

    N-methyl-d-aspartate receptor (NMDAR) activation can trigger both long- and short-term plasticity, promote cell survival, and initiate cell death. A number of studies suggest that the consequences of NMDAR activation can vary widely depending on whether synaptic or extrasynaptic receptors are activated. Here we have examined the spatial distribution of NMDARs of CA1 pyramidal neurons in acutely dissected hippocampal slices. Using a physiological definition of extrasynaptic receptors as those not accessible to single release events, we find that extrasynaptic NMDARs comprise a substantial proportion of the dendritic NMDAR pool (36%). This pool of extrasynaptic NMDARs is stable and does not shuttle into the synaptic receptor pool, as we observe no recovery of synaptic current after MK-801 synaptic blockade and washout. The subunit composition of synaptic and extrasynaptic NMDA receptor pools is similar at 3 weeks of age, with NR2B subunits present in both compartments. NR2B receptors are not enriched in the extrasynaptic compartment. Our data suggest that any role played by extrasynaptic NMDARs in synaptic transmission is dictated by their subcellular location rather than their subunit composition or mobility. PMID:17717018

  9. Extrasynaptic and synaptic NMDA receptors form stable and uniform pools in rat hippocampal slices.

    PubMed

    Harris, Alexander Z; Pettit, Diana L

    2007-10-15

    N-methyl-d-aspartate receptor (NMDAR) activation can trigger both long- and short-term plasticity, promote cell survival, and initiate cell death. A number of studies suggest that the consequences of NMDAR activation can vary widely depending on whether synaptic or extrasynaptic receptors are activated. Here we have examined the spatial distribution of NMDARs of CA1 pyramidal neurons in acutely dissected hippocampal slices. Using a physiological definition of extrasynaptic receptors as those not accessible to single release events, we find that extrasynaptic NMDARs comprise a substantial proportion of the dendritic NMDAR pool (36%). This pool of extrasynaptic NMDARs is stable and does not shuttle into the synaptic receptor pool, as we observe no recovery of synaptic current after MK-801 synaptic blockade and washout. The subunit composition of synaptic and extrasynaptic NMDA receptor pools is similar at 3 weeks of age, with NR2B subunits present in both compartments. NR2B receptors are not enriched in the extrasynaptic compartment. Our data suggest that any role played by extrasynaptic NMDARs in synaptic transmission is dictated by their subcellular location rather than their subunit composition or mobility.

  10. Isolation of CA1 nuclear enriched fractions from hippocampal slices to study activity-dependent nuclear import of synapto-nuclear messenger proteins.

    PubMed

    Yuanxiang, Pingan; Bera, Sujoy; Karpova, Anna; Kreutz, Michael R; Mikhaylova, Marina

    2014-08-10

    Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings. The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

  11. Electrophysiological observations in hippocampal slices from rats treated with the ketogenic diet.

    PubMed

    Stafstrom, C E; Wang, C; Jensen, F E

    1999-11-01

    The electrophysiological effects of the high-fat, low-carbohydrate ketogenic diet (KD) were assessed in normal and epileptic [kainic-acid(KA)-treated] adult rats using hippocampal slices. In the first set of experiments, normal rats were fed the KD or a standard control diet for 6-8 weeks (beginning on postnatal day 56, P56), after which they were sacrificed for hippocampal slices. All rats on the KD became ketotic. The baseline effects of the KD were determined by comparing extracellular measures of synaptic transmission and responses to evoked stimulation, and hippocampal excitability was tested in Mg(2+)-free medium. There were no differences in EPSP slope, input/output relationship, responses to evoked stimulation or Mg(2+)-free burst frequency between slices from control and KD-fed rats. In another set of experiments, rats were made epileptic by intraperitoneal injection of kainic acid (KA) on P54, which caused status epilepticus followed by the development of spontaneous recurrent seizures (SRS) over the next few weeks. Two days after KA-induced status, rats were divided into a control-fed group and a KD-fed group. Animals on the KD had significantly fewer SRS over the ensuing 8 weeks. In hippocampal slices from KA-treated, KD-fed rats, there were fewer evoked CA1 population spikes than from slices of control-fed rats. These results suggest that the KD does not alter baseline electrophysiological parameters in normal rats. In rats made chronically epileptic by administration of KA, KD treatment was associated with fewer spontaneous seizures and reduced CA1 excitability in vitro. Therefore, at least part of the KD mechanism of action may involve long-term changes in network excitability.

  12. Direct excitation of inhibitory interneurons by extracellular ATP mediated by P2Y1 receptors in the hippocampal slice.

    PubMed

    Kawamura, Masahito; Gachet, Christian; Inoue, Kazuhide; Kato, Fusao

    2004-12-01

    ATP is an important cell-to-cell signaling molecule mediating the interactions between astrocytes and neurons in the CNS. In the hippocampal slices, ATP suppresses excitatory transmission mostly through activation of adenosine A1 receptors, because the ectoenzyme activity for the extracellular breakdown of ATP to adenosine is high in slice preparations in contrast to culture environments. Because the hippocampus is also rich in the expression of P2 receptors activated specifically by ATP, we examined whether ATP modulates neuronal excitability in the acute slice preparations independently of adenosine receptors. Although ATP decreased the frequency of spontaneously occurring EPSCs in the CA3 pyramidal neurons through activation of adenosine A1 receptors, ATP concurrently increased the frequency of IPSCs in a manner dependent on action potential generation. This effect was mediated by P2Y1 receptors because (1) 2-methylthio-ATP (2meSATP) was the most potent agonist, (2) 2'-deoxy-N6-methyladenosine-3',5'-bisphosphate diammonium (MRS2179) abolished this effect, and (3) this increase in IPSC frequency was not observed in the transgenic mice lacking P2Y1 receptor proteins. Application of 2meSATP elicited MRS2179-sensitive time- and voltage-dependent inward currents in the interneurons, which depolarized the cell to firing threshold. Also, it increased [Ca2+]i in both astrocytes and interneurons, but, unlike the former effect, the latter was entirely dependent on Ca2+ entry. Thus, in hippocampal slices, in addition to activating A1 receptors of the excitatory terminals after being converted to adenosine, ATP activates P2Y1 receptors in the interneurons, which is linked to activation of unidentified excitatory conductance, through mechanisms distinct from those in the astrocytes.

  13. Synaptic transmission despite severe hypoxia in hippocampal slices of the deep-diving hooded seal.

    PubMed

    Geiseler, Samuel J; Larson, John; Folkow, Lars P

    2016-10-15

    Brain neurons of the deep-diving hooded seal (Cystophora cristata) are known to be inherently hypoxia tolerant. Here, we have used in vitro field potential recordings in hippocampal slices to compare effects of severe hypoxia on synaptic transmission in hooded seals vs. non-diving mammals. Synaptic responses of mice (Mus musculus) to hypoxia were in accordance with previously published data. Hippocampal slices of reindeer (Rangifer tarandus), an alternative large-mammal non-diving model, behaved in a similar way as mouse slices, in that synaptic activity disappeared rapidly without recovery after >20min in hypoxia. The synaptic activity of hooded seal slices decreased in hypoxia, but unlike mice and reindeer, it remained at >30% of the normoxic amplitude throughout 3h of severe hypoxia. Also, upon reoxygenation, the signal recovered to ∼50% of the pre-challenge (normoxic) amplitude. The AMPA-type glutamate receptor antagonist CNQX eliminated this signal, showing that it was not an artifact. Paired pulse facilitation (PPF), typically associated with increased presynaptic calcium (Ca(2+)) levels, was significantly reduced in the seal slices. We propose that the build-up of Ca(2+) concentration is limited in seal presynaptic terminals, possibly due to a high Ca(2+) buffering capacity, which could explain both the attenuated PPF and the remarkable neural hypoxia tolerance of this species. Although we found no significant hypoxia-induced upregulation of mRNA for the Ca(2+) binding proteins calbindin d28k or parvalbumin in hooded seal hippocampal slices, a recent study reports very high transcript levels of the Ca(2+) binding protein S100B in this species, which is in support of the hypothesis. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  14. Dithiothreitol elicits epileptiform activity in CA1 of the guinea pig hippocampal slice

    SciTech Connect

    Tolliver, J.M.; Pellmar, T.C.

    1987-01-01

    Dithiothreitol (DTT) is a sulfhydryl reducing agent used as a radioprotectant. Exposure of hippocampal slices, for 30 min to 0.5 micromoles DTT irreversibly increased the orthodromic population spike amplitude, promoted repetitive firing and induced spontaneous epileptiform activity in the CA1 subfield. The same concentration of the oxidized form of DTT did not increase hippocampal excitability. Although the slope of the population synaptic response to afferent stimulation (popPSP) was unchanged by DTT, the duration of the popPSP was prolonged. Recurrent inhibition was unaffected. DTT probably exerts its effects through an irreversible chemical reaction with cellular components. Possible mechanisms of DTT-induced epileptiform activity are discussed.

  15. Cell death and proliferation in acute slices and organotypic cultures of mammalian CNS.

    PubMed

    Lossi, Laura; Alasia, Silvia; Salio, Chiara; Merighi, Adalberto

    2009-08-01

    Analysis of the interplay between cell proliferation and death has been greatly advantaged by the development of CNS slice preparations. In slices, interactions between neurons and neurons and the glial cells are fundamentally preserved in a fashion close to the in vivo situation. In parallel, these preparations offer the possibility of an easy experimental manipulation. Two main types of slices are currently in use: the acute slices, which are short living preparations where the major functions of the intact brain (including neurogenesis) are maintained, and the organotypic cultures, where the maturation and plasticity of neuronal circuitries in relation to naturally occurring neuronal death and/or experimental insults can be followed over several weeks in vitro. We will discuss here the main advantages/disadvantages linked to the use of CNS slices for histological analysis of neuronal proliferation and death, as well as the main findings obtained in the most popular types of preparations, i.e. the cortical, hippocampal, cerebellar and retinal slices.

  16. Endogenous 24S-hydroxycholesterol modulates NMDAR-mediated function in hippocampal slices

    PubMed Central

    Sun, Min-Yu; Izumi, Yukitoshi; Benz, Ann; Zorumski, Charles F.

    2015-01-01

    N-methyl-d-aspartate receptors (NMDARs), a major subtype of glutamate receptors mediating excitatory transmission throughout the central nervous system (CNS), play critical roles in governing brain function and cognition. Because NMDAR dysfunction contributes to the etiology of neurological and psychiatric disorders including stroke and schizophrenia, NMDAR modulators are potential drug candidates. Our group recently demonstrated that the major brain cholesterol metabolite, 24S-hydroxycholesterol (24S-HC), positively modulates NMDARs when exogenously administered. Here, we studied whether endogenous 24S-HC regulates NMDAR activity in hippocampal slices. In CYP46A1−/− (knockout; KO) slices where endogenous 24S-HC is greatly reduced, NMDAR tone, measured as NMDAR-to-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) excitatory postsynaptic current (EPSC) ratio, was reduced. This difference translated into more NMDAR-driven spiking in wild-type (WT) slices compared with KO slices. Application of SGE-301, a 24S-HC analog, had comparable potentiating effects on NMDAR EPSCs in both WT and KO slices, suggesting that endogenous 24S-HC does not saturate its NMDAR modulatory site in ex vivo slices. KO slices did not differ from WT slices in either spontaneous neurotransmission or in neuronal intrinsic excitability, and exhibited LTP indistinguishable from WT slices. However, KO slices exhibited higher resistance to persistent NMDAR-dependent depression of synaptic transmission induced by oxygen-glucose deprivation (OGD), an effect restored by SGE-301. Together, our results suggest that loss of positive NMDAR tone does not elicit compensatory changes in excitability or transmission, but it protects transmission against NMDAR-mediated dysfunction. We expect that manipulating this endogenous NMDAR modulator may offer new treatment strategies for neuropsychiatric dysfunction. PMID:26745248

  17. In vitro measurements of extracellular L-glutamate level in region CA3 of mouse hippocampal slices under chemical stimulation.

    PubMed

    Chiba, Hiromi; Deguchi, Yukari; Kanazawa, Ena; Kawai, Jun; Nozawa, Keiichiro; Shoji, Atsushi; Sugawara, Masao

    2010-01-01

    The concentration level of extracellular L-glutamate released from region CA3 of mouse hippocampal slices under tetraethylammonium (TEA) chloride and KCl stimulation was measured with independent methods, i.e., a capillary-based enzyme sensor, a patch sensor, and an enzyme-based imaging method. The L-glutamate level was compared with those at regions CA1 and DG. It was found that the enhanced concentration level at CA3 by TEA stimulation is very similar to that at CA1, but it is much lower than that at DG. The order of the regional distribution of L-glutamate, i.e., DG > CA1 ≈ CA3, was the same as that obtained by K(+) stimulation. However, in the presence of an uptake inhibitor, DL-TBOA, KCl stimulation showed the strongest L-glutamate flux at CA1, while TEA stimulation exhibited the strongest flux at CA3. The usefulness of the present approach for knowing the extracellular L-glutamate level in acute hippocampal slices is discussed.

  18. Parkia biglobosa Improves Mitochondrial Functioning and Protects against Neurotoxic Agents in Rat Brain Hippocampal Slices

    PubMed Central

    Komolafe, Kayode; Olaleye, Tolulope M.; Seeger, Rodrigo L.; Carvalho, Fabiano B.; Boligon, Aline A.; Athayde, Margareth L.; Klimaczewski, Claudia V.; Akindahunsi, Akintunde A.; Rocha, Joao B. T.

    2014-01-01

    Objective. Methanolic leaf extracts of Parkia biglobosa, PBE, and one of its major polyphenolic constituents, catechin, were investigated for their protective effects against neurotoxicity induced by different agents on rat brain hippocampal slices and isolated mitochondria. Methods. Hippocampal slices were preincubated with PBE (25, 50, 100, or 200 µg/mL) or catechin (1, 5, or 10 µg/mL) for 30 min followed by further incubation with 300 µM H2O2, 300 µM SNP, or 200 µM PbCl2 for 1 h. Effects of PBE and catechin on SNP- or CaCl2-induced brain mitochondrial ROS formation and mitochondrial membrane potential (ΔΨm) were also determined. Results. PBE and catechin decreased basal ROS generation in slices and blunted the prooxidant effects of neurotoxicants on membrane lipid peroxidation and nonprotein thiol contents. PBE rescued hippocampal cellular viability from SNP damage and caused a significant boost in hippocampus Na+, K+-ATPase activity but with no effect on the acetylcholinesterase activity. Both PBE and catechin also mitigated SNP- or CaCl2-dependent mitochondrial ROS generation. Measurement by safranine fluorescence however showed that the mild depolarization of the ΔΨm by PBE was independent of catechin. Conclusion. The results suggest that the neuroprotective effect of PBE is dependent on its constituent antioxidants and mild mitochondrial depolarization propensity. PMID:25177688

  19. Cariprazine delays ouabain-evoked epileptiform spikes and loss of activity in rat hippocampal slices.

    PubMed

    El-Mallakh, Rif S; Payne, Ralphiel S; Schurr, Avital; Gao, Yonglin; Lei, Zhemin; Kiss, Béla; Gyertyán, István; Adham, Nika

    2015-09-30

    In the only bipolar cycling in vitro model, rat hippocampal slices are treated with the sodium pump inhibitor ouabain, which induces epileptiform activity, followed by refractory activity loss that recovers and cycles back to epileptiform activity. Thus, clinical cycling seen in patients with bipolar disorder is modeled on a cellular level as alternating hyperactivity and hypoactivity interspersed with normal activity. In this study, we tested the ability of cariprazine a new antipsychotic candidate to block ouabain-induced changes in rat hippocampal slices. Cycling of population spikes and epileptiform bursts was evoked using an extracellular stimulation electrode located in the Schaeffer collaterals of 400-µm-thick rat hippocampal slices treated with ouabain (3.3μM) alone or in combination with cariprazine (1, 5, 25, and 50µM). Responses were recorded using an extracellular electrode placed in the cell body layer of the CA1 region. Cariprazine 25 and 50µM delayed ouabain-induced epileptiform burst onset and subsequent activity loss. Lower cariprazine concentrations were ineffective. Cariprazine delays the onset of ouabain-induced epileptiform bursts and the loss of spiking activity similarly to that previously demonstrated with the mood stabilizer lithium. These results suggest that cariprazine may have therapeutic potential for treatment of bipolar disorder. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  20. In Vitro Manganese Exposure Disrupts MAPK Signaling Pathways in Striatal and Hippocampal Slices from Immature Rats

    PubMed Central

    Peres, Tanara Vieira; Pedro, Daniela Zótico; de Cordova, Fabiano Mendes; Lopes, Mark William; Gonçalves, Filipe Marques; Mendes-de-Aguiar, Cláudia Beatriz Nedel; Walz, Roger; Farina, Marcelo; Aschner, Michael; Leal, Rodrigo Bainy

    2013-01-01

    The molecular mechanisms mediating manganese (Mn)-induced neurotoxicity, particularly in the immature central nervous system, have yet to be completely understood. In this study, we investigated whether mitogen-activated protein kinases (MAPKs) and tyrosine hydroxylase (TH) could represent potential targets of Mn in striatal and hippocampal slices obtained from immature rats (14 days old). The aim of this study was to evaluate if the MAPK pathways are modulated after subtoxic Mn exposure, which do not significantly affect cell viability. The concentrations of manganese chloride (MnCl2; 10–1,000 μM) caused no change in cell viability in slices exposed for 3 or 6 hours. However, Mn exposure significantly increased extracellular signal-regulated kinase (ERK) 1/2, as well as c-Jun N-terminal kinase (JNK) 1/2/3 phosphorylation at both 3 and 6 hours incubations, in both brain structures. Furthermore, Mn exposure did not change the total content or phosphorylation of TH at the serine 40 site in striatal slices. Thus, Mn at concentrations that do not disrupt cell viability causes activation of MAPKs (ERK1/2 and JNK1/2/3) in immature hippocampal and striatal slices. These findings suggest that altered intracellular MAPKs signaling pathways may represent an early event concerning the effects of Mn in the immature brain. PMID:24324973

  1. Simultaneous activation of gamma and theta network oscillations in rat hippocampal slice cultures.

    PubMed

    Fischer, Yacov; Wittner, Lucia; Freund, Tamas F; Gähwiler, Beat H

    2002-03-15

    Hippocampal activity in vivo is characterized by concurrent oscillations at theta (4-15 Hz) and gamma (20-80 Hz) frequencies. Here we show that cholinergic receptor activation (methacholine 10-20 nm) in hippocampal slice cultures induces an oscillatory mode of activity, in which the intrinsic network oscillator (located in the CA3 area) expresses simultaneous theta and gamma network oscillations. Pyramidal cells display synaptic theta oscillations, characterized by cycles consisting of population EPSP-IPSP sequences that are dominated by population IPSPs. These rhythmic IPSPs most probably result from theta-modulated spiking activity of several interneurons. At the same time, the majority of interneurons consistently display synaptic gamma oscillations. These oscillatory cycles consist of fast depolarizing rhythmic events that are likely to reflect excitatory input from CA3 pyramidal cells. Interneurons comprising this functional group were identified morphologically. They include four known types of interneurons (basket, O-LM, bistratified and str. lucidum-specific cells) and one new type of CA3 interneuron (multi-subfield cell). The oscillatory activity of these interneurons is only weakly correlated between neighbouring cells, and in about half of these (44 %) is modulated by depolarizing theta rhythmicity. The overall characteristics of acetylcholine-induced oscillations in slice cultures closely resemble the rhythmicity observed in hippocampal field and single cell recordings in vivo. Both rhythmicities depend on intrinsic synaptic interactions, and are expressed by different cell types. The fact that these oscillations persist in a network lacking extra-hippocampal connections emphasizes the importance of intrinsic mechanisms in determining this form of hippocampal activity.

  2. Cutting of living hippocampal slices by a highly pressurised water jet (macromingotome).

    PubMed

    Bingmann, D; Wiemann, M; Speckmann, E J; Köhling, R; Straub, H; Dunze, K; Wittkowski, W

    2000-10-15

    Living brain slices are usually cut with razor blades, which compress a ca. 50-microm-thick layer of tissue. This results in cell debris and lesioned cells which, e.g. form diffusion barriers between the bath and living neurons underneath, thereby prolonging response times of neurons to drugs in the bath saline and impeding the experimental access to intact neurons. To avoid such drawbacks, a macromingotome was developed which cuts nervous tissue with water jets. Physiological saline under pressures of 100-1800 bar was ejected through nozzles of 35-100 microm to cut 300-500-microm-thick hippocampal slices. Systematic variations of pressure and nozzle diameter revealed best results at 400-600 bar and with nozzle diameters of 60-80 microm. Under these conditions, intact CA1- and CA3-neurons as well as granule cells were detected with infrared microscopy at less than 10 microm underneath the surface of the slice. Superficial neurons with intact fine structures were also seen when the slices were studied by light-microscopy. Intra- and extracellular recordings from superficial neurons showed normal membrane- and full action potentials and the development of stable epileptiform discharges in 0 Mg(2+)-saline. These results indicate that the macromingotome offers an alternative way of cutting slices which may facilitate electrophysiological/neuropharmacological or fluorometric studies on superficial neurons.

  3. Long-lasting desynchronization in rat hippocampal slice induced by coordinated reset stimulation

    NASA Astrophysics Data System (ADS)

    Tass, P. A.; Silchenko, A. N.; Hauptmann, C.; Barnikol, U. B.; Speckmann, E.-J.

    2009-07-01

    In computational models it has been shown that appropriate stimulation protocols may reshape the connectivity pattern of neural or oscillator networks with synaptic plasticity in a way that the network learns or unlearns strong synchronization. The underlying mechanism is that a network is shifted from one attractor to another, so that long-lasting stimulation effects are caused which persist after the cessation of stimulation. Here we study long-lasting effects of multisite electrical stimulation in a rat hippocampal slice rendered epileptic by magnesium withdrawal. We show that desynchronizing coordinated reset stimulation causes a long-lasting desynchronization between hippocampal neuronal populations together with a widespread decrease in the amplitude of the epileptiform activity. In contrast, periodic stimulation induces a long-lasting increase in both synchronization and amplitude.

  4. Reversible loss of dendritic spines and altered excitability after chronic epilepsy in hippocampal slice cultures.

    PubMed Central

    Müller, M; Gähwiler, B H; Rietschin, L; Thompson, S M

    1993-01-01

    The morphological and functional consequences of epileptic activity were investigated by applying the convulsants bicuculline and/or picrotoxin to mature rat hippocampal slice cultures. After 3 days, some cells in all hippocampal subfields showed signs of degeneration, including swollen somata, vacuolation, and dendritic deformities, whereas others displayed only a massive reduction in the number of their dendritic spines. Intracellular recordings from CA3 pyramidal cells revealed a decrease in the amplitude of evoked excitatory synaptic potentials. gamma-Aminobutyric acid-releasing interneurons and inhibitory synaptic potentials were unaffected. Seven days after withdrawal of convulsants, remaining cells possessed a normal number of dendritic spines, thus demonstrating a considerable capacity for recovery. The pathological changes induced by convulsants are similar to those found in the hippocampi of human epileptics, suggesting that they are a consequence, rather than a cause, of epilepsy. Images PMID:8093558

  5. Long-lasting desynchronization in rat hippocampal slice induced by coordinated reset stimulation

    SciTech Connect

    Tass, P. A.; Barnikol, U. B.; Silchenko, A. N.; Hauptmann, C.; Speckmann, E.-J.

    2009-07-15

    In computational models it has been shown that appropriate stimulation protocols may reshape the connectivity pattern of neural or oscillator networks with synaptic plasticity in a way that the network learns or unlearns strong synchronization. The underlying mechanism is that a network is shifted from one attractor to another, so that long-lasting stimulation effects are caused which persist after the cessation of stimulation. Here we study long-lasting effects of multisite electrical stimulation in a rat hippocampal slice rendered epileptic by magnesium withdrawal. We show that desynchronizing coordinated reset stimulation causes a long-lasting desynchronization between hippocampal neuronal populations together with a widespread decrease in the amplitude of the epileptiform activity. In contrast, periodic stimulation induces a long-lasting increase in both synchronization and amplitude.

  6. Metabolic Therapy for Temporal Lobe Epilepsy in a Dish: Investigating Mechanisms of Ketogenic Diet using Electrophysiological Recordings in Hippocampal Slices

    PubMed Central

    Kawamura, Masahito Jr.; Ruskin, David N.; Masino, Susan A.

    2016-01-01

    The hippocampus is prone to epileptic seizures and is a key brain region and experimental platform for investigating mechanisms associated with the abnormal neuronal excitability that characterizes a seizure. Accordingly, the hippocampal slice is a common in vitro model to study treatments that may prevent or reduce seizure activity. The ketogenic diet is a metabolic therapy used to treat epilepsy in adults and children for nearly 100 years; it can reduce or eliminate even severe or refractory seizures. New insights into its underlying mechanisms have been revealed by diverse types of electrophysiological recordings in hippocampal slices. Here we review these reports and their relevant mechanistic findings. We acknowledge that a major difficulty in using hippocampal slices is the inability to reproduce precisely the in vivo condition of ketogenic diet feeding in any in vitro preparation, and progress has been made in this in vivo/in vitro transition. Thus far at least three different approaches are reported to reproduce relevant diet effects in the hippocampal slices: (1) direct application of ketone bodies; (2) mimicking the ketogenic diet condition during a whole-cell patch-clamp technique; and (3) reduced glucose incubation of hippocampal slices from ketogenic diet–fed animals. Significant results have been found with each of these methods and provide options for further study into short- and long-term mechanisms including Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels, vesicular glutamate transporter (VGLUT), pannexin channels and adenosine receptors underlying ketogenic diet and other forms of metabolic therapy. PMID:27847463

  7. Metabolic Therapy for Temporal Lobe Epilepsy in a Dish: Investigating Mechanisms of Ketogenic Diet using Electrophysiological Recordings in Hippocampal Slices.

    PubMed

    Kawamura, Masahito Jr; Ruskin, David N; Masino, Susan A

    2016-01-01

    The hippocampus is prone to epileptic seizures and is a key brain region and experimental platform for investigating mechanisms associated with the abnormal neuronal excitability that characterizes a seizure. Accordingly, the hippocampal slice is a common in vitro model to study treatments that may prevent or reduce seizure activity. The ketogenic diet is a metabolic therapy used to treat epilepsy in adults and children for nearly 100 years; it can reduce or eliminate even severe or refractory seizures. New insights into its underlying mechanisms have been revealed by diverse types of electrophysiological recordings in hippocampal slices. Here we review these reports and their relevant mechanistic findings. We acknowledge that a major difficulty in using hippocampal slices is the inability to reproduce precisely the in vivo condition of ketogenic diet feeding in any in vitro preparation, and progress has been made in this in vivo/in vitro transition. Thus far at least three different approaches are reported to reproduce relevant diet effects in the hippocampal slices: (1) direct application of ketone bodies; (2) mimicking the ketogenic diet condition during a whole-cell patch-clamp technique; and (3) reduced glucose incubation of hippocampal slices from ketogenic diet-fed animals. Significant results have been found with each of these methods and provide options for further study into short- and long-term mechanisms including Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels, vesicular glutamate transporter (VGLUT), pannexin channels and adenosine receptors underlying ketogenic diet and other forms of metabolic therapy.

  8. Somatostatin immunohistochemistry of hippocampal slices with lucifer yellow-stained pyramidal neurons responding to somatostatin.

    PubMed

    Joëls, M; Madamba, S G; Moore, S D; Morrison, J H; Siggins, G R

    1990-04-24

    We have combined electrophysiology and immunohistochemistry to study the somatostatin (SS) innervation of neurons in the rat hippocampal slice. After recording the intracellular response of a pyramidal CA1 neuron in vitro to SS, Lucifer Yellow was injected into the cell and the slice fixed and processed for immunohistochemical localization of SS in the vicinity of the recorded neuron. Most pyramidal neurons (70%) responded to SS with a hyperpolarization associated with marked slowing of spontaneous discharge and reduced input resistance. SS-containing elements either crossed, ran parallel or seemingly terminated on the Lucifer Yellow-filled SS-responsive cell. These occurrences of close proximity of apparent pre- and postsynaptic elements were observed in all layers of the CA1 region and may represent synaptic terminations of SS elements on a pyramidal neuron that are likely to elicit membrane hyperpolarizations.

  9. Lactate Effectively Covers Energy Demands during Neuronal Network Activity in Neonatal Hippocampal Slices

    PubMed Central

    Ivanov, Anton; Mukhtarov, Marat; Bregestovski, Piotr; Zilberter, Yuri

    2011-01-01

    Although numerous experimental data indicate that lactate is efficiently used for energy by the mature brain, the direct measurements of energy metabolism parameters during neuronal network activity in early postnatal development have not been performed. Therefore, the role of lactate in the energy metabolism of neurons at this age remains unclear. In this study, we monitored field potentials and contents of oxygen and NAD(P)H in correlation with oxidative metabolism during intense network activity in the CA1 hippocampal region of neonatal brain slices. We show that in the presence of glucose, lactate is effectively utilized as an energy substrate, causing an augmentation of oxidative metabolism. Moreover, in the absence of glucose lactate is fully capable of maintaining synaptic function. Therefore, during network activity in neonatal slices, lactate can be an efficient energy substrate capable of sustaining and enhancing aerobic energy metabolism. PMID:21602909

  10. Ballistic labeling and dynamic imaging of astrocytes in organotypic hippocampal slice cultures.

    PubMed

    Benediktsson, Adrienne M; Schachtele, Scott J; Green, Steven H; Dailey, Michael E

    2005-01-30

    Protoplasmic astrocytes in mammalian CNS tissues in vivo have a highly complex 3D morphology, but in dissociated cell cultures they often assume a flattened, fibroblast-like morphology bearing only a few, simple processes. By fluorescent labeling and confocal reconstruction we show that many astrocytes in organotypic hippocampal slice cultures exhibit a more native complex cytoarchitecture. Although astrocytes at the surface of slice cultures show a reactive form with several thick glial fibrillary acidic protein (GFAP)-positive processes, astrocytes situated in deeper portions of tissue slices retain a highly complex 3D morphology with many fine spine- or veil-like protrusions. Dozens of astrocytes can be labeled in single slice cultures by gene gun-mediated ballistic delivery of gold or tungsten particles carrying cDNAs (Biolistics), lipophilic dyes (DiOlistics), or fluorescent intracellular calcium indicators (Calistics). Expression of a membrane-targeted form of eGFP (Lck-GFP) is superior to soluble eGFP for resolving fine astrocytic processes. Time-lapse confocal imaging of Lck-GFP transfected astrocytes or "calistically" labeled astrocytes show structural remodeling and calcium transients, respectively. This approach provides an in vitro system for investigating the functional architecture, development and dynamic remodeling of astrocytes and their relationships to neurons and glia in live mammalian brain tissues.

  11. The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

    PubMed Central

    Chip, Sophorn; Zhu, Xinzhou; Kapfhammer, Josef P.

    2014-01-01

    Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection. PMID:25408363

  12. De novo synthesized estradiol protects against methylmercury-induced neurotoxicity in cultured rat hippocampal slices.

    PubMed

    Yamazaki, Takeshi; Yamamoto, Megumi; Ishihara, Yasuhiro; Komatsu, Shota; Munetsuna, Eiji; Onizaki, Masahiro; Ishida, Atsuhiko; Kawato, Suguru; Mukuda, Takao

    2013-01-01

    Estrogen, a class of female sex steroids, is neuroprotective. Estrogen is synthesized in specific areas of the brain. There is a possibility that the de novo synthesized estrogen exerts protective effect in brain, although direct evidence for the neuroprotective function of brain-synthesized estrogen has not been clearly demonstrated. Methylmercury (MeHg) is a neurotoxin that induces neuronal degeneration in the central nervous system. The neurotoxicity of MeHg is region-specific, and the molecular mechanisms for the selective neurotoxicity are not well defined. In this study, the protective effect of de novo synthesized 17β-estradiol on MeHg-induced neurotoxicity in rat hippocampus was examined. Neurotoxic effect of MeHg on hippocampal organotypic slice culture was quantified by propidium iodide fluorescence imaging. Twenty-four-hour treatment of the slices with MeHg caused cell death in a dose-dependent manner. The toxicity of MeHg was attenuated by pre-treatment with exogenously added estradiol. The slices de novo synthesized estradiol. The estradiol synthesis was not affected by treatment with 1 µM MeHg. The toxicity of MeHg was enhanced by inhibition of de novo estradiol synthesis, and the enhancement of toxicity was recovered by the addition of exogenous estradiol. The neuroprotective effect of estradiol was inhibited by an estrogen receptor (ER) antagonist, and mimicked by pre-treatment of the slices with agonists for ERα and ERβ, indicating the neuroprotective effect was mediated by ERs. Hippocampus de novo synthesized estradiol protected hippocampal cells from MeHg-induced neurotoxicity via ERα- and ERβ-mediated pathways. The self-protective function of de novo synthesized estradiol might be one of the possible mechanisms for the selective sensitivity of the brain to MeHg toxicity.

  13. De Novo Synthesized Estradiol Protects against Methylmercury-Induced Neurotoxicity in Cultured Rat Hippocampal Slices

    PubMed Central

    Ishihara, Yasuhiro; Komatsu, Shota; Munetsuna, Eiji; Onizaki, Masahiro; Ishida, Atsuhiko; Kawato, Suguru; Mukuda, Takao

    2013-01-01

    Background Estrogen, a class of female sex steroids, is neuroprotective. Estrogen is synthesized in specific areas of the brain. There is a possibility that the de novo synthesized estrogen exerts protective effect in brain, although direct evidence for the neuroprotective function of brain-synthesized estrogen has not been clearly demonstrated. Methylmercury (MeHg) is a neurotoxin that induces neuronal degeneration in the central nervous system. The neurotoxicity of MeHg is region-specific, and the molecular mechanisms for the selective neurotoxicity are not well defined. In this study, the protective effect of de novo synthesized 17β-estradiol on MeHg-induced neurotoxicity in rat hippocampus was examined. Methodology/Principal Findings Neurotoxic effect of MeHg on hippocampal organotypic slice culture was quantified by propidium iodide fluorescence imaging. Twenty-four-hour treatment of the slices with MeHg caused cell death in a dose-dependent manner. The toxicity of MeHg was attenuated by pre-treatment with exogenously added estradiol. The slices de novo synthesized estradiol. The estradiol synthesis was not affected by treatment with 1 µM MeHg. The toxicity of MeHg was enhanced by inhibition of de novo estradiol synthesis, and the enhancement of toxicity was recovered by the addition of exogenous estradiol. The neuroprotective effect of estradiol was inhibited by an estrogen receptor (ER) antagonist, and mimicked by pre-treatment of the slices with agonists for ERα and ERβ, indicating the neuroprotective effect was mediated by ERs. Conclusions/Significance Hippocampus de novo synthesized estradiol protected hippocampal cells from MeHg-induced neurotoxicity via ERα- and ERβ-mediated pathways. The self-protective function of de novo synthesized estradiol might be one of the possible mechanisms for the selective sensitivity of the brain to MeHg toxicity. PMID:23405170

  14. Biocompatibility of silicon-based arrays of electrodes coupled to organotypic hippocampal brain slice cultures.

    PubMed

    Kristensen, B W; Noraberg, J; Thiébaud, P; Koudelka-Hep, M; Zimmer, J

    2001-03-30

    In this study we examined the passive biocompatibility of a three-dimensional microelectrode array (MEA), designed to be coupled to organotypic brain slice cultures for multisite recording of electrophysiological signals. Hippocampal (and corticostriatal) brain slices from 1-week-old (and newborn) rats were grown for 4-8 weeks on the perforated silicon chips with silicon nitride surfaces and 40 microm sized holes and compared with corresponding tissue slices grown on conventional semiporous membranes. In terms of preservation of the basic cellular and connective organization, as visualized by Nissl staining, Timm sulphide silver-staining, microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP) immunostaining, the slice cultures grown on chips did not differ from conventionally grown slice cultures. Neither were there any signs of astrogliosis or neurodegeneration around the upper recording part of the 47-microm-high platinum-tip electrodes. Slice cultures grown on a separate set of chips with platinum instead of silicon nitride surfaces also displayed normal MAP2 and GFAP immunostaining. The width of the GFAP-rich zone (glia limitans) at the bottom surface of the slice cultures was the same ( approximately 20 microm) in cultures grown on chips with silicon nitride and platinum surfaces and on conventional insert membranes. The slice cultures grown on chips maintained a normal, subfield differentiated susceptibility to the glutamate receptor agonist N-methyl-D-aspartate (NMDA) and the neurotoxin trimethyltin (TMT), as demonstrated by the cellular uptake of propidium iodide (PI), which was used as a reproducible and quantifiable marker for neuronal degeneration. We conclude that organotypic brain slice cultures can grow on silicon-based three-dimensional microelectrode arrays and develop normally with display of normal subfield differentiated susceptibilities to known excito- and neurotoxins. From this it is anticipated that the set

  15. Hyperexcitability in combined entorhinal/hippocampal slices of adult rat after exposure to brain-derived neurotrophic factor.

    PubMed

    Scharfman, H E

    1997-08-01

    Effects of brain-derived neurotrophic factor (BDNF) in area CA3, the dentate gyrus, and medial entorhinal cortex were examined electrophysiologically by bath application of BDNF in slices containing the hippocampus and entorhinal cortex. Bath application of 25-100 ng/ml BDNF for 30-90 min increased responses to single afferent stimuli in selective pathways in the majority of slices. In area CA3, responses to mossy fiber stimulation increased in 73% of slices and entorhinal cortex responses to white matter stimulation increased in 64% of slices. After exposure to BDNF, these areas also demonstrated evidence of hyperexcitability, because responses to repetitive stimulation (1-Hz paired pulses for several s) produced multiple population spikes in response to mossy fiber stimulation in CA3 or multiple field potentials in response to white matter stimulation in the entorhinal cortex. Repetitive field potentials persisted after repetitive stimulation ended and usually were followed by spreading depression. Enhancement of responses to single stimuli and hyperexcitability were never evoked in untreated slices or after bath application of boiled BDNF or cytochrome C. The tyrosine kinase antagonist K252a (2 microM) blocked the effects of BDNF. In area CA3, both the potentiation of responses to single stimuli and hyperexcitability showed afferent specificity, because responses to mossy fiber stimulation were affected but responses to fimbria or Schaffer collateral stimulation were not. In addition, regional specificity was demonstrated in that the dentate gyrus was much less affected. The effects of BDNF in area CA3 were similar to those produced by bath application of low doses of kainic acid, which is thought to modulate glutamate release from mossy fiber terminals by a presynaptic action. These results suggest that BDNF has acute effects on excitability in different areas of the hippocampal-entorhinal circuit. These effects appear to be greatest in areas that are highly

  16. DYNAMIC AND INTERACTING PROFILES OF •NO AND O2 IN RAT HIPPOCAMPAL SLICES

    PubMed Central

    Ledo, Ana; Barbosa, Rui; Cadenas, Enrique; Laranjinha, João

    2010-01-01

    Nitric oxide (•NO) is a ubiquitous signaling molecule that participates in the neuromolecular phenomena associated with memory formation. In the hippocampus, neuronal •NO production is coupled to the activation of the NMDA-type of glutamate receptor. Although, •NO-mediated signaling has been associated with soluble guanylate cyclase activation, cytochrome oxidase is also a target for this gaseous free radical, for which •NO competes with O2. Here, we show, for the first time in a model preserving tissue cytoarchitecture (rat hippocampal slices) and at a physiological O2 concentration, that endogenous NMDA-evoked •NO production inhibits tissue O2 consumption for submicromolar concentrations. The simultaneous real-time recordings reveal a direct correlation between the profiles of •NO and O2 in the CA1 subregion of the hippocampal slice. These results, obtained in a system close to in vivo models, strongly support the current paradigm for O2 and •NO interplay in the regulation of cellular respiration. PMID:20100565

  17. Effects of positive AMPA receptor modulators on calpain-mediated spectrin degradation in cultured hippocampal slices.

    PubMed

    Jourdi, Hussam; Yanagihara, Ted; Martinez, Ulises; Bi, Xiaoning; Lynch, Gary; Baudry, Michel

    2005-01-01

    Positive modulators of AMPA receptors (AMPAr), also known as ampakines, are allosteric effectors of the receptors and have been extensively studied in past years due to their potential use as treatment for various diseases and ailments of the central nervous system such as mild cognitive impairment, schizophrenia, and Alzheimer's disease. Ampakines have been shown to improve performance on memory tasks in animals and in human subjects, an effect linked to their ability to increase agonist-mediated ion influx through AMPAr, thus leading to enhanced synaptic responses and facilitation of long-term potentiation (LTP) induction at glutamatergic synapses. As LTP is associated with calpain activation and spectrin degradation, we determined the effects of ampakine treatment of cultured hippocampal slices on spectrin degradation. Calpain activation was evaluated by determining the levels of the 145-150kDa degradation products of spectrin. Our data indicated that incubation of hippocampal slices with some, but not all positive modulators of AMPA receptors resulted in enhanced spectrin degradation, an effect that was blocked by a calpain inhibitor. In addition, an antagonist of AMPAr but not of NMDAr blocked ampakine-induced spectrin degradation. These results indicate that prolonged treatment with selected ampakines leads to spectrin degradation mediated by activation of the calcium-dependent protease calpain.

  18. The Control of Seizure-Like Activity in the Rat Hippocampal Slice

    PubMed Central

    Khosravani, Houman; Carlen, Peter L.; Velazquez, Jose L. Perez

    2003-01-01

    The sudden and transient hypersynchrony of neuronal firing that characterizes epileptic seizures can be considered as the transitory stabilization of metastable states present within the dynamical repertoire of a neuronal network. Using an in vitro model of recurrent spontaneous seizures in the rat horizontal hippocampal slice preparation, we present an approach to characterize the dynamics of the transition to seizure, and to use this information to control the activity and avoid the occurrence of seizure-like events. The transition from the interictal activity (between seizures) to the seizure-like event is aborted by brief (20–50 s) low-frequency (0.5 Hz) periodic forcing perturbations, applied via an extracellular stimulating electrode to the mossy fibers, the axons of the dentate neurons that synapse onto the CA3 pyramidal cells. This perturbation results in the stabilization of an interictal-like low-frequency firing pattern in the hippocampal slice. The results derived from this work shed light on the dynamics of the transition to seizure and will further the development of algorithms that can be used in automated devices to stop seizure occurrence. PMID:12524321

  19. Rosiglitazone Suppresses In Vitro Seizures in Hippocampal Slice by Inhibiting Presynaptic Glutamate Release in a Model of Temporal Lobe Epilepsy

    PubMed Central

    Wong, Shi-Bing; Cheng, Sin-Jhong; Hung, Wei-Chen; Lee, Wang-Tso; Min, Ming-Yuan

    2015-01-01

    Peroxisomal proliferator-activated receptor gamma (PPARγ) is a nuclear hormone receptor whose agonist, rosiglitazone has a neuroprotective effect to hippocampal neurons in pilocarpine-induced seizures. Hippocampal slice preparations treated in Mg2+ free medium can induce ictal and interictal-like epileptiform discharges, which is regarded as an in vitro model of N-methyl-D-aspartate (NMDA) receptor-mediated temporal lobe epilepsy (TLE). We applied rosiglitazone in hippocampal slices treated in Mg2+ free medium. The effects of rosiglitazone on hippocampal CA1-Schaffer collateral synaptic transmission were tested. We also examined the neuroprotective effect of rosiglitazone toward NMDA excitotoxicity on cultured hippocampal slices. Application of 10μM rosiglitazone significantly suppressed amplitude and frequency of epileptiform discharges in CA1 neurons. Pretreatment with the PPARγ antagonist GW9662 did not block the effect of rosiglitazone on suppressing discharge frequency, but reverse the effect on suppressing discharge amplitude. Application of rosiglitazone suppressed synaptic transmission in the CA1-Schaffer collateral pathway. By miniature excitatory-potential synaptic current (mEPSC) analysis, rosiglitazone significantly suppressed presynaptic neurotransmitter release. This phenomenon can be reversed by pretreating PPARγ antagonist GW9662. Also, rosiglitazone protected cultured hippocampal slices from NMDA-induced excitotoxicity. The protective effect of 10μM rosiglitazone was partially antagonized by concomitant high dose GW9662 treatment, indicating that this effect is partially mediated by PPARγ receptors. In conclusion, rosiglitazone suppressed NMDA receptor-mediated epileptiform discharges by inhibition of presynaptic neurotransmitter release. Rosiglitazone protected hippocampal slice from NMDA excitotoxicity partially by PPARγ activation. We suggest that rosiglitazone could be a potential agent to treat patients with TLE. PMID:26659605

  20. Effect of the nootropic drug oxiracetam on field potentials of rat hippocampal slices.

    PubMed Central

    Pugliese, A. M.; Corradetti, R.; Ballerini, L.; Pepeu, G.

    1990-01-01

    1. The effect of the nootropic drug oxiracetam on hippocampal neurotransmission was investigated in the CA1 region of the rat hippocampal slice in vitro by use of extracellular recordings. 2. Superfusion of oxiracetam (0.1-100 microM) produced a concentration-dependent, wash-resistant (greater than 90 min), increase in initial slope and amplitude of the dendritic field excitatory postsynaptic potential (e.p.s.p.). This increase was maximal at a concentration of 1 microM (70%). 3. Input-output curves relating the initial slope to the amplitude of the afferent volley were significantly (P less than 0.05) steeper and showed a greater maximal response in the presence of 1 microM oxiracetam than in control conditions. 4. Two trains of high frequency stimulation (100 Hz, 0.4 s, 5 min apart) delivered in the stratum radiatum 30 min after washout of oxiracetam (1 microM) still elicited a long-term potentiation (LTP) of the field e.p.s.p. However, the absolute magnitude of the LTP produced did not differ from that obtained in untreated slices. 5. After induction and establishment of LTP, oxiracetam (1 microM) had a smaller (27%) and reversible effect on the evoked field e.p.s.p. 6. D-2-Amino-5-phosphonopentanoic acid (AP-5), at the same concentration (50 microM) which in our conditions prevented the induction of LTP, blocked the action of 1 microM oxiracetam and strongly depressed the effect of higher concentrations of the nootropic drug. 7. It is concluded that oxiracetam provokes an enduring increase of neurotransmission in the CA1 rat hippocampal region.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1970492

  1. Significant glial alterations in response to iron loading in a novel organotypic hippocampal slice culture model

    PubMed Central

    Healy, Sinead; McMahon, Jill; Owens, Peter; FitzGerald, Una

    2016-01-01

    Aberrant iron deposition in the brain is associated with neurodegenerative disorders including Multiple Sclerosis, Alzheimer’s disease and Parkinson’s disease. To study the collective response to iron loading, we have used hippocampal organotypic slices as a platform to develop a novel ex vivo model of iron accumulation. We demonstrated differential uptake and toxicity of iron after 12 h exposure to 10 μM ferrous ammonium sulphate, ferric citrate or ferrocene. Having established the supremacy of ferrocene in this model, the cultures were then loaded with 0.1–100 μM ferrocene for 12 h. One μM ferrocene exposure produced the maximal 1.6-fold increase in iron compared with vehicle. This was accompanied by a 1.4-fold increase in ferritin transcripts and mild toxicity. Using dual-immunohistochemistry, we detected ferritin in oligodendrocytes, microglia, but rarely in astrocytes and never in neurons in iron-loaded slice cultures. Moreover, iron loading led to a 15% loss of olig2-positive cells and a 16% increase in number and greater activation of microglia compared with vehicle. However, there was no appreciable effect of iron loading on astrocytes. In what we believe is a significant advance on traditional mono- or dual-cultures, our novel ex vivo slice-culture model allows characterization of the collective response of brain cells to iron-loading. PMID:27808258

  2. Altered Network Timing in the CA3-CA1 Circuit of Hippocampal Slices from Aged Mice

    PubMed Central

    Kanak, Daniel J.; Rose, Gregory M.; Zaveri, Hitten P.; Patrylo, Peter R.

    2013-01-01

    Network patterns are believed to provide unique temporal contexts for coordinating neuronal activity within and across different regions of the brain. Some of the characteristics of network patterns modeled in vitro are altered in the CA3 or CA1 subregions of hippocampal slices from aged mice. CA3–CA1 network interactions have not been examined previously. We used slices from aged and adult mice to model spontaneous sharp wave ripples and carbachol-induced gamma oscillations, and compared measures of CA3–CA1 network timing between age groups. Coherent sharp wave ripples and gamma oscillations were evident in the CA3–CA1 circuit in both age groups, but the relative timing of activity in CA1 stratum pyramidale was delayed in the aged. In another sample of aged slices, evoked Schaffer collateral responses were attenuated in CA3 (antidromic spike amplitude) and CA1 (orthodromic field EPSP slope). However, the amplitude and timing of spontaneous sharp waves recorded in CA1 stratum radiatum were similar to adults. In both age groups unit activity recorded juxtacellularly from unidentified neurons in CA1 stratum pyramidale and stratum oriens was temporally modulated by CA3 ripples. However, aged neurons exhibited reduced spike probability during the early cycles of the CA3 ripple oscillation. These findings suggest that aging disrupts the coordination of patterned activity in the CA3–CA1 circuit. PMID:23593474

  3. Imaging cell volume changes and neuronal excitation in the hippocampal slice.

    PubMed

    Andrew, R D; MacVicar, B A

    1994-09-01

    Brain cell swelling is a consequence of seizure, ischemia or excitotoxicity. Changes in light reflectance from cortical surface are now used to monitor brain activity but these intrinsic signals are poorly understood. The objectives of this study were first, to show that changes in light transmittance were correlated with cell volume and second, to image increases in light transmittance as they related to neuronal activation. Transverse hippocampal slices from the rat were used for the study. Brief exposure (4-6 min) to hypo-osmotic artificial cerebrospinal fluid (-40 mOsm) elevated light transmittance consistently and reversibly in most regions of the slice and particularly in CA1 dendritic regions. Neither zero-Ca2+ artificial cerebrospinal fluid nor tetrodotoxin altered the transmittance increase and its subsequent reversal, suggesting that it was dependent on osmolality but independent of synaptic transmission and neuronal firing. The amplitude of the CA1 population spike evoked from Schaffer collaterals increased concomitantly with the hypo-osmotic increase in light transmittance, providing evidence that the extracellular tissue resistance increased. Hyper-osmotic artificial cerebrospinal fluid (+40 mOsm) containing impermeant mannitol consistently lowered light transmittance and the amplitude of the population spike. Glycerol (+40 mOsm), which is cell permeant, did not have an affect. Taken together these observations indicate that osmotic challenge alters light transmittance by inducing changes in cell volume. Transmittance increases induced by hypo-osmotic artificial cerebrospinal fluid or 10 microM kainate were small in the CA1 cell body region compared to dendritic regions. Similarly, orthodromic stimulation of axons terminating in stratum oriens or in stratum radiatum evoked transmittance increases only in their respective postsynaptic areas. In contrast, the cell body region and its adjacent proximal-apical dendrites (both sites of action potential

  4. Heterogeneous spatial patterns of long-term potentiation in rat hippocampal slices

    PubMed Central

    Chang, Payne Y; Jackson, Meyer B

    2006-01-01

    Although LTP (long-term potentiation) of synaptic transmission has received much attention as a model for learning and memory, its function within a neural circuit context remains poorly understood. To monitor LTP over an extensive circuit, we imaged responses in hippocampal slices using a voltage-sensitive dye. Following theta-burst stimulation, evoked optical signals showed an increase that lasted 40 min or more. Weak stimuli only potentiated the local area around the stimulating electrode, but stronger stimuli induced LTP over a wide area with a complex and non-uniform spatial pattern. The expression of LTP showed distinct peaks and valleys that depended on which axons were activated. Interestingly, the spatial distribution of LTP bore no relation to the spatial distribution of single-shock responses, but closely resembled the distribution of postsynaptic spikes evoked by theta bursts. Thus, postsynaptic spikes during induction constitute a critical determinant for the expression of LTP in intact circuits. PMID:16873414

  5. Anisomycin inhibits the late maintenance of long-term depression in rat hippocampal slices in vitro.

    PubMed

    Sajikumar, Sreedharan; Frey, Julietta U

    2003-02-27

    Studies were performed to investigate whether electrically-induced long-term depression (LTD) within rat hippocampal slices in vitro shares any common cellular features with LTD in the intact animal, with particular emphasis being placed on mechanisms required for its late maintenance. Our initial studies have led to the development of stimulation protocols which are able to reliably produce different forms of LTD. Depending on the induction protocol applied, we are able to demonstrate a transient protein synthesis-independent early-LTD with a duration of up to 3-4 h, together with a de novo protein synthesis-dependent late-LTD lasting for at least 8 h. Furthermore, we are able to show input-specific LTD within the CA1 region, with expression shown only by those synapses specifically stimulated by a low-frequency protocol. These studies are important pre-requisites to investigate mechanisms of 'synaptic tagging' and 'late-associativity' during LTD.

  6. Extracellular adenosine concentrations during in vitro ischaemia in rat hippocampal slices

    PubMed Central

    Latini, Serena; Bordoni, Francesca; Pedata, Felicita; Corradetti, Renato

    1999-01-01

    The application of an ischaemic insult in hippocampal slices results in the depression of synaptic transmission, mainly attributed to the activation of A1 adenosine receptors by adenosine released in the extracellular space. To estimate the concentration of endogenous adenosine acting at the receptor level during an ischaemic episode, we recorded field e.p.s.ps (fe.p.s.ps) from hippocampal slices, and evaluated the ability of the selective A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), to reverse the fe.p.s.p. depression induced by in vitro ischaemia. A relationship between the IC50 of an antagonist and the endogenous concentration of a neurotransmitter has been used for pharmacological analysis. The complete and reversible depression of fe.p.s.p. in the CA1 region induced by 5 min ischaemia was decreased in the presence of DPCPX (50–500 nM). 8-Phenyltheophylline (10 μM) abolished the depression of fe.p.s.ps during the ischaemic period, while a small (peak effect 12±4%) decrease in fe.p.s.ps was observed during the initial phase of reperfusion. In the time-interval of maximal depression of fe.p.s.ps., IC50 and adenosine concentration changed as function of time with a good degree of correlation. The maximal value of adenosine concentration was 30 μM. Our data provide an estimation of the adenosine concentration reached at the receptor level during an ischaemic episode, with a higher time discrimination (15 s) than that achieved with any biochemical approach. This estimation may be useful in order to establish appropriate concentrations of purinergic compounds to be tested for their pharmacological effects during an ischaemic episode. PMID:10401564

  7. Ethanol impairs muscarinic receptor-induced neuritogenesis in rat hippocampal slices: role of astrocytes and extracellular matrix proteins

    PubMed Central

    Giordano, Gennaro; Guizzetti, Marina; Dao, Khoi; Mattison, Hayley A.; Costa, Lucio G.

    2011-01-01

    In an in vitro co-culture system of astrocytes and neurons, stimulation of cholinergic muscarinic receptors in astrocytes had been shown to cause neuritogenesis in hippocampal neurons, and this effect was inhibited by ethanol. The present study sought to confirm these earlier findings in a more complex system, in vitro rat hippocampal slices in culture. Exposure of hippocampal slices to the cholinergic agonist carbachol (1 mM for 24 h) induced neurite outgrowth in hippocampal pyramidal neurons, which was mediated by activation of muscarinic M3 receptors. Specifically, carbachol induced a >4-fold increase in the length of the longest neurite, and a 4-fold increase in the length of minor neurites and in the number of branches. Co-incubation of carbachol with ethanol (50 mM) resulted in significant inhibition of the effects induced by carbachol on all parameters measured. Neurite outgrowth in CNS neurons is dependent on various permissive factors that are produced and released by glial cells. In hippocampal slices carbachol increased the levels of two extracellular matrix protein, fibronectin and laminin-1, by 1.6-fold, as measured by Western blot. Co-incubation of carbachol with ethanol significantly inhibited these increases. Carbachol-induced increases in levels of extracellular matrix proteins were antagonized by a M3 muscarinic receptor antagonist. Furthermore, function-blocking fibronectin or laminin-1 antibodies antagonized the effect of carbachol on neurite outgrowth. These results indicate that in hippocampal slices stimulation of muscarinic M3 receptors induces neurite outgrowth, which is mediated by fibronectin and laminin-1, two extracellular matrix proteins released by astrocytes. By decreasing fibronectin and laminin levels ethanol prevents carbachol-induced neuritogenesis. These findings highlight the importance of glial-neuronal interactions as important targets in the developmental neurotoxicity of alcohol. PMID:21884684

  8. Atorvastatin enhances kainate-induced gamma oscillations in rat hippocampal slices.

    PubMed

    Li, Chengzhang; Wang, Jiangang; Zhao, Jianhua; Wang, Yali; Liu, Zhihua; Guo, Fang Li; Wang, Xiao Fang; Vreugdenhil, Martin; Lu, Cheng Biao

    2016-09-01

    Atorvastatin has been shown to affect cognitive functions in rodents and humans. However, the underlying mechanism is not fully understood. Because hippocampal gamma oscillations (γ, 20-80 Hz) are associated with cognitive functions, we studied the effect of atorvastatin on persistent kainate-induced γ oscillation in the CA3 area of rat hippocampal slices. The involvement of NMDA receptors and multiple kinases was tested before and after administration of atorvastatin. Whole-cell current-clamp and voltage-clamp recordings were made from CA3 pyramidal neurons and interneurons before and after atorvastatin application. Atorvastatin increased γ power by ~ 50% in a concentration-dependent manner, without affecting dominant frequency. Whereas atorvastatin did not affect intrinsic properties of both pyramidal neurons and interneurons, it increased the firing frequency of interneurons but not that of pyramidal neurons. Furthermore, whereas atorvastatin did not affect synaptic current amplitude, it increased the frequency of spontaneous inhibitory post-synaptic currents, but did not affect the frequency of spontaneous excitatory post-synaptic currents. The atorvastatin-induced enhancement of γ oscillations was prevented by pretreatment with the PKA inhibitor H89, the ERK inhibitor U0126, or the PI3K inhibitor wortmanin, but not by the NMDA receptor antagonist D-AP5. Taken together, these results demonstrate that atorvastatin enhanced the kainate-induced γ oscillation by increasing interneuron excitability, with an involvement of multiple intracellular kinase pathways. Our study suggests that the classical cholesterol-lowering agent atorvastatin may improve cognitive functions compromised in disease, via the enhancement of hippocampal γ oscillations.

  9. Base excision repair activities in organotypic hippocampal slice cultures exposed to oxygen and glucose deprivation.

    PubMed

    Rolseth, Veslemøy; Rundén-Pran, Elise; Neurauter, Christine Gran; Yndestad, Arne; Luna, Luisa; Aukrust, Pål; Ottersen, Ole Petter; Bjørås, Magnar

    2008-06-01

    The capacity for DNA repair is likely to be one of the factors that determine the vulnerability of neurons to ischemic stress and may influence the pathological outcome of stroke. In this report, initiation of base excision repair (BER) was assessed by analysis of enzyme activity and gene expression level of DNA glycosylases and AP-endonucleases in rat organotypic hippocampal slice cultures exposed to oxygen and glucose deprivation (OGD) - an in vitro model of stroke. Under basal conditions, AP-endonuclease activity and base removal of ethenoadenine and 8-oxoguanine (8-oxoG) were higher (by approximately 20-35 %) in CA3/fascia dentata (FD) than in CA1. Base removal of uracil did not differ between the two hippocampal regions, while removal of 5-hydroxyuracil (5-OHU) was slightly less efficient in CA3/FD than in CA1. Analyses performed immediately after 30 min of OGD revealed a decreased AP-endonuclease activity (by approximately 20%) in CA1 as well as CA3/FD, and an increased ethenoadenine activity (by approximately 25%) in CA1. Activities for 8-oxoG, 5-OHU and uracil showed no significant changes at this time point. At 8h after OGD, none of the enzyme activities differed from control values. Real-time RT-PCR showed that transcription of DNA glycosylases, including Ogg1, Nth1, Ung, Aag, Neil1 and Neil2 were not changed in response to OGD treatment (t=0 h). The hippocampal expression of Neil2 was low compared with the other DNA glycosylases. These data indicate that CA1 has a lower capacity than CA3/FD for removal of base lesions under basal conditions. The relatively low capacity for BER in basal conditions and the apparent failure to upregulate repair of oxidative damage after OGD might contribute to the high vulnerability of CA1 to ischemic injury.

  10. Active decay of composite excitatory postsynaptic potentials in hippocampal slices from young rats.

    PubMed

    Dozmorov, Mikhail; Niu, Yin-Ping; Xu, Hui-Ping; Xiao, Min-Yi; Li, Rui; Sandberg, Mats; Wigström, Holger

    2003-05-23

    NMDA receptor dependent synaptic plasticity was examined in hippocampal slices using a novel pharmacological pairing procedure. Field excitatory postsynaptic potentials (EPSPs) were recorded from the CA1 area of slices maintained in a low Mg(2+) solution using a stimulus rate of 0.1-0.2 Hz. The NMDA receptor antagonist 2-amino-5-phosphonovalerate (AP5) was initially included in the perfusion solution to establish baseline recording of isolated AMPA EPSPs. Washing out AP5 led to the expression of composite EPSPs, containing both AMPA and NMDA receptor mediated components. Following an initial, transient potentiation of the AMPA component, the composite responses gradually decayed for several hours, involving AMPA and NMDA components to a similar extent. This decay was input specific and could be terminated at any stage by reapplication of AP5. Subsequent long-term potentiation (LTP) reversed the effect to an extent inversely related to the degree of depression. Experiments to test the interaction with long-term depression (LTD) revealed a significant but incomplete overlap between the two depression processes. In conclusion, pairing synaptic activation at test stimulus frequency with pharmacological unblocking of NMDA receptors allows for expression of composite EPSPs that decay substantially, due to an active mechanism. The underlying process appears to be at least partly distinct from those involved in homosynaptic LTP and LTD.

  11. Identification and two-photon imaging of oligodendrocyte in CA1 region of hippocampal slices

    SciTech Connect

    Zhou Wei; Ge Wooping; Zeng Shaoqun; Duan Shumin; Luo Qingming . E-mail: qluo@mail.hust.edu.cn

    2007-01-19

    Oligodendrocyte (OL) plays a critical role in myelination and axon maintenance in central nervous system. Recent studies show that OL can also express NMDA receptors in development and pathological situations in white matter. There is still lack of studies about OL properties and function in gray matter of brain. Here we reported that some glial cells in CA1 region of rat hippocampal slices (P15-23) had distinct electrophysiological characteristics from the other glia cells in this region, while they displayed uniform properties with OL from white matter in previous report; therefore, they were considered as OL in hippocampus. By loading dye in recording pipette and imaging with two-photon laser scanning microscopy, we acquired the high spatial resolution, three-dimension images of these special cells in live slices. The OL in hippocampus shows a complex process-bearing shape and the distribution of several processes is parallel to Schaffer fiber in CA1 region. When stimulating Schaffer fiber, OL displays a long duration depolarization mediated by inward rectifier potassium channel. This suggested that the OL in CA1 region could sense the neuronal activity and contribute to potassium clearance.

  12. Desflurane impairs outcome of organotypic hippocampal slices in an in vitro model of traumatic brain injury

    PubMed Central

    Krings, Matthias; Höllig, Anke; Liu, Jingjin; Grüsser, Linda; Rossaint, Rolf; Coburn, Mark

    2016-01-01

    Decreased mortality and disability after traumatic brain injury is a significant medical challenge. Desflurane, a widely used volatile anesthetic has proven to be neuroprotective in a variety of in vitro and in vivo models of ischemic brain injury. The aim of this study was to investigate whether desflurane exhibits neuroprotective properties in an in vitro model of traumatic brain injury. Organotypic hippocampal slice cultures were prepared from brains of 5–7-day-old C57/BL6 mouse pups. After 14 days of culture, the slices were subjected to a focal mechanical trauma and thereafter incubated with three different concentrations of desflurane (2, 4 and 6%) for 2, 24 and 72 hours. Cell injury was assessed with propodium iodide uptake. Our results showed that after 2 hours of desflurane exposure, no significant change in trauma intensity was observed. However, 2% and 4% desflurane could reduce the trauma intensity significantly in the no trauma group than in the no desflurane and trauma group. Incubation with 4% desflurane for 24 hours doubled the trauma intensity in comparison to the trauma control group and the trauma intensity further increased after 72 hours of incubation. Furthermore, a dose-dependent increase of trauma intensity after 24 hours exposure was observed. Our results suggest that a general neuroprotective attribute of desflurane in an in vitro model of traumatic brain injury was not observed. PMID:27826417

  13. Termination of epileptiform activity by cooling in rat hippocampal slice epilepsy models.

    PubMed

    Motamedi, Gholam K; Salazar, Patricia; Smith, Eric L; Lesser, Ronald P; Webber, William R S; Ortinski, Pavel I; Vicini, Stefano; Rogawski, Michael A

    2006-08-01

    Cooling has been shown to terminate experimentally induced epileptiform activity in models of epilepsy without causing injury to the cooled brain, suggesting that cooling could represent an approach to seizure control in intractable focal epilepsies. Here we sought to determine the most effective way to apply cooling to abort spontaneous epileptiform discharges in in vitro brain slice models. We induced spontaneous epileptiform activity in rat brain slices by exposure to 4-aminopyridine (4-AP), 4-AP plus bicuculline, and Mg(2+)-free artificial CSF (aCSF) at 28-34 degrees C. Extracellular field recordings were made at hippocampal or neocortical sites. Slice temperature was reduced by perfusion with cold aCSF. Rapid cooling at rates of 2-5 degrees C/s was compared to cooling at slower rates of 0.1-1 degrees C/s. Cooling at both rates reversibly aborted epileptiform discharges in all three models and at all recording sites. With rapid cooling, small temperature drops were highly effective in terminating discharges, an effect that was sustained for as long as the reduced temperature level was maintained. In contrast, slow cooling required much larger temperature drops to inhibit discharges. With slow cooling, absolute temperature drops to 21-22 degrees C caused a 90% reduction in event frequency, but cooling to 14-15 degrees C was required to terminate discharges. We conclude that rapid cooling as effectively aborts discharges in in vitro epilepsy models as does slow cooling, but the magnitude of the temperature change required is less. Practical devices to inhibit seizure activity may only need to induce small temperature drops, if the cooling can be applied sufficiently rapidly.

  14. Chloride-cotransport blockade desynchronizes neuronal discharge in the "epileptic" hippocampal slice.

    PubMed

    Hochman, D W; Schwartzkroin, P A

    2000-01-01

    Antagonism of the chloride-cotransport system in hippocampal slices has been shown to block spontaneous epileptiform (i.e., hypersynchronized) discharges without diminishing excitatory synaptic transmission. Here we test the hypotheses that chloride-cotransport blockade, with furosemide or low-chloride (low-[Cl(-)](o)) medium, desynchronizes the firing activity of neuronal populations and that this desynchronization is mediated through nonsynaptic mechanisms. Spontaneous epileptiform discharges were recorded from the CA1 and CA3 cell body layers of hippocampal slices. Treatment with low-[Cl(-)](o) medium led to cessation of spontaneous synchronized bursting in CA1 >/=5-10 min before its disappearance from CA3. During the time that CA3 continued to burst spontaneously but CA1 was silent, electrical stimulation of the Schaffer collaterals showed that hyperexcited CA1 synaptic responses were maintained. Paired intracellular recordings from CA1 pyramidal cells showed that during low-[Cl(-)](o) treatment, the timing of action potential discharges became desynchronized; desynchronization was identified with phase lags in firing times of action potentials between pairs of neurons as well as a with a broadening and diminution of the CA1 field amplitude. Continued exposure to low-[Cl(-)](o) medium increased the degree of the firing-time phase shifts between pairs of CA1 pyramidal cells until the epileptiform CA1 field potential was abolished completely. Intracellular recordings during 4-aminopyridine (4-AP) treatment showed that prolonged low-[Cl(-)](o) exposure did not diminish the frequency or amplitude of spontaneous postsynaptic potentials. CA3 antidromic responses to Schaffer collateral stimulation were not significantly affected by prolonged low-[Cl(-)](o) exposure. In contrast to CA1, paired intracellular recordings from CA3 pyramidal cells showed that chloride-cotransport blockade did not cause a significant desynchronization of action potential firing times in the

  15. Allopregnanolone-mediated protective effects of progesterone on tributyltin-induced neuronal injury in rat hippocampal slices.

    PubMed

    Ishihara, Yasuhiro; Kawami, Tomohito; Ishida, Atsuhiko; Yamazaki, Takeshi

    2013-05-01

    Increasing evidence shows that progesterone, a neuroactive steroid, has protective actions in central nervous system, but there is little evidence to show the protective mechanism of progesterone on neurotoxicity induced by environmental chemicals. In this study, we examined the effects of progesterone on neuronal injury induced by tributyltin (TBT) in rat hippocampal slices. Treatment with progesterone dose-dependently suppressed hippocampal neuronal injury induced by TBT. The neuroprotective action of progesterone was completely canceled with pretreatment by finasteride, a 5α-reductase inhibitor, but it was not affected by mifepristone, a progesterone receptor antagonist, or by SU-10603, a cytochrome P450 17α inhibitor. The content of allopregnanolone in the slices was significantly increased by treatment with progesterone, and this increment was greatly suppressed with a pretreatment of finasteride. Treatment with allopregnanolone attenuated neuronal injury induced by TBT in a dose-dependent manner. The neuroprotective effects not only of progesterone but also of allopregnanolone were canceled by bicuculline, a potent gamma-aminobutyric acid A (GABAA) receptor antagonist. Pretreatment with muscimol, a GABAA receptor agonist, attenuated hippocampal neuronal injury elicited by TBT. Taken together, allopregnanolone converted from progesterone in hippocampal slices could protect neurons from TBT-induced neurotoxicity due to a GABAA receptor-dependent mechanism. One of the physiological roles of neuroactive steroids might be neuroprotection from environmental chemicals. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Synchronization of GABAergic interneuronal network in CA3 subfield of neonatal rat hippocampal slices.

    PubMed

    Khazipov, R; Leinekugel, X; Khalilov, I; Gaiarsa, J L; Ben-Ari, Y

    1997-02-01

    1. Cell-attached and whole-cell recordings from interneurons localized in the stratum radiatum of the CA3 subfield (SR-CA3) of neonatal (postnatal days 2-5) rat hippocampal slices were performed to study their activity during the generation of GABAergic giant depolarizing potentials (GDPs) in CA3 pyramidal cells. 2. Dual recordings revealed that during the generation of GDPs in CA3 pyramidal cells, the interneurons fire bursts of spikes, on average 4.5 +/- 1.4 spikes per burst (cell-attached mode). There bursts were induced by periodical large inward currents (interneuronal GDPs) recorded in whole-cell mode. 3. Interneuronal GDPs revealed typical features of polysynaptic neuronal network-driven events: they were blocked by TTX and by high divalent cation medium and they could be evoked in an all-or-none manner by electrical stimulation in different regions of the hippocampus. The network elements required for the generation of GDPs are present in local CA3 circuits since spontaneous GDPs were present in the isolated CA3 subfield of the hippocampal slice. 4. Interneuronal GDPs were mediated by GABAA and glutamate receptors, since: (i) their reversal potential strongly depended on [Cl-]i; (ii) at the reversal potential of GABAA postsynaptic currents an inward component of GDPs was composed of events with the same kinetics as alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-mediated EPSCs; and (iii) once GABAA receptors were blocked intracellularly by dialysis with F(-)-MgATP-free solution, the remaining component of interneuronal GDPs reversed near 0 mV and rectified at membrane potentials more negative than -20 mV, suggesting an important contribution of NMDA receptors in addition to AMPA receptors. 5. In cell-attached recordings from interneurons, electrical stimulation in the stratum radiatum evoked a burst of spikes that corresponded to evoked GDPs. Pharmacological study of this response revealed that excitation of SR-CA3 interneurons during

  17. Synchronization of GABAergic interneuronal network in CA3 subfield of neonatal rat hippocampal slices.

    PubMed Central

    Khazipov, R; Leinekugel, X; Khalilov, I; Gaiarsa, J L; Ben-Ari, Y

    1997-01-01

    1. Cell-attached and whole-cell recordings from interneurons localized in the stratum radiatum of the CA3 subfield (SR-CA3) of neonatal (postnatal days 2-5) rat hippocampal slices were performed to study their activity during the generation of GABAergic giant depolarizing potentials (GDPs) in CA3 pyramidal cells. 2. Dual recordings revealed that during the generation of GDPs in CA3 pyramidal cells, the interneurons fire bursts of spikes, on average 4.5 +/- 1.4 spikes per burst (cell-attached mode). There bursts were induced by periodical large inward currents (interneuronal GDPs) recorded in whole-cell mode. 3. Interneuronal GDPs revealed typical features of polysynaptic neuronal network-driven events: they were blocked by TTX and by high divalent cation medium and they could be evoked in an all-or-none manner by electrical stimulation in different regions of the hippocampus. The network elements required for the generation of GDPs are present in local CA3 circuits since spontaneous GDPs were present in the isolated CA3 subfield of the hippocampal slice. 4. Interneuronal GDPs were mediated by GABAA and glutamate receptors, since: (i) their reversal potential strongly depended on [Cl-]i; (ii) at the reversal potential of GABAA postsynaptic currents an inward component of GDPs was composed of events with the same kinetics as alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-mediated EPSCs; and (iii) once GABAA receptors were blocked intracellularly by dialysis with F(-)-MgATP-free solution, the remaining component of interneuronal GDPs reversed near 0 mV and rectified at membrane potentials more negative than -20 mV, suggesting an important contribution of NMDA receptors in addition to AMPA receptors. 5. In cell-attached recordings from interneurons, electrical stimulation in the stratum radiatum evoked a burst of spikes that corresponded to evoked GDPs. Pharmacological study of this response revealed that excitation of SR-CA3 interneurons during

  18. Fiber Tract Stimulation Can Reduce Epileptiform Activity in an in-vitro Bilateral Hippocampal Slice Preparation

    PubMed Central

    Toprani, Sheela; Durand, Dominique

    2012-01-01

    Mesial temporal lobe epilepsy (MTLE) is a common medically refractory neurological disease that has been treated with electrical stimulation of gray matter with limited success. However, stimulation of a white matter tract connecting the hippocampi could maximize treatment efficacy and extent. We tested low-frequency stimulation (LFS) of a novel target that enables simultaneous targeting of bilateral hippocampi: the ventral hippocampal commissure (VHC) with a novel in-vitro slice preparation containing bilateral hippocampi connected by the VHC. The goal of this study is to understand the role of hippocampal interplay in seizure propagation and reduction by commissural fiber tract stimulation. LFS is applied to the VHC as extracellular and intracellular recording techniques are combined with signal processing to estimate several metrics of epilepsy including: (1) total time occupied by seizure activity (%); (2) seizure duration (s); (3) seizures per minute (#); and (4) power in the ictal (V2Hz−1); as well as (5) interictal spectra (V2Hz−1). Bilateral epileptiform activity in this preparation is highly correlated between hippocampi. Application of LFS to the VHC reduces all metrics of epilepsy during treatment in an amplitude and frequency dependent manner. This study lends several insights into the mechanisms of bilateral seizure reduction by LFS of the VHC, including that depolarization blocking, LTD/LTP and GABAA are not involved. Importantly, enhanced post-stimulation 1-Hz spiking correlates with long-lasting seizure reduction and both are heightened by targeting bilateral hippocampi via the VHC. Therefore, stimulating bilateral hippocampi via a single electrode in the VHC may provide an effective MTLE treatment. PMID:23123405

  19. Modulation by adenine nucleotides of epileptiform activity in the CA3 region of rat hippocampal slices

    PubMed Central

    Ross, F M; Brodie, M J; Stone, T W

    1998-01-01

    Hippocampal slices (450 μm) generate epileptiform bursts of an interictal nature when perfused with a zero magnesium medium containing 4-aminopyridine (50 μM). The effect of adenine nucleotides on this activity was investigated.ATP and adenosine depressed this epileptiform activity in a concentration-dependent manner, with both purines being equipotent at concentrations above 10 μM.Adenosine deaminase 0.2 u ml−1, a concentration that annuls the effect of adenosine (50 μM), did not significantly alter the depression of activity caused by ATP (50 μM).8-Cyclopentyl-1, 3-dimethylxanthine (CPT), an A1 receptor antagonist, enhanced the discharge rate significantly and inhibited the depressant effect of both ATP and adenosine such that the net effect of ATP or adenosine plus CPT was excitatory.Several ATP analogues were also tested: α, β-methyleneATP (α, β-meATP), 2-methylthioATP (2-meSATP) and uridine triphosphate (UTP). Only α, β-meATP (10 μM) produced an increase in the frequency of spontaneous activity which suggests a lack of involvement of P2Y or P2U receptors.Suramin and pyridoxalphosphate-6-azophenyl-2′, 4′-disulphonic acid (PPADS), P2 receptor antagonists, failed to inhibit the depression produced by ATP (50 μM). The excitatory effect of α, β-meATP (10 μM) was inhibited by suramin (50 μM) and PPADS (5 μM).ATP therefore depresses epileptiform activity in this model in a manner which is not consistent with the activation of known P1 or P2 receptors, suggesting the involvement of a xanthine-sensitive nucleotide receptor. The results are also indicative of an excitatory P2X receptor existing in the hippocampal CA3 region. PMID:9484856

  20. Neuronal and Astroglial Correlates Underlying Spatiotemporal Intrinsic Optical Signal in the Rat Hippocampal Slice

    PubMed Central

    Pál, Ildikó; Nyitrai, Gabriella; Kardos, Julianna; Héja, László

    2013-01-01

    Widely used for mapping afferent activated brain areas in vivo, the label-free intrinsic optical signal (IOS) is mainly ascribed to blood volume changes subsequent to glial glutamate uptake. By contrast, IOS imaged in vitro is generally attributed to neuronal and glial cell swelling, however the relative contribution of different cell types and molecular players remained largely unknown. We characterized IOS to Schaffer collateral stimulation in the rat hippocampal slice using a 464-element photodiode-array device that enables IOS monitoring at 0.6 ms time-resolution in combination with simultaneous field potential recordings. We used brief half-maximal stimuli by applying a medium intensity 50 Volt-stimulus train within 50 ms (20 Hz). IOS was primarily observed in the str. pyramidale and proximal region of the str. radiatum of the hippocampus. It was eliminated by tetrodotoxin blockade of voltage-gated Na+ channels and was significantly enhanced by suppressing inhibitory signaling with gamma-aminobutyric acid(A) receptor antagonist picrotoxin. We found that IOS was predominantly initiated by postsynaptic Glu receptor activation and progressed by the activation of astroglial Glu transporters and Mg2+-independent astroglial N-methyl-D-aspartate receptors. Under control conditions, role for neuronal K+/Cl− cotransporter KCC2, but not for glial Na+/K+/Cl− cotransporter NKCC1 was observed. Slight enhancement and inhibition of IOS through non-specific Cl− and volume-regulated anion channels, respectively, were also depicted. High-frequency IOS imaging, evoked by brief afferent stimulation in brain slices provide a new paradigm for studying mechanisms underlying IOS genesis. Major players disclosed this way imply that spatiotemporal IOS reflects glutamatergic neuronal activation and astroglial response, as observed within the hippocampus. Our model may help to better interpret in vivo IOS and support diagnosis in the future. PMID:23469218

  1. Neuronal and astroglial correlates underlying spatiotemporal intrinsic optical signal in the rat hippocampal slice.

    PubMed

    Pál, Ildikó; Nyitrai, Gabriella; Kardos, Julianna; Héja, László

    2013-01-01

    Widely used for mapping afferent activated brain areas in vivo, the label-free intrinsic optical signal (IOS) is mainly ascribed to blood volume changes subsequent to glial glutamate uptake. By contrast, IOS imaged in vitro is generally attributed to neuronal and glial cell swelling, however the relative contribution of different cell types and molecular players remained largely unknown. We characterized IOS to Schaffer collateral stimulation in the rat hippocampal slice using a 464-element photodiode-array device that enables IOS monitoring at 0.6 ms time-resolution in combination with simultaneous field potential recordings. We used brief half-maximal stimuli by applying a medium intensity 50 Volt-stimulus train within 50 ms (20 Hz). IOS was primarily observed in the str. pyramidale and proximal region of the str. radiatum of the hippocampus. It was eliminated by tetrodotoxin blockade of voltage-gated Na(+) channels and was significantly enhanced by suppressing inhibitory signaling with gamma-aminobutyric acid(A) receptor antagonist picrotoxin. We found that IOS was predominantly initiated by postsynaptic Glu receptor activation and progressed by the activation of astroglial Glu transporters and Mg(2+)-independent astroglial N-methyl-D-aspartate receptors. Under control conditions, role for neuronal K(+)/Cl(-) cotransporter KCC2, but not for glial Na(+)/K(+)/Cl(-) cotransporter NKCC1 was observed. Slight enhancement and inhibition of IOS through non-specific Cl(-) and volume-regulated anion channels, respectively, were also depicted. High-frequency IOS imaging, evoked by brief afferent stimulation in brain slices provide a new paradigm for studying mechanisms underlying IOS genesis. Major players disclosed this way imply that spatiotemporal IOS reflects glutamatergic neuronal activation and astroglial response, as observed within the hippocampus. Our model may help to better interpret in vivo IOS and support diagnosis in the future.

  2. Spatiotemporal evidence of apoptosis-mediated ischemic injury in organotypic hippocampal slice cultures.

    PubMed

    Cho, Seongeun; Liu, Danni; Fairman, Denise; Li, Ping; Jenkins, Lorayne; McGonigle, Paul; Wood, Andrew

    2004-07-01

    Oxygen-glucose deprivation (OGD) induced neuron-specific cell death in organotypic hippocampal slice cultures. Neuronal death was first evident in the CA1 region 24 h after the injury as assessed by propidium iodide (PI) labeling, and continued to extend to the CA3/4 region up to 72 h. At 6 days post-OGD, PI labeling was weak and diffuse with no clear demarcation of pyknotic nuclei. To characterize biochemical changes produced by OGD, cellular efflux of three key amino acid neurotransmitters was evaluated. OGD elicited large increases in the release of GABA and aspartate (55- and 4.5-fold increase over basal, respectively), while there were no detectable changes in extracellular glutamate levels. In order to ascertain the existence of the synaptic pool of glutamate, sister cultures were treated with sodium azide. This evoked a strong increase in glutamate release, suggesting the intactness of the glutamate system. Further studies revealed a time-dependent activation of caspase 3 following OGD, shown by immunoblot analysis as well as by confocal laser scanning microscopy. While we did not observe the activation of caspases 1, 2, or 8 in our model, the activation of caspase 9 was evident, peaking at 12 h post-OGD. Despite no apparent increase in glutamate release by ischemic slices, treatment with a N-methyl-D-aspartate (NMDA) antagonist or an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) antagonist significantly reduced neuronal death. Furthermore, a pan-caspase inhibitor (zVAD-fmk), but not the caspase 3 inhibitor (DEVD-fmk), provided partial neuroprotection. Inhibition of a Ca(2+)-dependent cysteine protease, calpain, by MDL28170 also elicited partial neuroprotective effects.

  3. Efficacy of a new charge-balanced biphasic electrical stimulus in the isolated sciatic nerve and the hippocampal slice.

    PubMed

    Cappaert, Natalie L M; Ramekers, Dyan; Martens, Hubert C F; Wadman, Wytse J

    2013-02-01

    Most deep brain stimulators apply rectangular monophasic voltage pulses. By modifying the stimulus shape, it is possible to optimize stimulus efficacy and find the best compromise between clinical effect, minimal side effects and power consumption of the stimulus generator. In this study, we compared the efficacy of three types of charge-balanced biphasic pulses (CBBPs, nominal duration 100 μs) in isolated sciatic nerves and in in vitro hippocampal brain slices of the rat. Using these two models, we tested the efficacy of several stimulus shapes exclusively on axons (in the sciatic nerve) and compared the effect with that of stimuli in the more complex neuronal network of the hippocampal slice by considering the stimulus-response relation. We showed that (i) adding an interphase gap (IPG, range 100-500 μs) to the CBBP enhances stimulus efficacy in the rat sciatic nerve and (ii) that this type of stimuli (CBBP with IPG) is also more effective in hippocampal slices. This benefit was similar for both models of voltage and current stimulation. In our two models, asymmetric CBBPs were less beneficial. Therefore, CBBPs with IPG appear to be well suited for application to DBS, since they enhance efficacy, extend battery life and potentially reduce harmful side effects.

  4. Melatonin protects against oxygen and glucose deprivation by decreasing extracellular glutamate and Nox-derived ROS in rat hippocampal slices.

    PubMed

    Patiño, Paloma; Parada, Esther; Farré-Alins, Victor; Molz, Simone; Cacabelos, Ramón; Marco-Contelles, José; López, Manuela G; Tasca, Carla I; Ramos, Eva; Romero, Alejandro; Egea, Javier

    2016-12-01

    Therapeutic interventions on pathological processes involved in the ischemic cascade, such as oxidative stress, neuroinflammation, excitotoxicity and/or apoptosis, are of urgent need for stroke treatment. Melatonin regulates a large number of physiological actions and its beneficial properties have been reported. The aim of this study was to investigate whether melatonin mediates neuroprotection in rat hippocampal slices subjected to oxygen-glucose-deprivation (OGD) and glutamate excitotoxicity. Thus, we describe here that melatonin significantly reduced the amount of lactate dehydrogenase released in the OGD-treated slices, reverted neuronal injury caused by OGD-reoxygenation in CA1 and CA3 hippocampal regions, restored the reduction of GSH content of the hippocampal slices induced by OGD, and diminished the oxidative stress produced in the reoxygenation period. Furthermore, melatonin afforded maximum protection against glutamate-induced toxicity and reversed the glutamate released almost basal levels, at 10 and 30μM concentration, respectively. Consequently, we propose that melatonin might strongly and positively influence the outcome of brain ischemia/reperfusion.

  5. Electroosmotic sampling. Application to determination of ectopeptidase activity in organotypic hippocampal slice cultures.

    PubMed

    Xu, Hongjuan; Guy, Yifat; Hamsher, Amy; Shi, Guoyue; Sandberg, Mats; Weber, Stephen G

    2010-08-01

    We hypothesize that peptide-containing solutions pulled through tissue should reveal the presence and activity of peptidases in the tissue. Using the natural zeta-potential in the organotypic hippocampal slice culture (OHSC), physiological fluids can be pulled through the tissue with an electric field. The hydrolysis of the peptides present in the fluid drawn through the tissue can be determined using capillary HPLC with electrochemical detection of the biuret complexes of the peptides following a postcolumn reaction. We have characterized this new sampling method by measuring the flow rate, examining the use of internal standards, and examining cell death caused by sampling. The sampling flow rate ranges from 60 to 150 nL/min with a 150 microm (ID) sampling capillary with an electric field (at the tip of the capillary) from 30 to 60 V/cm. Cell death can be negligible with controlled sampling conditions. Using this sampling approach, we have electroosmotically pulled Leu-enkephalin through OHSCs to identify ectopeptidase activity in the CA3 region. These studies show that a bestatin-sensitive aminopeptidase may be critical for the hydrolysis of exogenous Leu-enkephalin, a neuropeptide present in the CA3 region of OHSCs.

  6. Short-term environmental enrichment enhances synaptic plasticity in hippocampal slices from aged rats.

    PubMed

    Stein, Liana R; O'Dell, Kazuko A; Funatsu, Michiyo; Zorumski, Charles F; Izumi, Yukitoshi

    2016-08-04

    Age-associated changes in cognition are mirrored by impairments in cellular models of memory and learning, such as long-term potentiation (LTP) and long-term depression (LTD). In young rodents, environmental enrichment (EE) can enhance memory, alter LTP and LTD, as well as reverse cognitive deficits induced by aging. Whether short-term EE can benefit cognition and synaptic plasticity in aged rodents is unclear. Here, we tested if short-term EE could overcome age-associated impairments in induction of LTP and LTD. LTP and LTD could not be induced in the CA1 region of hippocampal slices in control, aged rats using standard stimuli that are highly effective in young rats. However, exposure of aged littermates to EE for three weeks enabled successful induction of LTP and LTD. EE-facilitated LTP was dependent upon N-methyl-d-aspartate receptors (NMDARs). These alterations in synaptic plasticity occurred with elevated levels of phosphorylated cAMP response element-binding protein and vascular endothelial growth factor, but in the absence of changes in several other synaptic and cellular markers. Importantly, our study suggests that even a relatively short period of EE is sufficient to alter synaptic plasticity and molecular markers linked to cognitive function in aged animals. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  7. Simultaneous monitoring of excitatory postsynaptic potentials and extracellular L-glutamate in mouse hippocampal slices.

    PubMed

    Hozumi, Shizuko; Ikezawa, Kana; Shoji, Atushi; Hirano-Iwata, Ayumi; Bliss, Tim; Sugawara, Masao

    2011-02-15

    Simultaneous monitoring of amperometric currents at a glass capillary sensor based on recombinant GluOx and field excitatory postsynaptic potentials (fEPSPs) were performed in region CA1 of mouse hippocampal slices. A transient increase in the glutamate current relative to the basal one at control stimulation (0.052Hz) was evoked by stimulation at 2 Hz for 2 min. The magnitude of the glutamate current was dependent on the intensity (current) of a 2 Hz stimulus and reflected the slope of the fEPSP. The in situ calibration of the L-glutamate sensor revealed that the extracellular concentration of L-glutamate released by 2 Hz stimulation before tetanus is in the range from 0.8 to 2.2 μM and it is enhanced after tetanic stimulation. The L-glutamate level at a test stimulus (0.052 Hz) was estimated to be 32 nM. The recombinant GluOx-based sensor exhibited weak responses to glutamine above 300 μM and L-aspartic acid above 200 μM. The potential use of a glass capillary sensor in combination with fEPSP measurements for electrophysiological study is discussed.

  8. Effects of uniform extracellular DC electric fields on excitability in rat hippocampal slices in vitro

    PubMed Central

    Bikson, Marom; Inoue, Masashi; Akiyama, Hiroki; Deans, Jackie K; Fox, John E; Miyakawa, Hiroyoshi; Jefferys, John G R

    2004-01-01

    The effects of uniform steady state (DC) extracellular electric fields on neuronal excitability were characterized in rat hippocampal slices using field, intracellular and voltage-sensitive dye recordings. Small electric fields (<|40| mV mm−1), applied parallel to the somato-dendritic axis, induced polarization of CA1 pyramidal cells; the relationship between applied field and induced polarization was linear (0.12 ± 0.05 mV per mV mm−1 average sensitivity at the soma). The peak amplitude and time constant (15–70 ms) of membrane polarization varied along the axis of neurons with the maximal polarization observed at the tips of basal and apical dendrites. The polarization was biphasic in the mid-apical dendrites; there was a time-dependent shift in the polarity reversal site. DC fields altered the thresholds of action potentials evoked by orthodromic stimulation, and shifted their initiation site along the apical dendrites. Large electric fields could trigger neuronal firing and epileptiform activity, and induce long-term (>1 s) changes in neuronal excitability. Electric fields perpendicular to the apical–dendritic axis did not induce somatic polarization, but did modulate orthodromic responses, indicating an effect on afferents. These results demonstrate that DC fields can modulate neuronal excitability in a time-dependent manner, with no clear threshold, as a result of interactions between neuronal compartments, the non-linear properties of the cell membrane, and effects on afferents. PMID:14978199

  9. Guanosine controls inflammatory pathways to afford neuroprotection of hippocampal slices under oxygen and glucose deprivation conditions.

    PubMed

    Dal-Cim, Tharine; Ludka, Fabiana K; Martins, Wagner C; Reginato, Charlise; Parada, Esther; Egea, Javier; López, Manuela G; Tasca, Carla I

    2013-08-01

    Guanosine (GUO) is an endogenous modulator of glutamatergic excitotoxicity and has been shown to promote neuroprotection in in vivo and in vitro models of neurotoxicity. This study was designed to understand the neuroprotective mechanism of GUO against oxidative damage promoted by oxygen/glucose deprivation and reoxygenation (OGD). GUO (100 μM) reduced reactive oxygen species production and prevented mitochondrial membrane depolarization induced by OGD. GUO also exhibited anti-inflammatory actions as inhibition of nuclear factor kappa B activation and reduction of inducible nitric oxide synthase induction induced by OGD. These GUO neuroprotective effects were mediated by adenosine A1 receptor, phosphatidylinositol-3 kinase and MAPK/ERK. Furthermore, GUO recovered the impairment of glutamate uptake caused by OGD, an effect that occurred via a Pertussis toxin-sensitive G-protein-coupled signaling, blockade of adenosine A2A receptors (A2A R), but not via A1 receptor. The modulation of glutamate uptake by GUO also involved MAPK/ERK activation. In conclusion, GUO, by modulating adenosine receptor function and activating MAPK/ERK, affords neuroprotection of hippocampal slices subjected to OGD by a mechanism that implicates the following: (i) prevention of mitochondrial membrane depolarization, (ii) reduction of oxidative stress, (iii) regulation of inflammation by inhibition of nuclear factor kappa B and inducible nitric oxide synthase, and (iv) promoting glutamate uptake.

  10. Ischaemia differentially regulates GABAB receptor subunits in organotypic hippocampal slice cultures

    PubMed Central

    Cimarosti, Helena; Kantamneni, Sriharsha; Henley, Jeremy M.

    2012-01-01

    Reduced synaptic inhibition due to dysfunction of ionotropic GABAA receptors has been proposed as one factor in cerebral ischaemia-induced excitotoxic cell death. However, the participation of the inhibitory metabotropic GABAB receptors in these pathological processes has not been extensively investigated. We used oxygen–glucose deprivation (OGD) and NMDA-induced excitotoxicity as models to investigate whether ischaemia-like challenges alter the protein levels of GABAB1 and GABAB2 receptor subunits in rat organotypic hippocampal slice cultures. Twenty-four hours after the insult both OGD and NMDA produced a marked decrease in the total levels of GABAB2 (~75%), while there was no significant change in the levels of GABAB1 after OGD, but an increase after NMDA treatment (~100%). The GABAB receptor agonist baclofen (100 μM) was neuroprotective following OGD or NMDA treatment if added before or during the insult. GABAB receptors comprise heterodimers of GABAB1 and GABAB2 subunits and our results suggest that the separate subunits are independently regulated in response to extreme neuronal stress. However, because GABAB2 is required for functional surface expression, down-regulation of this subunit removes an important inhibitory feedback mechanism under pathological conditions. PMID:19328818

  11. Hybrid voltage sensor imaging of electrical activity from neurons in hippocampal slices from transgenic mice

    PubMed Central

    Wang, Dongsheng; McMahon, Shane; Zhang, Zhen

    2012-01-01

    Gene targeting with genetically encoded optical voltage sensors brings the methods of voltage imaging to genetically defined neurons and offers a method of studying circuit activity in these selected populations. The present study reports the targeting of genetically encoded hybrid voltage sensors (hVOS) to neurons in transgenic mice. The hVOS family of probes employs a membrane-targeted fluorescent protein, which generates voltage-dependent fluorescence changes in the presence of dipicrylamine (DPA) as the result of a voltage-dependent optical interaction between the two molecules. We generated transgenic mice with two different high-performance hVOS probes under control of a neuron-specific thy-1 promoter. Hippocampal slices from these animals present distinct spatial patterns of expression, and electrical stimulation evoked fluorescence changes as high as 3%. Glutamate receptor and Na+ channel antagonists blocked these responses. One hVOS probe tested here harbors an axonal targeting motif (from GAP-43) and shows preferential expression in axons; this probe can thus report axonal voltage changes. Voltage imaging in transgenic mice expressing hVOS probes opens the door to the study of functional activity in genetically defined populations of neurons in intact neural circuits. PMID:22993267

  12. Histamine H1 and endothelin ETB receptors mediate phospholipase D stimulation in rat brain hippocampal slices.

    PubMed

    Sarri, E; Picatoste, F; Claro, E

    1995-08-01

    Different neurotransmitter receptor agonists [carbachol, serotonin, noradrenaline, histamine, endothelin-1, and trans-(1S,3R)-aminocyclopentyl-1,3-dicarboxylic acid (trans-ACPD)], known as stimuli of phospholipase C in brain tissue, were tested for phospholipase D stimulation in [32P]Pi-prelabeled rat brain cortical and hippocampal slices. The accumulation of [32P]phosphatidylethanol was measured as an index of phospholipase D-catalyzed transphosphatidylation in the presence of ethanol. Among the six neurotransmitter receptor agonists tested, only noradrenaline, histamine, endothelin-1, and trans-ACPD stimulated phospholipase D in hippocampus and cortex, an effect that was strictly dependent of the presence of millimolar extracellular calcium concentrations. The effect of histamine (EC50 18 microM) was inhibited by the H1 receptor antagonist mepyramine with a Ki constant of 0.7 nM and was resistant to H2 and H3 receptor antagonists (ranitidine and tioperamide, respectively). Endothelin-1-stimulated phospholipase D (EC50 44 nM) was not blocked by BQ-123, a specific antagonist of the ETA receptor. Endothelin-3 and the specific ETB receptor agonist safarotoxin 6c were also able to stimulate phospholipase D with efficacies similar to that of endothelin-1, and EC50 values of 16 and 3 nM, respectively. These results show that histamine and endothelin-1 stimulate phospholipase D in rat brain through H1 and ETB receptors, respectively.

  13. Taurine release modified by GABAergic agents in hippocampal slices from adult and developing mice.

    PubMed

    Saransaari, P; Oja, S S

    2000-01-01

    In order to characterize the possible regulation of taurine release by GABAergic terminals, the effects of several agonists and antagonists of GABA receptors on the basal and K+-stimulated release of [3H]taurine were investigated in hippocampal slices from adult (3-month-old) and developing (7-day-old) mice using a superfusion system. Taurine release was concentration-dependently potentiated by GABA, which effect was reduced by phaclofen, saclofen and (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) at both ages, suggesting regulation by both GABA(B) and GABA(C) receptors. The involvement of GABA(A) receptors could not be excluded since the antagonist bicuculline was able to affect both basal and K+-evoked taurine release. Furthermore, several GABA(B) receptor effectors were able to inhibit K+-stimulated taurine release in the adults, while the GABA(C) receptor agonists trans-4-aminocrotonic acid (TACA) and cis-4-aminocrotonic acid (CACA) potentiated this release. The potentiation of taurine release by agents acting on the three types of GABA receptors in both adult and developing hippocampus further indicates the involvement of transporters operating in an outward direction. This inference is corroborated by the moderate but significant inhibition of taurine uptake by the same compounds.

  14. Chondroitin Sulfate Induces Depression of Synaptic Transmission and Modulation of Neuronal Plasticity in Rat Hippocampal Slices.

    PubMed

    Albiñana, Elisa; Gutierrez-Luengo, Javier; Hernández-Juarez, Natalia; Baraibar, Andrés M; Montell, Eulalia; Vergés, Josep; García, Antonio G; Hernández-Guijo, Jesus M

    2015-01-01

    It is currently known that in CNS the extracellular matrix is involved in synaptic stabilization and limitation of synaptic plasticity. However, it has been reported that the treatment with chondroitinase following injury allows the formation of new synapses and increased plasticity and functional recovery. So, we hypothesize that some components of extracellular matrix may modulate synaptic transmission. To test this hypothesis we evaluated the effects of chondroitin sulphate (CS) on excitatory synaptic transmission, cellular excitability, and neuronal plasticity using extracellular recordings in the CA1 area of rat hippocampal slices. CS caused a reversible depression of evoked field excitatory postsynaptic potentials in a concentration-dependent manner. CS also reduced the population spike amplitude evoked after orthodromic stimulation but not when the population spikes were antidromically evoked; in this last case a potentiation was observed. CS also enhanced paired-pulse facilitation and long-term potentiation. Our study provides evidence that CS, a major component of the brain perineuronal net and extracellular matrix, has a function beyond the structural one, namely, the modulation of synaptic transmission and neuronal plasticity in the hippocampus.

  15. Isolated Primary Blast Inhibits Long-Term Potentiation in Organotypic Hippocampal Slice Cultures.

    PubMed

    Vogel, Edward W; Effgen, Gwen B; Patel, Tapan P; Meaney, David F; Bass, Cameron R Dale; Morrison, Barclay

    2016-04-01

    Over the last 13 years, traumatic brain injury (TBI) has affected over 230,000 U.S. service members through the conflicts in Iraq and Afghanistan, mostly as a result of exposure to blast events. Blast-induced TBI (bTBI) is multi-phasic, with the penetrating and inertia-driven phases having been extensively studied. The effects of primary blast injury, caused by the shockwave interacting with the brain, remain unclear. Earlier in vivo studies in mice and rats have reported mixed results for primary blast effects on behavior and memory. Using a previously developed shock tube and in vitro sample receiver, we investigated the effect of isolated primary blast on the electrophysiological function of rat organotypic hippocampal slice cultures (OHSC). We found that pure primary blast exposure inhibited long-term potentiation (LTP), the electrophysiological correlate of memory, with a threshold between 9 and 39 kPa·ms impulse. This deficit occurred well below a previously identified threshold for cell death (184 kPa·ms), supporting our previously published finding that primary blast can cause changes in brain function in the absence of cell death. Other functional measures such as spontaneous activity, network synchronization, stimulus-response curves, and paired-pulse ratios (PPRs) were less affected by primary blast exposure, as compared with LTP. This is the first study to identify a tissue-level tolerance threshold for electrophysiological changes in neuronal function to isolated primary blast.

  16. Electroosmotic Sampling. Application to Determination of Ectopeptidase Activity in Organotypic Hippocampal Slice Cultures

    PubMed Central

    Xu, Hongjuan; Guy, Yifat; Hamsher, Amy; Shi, Guoyue; Sandberg, Mats; Weber, Stephen G.

    2010-01-01

    We hypothesize that peptide-containing solutions pulled through tissue should reveal the presence and activity of peptidases in the tissue. Using the natural ζ-potential in the organotypic hippocampal slice culture (OHSC), physiological fluids can be pulled through the tissue with an electric field. The hydrolysis of the peptides present in the fluid drawn through the tissue can be determined using capillary HPLC with electrochemical detection of the biuret complexes of the peptides following a postcolumn reaction. We have characterized this new sampling method by measuring the flow rate, examining the use of internal standards, and examining cell death caused by sampling. The sampling flow rate ranges from 60 to 150 nL/min with a 150 μm (ID) sampling capillary with an electric field (at the tip of the capillary) from 30 to 60 V/cm. Cell death can be negligible with controlled sampling conditions. Using this sampling approach, we have electroosmotically pulled Leu-enkephalin through OHSCs to identify ectopeptidase activity in the CA3 region. These studies show that a bestatin-sensitive aminopeptidase may be critical for the hydrolysis of exogenous Leu-enkephalin, a neuropeptide present in the CA3 region of OHSCs. PMID:20669992

  17. Multiple Single-Unit Long-Term Tracking on Organotypic Hippocampal Slices Using High-Density Microelectrode Arrays

    PubMed Central

    Gong, Wei; Senčar, Jure; Bakkum, Douglas J.; Jäckel, David; Obien, Marie Engelene J.; Radivojevic, Milos; Hierlemann, Andreas R.

    2016-01-01

    A novel system to cultivate and record from organotypic brain slices directly on high-density microelectrode arrays (HD-MEA) was developed. This system allows for continuous recording of electrical activity of specific individual neurons at high spatial resolution while monitoring at the same time, neuronal network activity. For the first time, the electrical activity patterns of single neurons and the corresponding neuronal network in an organotypic hippocampal slice culture were studied during several consecutive weeks at daily intervals. An unsupervised iterative spike-sorting algorithm, based on PCA and k-means clustering, was developed to assign the activities to the single units. Spike-triggered average extracellular waveforms of an action potential recorded across neighboring electrodes, termed “footprints” of single-units were generated and tracked over weeks. The developed system offers the potential to study chronic impacts of drugs or genetic modifications on individual neurons in slice preparations over extended times. PMID:27920665

  18. Ethanol alters the frequency, amplitude, and decay kinetics of Sr2+-supported, asynchronous NMDAR mEPSCs in rat hippocampal slices.

    PubMed

    Hendricson, Adam W; Sibbald, John R; Morrisett, Richard A

    2004-06-01

    To discriminate between pre- and postsynaptic effects of ethanol on N-methyl-d-aspartate receptor (NMDAR) signaling in hippocampus, we adapted the technique of Sr(2+) substitution to the hippocampal blind slice patch-clamp preparation. Hippocampal slices were isolated from 12- to 20-day-old rats that were killed in accordance with University of Texas Institutional Animal Care and Use Committee guidelines. NMDAR miniature excitatory postsynaptic currents (mEPSCs) were evoked from CA1 pyramidal neurons in the presence of Sr(2+) (4 mM), causing the synchronous EPSC observed in the presence of Ca(2+) to be supplanted by asynchronous mEPSCs. Amplitudes typically ranged from 5 to 40 pA and responded to the NMDAR antagonist (DL)-APV (50 microM), with a statistically significant reduction in mean amplitude. Ethanol (25, 50, and 75 mM) exerted dose-dependent effects on mEPSC amplitude and frequency. Peak amplitude inhibition was observed at 75 mM ethanol. Notably, ethanol significantly decreased event frequency at 50 and 75 mM ethanol. Ethanol (75 mM) also significantly increased the paired-pulse ratio of NMDAR EPSCs. Cumulative comparisons of decay time constants derived from single-exponential fitting of mEPSCs revealed significantly accelerated current decay kinetics in the presence of 75 mM ethanol. Taken together, these reductions in miniature event frequency and amplitude, concurrent with an increased rate of decay, suggest that the acute effects of ethanol on NMDAR signaling at hippocampal synapses are multifocal in nature. This finding of pre- and postsynaptic effects of ethanol on NMDAR signal strength in a brain region central to cognition is wholly consistent with previous reports of ethanol inhibition of NMDAR-long-term potentiation in vitro and with the profound cognitive deficits associated with binge-level intoxication in vivo.

  19. Light transmittance as an index of cell volume in hippocampal slices: optical differences of interfaced and submerged positions.

    PubMed

    Kreisman, N R; LaManna, J C; Liao, S C; Yeh, E R; Alcala, J R

    1995-09-25

    Light transmittance (T) in the CA1 region of hippocampal slices was measured during exposure to media of various osmolarities to determine the utility of optical measurements as an index of changes in cell volume. In slices positioned at the gas-liquid interface, hypo-osmotic medium consistently produced a decrease in T and hyperosmotic medium produced an increase in T. The magnitude of deltaT was graded as a function of the strength of osmotic change. All changes in T were reversible upon return to isosmotic medium. In contrast, osmotically induced changes in T in submerged slices were consistently opposite in direction to those observed in slices at the interface. The magnitude and direction of deltaT could be altered by systematic variation of the level of the bathing medium within the same chamber, indicating that both extrinsic optical properties of various interfaces, such as refraction and reflection, and intrinsic optical properties of the tissue contribute to the observed T. Spectral measurements eliminated the possibility that osmotically induced deltaT was the result of changes in light absorbance by intrinsic chromophores such as cytochromes or hemoglobin. The results show that measurements of deltaT can be a useful index of changes in cell volume in brain slices, provided that the level of the bath remains constant.

  20. Conditions sufficient for nonsynaptic epileptogenesis in the CA1 region of hippocampal slices.

    PubMed

    Bikson, Marom; Baraban, Scott C; Durand, Dominique M

    2002-01-01

    Nonsynaptic mechanisms exert a powerful influence on seizure threshold. It is well-established that nonsynaptic epileptiform activity can be induced in hippocampal slices by reducing extracellular Ca(2+) concentration. We show here that nonsynaptic epileptiform activity can be readily induced in vitro in normal (2 mM) Ca(2+) levels. Those conditions sufficient for nonsynaptic epileptogenesis in the CA1 region were determined by pharmacologically mimicking the effects of Ca(2+) reduction in normal Ca(2+) levels. Increasing neuronal excitability, by removing extracellular Mg(2+) and increasing extracellular K(+) (6-15 mM), induced epileptiform activity that was suppressed by postsynaptic receptor antagonists [D-(-)-2-amino-5-phosphonopentanoic acid, picrotoxin, and 6,7-dinitroquinoxaline-2,3-dione] and was therefore synaptic in nature. Similarly, epileptiform activity induced when neuronal excitability was increased in the presence of K(Ca) antagonists (verruculogen, charybdotoxin, norepinephrine, tetraethylammonium salt, and Ba(2+)) was found to be synaptic in nature. Decreases in osmolarity also failed to induce nonsynaptic epileptiform activity in the CA1 region. However, increasing neuronal excitability (by removing extracellular Mg(2+) and increasing extracellular K(+)) in the presence of Cd(2+), a nonselective Ca(2+) channel antagonist, or veratridine, a persistent sodium conductance enhancer, induced spontaneous nonsynaptic epileptiform activity in vitro. Both novel models were characterized using intracellular and ion-selective electrodes. The results of this study suggest that reducing extracellular Ca(2+) facilitates bursting by increasing neuronal excitability and inhibiting Ca(2+) influx, which might, in turn, enhance a persistent sodium conductance. Furthermore, these data show that nonsynaptic mechanisms can contribute to epileptiform activity in normal Ca(2+) levels.

  1. Interactions between adenosine and metabotropic glutamate receptors in the rat hippocampal slice

    PubMed Central

    Shahraki, Ali; Stone, Trevor W

    2003-01-01

    We have examined excitatory postsynaptic potentials and paired-pulse interactions in rat hippocampal slices to obtain more information about the site and mechanism of interactions between metabotropic glutamate receptors and adenosine receptors. The results show that the suppression of adenosine sensitivity is explained by a selectively reduced responsiveness to A1 receptor stimulation, and does not involve any facilitation of A2A adenosine receptors, since it can be obtained in the absence of endogenous adenosine and is not prevented by the A2A receptor blocker ZM241385. The glutamate receptors involved are of the group I class since the suppression of adenosine sensitivity is produced by ACPD and the group I selective compound DHPG. Furthermore, the effects of DHPG could be prevented by LY367385, a selective antagonist at the mGlu1a subtype of group I receptors. The selective antagonist at mGlu5 receptors, SIB1893, did not prevent the suppression of adenosine sensitivity by DHPG. Blockade of the DHPG/adenosine interaction was also obtained by superfusion with the protein kinasae C inhibitor chelerythrine. Since the suppression of adenosine responses by metabotropic receptor agonists was seen in the paired-pulse paradigm, we conclude that the observed interactions occur at the level of the presynaptic terminals. The interaction with adenosine receptors is not specific, but applies also to a suppression of responses mediated by the GABAB receptor agonist baclofen. We conclude that activation of the mGlu1a subtype of receptor can suppress responses mediated via adenosine A1 receptors, probably by activating protein kinase C. Since the changes induced by metabotropic glutamate receptor agonists last for at least 60 min, the data also imply that these interactions could play an important role in changes of synaptic function long after even transient increases of glutamate release in the CNS. PMID:12684261

  2. Effects of tetrahydrohyperforin in mouse hippocampal slices: neuroprotection, long-term potentiation and TRPC channels.

    PubMed

    Montecinos-Oliva, C; Schuller, A; Parodi, J; Melo, F; Inestrosa, N C

    2014-01-01

    Tetrahydrohyperforin (IDN5706) is a semi-synthetic compound derived from hyperforin (IDN5522) and is the main active principle of St. John's Wort. IDN5706 has shown numerous beneficial effects when administered to wild-type and double transgenic (APPswe/PSEN1ΔE9) mice that model Alzheimer's disease. However, its mechanism of action is currently unknown. Toward this end, we analysed field excitatory postsynaptic potentials (fEPSPs) in mouse hippocampal slices incubated with IDN5706 and in the presence of the TRPC3/6/7 activator 1-oleoyl-2-acetyl-sn-glycerol (OAG), the TRPC channel blocker SKF96365, and neurotoxic amyloid β-protein (Aβ) oligomers. To study spatial memory, Morris water maze (MWM) behavioural tests were conducted on wild-type mice treated with IDN5706 and SKF96365. In silico studies were conducted to predict a potential pharmacophore. IDN5706 and OAG had a similar stimulating effect on fEPSPs, which was inhibited by SKF96365. IDN5706 protected from reduced fEPSPs induced by Aβ oligomers. IDN5706 improved spatial memory in wild-type mice, an effect that was counteracted by co-administration of SKF96365. Our in silico studies suggest strong pharmacophore similarity of IDN5706 and other reported TRPC6 activators (IDN5522, OAG and Hyp9). We propose that the effect of IDN5706 is mediated through activation of the TRPC3/6/7 channel subfamily. The unveiling of the drug's mechanism of action is a necessary step toward the clinical use of IDN5706 in Alzheimer's disease.

  3. Standard antiepileptic drugs fail to block epileptiform activity in rat organotypic hippocampal slice cultures

    PubMed Central

    Albus, K; Wahab, A; Heinemann, U

    2008-01-01

    Background and purpose: Earlier studies had demonstrated that tonic–clonic seizure-like events (SLEs) resembling electrographic correlates of limbic seizures in animals and humans can be induced in organotypic hippocampal slice cultures (OHSCs). We have explored OHSCs for their suitability to serve as in vitro models of limbic seizures for studying seizure mechanisms and screening new antiepileptic compounds. Experimental approach: OHSCs were cultivated according to the interface method. Neuronal activity and extracellular potassium concentration were recorded under submerged conditions. SLEs were induced by lowering magnesium concentration or by applying the potassium channel blocker 4-aminopyridine. The effects of standard antiepileptic drugs (AEDs), carbamazepine, phenytoin, valproic acid, clonazepam, diazepam and phenobarbital sodium on SLEs were analysed. Key results: In more than 93% of OHSCs, AEDs did not prevent the induction of SLEs or stop ongoing seizure activity even when toxic concentrations were applied. This pharmacoresistance was independent of the method of seizure provocation, postnatal age at explantation (P2–P10) and cultivation time in vitro (2 months). SLEs were reversibly blocked by glutamate antagonists or the GABAA-agonist muscimol. Conclusions and implications: We present a simple to establish in vitro model of tonic–clonic SLEs that is a priori pharmacoresistant and thus has an advantage over animal models of pharmacoresistant seizures in which responders and non-responders can be sorted out only after an experiment. OHSCs could be suitable for exploring mechanisms of pharmacoresistant seizures and be used for the identification of new anticonvulsive compounds eventually effective in drug refractory epilepsy. PMID:18414393

  4. Adenosine actions on CA1 pyramidal neurones in rat hippocampal slices.

    PubMed

    Greene, R W; Haas, H L

    1985-09-01

    Intracellular recordings with a bridge amplifier of CA1 pyramidal neurones in vitro were employed to study the mechanisms of action of exogenously applied adenosine in the hippocampal slice preparation of the rat. Adenosine enhanced the calcium-dependent, long-duration after-hyperpolarization (a.h.p.) at least in part by a reduction in the rate of decay of the a.h.p. Both the reduced rate of decay and that of the control can be described with a single exponential. Antagonism of the calcium-dependent potassium current (and as a result, the a.h.p.) by bath application of CdCl2 or intracellular injection of EGTA (ethyleneglycolbis-(beta-aminoethyl ether)N,N'-tetraacetic acid) did not reduce the adenosine-evoked hyperpolarization or decrease in input resistance. Similarly, TEA (tetraethylammonium), which antagonizes both the voltage- and calcium-sensitive, delayed, outward rectification, had no effect on the adenosine-evoked changes in resting membrane properties. Adenosine did not affect the early, transient, outward rectification. During exposure to 4-aminopyridine (4-AP) in concentrations sufficient to antagonize this early rectification, the changes in resting membrane properties evoked by adenosine were unaffected. We conclude that the enhancement of the a.h.p. and accommodation by adenosine may be mediated by a change in the regulation of intracellular calcium. However, the mechanism responsible for the hyperpolarization and decrease in input resistance evoked by adenosine is both calcium and voltage insensitive. Thus, it appears distinct from that mediating the enhancement of the a.h.p. and accommodation.

  5. Adenosine actions on CA1 pyramidal neurones in rat hippocampal slices.

    PubMed Central

    Greene, R W; Haas, H L

    1985-01-01

    Intracellular recordings with a bridge amplifier of CA1 pyramidal neurones in vitro were employed to study the mechanisms of action of exogenously applied adenosine in the hippocampal slice preparation of the rat. Adenosine enhanced the calcium-dependent, long-duration after-hyperpolarization (a.h.p.) at least in part by a reduction in the rate of decay of the a.h.p. Both the reduced rate of decay and that of the control can be described with a single exponential. Antagonism of the calcium-dependent potassium current (and as a result, the a.h.p.) by bath application of CdCl2 or intracellular injection of EGTA (ethyleneglycolbis-(beta-aminoethyl ether)N,N'-tetraacetic acid) did not reduce the adenosine-evoked hyperpolarization or decrease in input resistance. Similarly, TEA (tetraethylammonium), which antagonizes both the voltage- and calcium-sensitive, delayed, outward rectification, had no effect on the adenosine-evoked changes in resting membrane properties. Adenosine did not affect the early, transient, outward rectification. During exposure to 4-aminopyridine (4-AP) in concentrations sufficient to antagonize this early rectification, the changes in resting membrane properties evoked by adenosine were unaffected. We conclude that the enhancement of the a.h.p. and accommodation by adenosine may be mediated by a change in the regulation of intracellular calcium. However, the mechanism responsible for the hyperpolarization and decrease in input resistance evoked by adenosine is both calcium and voltage insensitive. Thus, it appears distinct from that mediating the enhancement of the a.h.p. and accommodation. PMID:3932644

  6. Effectors of large-conductance calcium-activated potassium channel modulate glutamate excitotoxicity in organotypic hippocampal slice cultures.

    PubMed

    Piwońska, Marta; Szewczyk, Adam; Schröder, Ullrich H; Reymann, Klaus G; Bednarczyk, Iotr

    2016-01-01

    Mitochondria have been suggested as a potential target for cytoprotective strategies. It has been shown that increased K+ uptake mediate by mitochondrial ATP-regulated potassium channels (mitoKATP channel) or large-conductance Ca2+-activated potassium channels (mitoBKCa channel) may provide protection in different models of cell death. Since recent findings demonstrated the presence of BKCa channels in neuronal mitochondria, the goal of the present study was to test the potential neuroprotective effects of BKCa channel modulators. Using organotypic hippocampal slice cultures exposed to glutamate, we demonstrated that preincubation of the slices with the BKCa channel opener NS1619 resulted in decreased neuronal cell death measured as reduced uptake of propidium iodide. This neuroprotective effect was reversed by preincubation with the BKCa channel inhibitors paxilline and Iberiotoxin (IbTx). Moreover, mitochondrial respiration measurements revealed that NS1619 induced an IbTx-sensitive increase in state 2 respiration of isolated brain mitochondria. In addition, electrophysiological patch-clamp studies confirmed the presence of BKCa channels in mitoplasts isolated from embryonic hippocampal cells. Taken together, our results confirm presence of BKCa channel in rat hippocampal neurons mitochondria and suggest putative role for mitoBKCa in neuroprotection.

  7. A testbed to explore the optimal electrical stimulation parameters for suppressing inter-ictal spikes in human hippocampal slices.

    PubMed

    Min-Chi Hsiao; Pen-Ning Yu; Dong Song; Liu, Charles Y; Heck, Christi N; Millett, David; Berger, Theodore W

    2014-01-01

    New interventions using neuromodulatory devices such as vagus nerve stimulation, deep brain stimulation and responsive neurostimulation are available or under study for the treatment of refractory epilepsy. Since the actual mechanisms of the onset and termination of the seizure are still unclear, most researchers or clinicians determine the optimal stimulation parameters through trial-and-error procedures. It is necessary to further explore what types of electrical stimulation parameters (these may include stimulation frequency, amplitude, duration, interval pattern, and location) constitute a set of optimal stimulation paradigms to suppress seizures. In a previous study, we developed an in vitro epilepsy model using hippocampal slices from patients suffering from mesial temporal lobe epilepsy. Using a planar multi-electrode array system, inter-ictal activity from human hippocampal slices was consistently recorded. In this study, we have further transferred this in vitro seizure model to a testbed for exploring the possible neurostimulation paradigms to inhibit inter-ictal spikes. The methodology used to collect the electrophysiological data, the approach to apply different electrical stimulation parameters to the slices are provided in this paper. The results show that this experimental testbed will provide a platform for testing the optimal stimulation parameters of seizure cessation. We expect this testbed will expedite the process for identifying the most effective parameters, and may ultimately be used to guide programming of new stimulating paradigms for neuromodulatory devices.

  8. Dynamic confocal imaging in acute brain slices and organotypic slice cultures using a spectral confocal microscope with single photon excitation.

    PubMed

    Kasparov, S; Teschemacher, A G; Paton, J F R

    2002-11-01

    Confocal imaging in living brain slices allows the resolution of submicrometre structures of nerve cells, glia and brain vessels. Imaging living brain slices is in many respects different from conventional fixed histological preparations for which confocal microscopes were designed originally. Several problems (i.e. mechanical and thermal drift, and autofluorescence) resulting from the optical and structural properties of brain slices are discussed. Fluorescent indicators may be used to monitor numerous intracellular parameters such as pH and Ca(2+) concentration, but not all of them are equally suitable for this type of work. Genetically engineered fluorescent proteins can be used to visualise the fine dendritic structure of neurones or track particular intracellular structures and proteins. They have also been used to generate indicators for Ca(2+), cAMP and other molecules. While conventional chemical indicators can be either loaded into neurones via patch pipettes or as membrane-permeable esters, protein indicators can be expressed in various types of cells using adenoviral vectors. Adenoviral transgenesis can be performed in vitro in both acute slices and organotypic slice cultures. Organotypic slice cultures give excellent optical access to neurones loaded with either conventional fluorescent indicators or transfected with adenovirus to express fluorescent proteins. They are most suitable for experiments where both conventional and genetically engineered indicators are combined. Single photon imaging in brain slices is limited to the superficial layers (approximately

  9. Low-frequency electrical stimulation enhances the effectiveness of phenobarbital on GABAergic currents in hippocampal slices of kindled rats.

    PubMed

    Asgari, Azam; Semnanian, Saeed; Atapour, Nafiseh; Shojaei, Amir; Moradi-Chameh, Homeira; Ghafouri, Samireh; Sheibani, Vahid; Mirnajafi-Zadeh, Javad

    2016-08-25

    Low frequency stimulation (LFS) has been proposed as a new approach in the treatment of epilepsy. The anticonvulsant mechanism of LFS may be through its effect on GABAA receptors, which are the main target of phenobarbital anticonvulsant action. We supposed that co-application of LFS and phenobarbital may increase the efficacy of phenobarbital. Therefore, the interaction of LFS and phenobarbital on GABAergic inhibitory post-synaptic currents (IPSCs) in kindled and control rats was investigated. Animals were kindled by electrical stimulation of basolateral amygdala in a semi rapid manner (12 stimulations/day). The effect of phenobarbital, LFS and phenobarbital+LFS was investigated on GABAA-mediated evoked and miniature IPSCs in the hippocampal brain slices in control and fully kindled animals. Phenobarbital and LFS had positive interaction on GABAergic currents. In vitro co-application of an ineffective pattern of LFS (100 pulses at afterdischarge threshold intensity) and a sub-threshold dose of phenobarbital (100μM) which had no significant effect on GABAergic currents alone, increased the amplitude and area under curve of GABAergic currents in CA1 pyramidal neurons of hippocampal slices significantly. Interestingly, the sub-threshold dose of phenobarbital potentiated the GABAergic currents when applied on the hippocampal slices of kindled animals which received LFS in vivo. Post-synaptic mechanisms may be involved in observed interactions. Obtained results implied a positive interaction between LFS and phenobarbital through GABAA currents. It may be suggested that a combined therapy of phenobarbital and LFS may be a useful manner for reinforcing the anticonvulsant action of phenobarbital. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  10. Protective effect of 20-HETE inhibition in a model of oxygen-glucose deprivation in hippocampal slice cultures

    PubMed Central

    Renic, Marija; Kumar, Suresh N.; Gebremedhin, Debebe; Florence, Matthew A.; Gerges, Nashaat Z.; Falck, John R.; Harder, David R.

    2012-01-01

    Recent studies have indicated that inhibitors of the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) may have direct neuroprotective actions since they reduce infarct volume after ischemia reperfusion in the brain without altering blood flow. To explore this possibility, the present study used organotypic hippocampal slice cultures subjected to oxygen-glucose deprivation (OGD) and reoxygenation to examine whether 20-HETE is released by organotypic hippocampal slices after OGD and whether it contributes to neuronal death through the generation of ROS and activation of caspase-3. The production of 20-HETE increased twofold after OGD and reoxygenation. Blockade of the synthesis of 20-HETE with N-hydroxy-N′-(4-butyl-2-methylphenol)formamidine (HET0016) or its actions with a 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, reduced cell death, as measured by the release of lactate dehydrogenase and propidium iodide uptake. Administration of a 20-HETE mimetic, 20-hydroxyeicosa-5(Z),14(Z)-dienoic acid (5,14-20-HEDE), had the opposite effect and increased injury after OGD. The death of neurons after OGD was associated with an increase in the production of ROS and activation of caspase-3. These effects were attenuated by HET0016 and potentiated after the administration of 5,14-20-HEDE. These findings indicate that the production of 20-HETE by hippocampal slices is increased after OGD and that inhibitors of the synthesis or actions of 20-HETE protect neurons from ischemic cell death. The protective effect of 20-HETE inhibitors is associated with a decrease in superoxide production and activation of caspase-3. PMID:22245774

  11. Catuaba (Trichilia catigua) prevents against oxidative damage induced by in vitro ischemia-reperfusion in rat hippocampal slices.

    PubMed

    Kamdem, Jean Paul; Waczuk, Emily Pansera; Kade, Ige Joseph; Wagner, Caroline; Boligon, Aline Augusti; Athayde, Margareth Linde; Souza, Diogo Onofre; Rocha, João Batista Teixeira

    2012-12-01

    Oxidative stress is implicated in brain damage associated with ischemia-reperfusion. Natural antioxidants found in some plants used in folk medicine have been indicated as potential neuroprotective agents. Here we investigated whether Trichilia catigua, a traditional Brazilian herbal medicine alleged to exhibit a variety of neuropharmacological properties (antidepressant, anti-neurasthenic, anti-inflammatory etc.), could have neuroprotective properties in rat hippocampal slices subjected to 2 h oxygen and glucose deprivation (OGD) followed by 1 h reperfusion. Ischemia-reperfusion (I/R) significantly decreased mitochondrial viability, increased dichlorofluorescein oxidation above control both in the incubation medium and slices homogenates, increased lactate dehydrogenase into the incubation medium and decreased non-protein thiols. T. catigua (40-100 μg/mL) protected slices from the deleterious effects of OGD when present before OGD and during the reperfusion periods. Oxidative stress in the medium was also determined under different conditions and the results demonstrated that T. catigua could not protect slices from I/R when it was added to the medium after ischemic insult. Although the translation to a real in vivo situation of I/R is difficult to be done, the results indicated that T. catigua should be used as preventive and not as a curative agent against brain damage.

  12. Ampakine CX516 ameliorates functional deficits in AMPA receptors in a hippocampal slice model of protein accumulation.

    PubMed

    Kanju, Patrick M; Parameshwaran, Kodeeswaran; Sims, Catrina; Bahr, Ben A; Shonesy, Brian C; Suppiramaniam, Vishnu

    2008-11-01

    AMPAkines are positive modulators of AMPA receptors, and previous work has shown that these compounds can facilitate synaptic plasticity and improve learning and memory in both animals and humans; thus, their role in the treatment of cognitive impairment is worthy of investigation. In this study, we have utilized an organotypic slice model in which chloroquine-induced lysosomal dysfunction produces many of the pathogenic attributes of Alzheimer's disease. Our previous work demonstrated that synaptic AMPA receptor function is impaired in hippocampal slice cultures exhibiting lysosomal dysfunction leading to protein accumulation. The present study investigated the effect of the AMPAkine CX516 on AMPAR-mediated synaptic transmission as well as the CX516 induced modification of single channel AMPA receptor properties in this organotypic slice-culture model. In whole cell recordings from CA1 pyramidal neurons in chloroquine-treated slices we observed a significant decrease in AMPAR-mediated mEPSC frequency and amplitude indicating synaptic dysfunction. Following application of CX516, these parameters returned to nearly normal levels. Similarly, we report chloroquine-induced impairment of AMPAR single channel properties (decreased probability of opening and mean open time), and significant recovery of these properties following CX516 administration. These results suggest that AMPA receptors may be potential pharmaceutical targets for the treatment of neurodegenerative diseases, and highlights AMPAkines, in particular, as possible therapeutic agents.

  13. Increase of hypoxic tolerance in rat hippocampal slices following 3-nitropropionic acid is not mediated by endogenous nerve growth factor.

    PubMed

    Riepe, M W; Kasischke, K; Gericke, C A; Löwe, A; Hellweg, R

    1996-06-14

    Chemical preconditioning with low dose inhibition of succinic dehydrogenase by 3-nitropropionic acid (3-np) increases tolerance against succeeding hypoxia. Supraphysiological doses of nerve growth factor (NGF) repeatedly were shown to protect against ischemic damage. We investigated whether increased tolerance against hypoxia results from increased or accelerated production of endogenous NGF. Average recovery of population spike amplitude after 15 min of hypoxia and 45 min of reoxygenation was 31 +/- 9% (mean +/- SE) in control hippocampal slices. After pretreatment with 3-np (single i.p. injection of 20 mg/kg body weight 1 h to 3 days prior to slice preparation), recovery exceeded 90% (P < 0.01). However, NGF content did not increase upon slice preparation, hypoxia in vitro, and pretreatment with 3-np in vivo 1 h to 1 day prior to slice preparation with and without additional hypoxia in vitro. We conclude that early-onset tolerance to hypoxia induced by 3-np treatment is not caused by induction of endogenous NGF production.

  14. Ultrastructural investigation of microcalcification and the role of oxygen-glucose deprivation in cultured rat hippocampal slices.

    PubMed

    Riew, Tae-Ryong; Kim, Hong Lim; Shin, Yoo-Jin; Park, Joo-Hee; Pak, Ha-Jin; Lee, Mun-Yong

    2015-10-05

    Intracellular calcium accumulation is associated with cell death in several neuropathological disorders including brain ischemia, but the exact mechanisms of calcification need to be clarified. We used organotypic hippocampal slice culture - cultures subjected to oxygen-glucose deprivation (OGD) mimicking the in vivo situation to investigate the events underlying ectopic calcification. Alizarin red staining indicating calcium deposition was observed in the cornu ammonis (CA)1 and dentate gyrus regions in control hippocampal slices despite no specific labeling for cell death markers. Electron microscopy using the osmium/potassium dichromate method revealed scattered degenerated cells throughout the normally appearing CA1 region. They contained electron-dense precipitates within mitochondria, and electron probe microanalysis confirmed that they were calcifying mitochondria. Selective calcium deposition was noted within, but not beyond, mitochondria in these mineralized cells. They showed ultrastructural features of non-necrotic, non-apoptotic cell death and retained their compact ultrastructure, even after the majority of mitochondria were calcified. Unexpectedly, no intracellular calcification was noted in necrotic CA1 pyramidal cells after OGD, and there was no progression of calcification in OGD-lesioned slices. In addition, mineralized cells in both control and OGD-lesioned slices were closely associated with or completely engulfed by astrocytes but not microglia. These astrocytes were laden with heterogeneous cytoplasmic inclusions that appeared to be related with their phagocytic activity. These data demonstrate that microcalcification specifically associated with mitochondria might lead to a novel type of cell death and suggest that astrocytes may be involved in the phagocytosis of these mineralized cells and possibly in the regulation of ectopic calcification. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Effects of the endogeneous cannabinoid, anandamide, on neuronal activity in rat hippocampal slices.

    PubMed

    Ameri, A; Wilhelm, A; Simmet, T

    1999-04-01

    1. The arachidonic acid derivative arachidonylethanolamide (anandamide) is an endogeneous ligand of cannabinoid receptors that induces pharmacological actions similar to those of cannabinoids such as delta9-tetrahydrocannabinol (THC). We examined whether anandamide can influence excessive neuronal activity by investigating stimulation-induced population spikes and epileptiform activity in rat hippocampal slices. For this purpose, the effects of anandamide were compared with those of the synthetic cannabinoid agonist WIN 55,212-2 and its inactive S(-)-enantiomer WIN 55,212-3. 2. Both anandamide (1 and 10 microM) and WIN 55,212-2 (0.1 and 1 microM) decreased the amplitude of the postsynaptic population spike and the slope of the field excitatory postsynaptic potential (field e.p.s.p.) without affecting the presynaptic fibre spike of the afferents. At a concentration of 1 microM, WIN 55,212-2 completely suppressed the postsynaptic spike, whereas the S(-)-enantiomer WIN 55,212-3 produced only a slight depression. The CB1 receptor antagonist SR 141716 blocked the inhibition evoked by the cannabinoids. SR 141716 had a slight facilitatory effect on neuronal excitability by itself. 3. Anandamide shifted the input-output curve of the postsynaptic spike and the field e.p.s.p. to the right and increased the magnitude of paired-pulse facilitation indicating a presynaptic mechanism of action. 4. Anandamide and WIN 55,212-2, but not WIN 55,212-3, attenuated both stimulus-triggered epileptiform activity in CA1 elicited by omission of Mg2+ and spontaneously occurring epileptiform activity in CA3 elicited by omission of Mg2+ and elevation of K+ to 8 mM. The antiepileptiform effect of these cannabinoids was blocked by SR 141716. 5. In conclusion, cannabinoid receptors of the CB1 type as well as their endogeneous ligand, anandamide, are involved in the control of neuronal excitability, thus reducing excitatory neurotransmission at a presynaptic site, a mechanism which might be

  16. Effects of 2-arachidonylglycerol, an endogenous cannabinoid, on neuronal activity in rat hippocampal slices.

    PubMed

    Ameri, A; Simmet, T

    2000-03-01

    The monoacylglycerol 2-arachidonylglycerol is an endogenous ligand of cannabinoid receptors. We examined whether 2-arachidonylglycerol can influence excessive neuronal activity by investigating stimulation-induced population spikes and epileptiform activity in rat hippocampal slices. For this purpose, the effects of 2-arachidonylglycerol were compared with those of the synthetic cannabinoid agonist WIN 55,212-2. At concentrations of 10-50 microM, 2-arachidonylglycerol attenuated the amplitude of the orthodromic population spike and the slope of the field excitatory postsynaptic potential (field EPSP). However, the effect of the synthetic cannabinoid WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolol[ 1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone; 0.1 microM and 1 microM) was significantly higher than that of the endogenous ligand. At a concentration of 1 microM, WIN 55,212-2 completely suppressed the field EPSP. However, none of the investigated compounds did affect the presynaptic fiber spike of the afferents. The CB1 receptor antagonist SR 141716 (N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorphenyl)-4-methyl-3- pyrazole-carboxamide) blocked the inhibition evoked by the cannabinoids. Both 2-arachidonylglycerol (30 microM) and WIN 55,212-2 (100 nM) shifted the input-output curve of the postsynaptic spike and the field EPSP to the right and increased the magnitude of paired-pulse facilitation, indicating a presynaptic mechanism of action. 2-Arachidonylglycerol and WIN 55,212-2 attenuated the frequency of spontaneously occurring epileptiform burst discharges in CA3 elicited by omission of Mg2+ and elevation of K+ to 8 mM. The antiepileptiform effect of these cannabinoids was blocked by SR 141716. In conclusion, 2-arachidonylglycerol seems to limit neuronal excitability via cannabinoid receptors of the CB1 type. By acting predominantly at a presynaptic site, it is capable of reducing excitatory neurotransmission, a mechanism which might

  17. Effects of blast overpressure on neurons and glial cells in rat organotypic hippocampal slice cultures.

    PubMed

    Miller, Anna P; Shah, Alok S; Aperi, Brandy V; Budde, Matthew D; Pintar, Frank A; Tarima, Sergey; Kurpad, Shekar N; Stemper, Brian D; Glavaski-Joksimovic, Aleksandra

    2015-01-01

    Due to recent involvement in military conflicts, and an increase in the use of explosives, there has been an escalation in the incidence of blast-induced traumatic brain injury (bTBI) among US military personnel. Having a better understanding of the cellular and molecular cascade of events in bTBI is prerequisite for the development of an effective therapy that currently is unavailable. The present study utilized organotypic hippocampal slice cultures (OHCs) exposed to blast overpressures of 150 kPa (low) and 280 kPa (high) as an in vitro bTBI model. Using this model, we further characterized the cellular effects of the blast injury. Blast-evoked cell death was visualized by a propidium iodide (PI) uptake assay as early as 2 h post-injury. Quantification of PI staining in the cornu Ammonis 1 and 3 (CA1 and CA3) and the dentate gyrus regions of the hippocampus at 2, 24, 48, and 72 h following blast exposure revealed significant time dependent effects. OHCs exposed to 150 kPa demonstrated a slow increase in cell death plateauing between 24 and 48 h, while OHCs from the high-blast group exhibited a rapid increase in cell death already at 2 h, peaking at ~24 h post-injury. Measurements of lactate dehydrogenase release into the culture medium also revealed a significant increase in cell lysis in both low- and high-blast groups compared to sham controls. OHCs were fixed at 72 h post-injury and immunostained for markers against neurons, astrocytes, and microglia. Labeling OHCs with PI, neuronal, and glial markers revealed that the blast-evoked extensive neuronal death and to a lesser extent loss of glial cells. Furthermore, our data demonstrated activation of astrocytes and microglial cells in low- and high-blasted OHCs, which reached a statistically significant difference in the high-blast group. These data confirmed that our in vitro bTBI model is a useful tool for studying cellular and molecular changes after blast exposure.

  18. Effects of Blast Overpressure on Neurons and Glial Cells in Rat Organotypic Hippocampal Slice Cultures

    PubMed Central

    Miller, Anna P.; Shah, Alok S.; Aperi, Brandy V.; Budde, Matthew D.; Pintar, Frank A.; Tarima, Sergey; Kurpad, Shekar N.; Stemper, Brian D.; Glavaski-Joksimovic, Aleksandra

    2015-01-01

    Due to recent involvement in military conflicts, and an increase in the use of explosives, there has been an escalation in the incidence of blast-induced traumatic brain injury (bTBI) among US military personnel. Having a better understanding of the cellular and molecular cascade of events in bTBI is prerequisite for the development of an effective therapy that currently is unavailable. The present study utilized organotypic hippocampal slice cultures (OHCs) exposed to blast overpressures of 150 kPa (low) and 280 kPa (high) as an in vitro bTBI model. Using this model, we further characterized the cellular effects of the blast injury. Blast-evoked cell death was visualized by a propidium iodide (PI) uptake assay as early as 2 h post-injury. Quantification of PI staining in the cornu Ammonis 1 and 3 (CA1 and CA3) and the dentate gyrus regions of the hippocampus at 2, 24, 48, and 72 h following blast exposure revealed significant time dependent effects. OHCs exposed to 150 kPa demonstrated a slow increase in cell death plateauing between 24 and 48 h, while OHCs from the high-blast group exhibited a rapid increase in cell death already at 2 h, peaking at ~24 h post-injury. Measurements of lactate dehydrogenase release into the culture medium also revealed a significant increase in cell lysis in both low- and high-blast groups compared to sham controls. OHCs were fixed at 72 h post-injury and immunostained for markers against neurons, astrocytes, and microglia. Labeling OHCs with PI, neuronal, and glial markers revealed that the blast-evoked extensive neuronal death and to a lesser extent loss of glial cells. Furthermore, our data demonstrated activation of astrocytes and microglial cells in low- and high-blasted OHCs, which reached a statistically significant difference in the high-blast group. These data confirmed that our in vitro bTBI model is a useful tool for studying cellular and molecular changes after blast exposure. PMID:25729377

  19. Tumor necrosis factor receptor-1 is essential for LPS-induced sensitization and tolerance to oxygen-glucose deprivation in murine neonatal organotypic hippocampal slices.

    PubMed

    Markus, Tina; Cronberg, Tobias; Cilio, Corrado; Pronk, Cornelis; Wieloch, Tadeusz; Ley, David

    2009-01-01

    Inflammation and ischemia have a synergistic damaging effect in the immature brain. The role of tumor necrosis factor (TNF) receptors 1 and 2 in lipopolysaccharide (LPS)-induced sensitization and tolerance to oxygen-glucose deprivation (OGD) was evaluated in neonatal murine hippocampal organotypic slices. Hippocampal slices from balb/c, C57BL/6 TNFR1(-/-), TNFR2(-/-), and wild-type (WT) mice obtained at P6 were grown in vitro for 9 days. Preexposure to LPS immediately before OGD increased propidium iodide-determined cell death in regions CA1, CA3, and dentate gyrus from 4 up to 48 h after OGD (P<0.001). Extending the time interval between LPS exposure and OGD to 72 h resulted in tolerance, that is reduced neuronal cell death after OGD (P<0.05). Slices from TNFR1(-/-) mice showed neither LPS-induced sensitization nor LPS-induced tolerance to OGD, whereas both effects were present in slices from TNFR2(-/-) and WT mice. Cytokine secretion (TNFalpha and interleukin-6) during LPS exposure was decreased in TNFR1(-/-) slices and increased in TNFR2(-/-) as compared with WT slices. We conclude that LPS induces sensitization or tolerance to OGD depending on the time interval between exposure to LPS and OGD in murine hippocampal slice cultures. Both paradigms are dependent on signaling through TNFR1.

  20. Blast waves from detonated military explosive reduce GluR1 and synaptophysin levels in hippocampal slice cultures.

    PubMed

    Smith, Marquitta; Piehler, Thuvan; Benjamin, Richard; Farizatto, Karen L; Pait, Morgan C; Almeida, Michael F; Ghukasyan, Vladimir V; Bahr, Ben A

    2016-12-01

    Explosives create shockwaves that cause blast-induced neurotrauma, one of the most common types of traumatic brain injury (TBI) linked to military service. Blast-induced TBIs are often associated with reduced cognitive and behavioral functions due to a variety of factors. To study the direct effects of military explosive blasts on brain tissue, we removed systemic factors by utilizing rat hippocampal slice cultures. The long-term slice cultures were briefly sealed air-tight in serum-free medium, lowered into a 37°C water-filled tank, and small 1.7-gram assemblies of cyclotrimethylene trinitramine (RDX) were detonated 15cm outside the tank, creating a distinct shockwave recorded at the culture plate position. Compared to control mock-treated groups of slices that received equal submerge time, 1-3 blast impacts caused a dose-dependent reduction in the AMPA receptor subunit GluR1. While only a small reduction was found in hippocampal slices exposed to a single RDX blast and harvested 1-2days later, slices that received two consecutive RDX blasts 4min apart exhibited a 26-40% reduction in GluR1, and the receptor subunit was further reduced by 64-72% after three consecutive blasts. Such loss correlated with increased levels of HDAC2, a histone deacetylase implicated in stress-induced reduction of glutamatergic transmission. No evidence of synaptic marker recovery was found at 72h post-blast. The presynaptic marker synaptophysin was found to have similar susceptibility as GluR1 to the multiple explosive detonations. In contrast to the synaptic protein reductions, actin levels were unchanged, spectrin breakdown was not detected, and Fluoro-Jade B staining found no indication of degenerating neurons in slices exposed to three RDX blasts, suggesting that small, sub-lethal explosives are capable of producing selective alterations to synaptic integrity. Together, these results indicate that blast waves from military explosive cause signs of synaptic compromise without

  1. Analysis of Spine Motility of Newborn Granule Cells in Acute Brain Slices.

    PubMed

    Tashiro, Ayumu; Zhao, Chunmei; Suh, Hoonkyo; Gage, Fred H

    2015-10-01

    In this protocol, acute brain slices are prepared from mice in which newborn granule cells have been labeled using retroviral vector technology. Using a live-cell imaging stage and confocal microscopy coupled to imaging software, dendritic spines are analyzed.

  2. Effects of a naturally occurring neurosteroid on GABAA IPSCs during development in rat hippocampal or cerebellar slices

    PubMed Central

    Cooper, Elizabeth J; Johnston, Graham A R; Edwards, Frances A

    1999-01-01

    The effects of the naturally occurring neurosteroid tetrahydrodeoxycorticosterone (THDOC) on GABAA receptor-mediated miniature, spontaneous and evoked IPSCs was tested using patch-clamp techniques in slices of hippocampus and cerebellum from rats at two developmental stages (≈10 and ≈20 days postnatal). The cells studied were hippocampal granule cells and cerebellar Purkinje and granule cells. Most miniature GABAergic currents (mIPSCs) decayed with two exponentials and neurosteroids caused a ≈4-fold increase in the decay time constant of the second exponential at the highest concentration used (2 μm). Similar effects were seen at high concentrations of THDOC (1-2 μm) in all cell groups tested. No effects were seen on amplitude or rise time of mIPSCs. The effects of THDOC (1 μm) were shown to be stereoselective and rapidly reversible, indicating that the neurosteroid binds to the GABAA receptor, rather than acting genomically. At concentrations of THDOC likely to occur physiologically (50–100 nm), the decay time of IPSCs was also enhanced (25–50 %) in all cerebellar cell groups tested. In contrast, at 100 nm THDOC, seven of 11 hippocampal granule cells were sensitive from the 10 day group but the 20 day hippocampal granule cells showed no significant enhancement in the presence of these lower concentrations of THDOC. The differences in sensitivity of hippocampal and cerebellar cells to THDOC are compared to data reported in the literature on regional development of expression of different receptor subunits in the brain and it is suggested that the progressive relative insensitivity of the 20 day hippocampal cells may depend on increasing expression of the δ subunit of the GABAA receptor and possibly an increase in the α4 subunit. PMID:10581314

  3. Effect of acute and fractionated irradiation on hippocampal neurogenesis.

    PubMed

    Park, Min-Kyoung; Kim, Seolhwa; Jung, Uhee; Kim, Insub; Kim, Jin Kyu; Roh, Changhyun

    2012-08-08

    Ionizing radiation has become an inevitable health concern emanating from natural sources like space travel and from artificial sources like medical therapies. In general, exposure to ionizing radiation such as γ-rays is one of the methods currently used to stress specific model systems. In this study, we elucidated the long-term effect of acute and fractionated irradiation on DCX-positive cells in hippocampal neurogenesis. Groups of two-month-old C57BL/6 female mice were exposed to whole-body irradiation at acute dose (5 Gy) or fractional doses (1 Gy × 5 times and 0.5 Gy × 10 times). Six months after exposure to γ-irradiation, the hippocampus was analyzed. Doublecortin (DCX) immunohistochemistry was used to measure changes of neurogenesis in the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG). The number of DCX-positive cells was significantly decreased in all acute and fractionally irradiation groups. The long-term changes in DCX-positive cells triggered by radiation exposure showed a very different pattern to the short-term changes which tended to return to the control level in previous studies. Furthermore, the number of DCX-positive cells was relatively lower in the acute irradiation group than the fractional irradiation groups (approximately 3.6-fold), suggesting the biological change on hippocampal neurogenesis was more susceptible to being damaged by acute than fractional irradiation. These results suggest that the exposure to γ-irradiation as a long-term effect can trigger biological responses resulting in the inhibition of hippocampal neurogenesis.

  4. Selective Inhibition of KCC2 Leads to Hyperexcitability and Epileptiform Discharges in Hippocampal Slices and In Vivo

    PubMed Central

    Sivakumaran, Sudhir; Cardarelli, Ross A.; Maguire, Jamie; Kelley, Matt R.; Silayeva, Liliya; Morrow, Danielle H.; Mukherjee, Jayanta; Moore, Yvonne E.; Mather, Robert J.; Duggan, Mark E.; Brandon, Nicholas J.; Dunlop, John; Zicha, Stephen

    2015-01-01

    GABAA receptors form Cl− permeable channels that mediate the majority of fast synaptic inhibition in the brain. The K+/Cl− cotransporter KCC2 is the main mechanism by which neurons establish low intracellular Cl− levels, which is thought to enable GABAergic inhibitory control of neuronal activity. However, the widely used KCC2 inhibitor furosemide is nonselective with antiseizure efficacy in slices and in vivo, leading to a conflicting scheme of how KCC2 influences GABAergic control of neuronal synchronization. Here we used the selective KCC2 inhibitor VU0463271 [N-cyclopropyl-N-(4-methyl-2-thiazolyl)-2-[(6-phenyl-3-pyridazinyl)thio]acetamide] to investigate the influence of KCC2 function. Application of VU0463271 caused a reversible depolarizing shift in EGABA values and increased spiking of cultured hippocampal neurons. Application of VU0463271 to mouse hippocampal slices under low-Mg2+ conditions induced unremitting recurrent epileptiform discharges. Finally, microinfusion of VU0463271 alone directly into the mouse dorsal hippocampus rapidly caused epileptiform discharges. Our findings indicated that KCC2 function was a critical inhibitory factor ex vivo and in vivo. PMID:26019342

  5. Human Dental Pulp Cells Differentiate toward Neuronal Cells and Promote Neuroregeneration in Adult Organotypic Hippocampal Slices In Vitro.

    PubMed

    Xiao, Li; Ide, Ryoji; Saiki, Chikako; Kumazawa, Yasuo; Okamura, Hisashi

    2017-08-11

    The adult mammalian central nerve system has fundamental difficulties regarding effective neuroregeneration. The aim of this study is to investigate whether human dental pulp cells (DPCs) can promote neuroregeneration by (i) being differentiated toward neuronal cells and/or (ii) stimulating local neurogenesis in the adult hippocampus. Using immunostaining, we demonstrated that adult human dental pulp contains multipotent DPCs, including STRO-1, CD146 and P75-positive stem cells. DPC-formed spheroids were able to differentiate into neuronal, vascular, osteogenic and cartilaginous lineages under osteogenic induction. However, under neuronal inductive conditions, cells in the DPC-formed spheroids differentiated toward neuronal rather than other lineages. Electrophysiological study showed that these cells consistently exhibit the capacity to produce action potentials, suggesting that they have a functional feature in neuronal cells. We further co-cultivated DPCs with adult mouse hippocampal slices on matrigel in vitro. Immunostaining and presto blue assay showed that DPCs were able to stimulate the growth of neuronal cells (especially neurons) in both the CA1 zone and the edges of the hippocampal slices. Brain-derived neurotrophic factor (BDNF), was expressed in co-cultivated DPCs. In conclusion, our data demonstrated that DPCs are well-suited to differentiate into the neuronal lineage. They are able to stimulate neurogenesis in the adult mouse hippocampus through neurotrophic support in vitro.

  6. Novel sulfoglycolipid IG20 causes neuroprotection by activating the phase II antioxidant response in rat hippocampal slices.

    PubMed

    Punzón, Eva; García-Alvarado, Fernanda; Maroto, Marcos; Fernández-Mendívil, Cristina; Michalska, Patrycja; García-Álvarez, Isabel; Arranz-Tagarro, Juan Alberto; Buendia, Izaskun; López, Manuela G; León, Rafael; Gandía, Luis; Fernández-Mayoralas, Alfonso; García, Antonio G

    2017-04-01

    Compound IG20 is a newly synthesised sulphated glycolipid that promotes neuritic outgrowth and myelinisation, at the time it causes the inhibition of glial proliferation and facilitates exocytosis in chromaffin cells. Here we have shown that IG20 at 0.3-10 μM afforded neuroprotection in rat hippocampal slices stressed with veratridine, glutamate or with oxygen plus glucose deprivation followed by reoxygenation (OGD/reox). Excess production of reactive oxygen species (ROS) elicited by glutamate or ODG/reox was prevented by IG20 that also restored the depressed tissue levels of GSH and ATP in hippocampal slices subjected to OGD/reox. Furthermore, the augmented iNOS expression produced upon OGD/reox exposure was also counteracted by IG20. Additionally, the IG20 elicited neuroprotection was prevented by the presence of inhibitors of the signalling pathways Jak2/STAT3, MEK/ERK1/2, and PI3K/Akt, consistent with the ability of the compound to increase the phosphorylation of Jak2, ERK1/2, and Akt. Thus, the activation of phase II response and the Nrf2/ARE pathway could explain the antioxidant and anti-inflammatory effects and the ensuing neuroprotective actions of IG20. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Neuronal field potential in acute hippocampus slice recorded with transistor and micropipette electrode.

    PubMed

    Stangl, Christian; Fromherz, Peter

    2008-02-01

    Arrays of planar electrodes are often applied to record spatial patterns of neuronal field potentials in acute brain slices. The approach is hampered by layers of inactive tissue caused by the cutting process and also by a film of bath electrolyte that may exist between the slice and the substrate. To address this issue, we used a micropipette electrode to measure the vertical profile of evoked field potentials across acute slices from mouse hippocampus. In this way, we found that the signal due to an excitatory postsynaptic potential (EPSP) at the bottom of the slice was about 40% of the maximum at its centre. The vertical profile was matched by a volume-conductor model with proper boundary conditions. Simultaneously, voltage transients caused by EPSPs were measured with a field-effect transistor in the substrate. The transistor signals were in agreement with the evoked field potentials at the bottom of the slice. The study demonstrates: (i) that the loss of signal amplitude from the centre of a slice to the bottom is modest, despite an inactive tissue layer; and (ii) that in principle, planar sensors are able to record the field potential at the bottom of a slice. The results raise questions about the small voltages that are often observed with planar metal electrodes and about the reconstruction of the neuronal activity from field potentials at the bottom of acute slices using current-source density analysis.

  8. Dexmedetomidine promotes the recovery of the field excitatory postsynaptic potentials (fEPSPs) in rat hippocampal slices exposed to oxygen-glucose deprivation.

    PubMed

    Kim, Sung-Eun; Ko, Il-Gyu; Kim, Chang-Ju; Chung, Jun-Young; Yi, Jae-Woo; Choi, Jeong-Hyun; Jang, Myung-Soo; Han, Jin-Hee

    2016-09-19

    Dexmedetomidine (DEX), a selective α2 adrenergic agonist, is an anesthetic and sedative agent, and is reported to exert neuroprotective effects after hypoxic ischemia. However, there are few studies on the electrophysiological effect of DEX in hippocampal slices under ischemic conditions. The effects of DEX on field potential in hippocampal slices exposed to oxygen-glucose deprivation (OGD) were evaluated. Hippocampal slices were prepared from rats, and the evoked field excitatory postsynaptic potentials (fEPSPs) were recorded using the MED 64 system. Hypoxic-ischemia was induced by perfusion with glucose-free artificial cerebrospinal fluid (aCSF) bubbled with 95% N2 and 5% CO2, and hippocampal slices were perfused with DEX-added aCSF before, during, and after OGD induction. In the normal hippocampal slices, perfusion with 1 and 10μM DEX did not significantly decrease the normalized fEPSP amplitude, but 100μM DEX significantly reduced the fEPSP amplitude compared with its baseline control. The induction of OGD remarkably decreased the fEPSP amplitude, whereas the pre-, co-, and post-treatment of 10μM DEX gradually promoted recovery after washing out, and consequently the amplitude of fEPSP in DEX pre-, co-, and post-treated OGD slices were significantly higher than that in the untreated OGD slices at 10min and 60min after washing out. In particular, co-treatment with DEX conspicuously promoted the recovery of the fEPSP amplitude at the beginning of washing out. These results suggest the possibility of DEX as a therapeutic agent to prevent hypoxic-ischemic brain damage and promote functional recovery after ischemia.

  9. Electrophysiological Measurement of Cannabinoid-Mediated Synaptic Modulation in Acute Mouse Brain Slices

    PubMed Central

    Báldi, Rita; Ghose, Dipanwita; Grueter, Brad A.

    2016-01-01

    Endocannabinoids (eCBs) are a class of bioactive lipids that mediate retrograde synaptic modulation at central and peripheral synapses. The highly lipophilic nature of eCBs and the pharmacological tools available to interrogate this system require unique methodological consideration, especially when applied to ex vivo systems such as electrophysiological analysis in acute brain slices. Here we discuss protocols for measuring cannabinoid and eCB-mediated synaptic signaling in mouse brain slices including analysis of short-term, long-term, and tonic eCB signaling modes, and the unique considerations for working with eCBs and TRPV1/cannabinoid ligands in acute brain slices. PMID:27063786

  10. Biocompatibility of very small superparamagnetic iron oxide nanoparticles in murine organotypic hippocampal slice cultures and the role of microglia.

    PubMed

    Pohland, Martin; Glumm, Robert; Wiekhorst, Frank; Kiwit, Jürgen; Glumm, Jana

    2017-01-01

    Superparamagnetic iron oxide nanoparticles (SPIO) are applied as contrast media for magnetic resonance imaging (MRI) and treatment of neurologic diseases despite the fact that important information concerning their local interactions is still lacking. Due to their small size, SPIO have great potential for magnetically labeling different cell populations, facilitating their MRI tracking in vivo. Before SPIO are applied, however, their effect on cell viability and tissue homoeostasis should be studied thoroughly. We have previously published data showing how citrate-coated very small superparamagnetic iron oxide particles (VSOP) affect primary microglia and neuron cell cultures as well as neuron-glia cocultures. To extend our knowledge of VSOP interactions on the three-dimensional multicellular level, we further examined the influence of two types of coated VSOP (R1 and R2) on murine organotypic hippocampal slice cultures. Our data show that 1) VSOP can penetrate deep tissue layers, 2) long-term VSOP-R2 treatment alters cell viability within the dentate gyrus, 3) during short-term incubation VSOP-R1 and VSOP-R2 comparably modify hippocampal cell viability, 4) VSOP treatment does not affect cytokine homeostasis, 5) microglial depletion decreases VSOP uptake, and 6) microglial depletion plus VSOP treatment increases hippocampal cell death during short-term incubation. These results are in line with our previous findings in cell coculture experiments regarding microglial protection of neurite branching. Thus, we have not only clarified the interaction between VSOP, slice culture, and microglia to a degree but also demonstrated that our model is a promising approach for screening nanoparticles to exclude potential cytotoxic effects.

  11. Biocompatibility of very small superparamagnetic iron oxide nanoparticles in murine organotypic hippocampal slice cultures and the role of microglia

    PubMed Central

    Pohland, Martin; Glumm, Robert; Wiekhorst, Frank; Kiwit, Jürgen; Glumm, Jana

    2017-01-01

    Superparamagnetic iron oxide nanoparticles (SPIO) are applied as contrast media for magnetic resonance imaging (MRI) and treatment of neurologic diseases despite the fact that important information concerning their local interactions is still lacking. Due to their small size, SPIO have great potential for magnetically labeling different cell populations, facilitating their MRI tracking in vivo. Before SPIO are applied, however, their effect on cell viability and tissue homoeostasis should be studied thoroughly. We have previously published data showing how citrate-coated very small superparamagnetic iron oxide particles (VSOP) affect primary microglia and neuron cell cultures as well as neuron-glia cocultures. To extend our knowledge of VSOP interactions on the three-dimensional multicellular level, we further examined the influence of two types of coated VSOP (R1 and R2) on murine organotypic hippocampal slice cultures. Our data show that 1) VSOP can penetrate deep tissue layers, 2) long-term VSOP-R2 treatment alters cell viability within the dentate gyrus, 3) during short-term incubation VSOP-R1 and VSOP-R2 comparably modify hippocampal cell viability, 4) VSOP treatment does not affect cytokine homeostasis, 5) microglial depletion decreases VSOP uptake, and 6) microglial depletion plus VSOP treatment increases hippocampal cell death during short-term incubation. These results are in line with our previous findings in cell coculture experiments regarding microglial protection of neurite branching. Thus, we have not only clarified the interaction between VSOP, slice culture, and microglia to a degree but also demonstrated that our model is a promising approach for screening nanoparticles to exclude potential cytotoxic effects. PMID:28280327

  12. Imbalance between excitation and inhibition among synaptic connections of CA3 pyramidal neurons in cultured hippocampal slices.

    PubMed

    Cruz-Martín, Alberto; Schweizer, Felix E

    2008-03-01

    A fundamental property of small neuronal ensembles is their ability to be selectively activated by distinct stimuli. One cellular mechanism by which neurons achieve this input selectivity is by modulating the temporal dynamics of excitation and inhibition. We explored the interplay of excitation and inhibition in synapses between pyramidal neurons of cornu ammonis field 3 of the hippocampal formation (CA3) in cultured rat hippocampal slices, where activation of a single excitatory cell can readily recruit local interneurons. Simultaneous whole-cell recordings from pairs of CA3 pyramidal neurons revealed that the strength of connections was neither uniform nor balanced. Rather, stimulation of presynaptic neurons elicited distinct combinations of excitatory postsynaptic current-inhibitory postsynaptic current (EPSC-IPSC) amplitudes in the postsynaptic neurons. EPSC-IPSC sequences with small EPSCs had large IPSCs and sequences that contained large EPSCs had small IPSCs. In addition to differences in the amplitudes of the responses, the kinetics of the EPSCs were also different, creating distinct temporal dynamics of excitation and inhibition. Weaker EPSCs had significantly slower kinetics and were efficiently occluded by IPSCs, thereby further limiting their contribution to depolarizing the postsynaptic membrane. Our data suggest that hippocampal pyramidal cells may use an imbalance between excitation and inhibition as a filter to enhance selectivity toward preferential excitatory connections.

  13. Changes in intrinsic inhibition in isolated hippocampal slices during ethanol withdrawal; lack of correlation with withdrawal hyperexcitability.

    PubMed Central

    Whittington, M. A.; Little, H. J.; Lambert, J. D.

    1992-01-01

    1. Intracellular recordings were made from pyramidal cells in area CA1 in mouse isolated hippocampal slices, after chronic ethanol treatment in vivo. 2. Fast i.p.s.ps were isolated by injection of the impaled neurones with QX314 (to block fast sodium currents and the slow i.p.s.p.) and stimulating the interneurones in the presence of the glutamatergic blockers, CNQX and APV. 3. The isolated fast-inhibitory postsynaptic potential (f.-i.p.s.p.) was measured at intervals during the 7 h withdrawal period. The reversal potential and sensitivity to bicuculline suggested that the isolated f.-i.p.s.p. was mediated by activation of the GABAA receptor-chloride ionophore complex. 4. Measurement of stimulus-response relationships for the f.-i.p.s.ps revealed an initial increase in the maximum size of the i.p.s.p., evoked from a membrane potential of -50 mV, seen at 2 h into ethanol withdrawal. This was attributed to a negative shift in the reversal potential, Ei.p.s.p., with no observed change in conductance, Gi.p.s.p. 5. No differences in f.-i.p.s.ps evoked during ethanol withdrawal or in control slices were seen at 4 h or 6 h. At these times, epileptiform activity was seen in previous field potential recordings. 6. Paired pulse depression of the f.-i.p.s.p. was significantly increased at 2 h into withdrawal, when a 150 ms pulse interval was used. No differences were seen at later times in the ethanol withdrawal period. 7. The results suggest that ethanol withdrawal hyperexcitability in isolated hippocampal slices is not caused by primary decreases in inhibition mediated by the GABAA receptor-chloride ionophore complex.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1330182

  14. Homo- and heteroexchange of adenine nucleotides and nucleosides in rat hippocampal slices by the nucleoside transport system

    PubMed Central

    Sperlágh, Beáta; Szabó, Gábor; Erdélyi, Ferenc; Baranyi, Mária; Sylvester Vizi, E

    2003-01-01

    Here, we investigated how nucleotides and nucleosides affect the release of tritiated purines and endogenous adenosine 5′-triphosphate (ATP) from superfused rat hippocampal slices. ATP elicited concentration-dependent [3H]purine efflux from slices preloaded with [3H]adenosine. High-performance liquid chromatography analysis of the effluent showed that the tritium label represented the whole set of adenine nucleotides and nucleosides, and ATP significantly increased the outflow of [3H]ATP. Adenosine 5′-diphosphate, adenosine, uridine, uridine 5′-triphosphate, α,β-methylene-ATP and 3′-O-(4-benzoylbenzoyl)-ATP were also active in eliciting [3H]purine release. Adenosine (300 μM) also evoked endogenous ATP efflux from the hippocampal slices. Reverse transcription-coupled-polymerase chain reaction analysis revealed that mRNAs encoding a variety of P2X and P2Y receptor proteins are expressed in the rat hippocampus. Nevertheless, neither P2 receptor (i.e. pyridoxal-5-phosphate-6-azophenyl-2′,4′-disulphonic acid, 30 μM, suramin, 300 μM and reactive blue 2, 10 μM), nor adenosine receptor (8-cyclopentyl-1,3-dipropylxanthine, 250 nM and dimethyl-1-propargylxanthine, 250 nM) antagonists modified the effect of ATP (300 μM) to evoke [3H]purine release. The nucleoside transport inhibitors, dipyridamole (10 μM), nitrobenzylthioinosine (10 μM) and adenosine deaminase (2–10 U ml−1), but not the ecto-adenylate kinase inhibitor diadenosine pentaphosphate (200 μM) significantly reduced ATP-evoked [3H]purine efflux. In summary, we found that ATP and other nucleotides and nucleosides promote the release of one another and themselves by the nucleoside transport system. This action could have relevance during physiological and pathological elevation of extracellular purine levels high enough to reverse the nucleoside transporter. PMID:12788822

  15. PROPYLTHIOURACIL (PTU)-INDUCED HYPOTHYROIDISM: EFFECTS ON SYNAPTIC TRANSMISSION AND LONG TERM POTENTIATION IN HIPPOCAMPAL SLICES.

    EPA Science Inventory

    Concern has been raised over endocrine effects of some classes of environmental chemicals. Severe hypothyroidism during critical periods of brain developmental leads to alterations in hippocampal structure, learning deficits, yet neurophysiological properties of the hippocampus...

  16. PROPYLTHIOURACIL (PTU)-INDUCED HYPOTHYROIDISM: EFFECTS ON SYNAPTIC TRANSMISSION AND LONG TERM POTENTIATION IN HIPPOCAMPAL SLICES.

    EPA Science Inventory

    Concern has been raised over endocrine effects of some classes of environmental chemicals. Severe hypothyroidism during critical periods of brain developmental leads to alterations in hippocampal structure, learning deficits, yet neurophysiological properties of the hippocampus...

  17. Low-calcium field burst discharges of CA1 pyramidal neurones in rat hippocampal slices.

    PubMed Central

    Haas, H L; Jefferys, J G

    1984-01-01

    Incubation of rat hippocampal slices in solutions containing low Ca2+ and increased Mg2+ rapidly blocked synaptic responses and increased spontaneous firing of all the principal neurones. More remarkably, a rhythmic and synchronous bursting discharge developed, which was restricted to the CA1 population of pyramidal neurones. These 'field bursts' or 'spreading excitation' were rapidly abolished by restoring the Ca2+ to 2 mM, by increasing the Mg2+ to 6 mM or by decreasing K+ from 6 to 3 mM. The CA1 pyramidal cells depolarized after the change to the low-Ca2+ solution by about 10-20 mV. Individual field bursts were associated with a further depolarization of 10-12 mV surmounted by a burst of action potentials at about 20/s. This transient depolarization shift, recorded extracellularly as a negative field, could be attributed to the increase of [K+]o during the bursts, reaching 9-10 mM as measured by ion-sensitive electrodes. The bursts were followed by a hyperpolarization, seen extracellularly as a small soma-positive field, which was attributed to an electrogenic pump and/or a Ca2+-activated K+ conductance. Stimulation of the tightly packed pyramidal cell axons in the alveus elicited a train of population spikes, instead of the single spike normally seen, and could trigger a full field burst. Recordings of the alvear tract volley suggested that the repeated spikes arose within the pyramidal cells. Multiple recordings from CA1 revealed that field bursts usually, but by no means always, started near the caudal (subicular) end of the area. They spread through the cell layer at 0.04-0.12 m/s. The most rapid propagation was seen when the bursts had an abrupt onset; slower propagation (1-10 mm/s) occurred when the bursts started gradually, which generally was the case near the sites of burst initiation and termination. Usually the action potentials within each burst were synchronized into population spikes which spread across CA1 at 0.04-0.15 m/s. The site of initiation

  18. Simulation and experimental study of DC electric field distribution characteristics of rat hippocampal slices in vitro

    NASA Astrophysics Data System (ADS)

    Zheng, Yu; Dong, Lei; Gao, Yang; Qiu, Qian; Li, Ze-yan; Zhao, Zhe; Chen, Rui-juan; Wang, Hui-quan

    2016-06-01

    Direct current (DC) electric field is a noninvasive neuromodulation tool that can inhibit or facilitate excitability of neurons. Despite its efficacy, the dielectric constant of artificial cerebrospinal fluid and the position and direction of brain slices and other factors can affect the field intensity and distribution acting on the surface of rat hippocampus slices, thus causing errors. In this study, we describe a new analytical method optimized for DC electric fields acting on brain slices, and the design of an external DC electric field stimulator to allow scientific evaluation of brain slices. We investigated parameters regarding the uniformity of electric field distribution and identified the maximal parameters using the finite element method. Then, we selected and simplified slice images using magnetic resonance imaging data and calculated the electric field intensity of the original and simplified models. The electric field simulator induced action potential and excitatory postsynaptic current with intensities of 1, 5, and 10 V/m. This study describes the development of a new electric field stimulator and successfully demonstrates its practicability for scientific evaluation of tissue slices.

  19. Effects of monomethylarsonic and monomethylarsonous acid on evoked synaptic potentials in hippocampal slices of adult and young rats

    SciTech Connect

    Krueger, Katharina Straub, Heidrun; Hirner, Alfred V.; Hippler, Joerg; Binding, Norbert; Musshoff, Ulrich

    2009-04-01

    Arsenite and its metabolites, dimethylarsinic or dimethylarsinous acid, have previously been shown to disturb synaptic transmission in hippocampal slices of rats (Krueger, K., Gruner, J., Madeja, M., Hartmann, L.M., Hirner, A.V., Binding, N., Mu{beta}hoff, U., 2006a. Blockade and enhancement of glutamate receptor responses in Xenopus oocytes by methylated arsenicals. Arch. Toxicol. 80, 492-501, Krueger, K., Straub, H., Binding, N., Mu{beta}hoff, U., 2006b. Effects of arsenite on long-term potentiation in hippocampal slices from adult and young rats. Toxicol. Lett. 165, 167-173, Krueger, K., Repges, H., Hippler, J., Hartmann, L.M., Hirner, A.V., Straub, H., Binding, N., Mu{beta}hoff, U., 2007. Effects of dimethylarsinic and dimethylarsinous acid on evoked synaptic potentials in hippocampal slices of young and adult rats. Toxicol. Appl. Pharmacol. 225, 40-46). The present experiments investigate, whether the important arsenic metabolites monomethylarsonic acid (MMA{sup V}) and monomethylarsonous acid (MMA{sup III}) also influence the synaptic functions of the hippocampus. In hippocampal slices of young (14-21 days-old) and adult (2-4 months-old) rats, evoked synaptic field potentials from the Schaffer collateral-CA1 synapse were measured under control conditions and during and after 30 and 60 min of application of the arsenic compounds. MMA{sup V} had no effect on the synapse functions neither in slices of adult nor in those from young rats. However, MMA{sup III} strongly influenced the synaptic transmission: it totally depressed the amplitudes of fEPSPs at concentrations of 50 {mu}mol/l (adult rats) and 25 {mu}mol/l (young rats) and LTP amplitudes at concentrations of 25 {mu}mol/l (adult rats) and 10 {mu}mol/l (young rats), respectively. In contrast, application of 1 {mu}mol/l MMA{sup III} led to an enhancement of the LTP amplitude in young rats, which is interpretable by an enhancing effect on NMDA receptors and a lack of the blocking effect on AMPA receptors at

  20. Effects of monomethylarsonic and monomethylarsonous acid on evoked synaptic potentials in hippocampal slices of adult and young rats.

    PubMed

    Krüger, Katharina; Straub, Heidrun; Hirner, Alfred V; Hippler, Jörg; Binding, Norbert; Musshoff, Ulrich

    2009-04-01

    Arsenite and its metabolites, dimethylarsinic or dimethylarsinous acid, have previously been shown to disturb synaptic transmission in hippocampal slices of rats (Krüger, K., Gruner, J., Madeja, M., Hartmann, L.M., Hirner, A.V., Binding, N., Mubetahoff, U., 2006a. Blockade and enhancement of glutamate receptor responses in Xenopus oocytes by methylated arsenicals. Arch. Toxicol. 80, 492-501, Krüger, K., Straub, H., Binding, N., Mubetahoff, U., 2006b. Effects of arsenite on long-term potentiation in hippocampal slices from adult and young rats. Toxicol. Lett. 165, 167-173, Krüger, K., Repges, H., Hippler, J., Hartmann, L.M., Hirner, A.V., Straub, H., Binding, N., Mubetahoff, U., 2007. Effects of dimethylarsinic and dimethylarsinous acid on evoked synaptic potentials in hippocampal slices of young and adult rats. Toxicol. Appl. Pharmacol. 225, 40-46). The present experiments investigate, whether the important arsenic metabolites monomethylarsonic acid (MMA(V)) and monomethylarsonous acid (MMA(III)) also influence the synaptic functions of the hippocampus. In hippocampal slices of young (14-21 days-old) and adult (2-4 months-old) rats, evoked synaptic field potentials from the Schaffer collateral-CA1 synapse were measured under control conditions and during and after 30 and 60 min of application of the arsenic compounds. MMA(V) had no effect on the synapse functions neither in slices of adult nor in those from young rats. However, MMA(III) strongly influenced the synaptic transmission: it totally depressed the amplitudes of fEPSPs at concentrations of 50 micromol/l (adult rats) and 25 micromol/l (young rats) and LTP amplitudes at concentrations of 25 micromol/l (adult rats) and 10 micromol/l (young rats), respectively. In contrast, application of 1 micromol/l MMA(III) led to an enhancement of the LTP amplitude in young rats, which is interpretable by an enhancing effect on NMDA receptors and a lack of the blocking effect on AMPA receptors at this concentration (Kr

  1. Changes in extracellular adenosine levels and population spike amplitude during graded hypoxia in the rat hippocampal slice.

    PubMed

    Fowler, J C

    1993-01-01

    Concentrations of adenosine and inosine in the incubation media of baths containing rat hippocampal slices were measured during graded hypoxia. Slices were exposed to atmospheres containing 95%, 71%, 48%, 24%, or 0% oxygen, with 5% CO2 and a balance of N2. Absorbance HPLC measurements were made with samples drawn from static tissue baths each containing four slices supported on a net at the interface between the medium and the atmosphere. Concentrations of adenosine and inosine were proportionate to the fractional oxygen content. They were significantly higher in atmospheres of 24% and 0% O2. Introducing a 95% N2/5% O2 atmosphere in place of 95% O2/5% CO2 resulted in a roughly 4-fold increase in the adenosine concentration in the bath. The adenosine transport blocker dipyridamole (200 microM), and the convulsant drug picrotoxinin (300 microM), had little effect on basal levels of adenosine measured at 95% O2 but significantly augmented the responses seen at 48%, 24% and 0% O2. Picrotoxinin, while increasing the adenosine concentration did not change the ratio of adenosine to inosine. In contrast, dipyridamole significantly increased the ratio of adenosine to inosine. Evoked population spikes were recorded from the CA1 layer. The population spike amplitude was depressed as the fractional oxygen content was reduced. It is concluded that adenosine regulation in the slice preparation is similar to that seen in the intact animal. In particular, the amounts of adenosine released into the incubation medium are related to oxygen availability.

  2. BDNF mediates the neuroprotective effects of positive AMPA receptor modulators against MPP+-induced toxicity in cultured hippocampal and mesencephalic slices.

    PubMed

    Jourdi, H; Hamo, L; Oka, T; Seegan, A; Baudry, M

    2009-04-01

    Neurotoxicity is involved in various neurodegenerative diseases including Parkinson's disease (PD), which affects mesencephalic dopaminergic neurons of the substantia nigra (SN). Positive alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor modulators (PARMs, a.k.a. Ampakines, such as CX614) increase brain-derived neurotrophic factor (BDNF) protein levels in vivo and in cultured hippocampal slices. BDNF is a survival factor for various neuronal cell types including mesencephalic dopaminergic neurons. Using cultured mesencephalic and hippocampal slices, we investigated whether preincubation with CX614 could provide neuroprotection against MPP(+) toxicity and whether such neuroprotection was mediated by BDNF. Various treatment protocols were tested to demonstrate CX614-induced neuroprotection against MPP(+). Pretreatment with CX614 significantly reduced MPP(+)-induced toxicity and increased BDNF levels in both hippocampal and mesencephalic cultured slices; CX614 pretreatment for 6 h in hippocampal slices and 24 h in mesencephalic slices was sufficient to produce significant neuroprotection as assessed with lactate dehydrogenase release in slice medium and propidium iodide uptake in slices. Both a BDNF scavenger and an inhibitor of the BDNF receptor TrkB, abrogated CX614-mediated reduction of MPP(+)-induced toxicity. Inhibition of Ca(2+)-activated proteases, calpains, was also protective against MPP(+)-induced toxicity. However, co-application of calpain inhibitor with CX614 abolished CX614-mediated protection, suggesting a dual action of calpains in this model. We conclude that CX614 is neuroprotective against MPP(+)-induced toxicity, an effect mediated by increased BDNF expression and activation of BDNF-dependent signaling pathways. Our results provide support for using PARMs as a new therapy for neurodegenerative disorders, including PD.

  3. Maintaining network activity in submerged hippocampal slices: importance of oxygen supply.

    PubMed

    Hájos, Norbert; Ellender, Tommas J; Zemankovics, Rita; Mann, Edward O; Exley, Richard; Cragg, Stephanie J; Freund, Tamás F; Paulsen, Ole

    2009-01-01

    Studies in brain slices have provided a wealth of data on the basic features of neurons and synapses. In the intact brain, these properties may be strongly influenced by ongoing network activity. Although physiologically realistic patterns of network activity have been successfully induced in brain slices maintained in interface-type recording chambers, they have been harder to obtain in submerged-type chambers, which offer significant experimental advantages, including fast exchange of pharmacological agents, visually guided patch-clamp recordings, and imaging techniques. Here, we investigated conditions for the emergence of network oscillations in submerged slices prepared from the hippocampus of rats and mice. We found that the local oxygen level is critical for generation and propagation of both spontaneously occurring sharp wave-ripple oscillations and cholinergically induced fast oscillations. We suggest three ways to improve the oxygen supply to slices under submerged conditions: (i) optimizing chamber design for laminar flow of superfusion fluid; (ii) increasing the flow rate of superfusion fluid; and (iii) superfusing both surfaces of the slice. These improvements to the recording conditions enable detailed studies of neurons under more realistic conditions of network activity, which are essential for a better understanding of neuronal network operation.

  4. Copper Inhibits NMDA Receptor-Independent LTP and Modulates the Paired-Pulse Ratio after LTP in Mouse Hippocampal Slices

    PubMed Central

    Salazar-Weber, Nina L.; Smith, Jeffrey P.

    2011-01-01

    Copper misregulation has been implicated in the pathological processes underlying deterioration of learning and memory in Alzheimer's disease and other neurodegenerative disorders. Supporting this, inhibition of long-term potentiation (LTP) by copper (II) has been well established, but the exact mechanism is poorly characterized. It is thought that an interaction between copper and postsynaptic NMDA receptors is a major part of the mechanism; however, in this study, we found that copper (II) inhibited NMDA receptor-independent LTP in the CA3 region of hippocampal slices. In addition, in the CA3 and CA1 regions, copper modulated the paired-pulse ratio (PPR) in an LTP-dependent manner. Combined, this suggests the involvement of a presynaptic mechanism in the modulation of synaptic plasticity by copper. Inhibition of the copper-dependent changes in the PPR with cyclothiazide suggested that this may involve an interaction with the presynaptic AMPA receptors that regulate neurotransmitter release. PMID:22028985

  5. Properties of Taurine Release in Glucose-Free Media in Hippocampal Slices from Developing and Adult Mice

    PubMed Central

    Oja, Simo S.; Saransaari, Pirjo

    2015-01-01

    The release of preloaded [3H]taurine from hippocampal slices from developing 7-day-old and young adult 3-month-old mice was studied in a superfusion system in the absence of glucose. These hypoglycemic conditions enhanced the release at both ages, the effect being markedly greater in developing mice. A depolarizing K+ concentration accentuated the release, which indicates that it was partially mediated by exocytosis. The anion channel blockers were inhibitory, witnessing the contribution of ion channels. NO-generating agents fomented the release as a sign of the participation of excitatory amino acid receptors. The other second messenger systems were apparently less efficient. The much greater taurine release could be a reason for the well-known greater tolerance of developing nervous tissue to lack of glucose. PMID:26347028

  6. Conversion of Synthetic Aβ to In Vivo Active Seeds and Amyloid Plaque Formation in a Hippocampal Slice Culture Model.

    PubMed

    Novotny, Renata; Langer, Franziska; Mahler, Jasmin; Skodras, Angelos; Vlachos, Andreas; Wegenast-Braun, Bettina M; Kaeser, Stephan A; Neher, Jonas J; Eisele, Yvonne S; Pietrowski, Marie J; Nilsson, K Peter R; Deller, Thomas; Staufenbiel, Matthias; Heimrich, Bernd; Jucker, Mathias

    2016-05-04

    The aggregation of amyloid-β peptide (Aβ) in brain is an early event and hallmark of Alzheimer's disease (AD). We combined the advantages of in vitro and in vivo approaches to study cerebral β-amyloidosis by establishing a long-term hippocampal slice culture (HSC) model. While no Aβ deposition was noted in untreated HSCs of postnatal Aβ precursor protein transgenic (APP tg) mice, Aβ deposition emerged in HSCs when cultures were treated once with brain extract from aged APP tg mice and the culture medium was continuously supplemented with synthetic Aβ. Seeded Aβ deposition was also observed under the same conditions in HSCs derived from wild-type or App-null mice but in no comparable way when HSCs were fixed before cultivation. Both the nature of the brain extract and the synthetic Aβ species determined the conformational characteristics of HSC Aβ deposition. HSC Aβ deposits induced a microglia response, spine loss, and neuritic dystrophy but no obvious neuron loss. Remarkably, in contrast to in vitro aggregated synthetic Aβ, homogenates of Aβ deposits containing HSCs induced cerebral β-amyloidosis upon intracerebral inoculation into young APP tg mice. Our results demonstrate that a living cellular environment promotes the seeded conversion of synthetic Aβ into a potent in vivo seeding-active form. In this study, we report the seeded induction of Aβ aggregation and deposition in long-term hippocampal slice cultures. Remarkably, we find that the biological activities of the largely synthetic Aβ aggregates in the culture are very similar to those observed in vivo This observation is the first to show that potent in vivo seeding-active Aβ aggregates can be obtained by seeded conversion of synthetic Aβ in a living (wild-type) cellular environment. Copyright © 2016 the authors 0270-6474/16/365084-10$15.00/0.

  7. Glutamate controls the induction of GABA-mediated giant depolarizing potentials through AMPA receptors in neonatal rat hippocampal slices.

    PubMed

    Bolea, S; Avignone, E; Berretta, N; Sanchez-Andres, J V; Cherubini, E

    1999-05-01

    Glutamate controls the induction of GABA-mediated giant depolarizing potentials through AMPA receptors in neonatal rat hippocampal slices. Giant depolarizing potentials (GDPs) are generated by the interplay of the depolarizing action of GABA and glutamate. In this study, single and dual whole cell recordings (in current-clamp configuration) were performed from CA3 pyramidal cells in hippocampal slices obtained from postnatal (P) days P1- to P6-old rats to evaluate the role of ionotropic glutamate receptors in GDP generation. Superfusion of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10-40 microM) completely blocked GDPs. However, in the presence of CNQX, it was still possible to re-induce the appearance of GDPs with GABA (20 microM) or (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxadepropionate (AMPA) (5 microM). This effect was prevented by the more potent and selective AMPA receptor antagonist GYKI 53655 (50-100 microM). In the presence of GYKI 53655, both kainic or domoic acid (0.1-1 microM) were unable to induce GDPs. In contrast, bath application of D-(-)-2-amino-5-phosphonopentanoic acid (50 microM) or (+)-3-(2carboxy-piperazin-4-yl)-propyl-L-phosphonic acid (20 microM) produced only a 37 +/- 9% (SE) and 36 +/- 11% reduction in GDPs frequency, respectively. Cyclothiazide, a selective blocker of AMPA receptor desensitization, increased GDP frequency by 76 +/- 14%. Experiments were also performed with an intracellular solution containing KF to block GABAA receptor-mediated responses. In these conditions, a glutamatergic component of GDP was revealed. GDPs could still be recorded synchronous with those detected simultaneously with KCl-filled electrodes, although their amplitude was smaller. Similar results were found in pair recordings obtained from minislices containing only a small portion of the CA3 area. These data suggest that GDP generation requires activation of AMPA receptors by local release of glutamate from recurrent collaterals.

  8. Effects of neurosteroids on epileptiform activity induced by picrotoxin and 4-aminopyridine in the rat hippocampal slice.

    PubMed

    Salazar, Patricia; Tapia, Ricardo; Rogawski, Michael A

    2003-01-01

    The neurosteroids allopregnanolone (5alpha-pregnan-3alpha-ol-20-one; 5alpha,3alpha-P) and its 5beta-epimer pregnanolone (5beta,3alpha-P), and pregnenolone sulfate (PS) were examined for effects on spontaneous epileptiform discharges induced by 100 microM picrotoxin (PTX) and 55 microM 4-aminopyridine (4-AP) in the CA3 region of the rat hippocampal slice. At a concentration of 10 microM, 5alpha,3alpha-P partially reduced PTX-induced bursting and at 30 and 90 microM completely suppressed bursting. In contrast, 100 microM 5beta,3alpha-P failed to alter the discharge frequency. 5alpha,3alpha-P depressed 4-AP-induced bursting with similar potency as in the PTX model; 100 microM 5beta,3alpha-P was also partially effective. In the 4-AP model, 5alpha,3alpha-P inhibited both the more frequent predominantly positive-going potentials as well as the less frequent negative-going potentials that may be generated by synchronous GABAergic interneuron firing. PS enhanced the PTX bursting frequency and, in the 4-AP model, increased the frequency of negative potentials but did not alter the frequency of positive potentials. By itself, PS did not induce bursting. The effects of the steroids in the in vitro seizure models largely correspond with their activities on GABA(A) receptors; suppression of discharges may occur as a result of direct activation of these receptors rather than modulation of GABA-mediated synaptic responses. PTX and 4-AP-induced bursting in the hippocampal slice are useful models for directly assessing neurosteroid effects on seizure susceptibility under conditions that eliminate the factor of brain bioavailability.

  9. Pharmacological characterization of the effects of methylmercury and mercuric chloride on spontaneous noradrenaline release from rat hippocampal slices.

    PubMed

    Gassó, S; Suñol, C; Sanfeliu, C; Rodríguez-Farré, E; Cristòfol, R M

    2000-01-01

    The environmental contaminants methylmercury (MeHg) and mercuric chloride (HgCl2) stimulated the spontaneous release of [3H]noradrenaline ([3H]NA) from hippocampal slices in a time- and concentration-dependent manner. Both MeHg and HgCl2 were similarly potent, with an EC50 of 88.4 microM and 75.9 microM, respectively. The releasing effects of MeHg and HgCl2 increased in the presence of desipramine, showing that the mechanism does not involve reversal of the transmitter transporter, and were completely blocked by reserpine preincubation, indicating a vesicular origin of [3H]NA release. The voltage-gated Na+ channel blocker tetrodotoxin (TTX) did not affect the response to mercury compounds. [3H]NA release elicited by MeHg was partially dependent on extracellular Ca2+, since it decreased significantly in a Ca2+-free EGTA-containing medium whereas HgCl2 induced a release of [3H]NA independent of extracellular Ca2+. Neither Ca2+-channels blockers, cobalt chloride (CoCl2) and (omega-conotoxin-GVIA, nor the Na+/Ca2+-exchanger inhibitor benzamil reduced MeHg-evoked [3H]NA release. Moreover, thapsigargin or caffeine, endoplasmic reticulum Ca2+-depletors, did not modify metal-evoked [3H]NA release, whereas ruthenium red, which inhibits the mitochondrial Ca2+ transport, decreased the effect of both MeHg and HgCl2. All these data indicate that, in hippocampal slices, mercury compounds release [3H]NA from the vesicular pool by a mechanism involving Ca2+ mobilization from mitochondrial stores.

  10. Effects of amide creatine derivatives in brain hippocampal slices, and their possible usefulness for curing creatine transporter deficiency.

    PubMed

    Garbati, Patrizia; Adriano, Enrico; Salis, Annalisa; Ravera, Silvia; Damonte, Gianluca; Millo, Enrico; Balestrino, Maurizio

    2014-01-01

    The creatine/phosphocreatine system carries ATP from production to consumption sites and buffers the intracellular content of ATP at times of energy deprivation. The creatine transporter deficiency syndrome is an X-linked disease caused by a defective creatine transporter into the central nervous system. This disease is presently untreatable because creatine lacking its carrier cannot cross neither the blood-brain barrier nor the cell plasma membranes. Possible strategies to cure this condition are to couple creatine to molecules which have their own carrier, to exploit the latter to cross biological membranes or to modify the creatine molecule to make it more lipophilic, in such a way that it may more easily cross lipid-rich biological membranes. Such molecules could moreover be useful for treatment of stroke or other ischemic brain syndromes of normal (transporter working) tissue. In this paper we tested four molecules in in vitro hippocampal slices experiments to investigate whether or not they had a neuroprotective effect similar to that of creatine. On two of them we also performed biochemical measurements to investigate whether or not they were able to increase the creatine and phosphocreatine content of the hippocampal slices with and without block of the transporter. We found that these molecules increase levels of creatine after block of the transporter, and significantly increased the levels of phosphocreatine. Both significantly increased the total creatine content in both conditions of active and blocked transporter. This shows that these molecules are capable of entering cells through biological membranes without using the creatine transporter. By contrast, neither of them was able to delay synaptic block during anoxia of normal (transporter functioning) tissue. We conclude that these compounds might possibly be useful for therapy of creatine transporter deficiency, but further research is needed to understand their possible role in anoxia/ischemia of

  11. Folic Acid Protects Against Glutamate-Induced Excitotoxicity in Hippocampal Slices Through a Mechanism that Implicates Inhibition of GSK-3β and iNOS.

    PubMed

    Budni, Josiane; Molz, Simone; Dal-Cim, Tharine; Martín-de-Saavedra, Maria Dolores; Egea, Javier; Lopéz, Manuela G; Tasca, Carla Ines; Rodrigues, Ana Lúcia Severo

    2017-02-10

    Folic acid (folate) is a vitamin of the B-complex group crucial for neurological function. Considering that excitotoxicity and cell death induced by glutamate are involved in many disorders, the potential protective effect of folic acid on glutamate-induced cell damage in rat hippocampal slices and the possible intracellular signaling pathway involved in such effect were investigated. The treatment of hippocampal slices with folic acid (100 μM) significantly abrogated glutamate (1 mM)-induced reduction of cell viability measured by MTT reduction assay and inhibited glutamate-induced D-[(3)H]-aspartate release. To investigate the putative intracellular signaling pathways implicated in the protective effect of folic acid, we used a PI3K inhibitor, LY294002, which abolished the protective effects of folic acid against glutamate-induced cell damage and D-[(3)H] aspartate release. Moreover, hippocampal slices incubated with folic acid alone for 30 min presented increased phosphorylation of GSK-3β at Ser9, indicating an inhibition of the activity of this enzyme. Furthermore, folic acid in the presence of glutamate insult in hippocampal slices maintained for an additional period of 6 h in fresh culture medium without glutamate and/or folic acid induced phosphorylation of GSK-3β and β-catenin expression. In addition, glutamate-treated hippocampal slices showed increased iNOS expression that was reversed by folic acid. In conclusion, the results of this study show that the protective effect of folic acid against glutamate-induced excitotoxicity may involve the modulation of PI3K/GSK-3β/β-catenin pathway and iNOS inhibition.

  12. Effects of the GABA-uptake blocker NNC-711 on spontaneous sharp wave-ripple complexes in mouse hippocampal slices.

    PubMed

    Viereckel, Thomas; Kostic, Milos; Bähner, Florian; Draguhn, Andreas; Both, Martin

    2013-05-01

    The precise temporal and spatial activity patterns of neurons in cortical networks are organized by different state-dependent types of network oscillations. GABAergic inhibition plays a key role in the underlying mechanisms of such oscillations and it has been suggested that the duration of widely distributed phasic inhibitory postsynaptic potentials (IPSPs) determines the frequency of the resulting network oscillation. Here, we test this hypothesis in an in vitro model of sharp wave-ripple (SPW-R) complexes, a particularly fast pattern of network oscillations at ∼200 Hz which is involved in memory consolidation. We recorded SPW-R in mouse hippocampal slices in the absence and presence of NCC-711, an inhibitor of GABA uptake. The resulting prolongation of IPSP resulted in reduced occurrence of SPW-R, whereas the superimposed fast oscillations as well as the precision of rhythmic cell synchronization remained stable. Application of Diazepam which is a positive modulator of the GABAA receptor led to consistent results. We conclude that phasic inhibition is a major regulator of network excitability in CA3 (where SPW-Rs are generated), but does not set the frequency of hippocampal ripples. Copyright © 2013 Wiley Periodicals, Inc.

  13. Excitatory and inhibitory synaptic transmission is differentially influenced by two ortho-substituted polychlorinated biphenyls in the hippocampal slice preparation

    SciTech Connect

    Kim, Kyung Ho; Inan, Salim Yalcin; Berman, Robert F.; Pessah, Isaac N.

    2009-06-01

    Exposure to polychlorinated biphenyls impairs cognition and behavior in children. Two environmental PCBs 2,2',3,3',4,4',5-heptachlorobiphenyl (PCB170) and 2,2',3,5',6-pentachlorobiphenyl (PCB95) were examined in vitro for influences on synaptic transmission in rat hippocampal slices. Field excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1 region using a multi-electrode array. Perfusion with PCB170 (10 nM) had no effect on fEPSP slope relative to baseline period, whereas (100 nM) initially enhanced then depressed fEPSP slope. Perfusion of PCB95 (10 or 100 nM) persistently enhanced fEPSP slope > 200%, an effect that could be inhibited by dantrolene, a drug that attenuates ryanodine receptor signaling. Perfusion with picrotoxin (PTX) to block GABA neurotransmission resulted in a modest increase in fEPSP slope, whereas PTX + PCB170 (1-100 nM) persistently enhanced fEPSP slope in a dose dependent manner. fEPSP slope reached > 250% of baseline period in the presence of PTX + 100 nM PCB170, conditions that evoked marked epileptiform after-potential discharges. PCB95 and PCB170 were found to differentially influence the Ca{sup 2+}-dependence of [{sup 3}H]ryanodine-binding to hippocampal ryanodine receptors. Non-coplanar PCB congeners can differentially alter neurotransmission in a manner suggesting they can elicit imbalances between inhibitory and excitatory circuits within the hippocampus. Differential sensitization of ryanodine receptors by Ca{sup 2+} appears to mediate, at least in part, hippocampal excitotoxicity by non-coplanar PCBs.

  14. Effects of dimethylarsinic and dimethylarsinous acid on evoked synaptic potentials in hippocampal slices of young and adult rats

    SciTech Connect

    Krueger, Katharina Repges, Hendrik; Hippler, Joerg; Hartmann, Louise M.; Hirner, Alfred V.; Straub, Heidrun; Binding, Norbert; Musshoff, Ulrich

    2007-11-15

    In this study, the effects of pentavalent dimethylarsinic acid ((CH{sub 3}){sub 2}AsO(OH); DMA{sup V}) and trivalent dimethylarsinous acid ((CH{sub 3}){sub 2}As(OH); DMA{sup III}) on synaptic transmission generated by the excitatory Schaffer collateral-CA1 synapse were tested in hippocampal slices of young (14-21 day-old) and adult (2-4 month-old) rats. Both compounds were applied in concentrations of 1 to 100 {mu}mol/l. DMA{sup V} had no effect on the amplitudes of evoked fEPSPs or the induction of LTP recorded from the CA1 dendritic region either in adult or in young rats. However, application of DMA{sup III} significantly reduced the amplitudes of evoked fEPSPs in a concentration-dependent manner with a total depression following application of 100 {mu}mol/l DMA{sup III} in adult and 10 {mu}mol/l DMA{sup III} in young rats. Moreover, DMA{sup III} significantly affected the LTP-induction. Application of 10 {mu}mol/l DMA{sup III} resulted in a complete failure of the postsynaptic potentiation of the fEPSP amplitudes in slices taken both from adult and young rats. The depressant effect was not reversible after a 30-min washout of the DMA{sup III}. In slices of young rats, the depressant effects of DMA{sup III} were more pronounced than in those taken from adult ones. Compared to the (absent) effect of DMA{sup V} on synaptic transmission, the trivalent compound possesses a considerably higher neurotoxic potential.

  15. Synaptic GABAA activation induces Ca2+ rise in pyramidal cells and interneurons from rat neonatal hippocampal slices.

    PubMed Central

    Leinekugel, X; Tseeb, V; Ben-Ari, Y; Bregestovski, P

    1995-01-01

    1. Changes in intracellular Ca2+ concentration ([Ca2+]i) induced by activation of GABAA receptors (synaptic stimulation or application of the GABAA agonist isoguvacine) were studied on pyramidal cells and interneurons from hippocampal slices of rats from two age groups (postnatal days (P) 2-5 and P12-13) using the fluorescent dye fluo-3 and a confocal laser scanning microscope. Cells were loaded with the dye either intracellularly, using patch pipettes containing fluo-3 in the internal solution, or extracellularly, using pressure pulses applied to an extracellular pipette containing the permeant dye fluo-3 AM. 2. Interneurons and pyramidal cells from P2-5 slices loaded with fluo-3 AM responded by an increase in [Ca2+]i to isoguvacine and to glutamate, in contrast to cells from P12-13 slices which responded to glutamate but not to isoguvacine. 3. The isoguvacine-induced rise in [Ca2+]i was reversibly blocked by bath application of the GABAA receptor antagonist bicuculline (20 microM), suggesting the specific involvement of GABAA receptors. The sodium channel blocker tetrodotoxin (TTX, 1 microM in the bath) did not prevent the isoguvacine-induced rise in [Ca2+]i. 4. The isoguvacine-induced rise in [Ca2+]i was reversibly blocked by bath application of the calcium channel blocker D600 (50 microM) suggesting the involvement of voltage-dependent Ca2+ channels. 5. Electrical stimulation of afferent fibres induced a transient increase in [Ca2+]i in neonatal pyramidal cells and interneurons (P5) loaded non-invasively with fluo-3 AM. This elevation of [Ca2+]i was reversibly blocked by bicuculline (20 microM) but not by APV (50 microM) and CNQX (10 microM). 6. During simultaneous electrophysiological recording in the current-clamp mode and [Ca2+]i monitoring from P5 pyramidal cells, electrical stimulation of afferent fibres, in the presence of APV (50 microM) and CNQX (10 microM), caused synaptic depolarization accompanied by a few action potentials and a transient increase

  16. Effects of acute hippocampal stimulation on EEG dynamics.

    PubMed

    Nair, Sandeep P; Sackellares, J Chris; Shiau, Deng-Shan; Norman, Wendy M; Dance, Linda K; Pardalos, Panos M; Principe, Jose C; Carney, Paul R

    2006-01-01

    Progressive preictal dynamical convergence and postictal divergence of dynamical EEG descriptors among brain regions has been reported in human temporal lobe epilepsy (TLE) and in a rodent model of TLE. There are also reports of anticonvulsant effects of high frequency stimulation of the hippocampus in humans. We postulate that this anticonvulsant effect is due to dynamical resetting by the electrical stimulation. The following study investigated the effects of acute hippocampal electrical stimulation on dynamical transitions in the brain of a spontaneously seizing animal model of TLE to test the hypothesis of divergence in dynamical values by electrical stimulation of the hippocampus.

  17. Modulator effects of interleukin-1beta and tumor necrosis factor-alpha on AMPA-induced excitotoxicity in mouse organotypic hippocampal slice cultures.

    PubMed

    Bernardino, Liliana; Xapelli, Sara; Silva, Ana P; Jakobsen, Birthe; Poulsen, Frantz R; Oliveira, Catarina R; Vezzani, Annamaria; Malva, João O; Zimmer, Jens

    2005-07-20

    The inflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha (TNF-alpha) have been identified as mediators of several forms of neurodegeneration in the brain. However, they can produce either deleterious or beneficial effects on neuronal function. We investigated the effects of these cytokines on neuronal death caused by exposure of mouse organotypic hippocampal slice cultures to toxic concentrations of AMPA. Either potentiation of excitotoxicity or neuroprotection was observed, depending on the concentration of the cytokines and the timing of exposure. A relatively high concentration of mouse recombinant TNF-alpha (10 ng/ml) enhanced excitotoxicity when the cultures were simultaneously exposed to AMPA and to this cytokine. Decreasing the concentration of TNF-alpha to 1 ng/ml resulted in neuroprotection against AMPA-induced neuronal death independently on the application protocol. By using TNF-alpha receptor (TNFR) knock-out mice, we demonstrated that the potentiation of AMPA-induced toxicity by TNF-alpha involves TNF receptor-1, whereas the neuroprotective effect is mediated by TNF receptor-2. AMPA exposure was associated with activation and proliferation of microglia as assessed by macrophage antigen-1 and bromodeoxyuridine immunohistochemistry, suggesting a functional recruitment of cytokine-producing cells at sites of neurodegeneration. Together, these findings are relevant for understanding the role of proinflammatory cytokines and microglia activation in acute and chronic excitotoxic conditions.

  18. Spatial performance in a complex maze is associated with persistent long-term potentiation enhancement in mouse hippocampal slices at early training stages.

    PubMed

    Lange-Asschenfeldt, C; Lohmann, P; Riepe, M W

    2007-06-29

    Long-term potentiation (LTP) and long-term depression (LTD) are principal reflections of synaptic plasticity that have been implicated in learning and memory. We have previously shown that spatial learning in a newly validated complex maze is accompanied by depression of hippocampal CA1 synaptic activity in hippocampal slices of trained mice ("behavioral LTD"). In the present study, we investigated whether behavioral LTD is accompanied by alterations of subsequent LTP induced by high-frequency stimulation (HFS). Moreover, we were interested in the time course of such alterations in relation to training stage. Animals underwent 1, 2, and 8 days of spatial training in the complex maze, respectively. Hippocampal slices were taken 24 h after the last training session. We found a simultaneous decrease of basal synaptic response and increase of HFS induced LTP magnitude compared with slices of untrained animals. Synaptic plasticity was not influenced by repeated running wheel exercise in an additional control group without spatial learning. The mentioned alterations occurred already after day 2 of maze exploration parallel to the most pronounced improvement of behavioral performance but did not change thereafter until day 8 despite further learning progress. They were also found when animals were trained for 2 days and kept at rest for a subsequent 6 days. In conclusion, spatial learning may be reflected by distinct and persistent measurable alterations of synaptic plasticity in hippocampal CA1 neurons at early training stages.

  19. Effects of the Aconitum alkaloid mesaconitine in rat hippocampal slices and the involvement of α- and β-adrenoceptors

    PubMed Central

    Ameri, Angela

    1998-01-01

    The effects of mesaconitine, the main alkaloid contained in Aconiti tuber, were investigated by use of extracellular recordings of stimulus-evoked population spikes and field excitatory postsynaptic potentials (e.p.s.ps) in the CA1 region of rat hippocampal slices.At a concentration of 10 nM, mesaconitine evoked excitations, which were manifested as an increase in the amplitude of the orthodromic spike and the appearance of multiple spikes following the first postsynaptic spike, without affecting the magnitude of paired-pulse facilitation. The increase in spike amplitude was persistent and was not reversed by up to 90 min of washout. At concentrations of 30 and 100 nM, the alkaloid produced a biphasic effect, that is an excitation followed by an inhibition without having any effect upon the field e.p.s.p. At concentrations above 100 nM, mesaconitine suppressed the orthodromic population spike and the field e.p.s.p.The excitatory effect was also observed when electrical stimulation was stopped completely during the application of mesaconitine (10 nM) and during the first 15 min of washout.The enhancement of the population spike and the appearance of multiple spikes induced by mesaconitine (10–100 nM) were blocked by pretreatment with the β-adrenoceptor antagonists propranolol (1 μM) and timolol (1 μM), whereas the inhibitory effect was blocked by the α-adrenoceptor antagonists yohimbine (1 μM) and phentolamine (10 μM). However, when the β-adrenoceptor antagonist timolol was added 10 min after the application of mesaconitine, it failed to block the long-lasting enhancement of the spike amplitude and the appearance of multiple population spikes.Application of the selective β-adrenoceptor agonist isoprenaline (500 nM) to the hippocampal slices induced an increase in the amplitude of the orthodromic population spike and elicited 2–3 additional spikes. Mesaconitine (10 nM) did not further potentiate this enhancement of the spike

  20. Fluoride Induces a Volume Reduction in CA1 Hippocampal Slices Via MAP Kinase Pathway Through Volume Regulated Anion Channels

    PubMed Central

    Lee, Jaekwang; Han, Young-Eun; Favorov, Oleg; Tommerdahl, Mark; Whitsel, Barry

    2016-01-01

    Regulation of cell volume is an important aspect of cellular homeostasis during neural activity. This volume regulation is thought to be mediated by activation of specific transporters, aquaporin, and volume regulated anion channels (VRAC). In cultured astrocytes, it was reported that swelling-induced mitogen-activated protein (MAP) kinase activation is required to open VRAC, which are thought to be important in regulatory volume decrease and in the response of CNS to trauma and excitotoxicity. It has been also described that sodium fluoride (NaF), a recognized G-protein activator and protein phosphatase inhibitor, leads to a significant MAP kinase activation in endothelial cells. However, NaF's effect in volume regulation in the brain is not known yet. Here, we investigated the mechanism of NaF-induced volume change in rat and mouse hippocampal slices using intrinsic optical signal (IOS) recording, in which we measured relative changes in intracellular and extracellular volume as changes in light transmittance through brain slices. We found that NaF (1~5 mM) application induced a reduction in light transmittance (decreased volume) in CA1 hippocampus, which was completely reversed by MAP kinase inhibitor U0126 (10 µM). We also observed that NaF-induced volume reduction was blocked by anion channel blockers, suggesting that NaF-induced volume reduction could be mediated by VRAC. Overall, our results propose a novel molecular mechanism of NaF-induced volume reduction via MAP kinase signaling pathway by activation of VRAC. PMID:27122993

  1. Methamphetamine exposure antagonizes N-methyl-D-aspartate receptor-mediated neurotoxicity in organotypic hippocampal slice cultures.

    PubMed

    Smith, Katherine J; Self, Rachel L; Butler, Tracy R; Mullins, Michael M; Ghayoumi, Layla; Holley, Robert C; Littleton, John M; Prendergast, Mark A

    2007-07-09

    Glutamatergic systems have been increasingly recognized as mediators of methamphetamine's (METH) pharmacological effects though little is known about the means by which METH interacts with glutamate receptors. The present studies examined effects of METH (0.1-100 microM) on [3H]MK-801 binding to membranes prepared from adult rat cortex, hippocampus and cerebellum, as well as the neurotoxicity produced by 24-h exposure to N-methyl-D-aspartate (5-10 microM; NMDA) employing organotypic hippocampal slice cultures of neonatal rat. Co-incubation of [3H]MK-801 with METH (0.1-100 microM) did not reduce dextromethorphan (1 mM)-displaceable ligand binding. Exposure of slice cultures to NMDA for 24-h produced increases in uptake of the non-vital fluorescent marker propidium iodide (PI) of 150-500% above control levels, most notably, in the CA1 region pyramidal cell layer. Co-exposure to METH (>1.0 microM) with NMDA (5 microM) reduced PI uptake by approximately 50% in each subregion, though the CA1 pyramidal cell layer was markedly more sensitive to the protective effects of METH exposure. In contrast, METH exposure did not reduce PI uptake stimulated by 24-h exposure to 10 microM NMDA. Co-exposure to the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid (20 microM) prevented toxicity produced by exposure to 5 or 10 microM NMDA. These findings indicate that the pharmacological effects of short-term METH exposure involve inhibition of NMDA receptor-mediated neuronal signaling, not reflective of direct channel inhibition at an MK-801-sensitive site.

  2. Impaired tolerance to repetitive hypoxia in hippocampal slices of Cu,Zn superoxide dismutase transgenic mice.

    PubMed

    Büchner, M; Li, H; Huber, R; Timmler, M; Sehrsam, I; Kasischke, K; Völkel, H; Ludolph, A C; Riepe, M W

    1999-12-03

    Energy metabolism is impaired in the Cu,Zn superoxide dismutase transgenic mouse model of amyotrophic lateral sclerosis. The goal was to investigate tolerance against single and repetitive hypoxia in C57B6SJL-TgN(SOD1-G93A)1GUR mice (G93A mice). Posthypoxic recovery (15 min hypoxia, 45 min recovery) of population spike amplitude in hippocampal region CA1 was 38 +/- 29% (mean +/- SD) in controls and 67 +/- 41% (ns) in G93A mice at day 40. Upon in vivo pretreatment with 20 mg/kg 3-nitropropionate posthypoxic recovery increased to 82 +/- 32% (P < 0.01) in controls and decreased to 35 +/- 33% in G93A mice (P < 0.05 to pretreated controls). Results at day 80 and 110 were similar. We conclude that G93A mice show a long-lasting impairment to sustain repetitive hypoxic episodes whereas tolerance to a single hypoxic episode is comparable to controls.

  3. Tamoxifen mediated estrogen receptor activation protects against early impairment of hippocampal neuron excitability in an oxygen/glucose deprivation brain slice ischemia model

    PubMed Central

    Zhang, Huaqiu; Xie, Minjie; Schools, Gary P.; Feustel, Paul F.; Wang, Wei; Lei, Ting; Kimelberg, Harold K.; Zhou, Min

    2009-01-01

    Pretreatment of ovarectomized rats with estrogen shows long-term protection via activation of the estrogen receptor (ER). However, it remains unknown whether activation of the ER can provide protection against early neuronal damage when given acutely, we simulated ischemic conditions by applying oxygen and glucose deprived (OGD) solution to acute male rat hippocampal slices and examined the neuronal electrophysiological changes. Pyramidal neurons and interneurons showed a time-dependent membrane potential depolarization and reduction in evoked action potential frequency and amplitude over a 10 to 15 minute OGD exposure. These changes were largely suppressed by 10 μM TAM. The TAM effect was neuron-specific as the OGD induced astrocytic membrane potential depolarization was not altered. The TAM effect was mediated through ER activation because it could be simulated by 17β-estradiol and was completely inhibited by the ER inhibitor ICI 182, 780, and is therefore an example of TAM’s selective estrogen receptor modulator (SERM) action. We further show that TAM effects on OGD- induced impairment of neuronal excitability was largely due to activation of neuroprotective BK channels, as the TAM effect was markedly attenuated by the BK channel inhibitor paxilline at10 μM. TAM also significantly reduced the frequency and amplitude of AMPA receptor mediated spontaneous excitatory postsynaptic currents (sEPSCs) in pyramidal neurons which is an early consequence of OGD. Altogether, this study demonstrates that both 17β-estradiol and TAM attenuate neuronal excitability impairment early on in simulated ischemia model via ER activation mediated potentiation of BK K+ channels and reduction in enhanced neuronal AMPA/NMDA receptor-mediated excitotoxicity. PMID:18992727

  4. Tamoxifen mediated estrogen receptor activation protects against early impairment of hippocampal neuron excitability in an oxygen/glucose deprivation brain slice ischemia model.

    PubMed

    Zhang, Huaqiu; Xie, Minjie; Schools, Gary P; Feustel, Paul F; Wang, Wei; Lei, Ting; Kimelberg, Harold K; Zhou, Min

    2009-01-09

    Pretreatment of ovarectomized rats with estrogen shows long-term protection via activation of the estrogen receptor (ER). However, it remains unknown whether activation of the ER can provide protection against early neuronal damage when given acutely. We simulated ischemic conditions by applying oxygen and glucose deprived (OGD) solution to acute male rat hippocampal slices and examined the neuronal electrophysiological changes. Pyramidal neurons and interneurons showed a time-dependent membrane potential depolarization and reduction in evoked action potential frequency and amplitude over a 10 to 15 min OGD exposure. These changes were largely suppressed by 10 microM TAM. The TAM effect was neuron-specific as the OGD-induced astrocytic membrane potential depolarization was not altered. The TAM effect was mediated through ER activation because it could be simulated by 17beta-estradiol and was completely inhibited by the ER inhibitor ICI 182, 780, and is therefore an example of TAM's selective estrogen receptor modulator (SERM) action. We further show that TAM's effects on OGD-induced impairment of neuronal excitability was largely due to activation of neuroprotective BK channels, as the TAM effect was markedly attenuated by the BK channel inhibitor paxilline at 10 microM. TAM also significantly reduced the frequency and amplitude of AMPA receptor mediated spontaneous excitatory postsynaptic currents (sEPSCs) in pyramidal neurons which is an early consequence of OGD. Altogether, this study demonstrates that both 17beta-estradiol and TAM attenuate neuronal excitability impairment early on in a simulated ischemia model via ER activation mediated potentiation of BK K(+) channels and reduction in enhanced neuronal AMPA/NMDA receptor-mediated excitotoxicity.

  5. Acute ethanol suppresses glutamatergic neurotransmission through endocannabinoids in hippocampal neurons.

    PubMed

    Basavarajappa, Balapal S; Ninan, Ipe; Arancio, Ottavio

    2008-11-01

    Ethanol exposure during fetal development is a leading cause of long-term cognitive impairments. Studies suggest that ethanol exposure have deleterious effects on the hippocampus, a brain region that is important for learning and memory. Ethanol exerts its effects, in part, via alterations in glutamatergic neurotransmission, which is critical for the maturation of neuronal circuits during development. The current literature strongly supports the growing evidence that ethanol inhibits glutamate release in the neonatal CA1 hippocampal region. However, the exact molecular mechanism responsible for this effect is not well understood. In this study, we show that ethanol enhances endocannabinoid (EC) levels in cultured hippocampal neurons, possibly through calcium pathways. Acute ethanol depresses miniature post-synaptic current (mEPSC) frequencies without affecting their amplitude. This suggests that ethanol inhibits glutamate release. The CB1 receptors (CB1Rs) present on pre-synaptic neurons are not altered by acute ethanol. The CB1R antagonist SR 141716A reverses ethanol-induced depression of mEPSC frequency. Drugs that are known to enhance the in vivo function of ECs occlude ethanol effects on mEPSC frequency. Chelation of post-synaptic calcium by EGTA antagonizes ethanol-induced depression of mEPSC frequency. The activation of CB1R with the selective agonist WIN55,212-2 also suppresses the mEPSC frequency. This WIN55,212-2 effect is similar to the ethanol effects and is reversed by SR141716A. In addition, tetani-induced excitatory post-synaptic currents (EPSCs) are depressed by acute ethanol. SR141716A significantly reverses ethanol effects on evoked EPSC amplitude in a dual recording preparation. These observations, taken together, suggest the participation of ECs as retrograde messengers in the ethanol-induced depression of synaptic activities.

  6. An enzyme-entrapped agarose gel for visualization of ischemia-induced L-glutamate fluxes in hippocampal slices in a flow system.

    PubMed

    Tanaka, Kazuhisa; Shoji, Atushi; Sugawara, Masao

    2015-01-01

    An agarose gel slip containing L-glutamate oxidase (GluOx), horseradish peroxidase (HRP) and a dye DA-64 is proposed as a tool for visualizing ischemia-induced L-glutamate release in hippocampal slices in a flow system. The agarose slip with a detection limit of 6.0 ± 0.8 μmol L(-1) for L-glutamate enabled us to visualize L-glutamate fluxes in a flow system. The leak of a dye from the agarose gel was negligible and a diffusion blur due to spreading of Bindshedler's Green (BG) within the gel was suppressed. Monitoring the time-dependent change of ischemia-induced L-glutamate fluxes at neuronal regions CA1, DG and CA3 of hippocampal slices is demonstrated.

  7. Neuroprotection Promoted by Guanosine Depends on Glutamine Synthetase and Glutamate Transporters Activity in Hippocampal Slices Subjected to Oxygen/Glucose Deprivation.

    PubMed

    Dal-Cim, Tharine; Martins, Wagner C; Thomaz, Daniel T; Coelho, Victor; Poluceno, Gabriela Godoy; Lanznaster, Débora; Vandresen-Filho, Samuel; Tasca, Carla I

    2016-05-01

    Guanosine (GUO) has been shown to act as a neuroprotective agent against glutamatergic excitotoxicity by increasing glutamate uptake and decreasing its release. In this study, a putative effect of GUO action on glutamate transporters activity modulation was assessed in hippocampal slices subjected to oxygen and glucose deprivation (OGD), an in vitro model of brain ischemia. Slices subjected to OGD showed increased excitatory amino acids release (measured by D-[(3)H]aspartate release) that was prevented in the presence of GUO (100 µM). The glutamate transporter blockers, DL-TBOA (10 µM), DHK (100 µM, selective inhibitor of GLT-1), and sulfasalazine (SAS, 250 µM, Xc(-) system inhibitor) decreased OGD-induced D-aspartate release. Interestingly, DHK or DL-TBOA blocked the decrease in glutamate release induced by GUO, whereas SAS did not modify the GUO effect. GUO protected hippocampal slices from cellular damage by modulation of glutamate transporters, however selective blockade of GLT-1 or Xc- system only did not affect this protective action of GUO. OGD decreased hippocampal glutamine synthetase (GS) activity and GUO recovered GS activity to control levels without altering the kinetic parameters of GS activity, thus suggesting GUO does not directly interact with GS. Additionally, the pharmacological inhibition of GS activity with methionine sulfoximine abolished the effect of GUO in reducing D-aspartate release and cellular damage evoked by OGD. Altogether, results in hippocampal slices subjected to OGD show that GUO counteracts the release of excitatory amino acids, stimulates the activity of GS, and decreases the cellular damage by modulation of glutamate transporters activity.

  8. Brain-derived neurotrophic factor, but not neurotrophin-3, prevents ischaemia-induced neuronal cell death in organotypic rat hippocampal slice cultures.

    PubMed

    Pringle, A K; Sundstrom, L E; Wilde, G J; Williams, L R; Iannotti, F

    1996-06-28

    We have investigated the neuroprotective actions of neurotrophins in a model of ischaemia using slice cultures. Ischaemia was induced in organotypic hippocampal cultures by simultaneous oxygen and glucose deprivation. Cell death was assessed 24 h later by propidium iodide fluorescence. Pre- but not post-ischaemic addition of brain-derived neurotrophic factor (BDNF) produced a concentration-dependent reduction in neuronal damage. Neurotrophin-3 was not neuroprotective. These data suggest that BDNF may form part of an endogenous neuroprotective mechanism.

  9. Tolerance of guinea pig hippocampal slice CA1 neurons to hyperthermia evaluated by orthodromic and antidromic responses.

    PubMed

    Fujii, S

    1998-01-01

    The tolerance of electrical responses in the CA1 neurons of guinea pig hippocampal slices to elevated temperatures was studied by recording orthodromic and antidromic responses of the population spike (PS). Increasing the temperature of the perfusing medium from 30 degrees C to 49 degrees C resulted in a decreased amplitude of both the orthodromic and antidromic PS, the former disappearing at 42.0 +/- 1.8 degrees C and the latter at 46.2 +/- 1.3 degrees C (n = 8 for both). When the temperature was increased to 44 degrees C, maintained at this level for less than 27 min, then lowered to 30 degrees C, both the orthodromic and antidromic PS recovered within 60 min. When the temperature was increased to 45-49 degrees C, marked irreversible effects were seen with the orthodromic PS, recovery being dependent on the maximum temperature and duration of exposure, the change becoming irreversible after 13 min at 45 degrees C, 6 min at 46 degrees C, 4 min at 47 degrees C or 2.5 min at 48 degrees C. In contrast, the antidromic PS, recorded simultaneously, recovered on lowering the temperature to 30 degrees C in all cases tested, except when the temperature was increased to 46 degrees C and maintained at this level for 25-27 min. These results indicate that, in CA1 neurons, temperatures above 44 degrees C have more potent irreversible effects on synaptic transmission than on axonal or somal function.

  10. Metaplastic LTP inhibition after LTD induction in CA1 hippocampal slices involves NMDA Receptor-mediated Neurosteroidogenesis

    PubMed Central

    Izumi, Yukitoshi; O'Dell, Kazuko A; Zorumski, Charles F

    2013-01-01

    Long-term depression (LTD) induced by low-frequency electrical stimulation (LFS) in the CA1 region of the hippocampus is a form of synaptic plasticity thought to contribute to learning and memory and to the pathophysiology of neuropsychiatric disorders. In naïve hippocampal slices from juvenile rats, we previously found that LTD induction can impair subsequent induction of long-term potentiation (LTP) via a form of N-methyl-d-aspartate receptor (NMDAR)-dependent metaplasticity, and have recently observed that pharmacologically induced NMDAR-dependent LTP inhibition involves 5α-reduced neurosteroids that augment the actions of γ-aminobutyric acid (GABA). In this study, we found that both LFS-induced LTD and subsequent inhibition of LTP induction involve neurosteroid synthesis via NMDAR activation. Furthermore, the timing of 5α-reductase inhibition relative to LFS can dissociate effects on LTD and metaplastic LTP inhibition. These findings indicate that 5α-reduced neurosteroids play an important role in synaptic plasticity and synaptic modulation in the hippocampus. PMID:24303196

  11. Transient depression of excitatory synapses on interneurons contributes to epileptiform bursts during gamma oscillations in the mouse hippocampal slice.

    PubMed

    Traub, Roger D; Pais, Isabel; Bibbig, Andrea; Lebeau, Fiona E N; Buhl, Eberhard H; Garner, Helen; Monyer, Hannah; Whittington, Miles A

    2005-08-01

    Persistent gamma frequency (30-70 Hz) network oscillations occur in hippocampal slices under conditions of metabotropic glutamate receptor (mGluR) activation. Excessive mGluR activation generated a bistable pattern of network activity during which epochs of gamma oscillations of increasing amplitude were terminated by synchronized bursts and very fast oscillations (>70 Hz). We provide experimental evidence that, during this behavior, pyramidal cell-to-interneuron synaptic depression takes place, occurring spontaneously during the gamma rhythm and associated with the onset of epileptiform bursts. We further provide evidence that excitatory postsynaptic potentials (EPSPs) in pyramidal cells are potentiated during the interburst gamma oscillation. When these two types of synaptic plasticity are incorporated, phenomenologically, into a network model previously shown to account for many features of persistent gamma oscillations, we find that epochs of gamma do indeed alternate with epochs of very fast oscillations and epileptiform bursts. Thus the same neuronal network can generate either gamma oscillations or epileptiform bursts, in a manner depending on the degree of network drive and network-induced fluctuations in synaptic efficacies.

  12. Neuroprotective effects of α-tocotrienol on kainic acid-induced neurotoxicity in organotypic hippocampal slice cultures.

    PubMed

    Jung, Na Young; Lee, Kyung Hee; Won, Ran; Lee, Bae Hwan

    2013-09-05

    Vitamin E, such as alpha-tocopherol (ATPH) and alpha-tocotrienol (ATTN), is a chain-breaking antioxidant that prevents the chain propagation step during lipid peroxidation. In the present study, we investigated the effects of ATTN on KA-induced neuronal death using organotypic hippocampal slice culture (OHSC) and compared the neuroprotective effects of ATTN and ATPH. After 15 h KA (5 µM) treatment, delayed neuronal death was detected in the CA3 region and reactive oxygen species (ROS) formation and lipid peroxidation were also increased. Both co-treatment and post-treatment of ATPH (100 µM) or ATTN (100 µM) significantly increased the cell survival and reduced the number of TUNEL-positive cells in the CA3 region. Increased dichlorofluorescein (DCF) fluorescence and levels of thiobarbiturate reactive substances (TBARS) were decreased by ATPH and ATTN treatment. These data suggest that ATPH and ATTN treatment have protective effects on KA-induced cell death in OHSC. ATTN treatment tended to be more effective than ATPH treatment, even though there was no significant difference between ATPH and ATTN in co-treatment or post-treatment.

  13. Adenosine triphosphate depletion reverses sodium-dependent, neuronal uptake of glutamate in rat hippocampal slices.

    PubMed

    Madl, J E; Burgesser, K

    1993-10-01

    Extracellular accumulations of excitatory amino acids (EAAs) may mediate ischemic neuronal damage. Metabolic insults can decrease Na+ and K+ plasma membrane gradients, thereby reducing the driving force for uptake of EAAs into cells by Na(+)-dependent EAA cotransporters. EAA accumulations could result from decreased uptake and increased release due to reversal of these cotransporters. ATP depletion, uptake, and release of EAAs were measured by HPLC in slices treated with metabolic inhibitors. Inhibition and reversal of cotransporters were determined by uptake or release of D,L-threo-beta-hydroxyaspartate (OH-Asp), an EAA analog with high affinity for cotransporters. Moderate ATP depletion (7 > ATP nmol/mg protein > 3) reduced uptake by cotransporters without increasing release of EAAs. When ATP was severely depleted (ATP < 2 nmol/mg protein), increased release of EAAs and preloaded OH-Asp occurred, consistent with reversal of cotransporters. Release of glutamine and asparagine was not increased, confirming that release was not primarily due to nonselective increased membrane permeability. ATP depletion and ouabain acted synergistically to produce EAA release, strongly suggesting release was largely mediated by inhibition of Na/K-ATPases. Severe ATP depletion decreased glutamate-like immunoreactivity primarily in axonal terminal-like structures, suggesting release occurred primarily from terminals. Moderate ATP depletion may increase extracellular EAAs by decreasing uptake. Severe ATP depletion may further increase EAAs by reversing uptake, thereby releasing cytosolic neuronal pools of EAAs.

  14. Electrical coupling of astrocytes in rat hippocampal slices under physiological and simulated ischemic conditions.

    PubMed

    Xu, Guangjin; Wang, Wei; Kimelberg, Harold K; Zhou, Min

    2010-03-01

    Mammalian protoplasmic astrocytes are extensively coupled through gap junction channels but the biophysical properties of these channels under physiological and ischemic conditions in situ are not well defined. Using confocal morphometric analysis of biocytin-filled astrocytic syncytia in rat hippocampal CA1 stratum radiatum we found that each astrocyte directly couples, on average, to 11 other astrocytes with a mean interastrocytic distance of 45 microm. Voltage-independent and bidirectional transjunctional currents were always measured between directly coupled astrocyte pairs in dual voltage-clamp recordings, but never from astrocyte-NG2 glia or astrocyte-interneuron pairs. The electrical coupling ratio varied considerably among astrocytes in developing postnatal day 14 rats (P14, 0.5-12.4%, mean = 3.6%), but became more constant in young adult P21 rats (0.18-3.9%, mean = 1.6%), and the coupling ratio declined exponentially with increasing pair distance. Electrical coupling was not affected by short-term oxygen-glucose deprivation (OGD) treatment, but showed delayed inhibition in an acidic extracellular pH of 6.4. Combination of acidic pH (6.4) and OGD, a condition that better represents cerebral ischemia in vivo, accelerated the inhibition of electrical coupling. Our results show that, under physiological conditions, 20.7-24.2% of K(+) induced currents can travel from any astrocytic soma in CA1 stratum radiatum to the gap junctions of the nearest neighbor astrocytes, but this should be severely inhibited as a consequence of the OGD and acidosis seen in the ischemic brain.

  15. How to record a million synaptic weights in a hippocampal slice.

    PubMed

    Bhalla, Upinder S

    2008-06-20

    A key step toward understanding the function of a brain circuit is to find its wiring diagram. New methods for optical stimulation and optical recording of neurons make it possible to map circuit connectivity on a very large scale. However, single synapses produce small responses that are difficult to measure on a large scale. Here I analyze how single synaptic responses may be detectable using relatively coarse readouts such as optical recording of somatic calcium. I model a network consisting of 10,000 input axons and 100 CA1 pyramidal neurons, each represented using 19 compartments with voltage-gated channels and calcium dynamics. As single synaptic inputs cannot produce a measurable somatic calcium response, I stimulate many inputs as a baseline to elicit somatic action potentials leading to a strong calcium signal. I compare statistics of responses with or without a single axonal input riding on this baseline. Through simulations I show that a single additional input shifts the distribution of the number of output action potentials. Stochastic resonance due to probabilistic synaptic release makes this shift easier to detect. With approximately 80 stimulus repetitions this approach can resolve up to 35% of individual activated synapses even in the presence of 20% recording noise. While the technique is applicable using conventional electrical stimulation and extracellular recording, optical methods promise much greater scaling, since the number of synapses scales as the product of the number of inputs and outputs. I extrapolate from current high-speed optical stimulation and recording methods, and show that this approach may scale up to the order of a million synapses in a single two-hour slice-recording experiment.

  16. Activation of dentate hilar neurons by stimulation of the fimbria in rat hippocampal slices

    PubMed Central

    Scharfman, Helen E.

    2012-01-01

    It is has been shown that the major afferent input to the dentate gyrus, the perforant path, excites dentate hilar neurons. However, little is known about the other inputs to hilar cells. Therefore, we examined the responses of hilar neurons to stimulation of the fimbria. We positioned our stimulating electrodes so that granule cells were not excited antidromically by fimbria stimulation, although action potentials were easily triggered in area CA3b and CA3c pyramidal cells by such stimulation. In these experiments, fimbria stimulation evoked responses from every hilar cell tested, including examples of both of the major cell types, the spiny hilar ‘mossy’ cells (n=15) and the relatively aspiny. ‘fast-spiking’ cells (putative interneurons, n=5). Hilar cell responses consisted primarily of EPSPs that could trigger action potentials, but small IPSPs were also evoked in some cases, particularly in the fast-spiking cells. Excitation was blocked by an antagonist of the AMPA/kainate receptor subtype of excitatory amino acid receptors, 6-cyano-7-nitroquinoxaline-2,3-dione(CNQX, 5μM, n=5), whereas the cholinergic antagonist atropine (10μM) had no effect (n=4). When sequential intracellular recordings were made from hilar cells and area CA3 pyramidal cells in the same slice, hilar cell EPSPs began after action potentials of CA3b pyramidal cells, and stimulus strengths required to evoke hilar cell EPSPs were above threshold for area CA3b pyramidal cells. Taken together with the evidence that area CA3 pyramidal cells use an excitatory amino acid as a neurotransmitter [7, 21], and the demonstrations of area CA3 axon collaterals in the hilus [11, 16], the results raise the possibility that some area CA3 pyramidal cells excite dentate hilar neurons. PMID:8105429

  17. Actions of cromakalim on outward currents of CA1 neurones in hippocampal slices.

    PubMed Central

    Erdemli, G; Krnjević, K

    1994-01-01

    1. Membrane effects of cromakalim (Crom; 50-300 microM) were examined in CA1 neurones recorded mainly by intracellular, single-electrode voltage-clamping in slices (from Sprague-Dawley rats) kept in an interface chamber at 33 degrees C. 2. In 14 cells held at -63 +/- 3.5 mV, in the presence of tetrodotoxin, kynurenic acid and (in most cases) bicuculline, bath applied Crom produced no consistent change in holding current (-59 +/- 66 pA) or input conductance (GN) (-3.9 +/- 5.2%). 3. Overall there were no significant changes in instantaneous inward rectification or in Q-current inward relaxations. 4. In 18 out of 22 cells, outward currents, evoked by 0.5 s pulses to voltages > -50 and < -20 mV, were depressed by Crom (by 42 +/- 11%, for n = 22). Because this effect was consistently seen in Ca current-blocking media, containing either Mn and low Ca, or Cd (and also carbachol), the K channels depressed by Crom were probably of the delayed rectifier (IDR) type. 5. The Crom-control difference current (ICrom), obtained with slow depolarizing ramps, had a biphasic character, inward in the voltage (V) range > -50 < -20 mV (where outward currents are depressed by Crom) and tending outward for V > or = -20 mV. 6. In 10 out of 11 cells, Crom potentiated a D-like, slowly-inactivating outward current (by 88 +/- 31%, for n = 11).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7530570

  18. Weak Sinusoidal Electric Fields Entrain Spontaneous Ca Transients in the Dendritic Tufts of CA1 Pyramidal Cells in Rat Hippocampal Slice Preparations

    PubMed Central

    Maeda, Kazuma; Maruyama, Ryuichi; Nagae, Toru; Inoue, Masashi; Aonishi, Toru; Miyakawa, Hiroyoshi

    2015-01-01

    Neurons might interact via electric fields and this notion has been referred to as ephaptic interaction. It has been shown that various types of ion channels are distributed along the dendrites and are capable of supporting generation of dendritic spikes. We hypothesized that generation of dendritic spikes play important roles in the ephaptic interactions either by amplifying the impact of electric fields or by providing current source to generate electric fields. To test if dendritic activities can be modulated by electric fields, we developed a method to monitor local Ca-transients in the dendrites of a neuronal population in acute rat hippocampal slices by applying spinning-disk confocal microscopy and multi-cell dye loading technique. In a condition in which the dendrites of CA1 pyramidal neurons show spontaneous Ca-transients due to added 50 μM 4-aminopyridine to the bathing medium and adjusted extracellular potassium concentration, we examined the impact of sinusoidal electric fields on the Ca-transients. We have found that spontaneously occurring fast-Ca-transients in the tufts of the apical dendrites of CA1 pyramidal neurons can be blocked by applying 1 μM tetrodotoxin, and that the timing of the transients become entrained to sub-threshold 1-4 Hz electric fields with an intensity as weak as 0.84 mV/mm applied parallel to the somato-dendritic axis of the neurons. The extent of entrainment increases with intensity below 5 mV/mm, but does not increase further over the range of 5-20 mV/mm. These results suggest that population of pyramidal cells might be able to detect electric fields with biologically relevant intensity by modulating the timing of dendritic spikes. PMID:25811836

  19. Mitochondrial calcium ion and membrane potential transients follow the pattern of epileptiform discharges in hippocampal slice cultures.

    PubMed

    Kovács, Richard; Kardos, Julianna; Heinemann, Uwe; Kann, Oliver

    2005-04-27

    Emerging evidence suggests that mitochondrial dysfunction contributes to the pathophysiology of epilepsy. Recurrent mitochondrial Ca2+ ion load during seizures might act on mitochondrial membrane potential (DeltaPsim) and proton motive force. By using electrophysiology and confocal laser-scanning microscopy, we investigated the effects of epileptiform activity, as induced by low-Mg2+ ion perfusion in hippocampal slice cultures, on changes in DeltaPsim and in mitochondrial Ca2+ ion concentration ([Ca2+]m). The mitochondrial compartment was identified by monitoring DeltaPsim in the soma and dendrites of patched CA3 pyramidal cells using the mitochondria-specific voltage-sensitive dye rhodamine-123 (Rh-123). Interictal activity was accompanied by localized mitochondrial depolarization that was restricted to a few mitochondria in small dendrites. In contrast, robust Rh-123 release into the cytosol was observed during seizure-like events (SLEs), indicating simultaneous depolarization of mitochondria. This was critically dependent on Ca2+ ion uptake and extrusion, because inhibition of the mitochondrial Ca2+ ion uniporter by Ru360 and the mitochondrial Na+/Ca2+ ion exchanger by 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one but not the inhibitor of mitochondrial permeability transition pore, cyclosporin A, decreased the SLE-associated mitochondrial depolarization. The Ca2+ ion dependence of simultaneous mitochondrial depolarization suggested enhanced Ca2+ ion cycling across mitochondrial membranes during epileptiform activity. Indeed, [Ca2+]m fluctuated during interictal activity in single dendrites, and these fluctuations spread over the entire mitochondrial compartment during SLEs, as revealed using mitochondria-specific dyes (rhod-2 and rhod-ff) and spatial frequency-based image analysis. These findings strengthen the hypothesis that epileptic activity results in Ca2+ ion-dependent changes in mitochondrial function that might contribute to the

  20. Kinetics of activity-evoked pH transients and extracellular pH buffering in rat hippocampal slices.

    PubMed

    Tong, Chi-Kun; Chen, Kevin; Chesler, Mitchell

    2006-06-01

    The kinetics of activity-dependent, extracellular alkaline transients, and the buffering of extracellular pH (pH(e)), were studied in rat hippocampal slices using a fluorescein-dextran probe. Orthodromic stimuli generated alkaline transients < or = 0.05 pH units that peaked in 273 +/- 26 ms and decayed with a half-time of 508 +/- 43 ms. Inhibition of extracellular carbonic anhydrase (ECA) with benzolamide increased the rate of rise by 25%, doubled peak amplitude, and prolonged the decay three- to fourfold. The slow decay in benzolamide allowed marked temporal summation, resulting in a severalfold increase in amplitude during long stimulus trains. Addition of exogenous carbonic anhydrase reduced the rate of rise, halved the peak amplitude, but had no effect on the normalized decay. A simulation of extracellular buffering kinetics generated recoveries from a base load consistent with the observed decay of the alkaline transient in the presence of benzolamide. Under control conditions, the model approximated the observed decays with an acceleration of the CO2 hydration-dehydration reactions by a factor of 2.5. These data suggest low endogenous ECA activity, insufficient to maintain equilibrium during the alkaline transients. Disequilibrium implies a time-dependent buffering capacity, with a CO2/HCO3- contribution that is small shortly after a base load. It is suggested that within 100 ms, extracellular buffering capacity is about 1% of the value at equilibrium and is provided mainly by phosphate. Accordingly, in the time frame of synaptic transmission, small base loads would generate relatively large changes in interstitial pH.

  1. Nodule excitability in an animal model of periventricular nodular heterotopia: c-fos activation in organotypic hippocampal slices

    PubMed Central

    Doisy, Emily T.; Wenzel, H. Jürgen; Mu, Yi; Nguyen, Danh V.; Schwartzkroin, Philip A.

    2015-01-01

    Objective Aberrations in brain development may lead to dysplasic structures such as periventricular nodules. While these abnormal collections of neurons are often associated with difficult-to-control seizure activity, there is little consensus regarding the epileptogenicity of the nodules themselves. Since one common treatment option is surgical resection of suspected epileptic nodules, it is important to determine whether these structures in fact give rise, or essentially contribute, to epileptic activities. Methods To study the excitability of aberrant nodules, we have examined c-fos activation in organotypic hippocampal slice cultures generated from an animal model of periventricular nodular heterotopia created by treating pregnant rats with methylazoxymethanol. Using this preparation, we have also attempted to assess tissue excitability when the nodule is surgically removed from the culture. We then compared c-fos activation in this in vitro preparation to c-fos activation generated in an intact rat treated with kainic acid. Results Quantitative analysis of c-fos activation failed to show enhanced nodule excitability compared to neocortex or CA1 hippocampus. However, when we compared cultures with and without a nodule, presence of a nodule did affect the excitability of CA1 and cortex, at least as reflected in c-fos labeling. Surgical removal of the nodule did not result in a consistent decrease in excitability as reflected in the c-fos biomarker. Significance Our results from the organotypic culture were generally consistent with our observations on excitability in the intact rat – as seen not only with c-fos but also in previous electrophysiological studies. At least in this model, the nodule does not appear to be responsible for enhanced excitability (or, presumably, seizure initiation). Excitability is different in tissue that contains a nodule, suggesting altered network function, perhaps reflecting the abnormal developmental pattern that gave rise to

  2. Controlled pulse delivery of electrical stimulation differentially reduces epileptiform activity in Mg2+-free-treated hippocampal slices.

    PubMed

    Albensi, Benedict C; Toupin, Justin D; Oikawa, Kensuke; Oliver, Derek R

    2008-08-21

    Electrical stimulation for applications in epilepsy has been attempted in multiple brain regions [corrected] using high- or low-frequency stimulation protocols. Data suggest that specific frequencies may have more benefit at controlling seizure activity. To this end, investigators have tested low-frequency stimulation (LFS) protocols (0.1 to 25 Hz) in both animal models and in human epileptic patients and reported reduced epileptiform synchronization, afterdischarge thresholds, and seizure activity in general. Collectively, these studies imply that LFS may have benefit in reducing epileptiform activity, however, the effectiveness of various electrical parameters still needs to be determined in specific targets. This study aimed to systematically control the total number of stimulation pulses when using primarily LFS protocols (0.5, 0.75, 1, 2, 5, 10, and 25 Hz) delivered for the suppression of seizure-like activity in the hippocampal brain slice using a Mg2+-free model of epilepsy. Fifty Hz was also tested as a reference higher frequency protocol. Regulating the total number of pulses also controlled the amount of electrical work delivered. Of the LFS protocols tested, 0.5 Hz, and 1 Hz were optimal and significantly (p<0.05) reduced several measures of epileptiform activity. However, the higher frequency protocol, 50 Hz was similarly effective at significantly (p < 0.05) suppressing several aspects of epileptiform activity (but not for reduction of population-spike amplitude). The data show that these protocols, which had a controlled number of pulses differentially reduced epileptiform activity in our model where increasing the frequency of stimulation did not result in increased attenuation.

  3. Electrogenic uptake contributes a major component of the depolarizing action of L-glutamate in rat hippocampal slices.

    PubMed Central

    Frenguelli, B. G.; Blake, J. F.; Brown, M. W.; Collingridge, G. L.

    1991-01-01

    1. A grease-gap technique has been used to measure d.c. potentials, in response to the application of excitatory amino acids and electrical stimulation of the Schaffer collateral-commissural pathway, in the CA1 region of rat hippocampal slices. The actions of L-glutamate (L-Glu) have been quantified and compared to those of structurally related compounds. 2. Perfusion of L-Glu (90s applications) depolarized the tissue with a threshold of approximately 50 microM and a maximum response in excess of 10 mM. L-Aspartate (L-Asp) produced a similar dose-response relationship. By comparison N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) were more potent excitants, producing dose-dependent depolarizations over the range 2-50 microM. 3. Application of the agonists depressed the amplitude of electrically-evoked synaptic responses; an effect that presumably reflects depolarization of neuronal tissue. However, for a given agonist-induced d.c. potential. L-Glu or L-Asp caused smaller depressions of synaptic responses than did either NMDA or AMPA. 4. The combined application of 50 microM D-2-amino-5-phosphonopentanoate (AP5) and 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) substantially depressed synaptic responses and antagonized responses to NMDA and AMPA producing mean (+/- s.e.) dose-ratios of 12.2 +/- 1.2 and 7.0 +/- 0.8, respectively. However, these compounds produced minimal antagonism of responses to L-Glu and L-Asp (dose-ratios of 1.5 +/- 0.1 and 1.5 +/- 0.2, respectively). 5. Responses to the stereoisomers of homocysteate (HCA) were compared over the range 50 microM to 10 mM.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1673070

  4. Specificity of exogenous acetate and glutamate as astrocyte substrates examined in acute brain slices from female mice using methionine sulfoximine (MSO) to inhibit glutamine synthesis.

    PubMed

    Andersen, Jens Velde; McNair, Laura Frendrup; Schousboe, Arne; Waagepetersen, Helle Sønderby

    2017-02-28

    Removal of endogenously released glutamate is mediated primarily by astrocytes and exogenous (13) C-labeled glutamate has been applied to study glutamate metabolism in astrocytes. Likewise, studies have clearly established the relevance of (13) C-labeled acetate as an astrocyte specific metabolic substrate. Recent studies have, however, challenged the arguments used to anchor this astrocyte specificity of acetate and glutamate. The aim of the current study was to evaluate the specificity of acetate and glutamate as astrocyte substrates in brain slices. Acutely isolated hippocampal and cerebral cortical slices from female NMRI mice were incubated in media containing [1,2-(13) C]acetate or [U-(13) C]glutamate, with or without methionine sulfoximine (MSO) to inhibit glutamine synthetase (GS). Tissue extracts were analyzed by gas chromatography-mass spectrometry. Blocking GS abolished the majority of glutamine (13) C-labeling from [1,2-(13) C]acetate as intended. However, (13) C-labeling of GABA was only 40-50% reduced by MSO, suggesting considerable neuronal uptake of acetate. Moreover, labeling of glutamate from [1,2-(13) C]acetate in the presence of MSO exceeded the level probable from exclusive labeling of the astrocytic pool, which likewise suggests neuronal acetate metabolism. Approximately 50% of glutamate was uniformly labeled in slices incubated with [U-(13) C]glutamate in the presence of MSO, suggesting that neurons exhibit substantial uptake of exogenously provided glutamate. © 2017 Wiley Periodicals, Inc.

  5. Acute Alterations of Somatodendritic Action Potential Dynamics in Hippocampal CA1 Pyramidal Cells after Kainate-Induced Status Epilepticus in Mice

    PubMed Central

    Minge, Daniel; Bähring, Robert

    2011-01-01

    Pathophysiological remodeling processes at an early stage of an acquired epilepsy are critical but not well understood. Therefore, we examined acute changes in action potential (AP) dynamics immediately following status epilepticus (SE) in mice. SE was induced by intraperitoneal (i.p.) injection of kainate, and behavioral manifestation of SE was monitored for 3–4 h. After this time interval CA1 pyramidal cells were studied ex vivo with whole-cell current-clamp and Ca2+ imaging techniques in a hippocampal slice preparation. Following acute SE both resting potential and firing threshold were modestly depolarized (2–5 mV). No changes were seen in input resistance or membrane time constant, but AP latency was prolonged and AP upstroke velocity reduced following acute SE. All cells showed an increase in AP halfwidth and regular (rather than burst) firing, and in a fraction of cells the notch, typically preceding spike afterdepolarization (ADP), was absent following acute SE. Notably, the typical attenuation of backpropagating action potential (b-AP)-induced Ca2+ signals along the apical dendrite was strengthened following acute SE. The effects of acute SE on the retrograde spread of excitation were mimicked by applying the Kv4 current potentiating drug NS5806. Our data unveil a reduced somatodendritic excitability in hippocampal CA1 pyramidal cells immediately after acute SE with a possible involvement of both Na+ and K+ current components. PMID:22039527

  6. β-Adrenoceptor activation depresses brain inflammation and is neuroprotective in lipopolysaccharide-induced sensitization to oxygen-glucose deprivation in organotypic hippocampal slices

    PubMed Central

    2010-01-01

    Background Inflammation acting in synergy with brain ischemia aggravates perinatal ischemic brain damage. The sensitizing effect of pro-inflammatory exposure prior to hypoxia is dependent on signaling by TNF-α through TNF receptor (TNFR) 1. Adrenoceptor (AR) activation is known to modulate the immune response and synaptic transmission. The possible protective effect of α˜ and β˜AR activation against neuronal damage caused by tissue ischemia and inflammation, acting in concert, was evaluated in murine hippocampal organotypic slices treated with lipopolysaccharide (LPS) and subsequently subjected to oxygen-glucose deprivation (OGD). Method Hippocampal slices from mice were obtained at P6, and were grown in vitro for 9 days on nitrocellulose membranes. Slices were treated with β1(dobutamine)-, β2(terbutaline)-, α1(phenylephrine)- and α2(clonidine)-AR agonists (5 and 50 μM, respectively) during LPS (1 μg/mL, 24 h) -exposure followed by exposure to OGD (15 min) in a hypoxic chamber. Cell death in the slice CA1 region was assessed by propidium iodide staining of dead cells. Results Exposure to LPS + OGD caused extensive cell death from 4 up to 48 h after reoxygenation. Co-incubation with β1-agonist (50 μM) during LPS exposure before OGD conferred complete protection from cell death (P < 0.001) whereas the β2-agonist (50 μM) was partially protective (p < 0.01). Phenylephrine was weakly protective while no protection was attained by clonidine. Exposure to both β1- and β2-agonist during LPS exposure decreased the levels of secreted TNF-α, IL-6 and monocyte chemoattractant protein-1 and prevented microglia activation in the slices. Dobutamine remained neuroprotective in slices exposed to pure OGD as well as in TNFR1-/- and TNFR2-/- slices exposed to LPS followed by OGD. Conclusions Our data demonstrate that activation of both β1- and β2-receptors is neuroprotective and may offer mechanistic insights valuable for development of neuro-protective strategies

  7. Alteration in NMDA receptor subunit mRNA expression in vulnerable and resistant regions of in vitro ischemic rat hippocampal slices.

    PubMed

    Small, D L; Poulter, M O; Buchan, A M; Morley, P

    1997-08-29

    Brain insults, including cerebral ischemia, can alter glutamate receptor subunit expression in vulnerable neurons. Understanding these post-ischemic changes in glutamate receptors could enhance our ability to identify specific, novel neuroprotective compounds. Reverse transcription-polymerase chain reaction (RT-PCR) amplification was used to quantify the altered expression of the N-methyl-D-aspartate (NMDA) NR2A, NR2B and NR2C subunits relative to one another in rat hippocampal slices in resistant and vulnerable regions following in vitro oxygen-glucose deprivation. Ninety minutes after re-oxygenation and return to 10 mM glucose, there was a significant increase in the expression of NR2C relative to NR2B and NR2A in the slice as a whole, as well as in the selectively vulnerable CA1 region and the resistant CA3 and dentate gyrus regions.

  8. The rostral migratory stream generates hippocampal CA1 pyramidal-like neurons in a novel organotypic slice co-culture model

    PubMed Central

    Singec, Ilyas; Knoth, Rolf; Vida, Imre; Frotscher, Michael

    2015-01-01

    ABSTRACT The mouse subventricular zone (SVZ) generates large numbers of neuroblasts, which migrate in a distinct pathway, the rostral migratory stream (RMS), and replace specific interneurons in the olfactory bulb (OB). Here, we introduce an organotypic slice culture model that directly connects the RMS to the hippocampus as a new destination. RMS neuroblasts widely populate the hippocampus and undergo cellular differentiation. We demonstrate that RMS cells give rise to various neuronal subtypes and, surprisingly, to CA1 pyramidal neurons. Pyramidal neurons are typically generated before birth and are lost in various neurological disorders. Hence, this unique slice culture model enables us to investigate their postnatal genesis under defined in vitro conditions from the RMS, an unanticipated source for hippocampal pyramidal neurons. PMID:26340944

  9. Targeting neurotransmitter receptors with nanoparticles in vivo allows single-molecule tracking in acute brain slices

    NASA Astrophysics Data System (ADS)

    Varela, Juan A.; Dupuis, Julien P.; Etchepare, Laetitia; Espana, Agnès; Cognet, Laurent; Groc, Laurent

    2016-03-01

    Single-molecule imaging has changed the way we understand many biological mechanisms, particularly in neurobiology, by shedding light on intricate molecular events down to the nanoscale. However, current single-molecule studies in neuroscience have been limited to cultured neurons or organotypic slices, leaving as an open question the existence of fast receptor diffusion in intact brain tissue. Here, for the first time, we targeted dopamine receptors in vivo with functionalized quantum dots and were able to perform single-molecule tracking in acute rat brain slices. We propose a novel delocalized and non-inflammatory way of delivering nanoparticles (NPs) in vivo to the brain, which allowed us to label and track genetically engineered surface dopamine receptors in neocortical neurons, revealing inherent behaviour and receptor activity regulations. We thus propose a NP-based platform for single-molecule studies in the living brain, opening new avenues of research in physiological and pathological animal models.

  10. Potassium currents in acutely isolated human hippocampal dentate granule cells.

    PubMed Central

    Beck, H; Clusmann, H; Kral, T; Schramm, J; Heinemann, U; Elger, C E

    1997-01-01

    1. Properties of voltage- and Ca(2+)-dependent K+ currents were investigated in thirty-four dentate granule cells acutely isolated from the resected hippocampus of eleven patients with therapy-refractory temporal lobe epilepsy (TLE). 2. When intracellular Ca2+ was strongly buffered with 11.5 mM EGTA-1 mM Ca2+ in the recording pipette, K+ currents (IK) with a slow activation and biexponential time-dependent decay could be elicited, which showed a threshold for activation around -30 mV. 3. A contribution of Ca(2+)-dependent K+ currents became apparent with intracellular solution containing 1 mM BAPTA-0.1 mM Ca2+. Superfusion of low-Ca2+ extracellular solution blocked 43% of outward currents in this recording configuration. Outward current components could also be blocked by substituting 5 mM Ba2+ for extracellular Ca2+ (78%), or by application of 100 microM Cd2+ (25%). 4. The Ca(2+)-dependent K+ currents could be pharmacologically subdivided into two components. One component was sensitive to 500 microM tetraethylammmonium (TEA; 41%) and 10 nM charybdotoxin (CTX; 47.2%). The blocking effects of 10 nM CTX and 500 microM TEA were not additive, suggesting that both agents block the same conductance. A second, smaller outward current component was blocked by 50 nM apamin (13%). 5. A transient A-type K+ current could be observed in six neurones and showed a fast monoexponential time-dependent inactivation with a steady-state voltage dependence that was distinct from that of IK. The A-type current was blocked by 4-aminopyridine (4-AP) but not by TEA or low-Ca2+ solution. 6. We conclude that outward currents in human hippocampal dentate granule cells can be separated into at least four types by their kinetic and pharmacological properties. These include at least one voltage-dependent current similar to those observed in mammalian hippocampal neurones, and two Ca(2+)-dependent K+ currents that most probably correspond to SK- and BK-type currents. A classical A-type current

  11. Intrinsic excitability changes induced by acute treatment of hippocampal CA1 pyramidal neurons with exogenous amyloid β peptide.

    PubMed

    Tamagnini, Francesco; Scullion, Sarah; Brown, Jon T; Randall, Andrew D

    2015-07-01

    Accumulation of beta-amyloid (Aβ) peptides in the human brain is a canonical pathological hallmark of Alzheimer's disease (AD). Recent work in Aβ-overexpressing transgenic mice indicates that increased brain Aβ levels can be associated with aberrant epileptiform activity. In line with this, such mice can also exhibit altered intrinsic excitability (IE) of cortical and hippocampal neurons: these observations may relate to the increased prevalence of seizures in AD patients. In this study, we examined what changes in IE are produced in hippocampal CA1 pyramidal cells after 2-5 h treatment with an oligomeric preparation of synthetic human Aβ 1-42 peptide. Whole cell current clamp recordings were compared between Aβ-(500 nM) and vehicle-(DMSO 0.05%) treated hippocampal slices obtained from mice. The soluble Aβ treatment did not produce alterations in sub-threshold intrinsic properties, including membrane potential, input resistance, and hyperpolarization activated "sag". Similarly, no changes were noted in the firing profile evoked by 500 ms square current supra-threshold stimuli. However, Aβ 500 nM treatment resulted in the hyperpolarization of the action potential (AP) threshold. In addition, treatment with Aβ at 500 nM depressed the after-hyperpolarization that followed both a single AP or 50 Hz trains of a number of APs between 5 and 25. These data suggest that acute exposure to soluble Aβ oligomers affects IE properties of CA1 pyramidal neurons differently from outcomes seen in transgenic models of amyloidopathy. However, in both chronic and acute models, the IE changes are toward hyperexcitability, reinforcing the idea that amyloidopathy and increased incidence in seizures might be causally related in AD patients.

  12. Mifepristone (RU486) inhibits lateral perforant path long-term potentiation in hippocampal slices from prenatally morphine-exposed female rats.

    PubMed

    Velísek, Libor; Vathy, Ilona

    2005-11-01

    In brain slices from prenatally saline-exposed female rats during proestrus and diestrus, long-term potentiation (LTP) can be induced in the lateral perforant pathway (LPP). Prenatal morphine exposure suppresses LTP induction in the LPP during proestrus. Here we studied synaptic plasticity in the LPP in slices from female rats prenatally exposed to morphine. Two additional factors were investigated: the role of the estrous cycle and role of glucocorticoid receptors. Hippocampal slices were prepared from adult, prenatally saline- or morphine-exposed female rats. One hour prior to decapitation, vaginal smears were obtained and the rats either in proestrus or diestrus were treated with a non-specific glucocorticoid receptor antagonist mifepristone (RU486) or with a vehicle. LPP was stimulated with high-frequency stimulation. Short-tem plasticity (STP) and the induction and maintenance of long-term potentiation (LTP) were assessed. In all groups of prenatally saline-exposed rats, LTP was induced and maintained with the exception of RU486-treated rats during proestrus where the LTP was induced but not maintained. In prenatally morphine-exposed females in diestrus, both STP and LTP were induced after postnatal vehicle treatment. In morphine-exposed, proestrous females, neither STP nor LTP were induced irrespective of the postnatal treatment. Thus, prenatal morphine exposure suppresses the induction of LTP in the LPP, except during diestrus. Data indicate that the induction and maintenance of LTP in the LPP in hippocampal slices from female rats is multifactorial: ovarian steroids and functionality of glucocorticoid receptors cooperation are necessary for induction and maintenance of the LTP, prenatal morphine exposure interferes with this process possibly by its long-term effects on synaptic plasticity.

  13. Paired Burst Stimulation Causes GABAA Receptor-Dependent Spike Firing Facilitation in CA1 of Rat Hippocampal Slices

    PubMed Central

    Tominaga, Takashi; Tominaga, Yoko

    2016-01-01

    The theta oscillation (4–8 Hz) is a pivotal form of oscillatory activity in the hippocampus that is intermittently concurrent with gamma (25–100 Hz) burst events. In in vitro preparation, a stimulation protocol that mimics the theta oscillation, theta burst stimulation (TBS), is used to induce long-term potentiation. Thus, TBS is thought to have a distinct role in the neural network of the hippocampal slice preparation. However, the specific mechanisms that make TBS induce such neural circuit modifications are still unknown. Using electrophysiology and voltage-sensitive dye imaging (VSDI), we have found that TBS induces augmentation of spike firing. The augmentation was apparent in the first couple of brief burst stimulation (100 Hz four pulses) on a TBS-train in a presence of NMDA receptor blocker (APV 50 μM). In this study, we focused on the characterizes of the NMDA independent augmentation caused by a pair of the brief burst stimulation (the first pair of the TBS; paired burst stimulation-PBS). We found that PBS enhanced membrane potential responses on VSDI signal and intracellular recordings while it was absent in the current recording under whole-cell clamp condition. The enhancement of the response accompanied the augmentation of excitatory postsynaptic potential (EPSP) to spike firing (E-S) coupling. The paired burst facilitation (PBF) reached a plateau when the number of the first burst stimulation (priming burst) exceeds three. The interval between the bursts of 150 ms resulted in the maximum PBF. Gabazine (a GABAA receptor antagonist) abolished PBF. The threshold for spike generation of the postsynaptic cells measured with a current injection to cells was not lowered by the priming burst of PBS. These results indicate that PBS activates the GABAergic system to cause short-term E-S augmentation without raising postsynaptic excitability. We propose that a GABAergic system of area CA1 of the hippocampus produce the short-term E-S plasticity that could

  14. Development of GABA-mediated, chloride-dependent inhibition in CA1 pyramidal neurones of immature rat hippocampal slices.

    PubMed Central

    Zhang, L; Spigelman, I; Carlen, P L

    1991-01-01

    1. gamma-Aminobutyric acid (GABA)-mediated, Cl(-)-dependent inhibitory postsynaptic potentials (IPSPs) and GABA currents in immature rat hippocampal CA1 neurones were studied using the whole-cell recording technique in brain slices. 2. IPSPs evoked by electrical stimulation were observed in postnatal 2- to 5- (PN2-5), 8- to 13-(PN8-13) and 15- to 20-(PN15-20)day-old CA1 neurones. In the presence of glutamate receptor blockers 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D-2-amino-5-phosphonovaleric acid (APV), the reversal potential for the IPSP (EIPSP) was near the resting membrane potential (RMP) in the PN2-5 neurones, but 13 and 25 mV more negative than the RMP in PN8-13 and PN15-20 neurones respectively. IPSPs and GABA currents were blocked by the GABAA-receptor antagonists bicuculline or picrotoxin. 3. The reversal potential for somatic GABA currents (EGABA) was examined in the presence of tetrodotoxin (TTX). There was a strong dependence of the EGABA upon the patch pipette [Cl-] ([Cl-]p). indicating that the GABA currents were mediated by a Cl- conductance. In PN2-5 neurones, EGABA agreed with the value predicted by the Goldman-Hodgkin-Katz equation at given concentrations of internal and external anions permeable through GABA-activated Cl- channels, whereas EGABA in older neurones was 8-18 mV more negative. 4. Examination of the relations between EGABA, holding potential, [Cl-]p and resting conductance indicated that the membrane of the PN2-5 neurones was readily permeable to Cl- which followed a passive Donnan equilibrium. Passive distribution of Cl- played a decreasing role in PN8-13 neurones and in PN15-20 neurones. 5. To assess the contribution of outward Cl- co-transport, bath applications of high K+ or furosemide were performed. High K+ and furosemide caused a reversible positive shift of EGABA in PN15-20 neurones. Raising the temperature moved EGABA to a more negative potential, with a Q10 of 5 mV. A similar change of EGABA in response to high K

  15. Synergistic activation by cis-fatty acid and diacylglycerol of protein kinase C and protein phosphorylation in hippocampal slices.

    PubMed

    Chen, S G; Murakami, K

    1995-10-01

    cis-Unsaturated fatty acid, which activates protein kinase C in vitro, stimulates protein phosphorylation in intact hippocampal slices. Two protein bands (44,000 and 47,000 mol. wt) are particularly sensitive to cis-fatty acid and are phosphorylated in a dose- and time-dependent manner. The cis-fatty acid-stimulated protein phosphorylation can be further potentiated with diacylglycerol or 12-O-tetradecanoylphorbol 13-acetate. Several lines of evidence indicate that the cis-fatty acid-stimulated phosphorylation of these proteins is mediated by protein kinase C. First, the cis-fatty acid effect is mimicked by other protein kinase C activators such as diacylglycerol. Second, the stimulation of the phosphorylation by these activators can be blocked by staurosporine, which potently inhibits protein kinase C. Third, a concomitant application of cis-fatty acid and diacylglycerol or 12-O-tetradecanoylphorbol 13-acetate enhances the 44,000 and 47,000 mol. wt phosphorylation in a synergistic manner, which is a novel activation mode for protein kinase C. Fourth, they can be phosphorylated by purified protein kinase C (type III: alpha). Moreover, the synergistic activation of purified protein kinase C by cis-fatty acid and diacylglycerol leads to a drastic increase in the phosphorylation of these two protein bands. Two-dimensional gel electrophoresis and immunoblot analysis revealed that they are both acidic proteins. The 47,000 mol. wt band consists of two protein components; one is found to be F1/growth-associated protein-43 (pI = 4.5), and the other 47,000 mol, wt protein has broad pI ranging from 4.6 to 4.9. The 44,000 mol. wt component is a major phosphoprotein with pI of 4.8-5.1. Our results strongly indicate that cis-fatty acid can act as a regulator of endogenous protein kinase C in concert with diacylglycerol, and stimulate protein phosphorylation of its substrates such as F1/growth-associated protein-43 in the hippocampus.

  16. Opening of Astrocytic Mitochondrial ATP-Sensitive Potassium Channels Upregulates Electrical Coupling between Hippocampal Astrocytes in Rat Brain Slices

    PubMed Central

    Wang, Jiangping; Li, Zhongxia; Feng, Mei; Ren, Keming; Shen, Guoxia; Zhao, Congying; Jin, Xiaoming; Jiang, Kewen

    2013-01-01

    Astrocytes form extensive intercellular networks through gap junctions to support both biochemical and electrical coupling between adjacent cells. ATP-sensitive K+ (KATP) channels couple cell metabolic state to membrane excitability and are enriched in glial cells. Activation of astrocytic mitochondrial KATP (mitoKATP) channel regulates certain astrocytic functions. However, less is known about its impact on electrical coupling between directly coupled astrocytes ex vivo. By using dual patch clamp recording, we found that activation of mitoKATP channel increased the electrical coupling ratio in brain slices. The electrical coupling ratio started to increase 3 min after exposure to Diazoxide, a mitoKATP channel activator, peaked at 5 min, and maintained its level with little adaptation until the end of the 10-min treatment. Blocking the mitoKATP channel with 5-hydroxydecanoate, inhibited electrical coupling immediately, and by 10-min, the ratio dropped by 71% of the initial level. Activation of mitoKATP channel also decreased the latency time of the transjunctional currents by 50%. The increase in the coupling ratio resulting from the activation of the mitoKATP channel in a single astrocyte was further potentiated by the concurrent inhibiting of the channel on the recipient astrocyte. Furthermore, Meclofenamic acid, a gap-junction inhibitor which completely blocked the tracer coupling, hardly reversed the impact of mitoKATP channel's activation on electrical coupling (by 7%). The level of mitochondrial Connexin43, a gap junctional subunit, significantly increased by 70% in astrocytes after 10-min Diazoxide treatment. Phospho-ERK signals were detected in Connexin43 immunoprecipitates in the Diazoxide-treated astrocytes, but not untreated control samples. Finally, inhibiting ERK could attenuate the effects of Diazoxide on electrical coupling by 61%. These findings demonstrate that activation of astrocytic mitoKATP channel upregulates electrical coupling between

  17. The effects of glucose, mannose, fructose and lactate on the preservation of neural activity in the hippocampal slices from the guinea pig.

    PubMed

    Wada, H; Okada, Y; Uzuo, T; Nakamura, H

    1998-03-30

    Using hippocampal slices from guinea pigs, we investigated the effect of different concentrations of glucose and replacement of glucose with mannose, fructose and lactate on neural activity. As an index of neural activity, the population spikes (PS) were recorded in the granule cell layer of the dentate gyrus (DG) and the pyramidal cell layer of the CA3 area in the hippocampal slices. Lowering the concentration of glucose from 10 mM to 5, 3, 2, 1 and 0 mM caused a reduction in the PS amplitude. There were differences in the decay times of the PS evoked in these two regions. PS evoked in CA3 region decayed faster even at a concentration of 3 mM glucose at which PS in granule cell layer was well maintained. The decay time of the PS in the CA3 region in the presence of glucose up to a concentration of 3 mM was shorter than that evoked in the DG. After the replacement of glucose with mannose, fructose or lactate, the PS disappeared within 35 min and there were no significant differences between the decay times in the two regions of slices incubated in the same medium. ATP, creatine phosphate (CrP) and lactate levels in each slice were determined. To investigate whether mannose and fructose could be metabolized or not in the tissue slice, anaerobic production of lactate from glucose, mannose and fructose were measured during oxygen and glucose deprivation. Under anaerobic conditions for 60 min, the levels of high-energy phosphates decreased to 50% of the initial level and lactate was produced from glucose, mannose or fructose. However, there were significant differences in the rate of lactate production between the DG and CA3 areas during application of 3 mM glucose, 10 mM mannose and 10 mM fructose. These results indicate that mannose, fructose and lactate can be metabolized and are available for maintaining the levels of high-energy phosphates but not for neural activity in the tissue slices and that the presence of glucose is indispensable for the maintenance of

  18. Induction of c-fos mRNA expression in an in vitro hippocampal slice model of adult rats after kainate but not gamma-aminobutyric acid or bicuculline treatment.

    PubMed

    Massamiri, T; Khrestchatisky, M; Ben-Ari, Y

    1994-01-17

    Levels of gene expression following in vitro treatment of rat hippocampal slices with kainate, gamma-aminobutyric acid (GABA), or bicuculline were measured by the reverse transcription-coupled polymerase chain reaction method. Following a short-term exposure to kainate, c-fos gene expression was induced by 12-fold in the adult, but not the newborn, hippocampus. Under the same experimental conditions, zifl268 and brain-derived neurotrophic factor (BDNF) gene expression were unchanged. Our results also demonstrate a lack of induction of c-fos, zifl268 and BDNF after short-time treatment of either adult or newborn hippocampal slices with GABA or bicuculline. The relevance of the differential induction of gene expression in the adult and newborn in an in vitro hippocampal slice model as compared to previously described in vivo models is discussed.

  19. Oxygen/Glucose Deprivation and Reperfusion Cause Modifications of Postsynaptic Morphology and Activity in the CA3 Area of Organotypic Hippocampal Slice Cultures.

    PubMed

    Jung, Yeon Joo; Suh, Eun Cheng; Lee, Kyung Eun

    2012-12-01

    Brain ischemia leads to overstimulation of N-methyl-D-aspartate (NMDA) receptors, referred as excitotoxicity, which mediates neuronal cell death. However, less attention has been paid to changes in synaptic activity and morphology that could have an important impact on cell function and survival following ischemic insult. In this study, we investigated the effects of reperfusion after oxygen/glucose deprivation (OGD) not only upon neuronal cell death, but also on ultrastructural and biochemical characteristics of postsynaptic density (PSD) protein, in the stratum lucidum of the CA3 area in organotypic hippocampal slice cultures. After OGD/reperfusion, neurons were found to be damaged; the organelles such as mitochondria, endoplasmic reticulum, dendrites, and synaptic terminals were swollen; and the PSD became thicker and irregular. Ethanolic phosphotungstic acid staining showed that the density of PSD was significantly decreased, and the thickness and length of the PSD were significantly increased in the OGD/reperfusion group compared to the control. The levels of PSD proteins, including PSD-95, NMDA receptor 1, NMDA receptor 2B, and calcium/calmodulin-dependent protein kinase II, were significantly decreased following OGD/reperfusion. These results suggest that OGD/reperfusion induces significant modifications to PSDs in the CA3 area of organotypic hippocampal slice cultures, both morphologically and biochemically, and this may contribute to neuronal cell death and synaptic dysfunction after OGD/reperfusion.

  20. The impact of aging, hearing loss, and body weight on mouse hippocampal redox state, measured in brain slices using fluorescence imaging.

    PubMed

    Stebbings, Kevin A; Choi, Hyun W; Ravindra, Aditya; Llano, Daniel Adolfo

    2016-06-01

    The relationships between oxidative stress in the hippocampus and other aging-related changes such as hearing loss, cortical thinning, or changes in body weight are not yet known. We measured the redox ratio in a number of neural structures in brain slices taken from young and aged mice. Hearing thresholds, body weight, and cortical thickness were also measured. We found striking aging-related increases in the redox ratio that were isolated to the stratum pyramidale, while such changes were not observed in thalamus or cortex. These changes were driven primarily by changes in flavin adenine dinucleotide, not nicotinamide adenine dinucleotide hydride. Multiple regression analysis suggested that neither hearing threshold nor cortical thickness independently contributed to this change in hippocampal redox ratio. However, body weight did independently contribute to predicted changes in hippocampal redox ratio. These data suggest that aging-related changes in hippocampal redox ratio are not a general reflection of overall brain oxidative state but are highly localized, while still being related to at least one marker of late aging, weight loss at the end of life. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. GnRH neuron firing and response to GABA in vitro depend on acute brain slice thickness and orientation.

    PubMed

    Constantin, Stephanie; Piet, Richard; Iremonger, Karl; Hwa Yeo, Shel; Clarkson, Jenny; Porteous, Robert; Herbison, Allan E

    2012-08-01

    The GnRH neurons exhibit long dendrites and project to the median eminence. The aim of the present study was to generate an acute brain slice preparation that enabled recordings to be undertaken from GnRH neurons maintaining the full extent of their dendrites or axons. A thick, horizontal brain slice was developed, in which it was possible to record from the horizontally oriented GnRH neurons located in the anterior hypothalamic area (AHA). In vivo studies showed that the majority of AHA GnRH neurons projected outside the blood-brain barrier and expressed c-Fos at the time of the GnRH surge. On-cell recordings compared AHA GnRH neurons in the horizontal slice (AHAh) with AHA and preoptic area (POA) GnRH neurons in coronal slices [POA coronal (POAc) and AHA coronal (AHAc), respectively]. AHAh GnRH neurons exhibited tighter burst firing compared with other slice orientations. Although α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) excited GnRH neurons in all preparations, γ-aminobutyric acid (GABA) was excitatory in AHAc and POAc but inhibitory in AHAh slices. GABA(A) receptor postsynaptic currents were the same in AHAh and AHAc slices. Intriguingly, direct activation of GABA(A) or GABA(B) receptors respectively stimulated and inhibited GnRH neurons regardless of slice orientation. Subsequent experiments indicated that net GABA effects were determined by differences in the ratio of GABA(A) and GABA(B) receptor-mediated effects in "long" and "short" dendrites of GnRH neurons in the different slice orientations. These studies document a new brain slice preparation for recording from GnRH neurons with their extensive dendrites/axons and highlight the importance of GnRH neuron orientation relative to the angle of brain slicing in studying these neurons in vitro.

  2. Dense arrays of micro-needles for recording and electrical stimulation of neural activity in acute brain slices

    NASA Astrophysics Data System (ADS)

    Gunning, D. E.; Beggs, J. M.; Dabrowski, W.; Hottowy, P.; Kenney, C. J.; Sher, A.; Litke, A. M.; Mathieson, K.

    2013-02-01

    Objective. This paper describes the design, microfabrication, electrical characterization and biological evaluation of a high-density micro-needle array. The array records from and electrically stimulates individual neurons simultaneously in acute slices of brain tissue. Approach. Acute slices, arguably the closest in-vitro model of the brain, have a damaged surface layer. Since electrophysiological recording methods rely heavily on electrode-cell proximity, this layer significantly attenuates the signal amplitude making the use of traditional planar electrodes unsuitable. To penetrate into the tissue, bypassing the tissue surface, and to record and stimulate neural activity in the healthy interior volume of the slice, an array of 61 micro-needles was fabricated. Main results. This device is shown to record extracellular action potentials from individual neurons in acute cortical slices with a signal to noise ratio of up to ˜15:1. Electrical stimulation of individual neurons is achieved with stimulation thresholds of 1.1-2.9 µA. Significance. The novelty of this system is the combination of close needle spacing (60 µm), needle heights of up to 250 µm and small (5-10 µm diameter) electrodes allowing the recording of single unit activity. The array is coupled to a custom-designed readout system forming a powerful electrophysiological tool that permits two-way electrode-cell communication with populations of neurons in acute brain slices.

  3. Acute subarachnoid hemorrhage: using 64-slice multidetector CT angiography to "triage" patients' treatment.

    PubMed

    Agid, R; Lee, S K; Willinsky, R A; Farb, R I; terBrugge, K G

    2006-11-01

    To evaluate the clinical role of CT angiography (CTA) in patients with acute subarachnoid hemorrhage (SAH) for treatment decision-making. Consecutive patients with acute SAH had CTA using a 64-slice scanner for initial clinical decision-making. Image processing included multiplanar volume reformatted (MPVR) maximum intensity projections (MIP) and 3D volume-rendered reconstructions. CTAs were used for (1) evaluating the cause of SAH, and (2) triaging aneurysm-bearing patients to the more appropriate management, either surgical clipping or endovascular coiling. CTA findings were confirmed by neurosurgical exploration or catheter angiography (digital subtraction angiography, DSA). Successful coiling provided evidence that triaging to endovascular treatment was correct. Included in the study were 73 patients. CTA findings were confirmed by DSA or neurosurgical operation in 65 patients, and of these 65, 47 had aneurysmal SAH, 3 had vasculitis, 1 had arterial dissection and 14 had no underlying arterial abnormality. The cause of SAH was detected with CTA in 62 out of the 65 patients (95.4%, sensitivity 94%, specificity 100%). CTA revealed the aneurysm in 46 of 47 patients (98%, sensitivity 98%, specificity 100%, positive predictive value 100%, negative predictive value 82.3%), 1 of 3 vasculitides and 1 of 1 dissection. Of the 46 patients with aneurysm, 44 (95.7%) were referred for treatment based on CTA. In 2 patients (2 of 46, 4.4%) CTA was not informative enough to choose treatment requiring DSA. Of the 44 patients, 27 (61.4%) were referred to endovascular treatment and successful coiling was achieved in 25 (25 of 27, 92.6%). CTA using a 64-slice scanner is an accurate tool for detecting and characterizing aneurysms in acute SAH. CTA is useful in the decision process whether to coil or clip an aneurysm.

  4. Neural activity and the levels of high energy phosphates during deprivation of oxygen and/or glucose in hippocampal slices of immature and adult rats.

    PubMed

    Nabetani, M; Okada, Y; Kawai, S; Nakamura, H

    1995-02-01

    To investigate the relationship between neural activity and cerebral energy metabolism during anoxia or ischemia in neural tissue of different ages, hippocampal slices were prepared from four-, seven- and 10-day-old and adult rats. For the index of the neural activity, the population spikes were recorded in the pyramidal cell layer of the CA3 area. ATP and phosphocreatine levels in the slices were measured during oxygen and/or glucose deprivation. After deprivation of both oxygen and glucose, population spikes of the slices from four, seven- and 10-day-old and adult rats ceased completely in 14.2, 11.8, 9.4 and 5.3 min, respectively. The level of ATP at the time of cessation of population spike in four-, seven- and 10-day-old and adult rats was 37.4, 30.2, 28.5 and 56.4% of the original concentrations. After deprivation of glucose only, the decay time of the population spikes of the slices from four-, seven- and 10-day-old and adult rats was 17.8, 14.5, 9.0 and 10.0 min and at the time of population spikes cessation the level of ATP was 99.8, 84.2, 79.3 and 49%, respectively. After deprivation of oxygen only, population spikes of the slices from four, seven- and 10-day old and adult rats ceased completely in 257, 283, 109 and 8.5 min, respectively. The level of ATP at the time of population spikes cessation was 50, 40, 36.6 and 94.4% of the initial values, respectively. These results indicate that the immature rat is extremely resistant to oxygen deprivation from a functional and a metabolic view, whereas in the adult rat, preservation of neural activity depends much on both oxygen and glucose. During glucose deprivation, population spikes of the slices of immature and mature rats ceased rapidly although the level of ATP is preserved at high levels. This suggests that glucose plays an important role in the preservation of neural activity in addition to its major function as an energy substrate especially in immature animals.

  5. Acute Korsakoff-like amnestic syndrome resulting from left thalamic infarction following a right hippocampal hemorrhage.

    PubMed

    Rahme, R; Moussa, R; Awada, A; Ibrahim, I; Ali, Y; Maarrawi, J; Rizk, T; Nohra, G; Okais, N; Samaha, E

    2007-04-01

    Korsakoff-like amnestic syndromes have been rarely described following structural lesions of the central nervous system. In this report, we describe a case of acute Korsakoff-like syndrome resulting from the combination of a left anteromedian thalamic infarct and a right hippocampal hemorrhage. We also review the literature relevant to the neuropathology and pathophysiology of Korsakoff syndrome and anterograde amnesia.

  6. Acute Melamine Affects Spatial Memory Consolidation via Inhibiting Hippocampal NMDAR-Dependent LTD in Rats.

    PubMed

    An, Lei; Sun, Wei

    2017-02-16

    Converging evidence supported that melamine could impair learning and memory and hippocampal function by mechanisms as yet unknown. The aim of this study was to obtain the first clues of how melamine affected spatial cognition, and how it may act on hippocampal function to modulate plasticity. Morris water maze test was used to probe spatial learning and memory. Pharmacological approaches were employed to modulate NMDAR and AMPAR-dependent long-term synaptic plasticity of rats' hippocampal CA1 region. Both systemic and intrahippocampal application of melamine impaired the formation of long-term spatial memory, particularly consolidation memory. The reduced expression of NMDA-NR1 and -NR2B subunits, but not AMPAR subunits, was presented. Meanwhile melamine inhibited inductions of long-term potentiation (LTP) and long-term depression (LTD) via mediating NMDARs. Notably, the specific role of hippocampal CA1 LTD in regulation of spatial consolidation has been observed in normal physiology. Moreover, the prevention of inhibited hippocampal CA1 LTD but not LTP could rescue the disruption of long-term spatial memory of the melamine-treated rats. Taken together, our findings suggested that acute melamine exposure impaired spatial memory consolidation via disrupting hippocampal NMDAR-dependent LTD. This provided an important insight into the neurophysiology of melamine-related and other psychiatric disorders. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  7. Atorvastatin prevents cell damage via modulation of oxidative stress, glutamate uptake and glutamine synthetase activity in hippocampal slices subjected to oxygen/glucose deprivation.

    PubMed

    Vandresen-Filho, Samuel; Martins, Wagner C; Bertoldo, Daniela B; Mancini, Gianni; Herculano, Bruno A; de Bem, Andreza F; Tasca, Carla I

    2013-06-01

    Oxygen-glucose deprivation (OGD) in brain cells increases extracellular glutamate concentration leading to excitotoxicity. Glutamate uptake from the synaptic cleft is carried out by glutamate transporters, which are likely to be modulated by oxidative stress. Therefore, oxidative stress is associated with reduced activity of glutamate transporters and glutamine synthetase, thus increasing extracellular glutamate levels that may aggravate damage to brain cells. Atorvastatin, a cholesterol-lowering agent, has been shown to exert neuroprotective effects. The aim of this study was to investigate if in vivo atorvastatin treatment would have protective effects against hippocampal slices subjected to OGD, ex vivo. Atorvastatin pretreatment promoted increased cell viability after OGD and reoxygenation of hippocampal slices. Atorvastatin-induced neuroprotection may be related to diminished oxidative stress, since it prevented OGD-induced decrement of non-proteic thiols (NPSH) levels and increase in the production of reactive oxygen species (ROS). Atorvastatin pretreatment also prevented the OGD-induced decrease in glutamate uptake and glutamine synthetase activity, although it had no effect on OGD-induced excitatory aminoacids release. Addition of cholesterol before OGD and reoxygenation, abolished the protective effect of atorvastatin on cellular viability as well as on glutamate uptake and glutamine synthetase activity. Therefore, atorvastatin is capable of preventing OGD-induced cell death, an effect achieved due to modulation of glutamate uptake and glutamine synthetase activity, and associated with diminished oxidative stress. Additionally, atorvastatin effects were dependent on its action on cholesterol synthesis inhibition. Thus, atorvastatin might be a useful strategy in the prevention of glutamate exitotoxicity involved in brain injuries such as vascular disorders.

  8. Evidence for direct and indirect mechanisms in the potent modulatory action of interleukin-2 on the release of acetylcholine in rat hippocampal slices

    PubMed Central

    Seto, David; Kar, Satyabrata; Quirion, Rémi

    1997-01-01

    The biphasic nature of the potent modulatory action of interleukin-2 (IL-2) on hippocampal acetylcholine (ACh) release was investigated by use of brain slice superfusion.Both the potentiating (10−13 M) and inhibitory (10−9 M) effects of IL-2 on hippocampal ACh release were stimulation-dependent and were blocked by a neutralizing IL-2 receptor antibody, suggesting the activation of typical IL-2 receptors in both cases.Tetrodotoxin (TTX; 10 μM) failed to block the potentiation of ACh release induced by a very low concentration of IL-2 (10−13M) suggesting a direct effect on cholinergic nerve terminals.In contrast, the inhibitory effect seen at a higher concentration (10−9 M) was TTX-sensitive, and hence indicative of an indirect action.To establish the nature of this intermediate mediator, blockers of nitric oxide synthesis, and of opioid and γ-aminobutyric acid (GABA) receptors were used. Only GABAA and GABAB receptor antagonists altered the inhibitory action of IL-2, suggesting the participation of GABA as mediator.Taken together, these results provide further evidence for the potent role of IL-2 in the modulation of cholinergic function in the rat hippocampus. PMID:9134229

  9. Time-lapse imaging reveals highly dynamic structural maturation of postnatally born dentate granule cells in organotypic entorhino-hippocampal slice cultures

    PubMed Central

    Radic, Tijana; Jungenitz, Tassilo; Singer, Mathias; Beining, Marcel; Cuntz, Hermann; Vlachos, Andreas; Deller, Thomas; Schwarzacher, Stephan W.

    2017-01-01

    Neurogenesis of hippocampal granule cells (GCs) persists throughout mammalian life and is important for learning and memory. How newborn GCs differentiate and mature into an existing circuit during this time period is not yet fully understood. We established a method to visualize postnatally generated GCs in organotypic entorhino-hippocampal slice cultures (OTCs) using retroviral (RV) GFP-labeling and performed time-lapse imaging to study their morphological development in vitro. Using anterograde tracing we could, furthermore, demonstrate that the postnatally generated GCs in OTCs, similar to adult born GCs, grow into an existing entorhino-dentate circuitry. RV-labeled GCs were identified and individual cells were followed for up to four weeks post injection. Postnatally born GCs exhibited highly dynamic structural changes, including dendritic growth spurts but also retraction of dendrites and phases of dendritic stabilization. In contrast, older, presumably prenatally born GCs labeled with an adeno-associated virus (AAV), were far less dynamic. We propose that the high degree of structural flexibility seen in our preparations is necessary for the integration of newborn granule cells into an already existing neuronal circuit of the dentate gyrus in which they have to compete for entorhinal input with cells generated and integrated earlier. PMID:28256620

  10. Forebrain microglia from wild-type but not adult 5xFAD mice prevent amyloid-β plaque formation in organotypic hippocampal slice cultures.

    PubMed

    Hellwig, Sabine; Masuch, Annette; Nestel, Sigrun; Katzmarski, Natalie; Meyer-Luehmann, Melanie; Biber, Knut

    2015-09-29

    The role of microglia in amyloid-β (Aβ) deposition is controversial. In the present study, an organotypic hippocampal slice culture (OHSC) system with an in vivo-like microglial-neuronal environment was used to investigate the potential contribution of microglia to Aβ plaque formation. We found that microglia ingested Aβ, thereby preventing plaque formation in OHSCs. Conversely, Aβ deposits formed rapidly in microglia-free wild-type slices. The capacity to prevent Aβ plaque formation was absent in forebrain microglia from young adult but not juvenile 5xFamilial Alzheimer's disease (FAD) mice. Since no loss of Aβ clearance capacity was observed in both wild-type and cerebellar microglia from 5xFAD animals, the high Aβ1-42 burden in the forebrain of 5xFAD animals likely underlies the exhaustion of microglial Aβ clearance capacity. These data may therefore explain why Aβ plaque formation has never been described in wild-type mice, and point to a beneficial role of microglia in AD pathology. We also describe a new method to study Aβ plaque formation in a cell culture setting.

  11. Choline induces opposite changes in pyramidal neuron excitability and synaptic transmission through a nicotinic receptor-independent process in hippocampal slices.

    PubMed

    Albiñana, E; Luengo, J G; Baraibar, A M; Muñoz, M D; Gandía, L; Solís, J M; Hernández-Guijo, J M

    2017-06-01

    Choline is present at cholinergic synapses as a product of acetylcholine degradation. In addition, it is considered a selective agonist for α5 and α7 nicotinic acetylcholine receptors (nAChRs). In this study, we determined how choline affects action potentials and excitatory synaptic transmission using extracellular and intracellular recording techniques in CA1 area of hippocampal slices obtained from both mice and rats. Choline caused a reversible depression of evoked field excitatory postsynaptic potentials (fEPSPs) in a concentration-dependent manner that was not affected by α7 nAChR antagonists. Moreover, this choline-induced effect was not mimicked by either selective agonists or allosteric modulators of α7 nAChRs. Additionally, this choline-mediated effect was not prevented by either selective antagonists of GABA receptors or hemicholinium, a choline uptake inhibitor. The paired pulse facilitation paradigm, which detects whether a substance affects presynaptic release of glutamate, was not modified by choline. On the other hand, choline induced a robust increase of population spike evoked by orthodromic stimulation but did not modify that evoked by antidromic stimulation. We also found that choline impaired recurrent inhibition recorded in the pyramidal cell layer through a mechanism independent of α7 nAChR activation. These choline-mediated effects on fEPSP and population spike observed in rat slices were completely reproduced in slices obtained from α7 nAChR knockout mice, which reinforces our conclusion that choline modulates synaptic transmission and neuronal excitability by a mechanism independent of nicotinic receptor activation.

  12. Ionotropic glutamate receptors and glutamate transporters are involved in necrotic neuronal cell death induced by oxygen-glucose deprivation of hippocampal slice cultures.

    PubMed

    Bonde, C; Noraberg, J; Noer, H; Zimmer, J

    2005-01-01

    Organotypic hippocampal slice cultures represent a feasible model for studies of cerebral ischemia and the role of ionotropic glutamate receptors in oxygen-glucose deprivation-induced neurodegeneration. New results and a review of existing data are presented in the first part of this paper. The role of glutamate transporters, with special reference to recent results on inhibition of glutamate transporters under normal and energy-failure (ischemia-like) conditions is reviewed in the last part of the paper. The experimental work is based on hippocampal slice cultures derived from 7 day old rats and grown for about 3 weeks. In such cultures we investigated the subfield neuronal susceptibility to oxygen-glucose deprivation, the type of induced cell death and the involvement of ionotropic glutamate receptors. Hippocampal slice cultures were also used in our studies on glutamate transporters reviewed in the last part of this paper. Neurodegeneration was monitored and/or shown by cellular uptake of propidium iodide, loss of immunocytochemical staining for microtubule-associated protein 2 and staining with Fluoro-Jade B. To distinguish between necrotic vs. apoptotic neuronal cell death we used immunocytochemical staining for active caspase-3 (apoptosis indicator) and Hoechst 33342 staining of nuclear chromatin. Our experimental studies on oxygen-glucose deprivation confirmed that CA1 pyramidal cells were the most susceptible to this ischemia-like condition. Judged by propidium iodide uptake, a selective CA1 lesion, with only minor affection on CA3, occurred in cultures exposed to oxygen-glucose deprivation for 30 min. Nuclear chromatin staining by Hoechst 33342 and staining for active caspase-3 showed that oxygen-glucose deprivation induced necrotic cell death only. Addition of 10 microM of the N-methyl-D-aspartate glutamate receptor antagonist MK-801, and 20 microM of the non-N-methyl-D-aspartate glutamate receptor antagonist 2,3-dihyroxy-6-nitro-7-sulfamoyl

  13. Magnetic Resonance Imaging Findings of Mumps Meningoencephalitis with Bilateral Hippocampal Lesions without Preceding Acute Parotitis: A Case Report.

    PubMed

    Woo, Ah Reum; Lee, Ha Young; Lim, Myung Kwan; Kang, Young Hye; Cho, Soon Gu; Choi, Seong Hye; Baek, Ji Hyeon

    2017-01-01

    Meningitis is a common central nervous system (CNS) complication of the mumps, a viral infection, but encephalitis and meningoencephalitis are less common in mumps. We describe magnetic resonance imaging findings of acute mumps meningoencephalitis in a 32-year-old male who showed bilateral hippocampal lesions without preceding parotitis. Although it is rare, hippocampal involvement should be considered a CNS complication of mumps infection.

  14. Microchromatographic study of hippocampal area CA3 proteins during prolonged post-tetanic potentiation in surviving slices

    SciTech Connect

    Pankova, T.M.; Mikichur, N.I.; Ratushayak, A.S.; Shtark, M.B.

    1985-06-01

    This paper studies the synthesis of proteins and, in particular, of brain-specific proteins in a homogeneous population of postsynaptic cells during the development of prolonged post-tetanic potentiation (PPTP). By using a system of synaptic connections incorporation of tritium-leucine into water-soluble protein of this zone has been investigated during the development of PPTP (in surviving slices after stimulation of mossy fibers). During statistical analysis of the results mean values of deviation of relative radioactivity compared with the control in each fraction was expressed as a percentage. The results were analyzed by Student's test, at the 95% level of significance.

  15. Nondestructive evaluation of progressive neuronal changes in organotypic rat hippocampal slice cultures using ultrahigh-resolution optical coherence microscopy.

    PubMed

    Li, Fengqiang; Song, Yu; Dryer, Alexandra; Cogguillo, William; Berdichevsky, Yevgeny; Zhou, Chao

    2014-10-01

    Three-dimensional tissue cultures have been used as effective models for studying different diseases, including epilepsy. High-throughput, nondestructive techniques are essential for rapid assessment of disease-related processes, such as progressive cell death. An ultrahigh-resolution optical coherence microscopy (UHR-OCM) system with [Formula: see text] axial resolution and [Formula: see text] transverse resolution was developed to evaluate seizure-induced neuronal injury in organotypic rat hippocampal cultures. The capability of UHR-OCM to visualize cells in neural tissue was confirmed by comparison of UHR-OCM images with confocal immunostained images of the same cultures. In order to evaluate the progression of neuronal injury, UHR-OCM images were obtained from cultures on 7, 14, 21, and 28 days in vitro (DIVs). In comparison to DIV 7, statistically significant reductions in three-dimensional cell count and culture thickness from UHR-OCM images were observed on subsequent time points. In cultures treated with kynurenic acid, significantly less reduction in cell count and culture thickness was observed compared to the control specimens. These results demonstrate the capability of UHR-OCM to perform rapid, label-free, and nondestructive evaluation of neuronal death in organotypic hippocampal cultures. UHR-OCM, in combination with three-dimensional tissue cultures, can potentially prove to be a promising tool for high-throughput screening of drugs targeting various disorders.

  16. Acute pentobarbital treatment impairs spatial learning and memory and hippocampal long-term potentiation in rats.

    PubMed

    Wang, Wei; Tan, Tao; Tu, Man; He, Wenting; Dong, Zhifang; Han, Huili

    2015-10-01

    Reports of the effects of pentobarbital on learning and memory are contradictory. Some studies have not shown any interference with learning and memory, whereas others have shown that pentobarbital impairs memory and that these impairments can last for long periods. However, it is unclear whether acute local microinjections of pentobarbital affect learning and memory, and if so, the potential mechanisms are also unclear. Here, we reported that the intra-hippocampal infusion of pentobarbital (8.0mM, 1μl per side) significantly impaired hippocampus-dependent spatial learning and memory retrieval. Moreover, in vitro electrophysiological recordings revealed that these behavioral changes were accompanied by impaired hippocampal CA1 long-term potentiation (LTP) and suppressed neuronal excitability as reflected by a decrease in the number of action potentials (APs). These results suggest that acute pentobarbital application causes spatial learning and memory deficits that might be attributable to the suppression of synaptic plasticity and neuronal excitability.

  17. Rubia cordifolia, Fagonia cretica linn and Tinospora cordifolia exert neuroprotection by modulating the antioxidant system in rat hippocampal slices subjected to oxygen glucose deprivation.

    PubMed

    Rawal, Avinash K; Muddeshwar, Manohar G; Biswas, Saibal K

    2004-08-13

    The major damaging factor during and after the ischemic/hypoxic insult is the generation of free radicals, which leads to apoptosis, necrosis and ultimately cell death. Rubia cordifolia (RC), Fagonia cretica linn (FC) and Tinospora cordifolia (TC) have been reported to contain a wide variety of antioxidants and have been in use in the eastern system of medicine for various disorders. However, their mechanism of action was largely unknown. We therefore selected these herbs for the present study to test their neuroprotective ability and the associated mechanism in rat hippocampal slices subjected to oxygen-glucose deprivation (OGD). Hippocampal Slices were subjected to OGD (oxygen glucose deprivation) and divided into 3 groups: control, OGD and OGD + drug treated. Cytosolic Cu-Zn superoxide dismutase (Cu-Zn SOD), reduced glutathione (GSH), glutathione peroxidase (GPx), nitric oxide (NO) was measured as nitrite (NO2) in the supernatant and protein assays were performed in the respective groups at various time intervals. EPR was used to establish the antioxidant effect of RC, FC and TC with respect to superoxide anion (O2.-), hydroxyl radicals (. OH), nitric oxide (NO) radical and peroxynitrite anion (ONOO) generated from pyrogallol, menadione, DETA-NO and Sin-1 respectively. RT-PCR was performed for the three groups for GCLC, iNOS, Cu-Zn SOD and GAPDH gene expression. All the three herbs were effective in elevating the GSH levels, expression of the gamma-glutamylcysteine ligase and Cu-Zn SOD genes. The herbs also exhibited strong free radical scavenging properties against reactive oxygen and nitrogen species as studied by electron paramagnetic resonance spectroscopy. In addition all the three herbs significantly diminished the expression of iNOS gene after 48 hours which plays a major role in neuronal injury during hypoxia/ischemia. RC, FC and TC therefore attenuate oxidative stress mediated cell injury during OGD and exert the above effects at both the cytosolic as

  18. The relationship between decorrelation time and sample thickness in acute rat brain tissue slices (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Brake, Joshua; Jang, Mooseok; Yang, Changhuei

    2016-03-01

    The optical opacity of biological tissue has long been a challenge in biomedical optics due to the strong scattering nature of tissue in the optical regime. While most conventional optical techniques attempt to gate out multiply scattered light and use only unscattered light, new approaches in the field of wavefront shaping exploit the time reversible symmetry of optical scattering in order to focus light inside or through scattering media. While these approaches have been demonstrated effectively on static samples, it has proven difficult to apply them to dynamic biological samples since even small changes in the relative positions of the scatterers within will cause the time symmetry that wavefront shaping relies upon to decorrelate. In this paper we investigate the decorrelation curves of acute rat brain slices for thicknesses in the range 1-3 mm (1/e decorrelation time on the order of seconds) using multi-speckle diffusing wave spectroscopy (MSDWS) and compare the results with theoretical predictions. The results of this study demonstrate that the 1/L^2 relationship between decorrelation time and thickness predicted by diffusing wave spectroscopy provides a good rule of thumb for estimating how the decorrelation of a sample will change with increasing thickness. Understanding this relationship will provide insight to guide the future development of biophotonic wavefront shaping tools by giving an estimate of how fast wavefront shaping systems need to operate to overcome the dynamic nature of biological samples.

  19. The adenosine A2A receptor antagonist ZM241385 enhances neuronal survival after oxygen-glucose deprivation in rat CA1 hippocampal slices

    PubMed Central

    Pugliese, AM; Traini, C; Cipriani, S; Gianfriddo, M; Mello, T; Giovannini, MG; Galli, A; Pedata, F

    2009-01-01

    Background and purpose: Activation of adenosine A2A receptors in the CA1 region of rat hippocampal slices during oxygen-glucose deprivation (OGD), a model of cerebral ischaemia, was investigated. Experimental approach: We made extracellular recordings of CA1 field excitatory postsynaptic potentials (fepsps) followed by histochemical and immunohistochemical techniques coupled to Western blots. Key results: OGD (7 or 30 min duration) elicited an irreversible loss of fepsps invariably followed by the appearance of anoxic depolarization (AD), an unambiguous sign of neuronal damage. The application of the selective adenosine A2A receptor antagonist, ZM241385 (4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a}{1,3,5}triazin-5-ylamino]ethyl)phenol; 100–500 nmol·L−1) prevented or delayed AD appearance induced by 7 or 30 min OGD and protected from the irreversible fepsp depression elicited by 7 min OGD. Two different selective adenosine A2A receptor antagonists, SCH58261 and SCH442416, were less effective than ZM241385 during 7 min OGD. The extent of CA1 cell injury was assessed 3 h after the end of 7 min OGD by propidium iodide. Substantial CA1 pyramidal neuronal damage occurred in untreated slices, exposed to OGD, whereas injury was significantly prevented by 100 nmol·L−1 ZM241385. Glial fibrillary acid protein (GFAP) immunostaining showed that 3 h after 7 min OGD, astrogliosis was appreciable. Western blot analysis indicated an increase in GFAP 30 kDa fragment which was significantly reduced by treatment with 100 nmol·L−1 ZM241385. Conclusions and implications: In the CA1 hippocampus, antagonism of A2A adenosine receptors by ZM241385 was protective during OGD (a model of cerebral ischaemia) by delaying AD appearance, decreasing astrocyte activation and improving neuronal survival. PMID:19422385

  20. Altered regulation of brain-derived neurotrophic factor protein in hippocampus following slice preparation.

    PubMed

    Danzer, S C; Pan, E; Nef, S; Parada, L F; McNamara, J O

    2004-01-01

    Brain-derived neurotrophic factor (BDNF) and its cognate receptor tyrosine kinase B (TrkB) play important roles in regulating survival, structure, and function of CNS neurons. One method of studying the functions of these molecules has utilized in vitro hippocampal slice preparations. An important caveat to using slices, however, is that slice preparation itself might alter the expression of BDNF, thereby confounding experimental results. To address this concern, BDNF immunoreactivity was examined in rodent slices using two different methods of slice preparation. Rapid and anatomically selective regulation of BDNF content followed slice preparation using both methodologies; however, different patterns of altered BDNF immunoreactivity were observed. First, in cultured slices, BDNF content decreased in the dentate molecular layer and increased in the CA3 pyramidal cell layer and the mossy fiber pathway of the hippocampus after 30 min. Furthermore, an initially "punctate" pattern of BDNF labeling observed in the mossy fiber pathway of control sections changed to homogenous labeling of the pathway in vitro. In contrast to these findings, slices prepared as for acute slice physiology exhibited no change in BDNF content in the molecular layer and mossy fiber pathway 30 min after slicing, but exhibited significant increases in the dentate granule and CA3 pyramidal cell layers. These findings demonstrate that BDNF protein content is altered following slice preparation, that different methods of slice preparation produce different patterns of BDNF regulation, and raise the possibility that BDNF release and TrkB activation may also be regulated. These consequences of hippocampal slice preparation may confound analyses of exogenous or endogenous BDNF on hippocampal neuronal structure or function.

  1. Guanosine is neuroprotective against oxygen/glucose deprivation in hippocampal slices via large conductance Ca²+-activated K+ channels, phosphatidilinositol-3 kinase/protein kinase B pathway activation and glutamate uptake.

    PubMed

    Dal-Cim, T; Martins, W C; Santos, A R S; Tasca, C I

    2011-06-02

    Guanine derivatives (GD) have been implicated in many relevant brain extracellular roles, such as modulation of glutamate transmission and neuronal protection against excitotoxic damage. GD are spontaneously released to the extracellular space from cultured astrocytes and during oxygen/glucose deprivation (OGD). The aim of this study has been to evaluate the potassium channels and phosphatidilinositol-3 kinase (PI3K) pathway involvement in the mechanisms related to the neuroprotective role of guanosine in rat hippocampal slices subjected to OGD. The addition of guanosine (100 μM) to hippocampal slices subjected to 15 min of OGD and followed by 2 h of re-oxygenation is neuroprotective. The presence of K+ channel blockers, glibenclamide (20 μM) or apamin (300 nM), revealed that neuroprotective effect of guanosine was not dependent on ATP-sensitive K+ channels or small conductance Ca²+-activated K+ channels. The presence of charybdotoxin (100 nM), a large conductance Ca²+-activated K+ channel (BK) blocker, inhibited the neuroprotective effect of guanosine. Hippocampal slices subjected to OGD and re-oxygenation showed a significant reduction of glutamate uptake. Addition of guanosine in the re-oxygenation period has blocked the reduction of glutamate uptake. This guanosine effect was inhibited when hippocampal slices were pre-incubated with charybdotoxin or wortmanin (a PI3K inhibitor, 1 μM) in the re-oxygenation period. Guanosine promoted an increase in Akt protein phosphorylation. However, the presence of charybdotoxin blocked such effect. In conclusion, the neuroprotective effect of guanosine involves augmentation of glutamate uptake, which is modulated by BK channels and the activation of PI3K pathway. Moreover, neuroprotection caused by guanosine depends on the increased expression of phospho-Akt protein.

  2. Bupropion inhibits nicotine-evoked [(3)H]overflow from rat striatal slices preloaded with [(3)H]dopamine and from rat hippocampal slices preloaded with [(3)H]norepinephrine.

    PubMed

    Miller, Dennis K; Sumithran, Sangeetha P; Dwoskin, Linda P

    2002-09-01

    Bupropion, an efficacious antidepressant and smoking cessation agent, inhibits dopamine and norepinephrine transporters (DAT and NET, respectively). Recently, bupropion has been reported to noncompetitively inhibit alpha3beta2, alpha3beta4, and alpha4beta2 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes or established cell lines. The present study evaluated bupropion-induced inhibition of native alpha3beta2* and alpha3beta4* nAChRs using functional neurotransmitter release assays, nicotine-evoked [(3)H]overflow from superfused rat striatal slices preloaded with [(3)H]dopamine ([(3)H]DA), and nicotine-evoked [(3)H]overflow from hippocampal slices preloaded with [(3)H]norepinephrine ([(3)H]NE). The mechanism of inhibition was evaluated using Schild analysis. To eliminate the interaction of bupropion with DAT or NET, nomifensine or desipramine, respectively, was included in the superfusion buffer. A high bupropion concentration (100 microM) elicited intrinsic activity in the [(3)H]DA release assay. However, none of the concentrations (1 nM-100 microM) examined evoked [(3)H]NE overflow and, thus, were without intrinsic activity in this assay. Moreover, bupropion inhibited both nicotine-evoked [(3)H]DA overflow (IC(50) = 1.27 microM) and nicotine-evoked [(3)H]NE overflow (IC(50) = 323 nM) at bupropion concentrations well below those eliciting intrinsic activity. Results from Schild analyses suggest that bupropion competitively inhibits nicotine-evoked [(3)H]DA overflow, whereas evidence for receptor reserve was obtained upon assessment of bupropion inhibition of nicotine-evoked [(3)H]NE overflow. Thus, bupropion acts as an antagonist at alpha3beta2* and alpha3beta4* nAChRs in rat striatum and hippocampus, respectively, across the same concentration range that inhibits DAT and NET function. The combination of nAChR and transporter inhibition produced by bupropion may contribute to its clinical efficacy as a smoking cessation agent.

  3. Effect of Rubia cordifolia, Fagonia cretica linn, and Tinospora cordifolia on free radical generation and lipid peroxidation during oxygen-glucose deprivation in rat hippocampal slices.

    PubMed

    Rawal, Avinash; Muddeshwar, Manohar; Biswas, Saibal

    2004-11-12

    The major damaging factor during and after the ischemic/hypoxic insult is the generation of free radicals, which leads to apoptosis, necrosis, and ultimately cell death. Rubia cordifolia (RC), Fagonia cretica linn (FC), and Tinospora cordifolia (TC) have been reported to contain a wide variety of antioxidants and have been in use in the eastern system of medicine for various disorders. Hippocampal slices were subjected to oxygen-glucose deprivation (OGD) and divided into three groups, control, OGD, and OGD+drug treated. Cytosolic reduced glutathione (GSH), nitric oxide [NO, measured as nitrite (NO2)]. EPR was used to establish the antioxidant effect of RC, FC, and TC with respect to superoxide anion (O*2-), hydroxyl radicals (*OH), nitric oxide (NO) radical, and peroxynitrite anion (ONOO-) generated from pyrogallol, menadione, DETA-NO, and Sin-1, respectively. RT-PCR was performed for the three herbs to assess their effect on the expression of gamma-glutamylcysteine ligase (GCLC), iNOS, and GAPDH gene expression. All the three herbs were effective in elevating the GSH levels and expression of the GCLC. The herbs also exhibited strong free radical scavenging properties against reactive oxygen and nitrogen species as revealed by electron paramagnetic resonance spectroscopy, diminishing the expression of iNOS gene. RC, FC, and TC therefore attenuate oxidative stress mediated cell injury during OGD and exert the above effects at both the cytosolic as well as at gene expression levels and may be effective therapeutic tool against ischemic brain damage.

  4. Effect of short-term exposure to dichlorvos on synaptic plasticity of rat hippocampal slices: Involvement of acylpeptide hydrolase and {alpha}{sub 7} nicotinic receptors

    SciTech Connect

    Olmos, Cristina; Sandoval, Rodrigo; Rozas, Carlos; Navarro, Sebastian; Wyneken, Ursula; Zeise, Marc; Morales, Bernardo; Pancetti, Floria

    2009-07-01

    Dichlorvos is the active molecule of the pro-drug metrifonate used to revert the cognitive deficits associated with Alzheimer's disease. A few years ago it was reported that dichlorvos inhibits the enzyme acylpeptide hydrolase at lower doses than those necessary to inhibit acetylcholinesterase to the same extent. Therefore, the aim of our investigation was to test the hypothesis that dichlorvos can enhance synaptic efficacy through a mechanism that involves acylpeptide hydrolase instead of acetylcholinesterase inhibition. We used long-term potentiation induced in rat hippocampal slices as a model of synaptic plasticity. Our results indicate that short-term exposures (20 min) to 50 {mu}M dichlorvos enhance long-term potentiation in about 200% compared to the control condition. This effect is correlated with approximately 60% inhibition of acylpeptide hydrolase activity, whereas acetylcholinesterase activity remains unaffected. Paired-pulse facilitation and inhibition experiments indicate that dichlorvos does not have any presynaptic effect in the CA3 {yields} CA1 pathway nor affect gabaergic interneurons. Interestingly, the application of 100 nM methyllicaconitine, an {alpha}{sub 7} nicotinic receptor antagonist, blocked the enhancing effect of dichlorvos on long-term potentiation. These results indicate that under the exposure conditions described above, dichlorvos enhances long-term potentiation through a postsynaptic mechanism that involves (a) the inhibition of the enzyme acylpeptide hydrolase and (b) the modulation of {alpha}{sub 7} nicotinic receptors.

  5. Aerobic Production and Utilization of Lactate Satisfy Increased Energy Demands Upon Neuronal Activation in Hippocampal Slices and Provide Neuroprotection Against Oxidative Stress

    PubMed Central

    Schurr, Avital; Gozal, Evelyne

    2012-01-01

    Ever since it was shown for the first time that lactate can support neuronal function in vitro as a sole oxidative energy substrate, investigators in the field of neuroenergetics have been debating the role, if any, of this glycolytic product in cerebral energy metabolism. Our experiments employed the rat hippocampal slice preparation with electrophysiological and biochemical methodologies. The data generated by these experiments (a) support the hypothesis that lactate, not pyruvate, is the end-product of cerebral aerobic glycolysis; (b) indicate that lactate plays a major and crucial role in affording neural tissue to respond adequately to glutamate excitation and to recover unscathed post-excitation; (c) suggest that neural tissue activation is accompanied by aerobic lactate and NADH production, the latter being produced when the former is converted to pyruvate by mitochondrial lactate dehydrogenase (mLDH); (d) imply that NADH can be utilized as an endogenous scavenger of reactive oxygen species (ROS) to provide neuroprotection against ROS-induced neuronal damage. PMID:22275901

  6. Effects of deprivation of oxygen or glucose on the neural activity in the guinea pig hippocampal slice--intracellular recording study of pyramidal neurons.

    PubMed

    Takata, T; Okada, Y

    1995-06-12

    The block of synaptic transmission and neural activity during deprivation of oxygen or glucose has been simply attributed to the lack of energy due to the disorder of energy production. To clarify the interrelation between neural activity and energy metabolism during hypoxia or glucose deprivation, we studied the changes in ATP levels and electrical events of pyramidal neurons in the CA3 region and [Ca2+]i mobilization of the dendritic and cellular region of CA3 area, using guinea pig hippocampal slices. The studies of field potentials and intracellular recording from the pyramidal cell of CA3 area during hypoxia or glucose deprivation revealed that the cessation of synaptic activity and the depolarization of resting potential occurred earlier than during glucose deprivation while the increase of [Ca2+]i was slow during hypoxia but rapid during glucose deprivation although the ATP level of CA3 area was maintained at its original level for 20 min during both conditions. When glucose was replaced by lactate, ATP concentration was not reduced but the electrical activity decayed and [Ca2+]i increased with the similar time course as observed during lack of glucose, only. These results suggest that different mechanisms underlie the block of synaptic transmission in the CA3 pyramidal neurons during hypoxia and glucose deprivation and that lactate cannot substitute for glucose in the maintenance of neural activity.

  7. Subfield-specific neurovascular remodeling in the entorhino-hippocampal-organotypic slice culture as a response to oxygen–glucose deprivation and excitotoxic cell death

    PubMed Central

    Chip, Sophorn; Nitsch, Cordula; Wellmann, Sven; Kapfhammer, Josef P

    2013-01-01

    Transient ischemia causes delayed neurodegeneration in selective brain areas, particularly in the CA1 field of the hippocampus. This is accompanied by neurovascular impairment. It is unknown whether neurodegeneration is the cause or consequence of vascular changes. In an entorhino-hippocampal-organotypic slice culture system with well-preserved blood vessels, we studied the interplay between neurodegeneration and neurovasculature. Short-term oxygen and glucose deprivation (OGD) resulted in upregulation of hypoxic markers and with a delay of 24 to 48 hours in selective nerve cell death in CA1. In parallel, local vessel density decreased as detected by markers of endothelial cells and of the extracellular matrix. Claudin-5, a tight junction protein and marker of the blood–brain barrier was reduced. Preventing neuronal death with tetrodotoxin or 6-cyano-7-nitroquinoxaline-2,3-dione rescued blood vessels, suggesting that vessel loss is not due to OGD per se but a consequence of neuronal death. Induction of excitotoxic neuronal death with AMPA caused widespread neurodegeneration, but vessel reduction was confined to CA1. In dentate gyrus without neuronal loss, vessel density increased. We propose that neuronal stress and death influence maintenance, loss and remodeling of the neurovasculature and that the type of vascular response is in addition determined by local factors within the hippocampus. PMID:23232944

  8. Electroosmotic perfusion of tissue: sampling the extracellular space and quantitative assessment of membrane-bound enzyme activity in organotypic hippocampal slice cultures

    PubMed Central

    Ou, Yangguang; Wu, Juanfang; Sandberg, Mats

    2014-01-01

    This review covers recent advances in sampling fluid from the extracellular space of brain tissue by electroosmosis (EO). Two techniques, EO sampling with a single fused-silica capillary and EO push–pull perfusion, have been developed. These tools were used to investigate the function of membrane-bound enzymes with outward-facing active sites, or ectoenzymes, in modulating the activity of the neuropeptides leu-enkephalin and galanin in organotypic-hippocampal-slice cultures (OHSCs). In addition, the approach was used to determine the endogenous concentration of a thiol, cysteamine, in OHSCs. We have also investigated the degradation of coenzyme A in the extracellular space. The approach provides information on ectoenzyme activity, including Michaelis constants, in tissue, which, as far as we are aware, has not been done before. On the basis of computational evidence, EO push–pull perfusion can distinguish ectoenzyme activity with a ~100 µm spatial resolution, which is important for studies of enzyme kinetics in adjacent regions of the rat hippocampus. PMID:25168111

  9. Effects of long-term lithium and desipramine treatment upon clonidine-induced inhibition of /sup 3/H-norepinephrine release from rat hippocampal slices

    SciTech Connect

    Spengler, R.N.; Hollingsworth, P.J.; Smith, C.B.

    1986-03-01

    Long-term treatment with antidepressant agents alters the specific binding of /sup 3/H-clonidine, an alpha/sub 2/ adrenoreceptor agonist, to neural membranes isolated from specific areas of the rat brain. The purpose of the present study was to determine whether these changes in binding of /sup 3/H-clonidine represent an alteration in the functional state of the presynaptic alpha/sub 2/ adrenorecepotr. Hippocampal slices were incubated with /sup 3/H-norepinephrine (/sup 3/H-NE, 330 nM0 for 20 min, washed with fresh buffer for 30 min and then stimulated (4 Hz, 2 msec duration, 2 min) at 12 min intervals. Cumulative concentration-effect curves were determined of /sup 3/H-NE. Rats were injected, i.p., twice daily for 14 days with lithium chloride (105 mg/kg), desipramine HCl (10 mg/kg) or saline. In controls, the EC50 for clonidine was 2.3 +/- 1.0 nM (n = 3). After lithium treatment, the clonidine concentration-effect curve was shifted to the right, and the EC50 as 12.1 +/- 4.3 nM. Desipramine treatment nearly abolished the inhibitory effect of clonidine upon the release of /sup 3/H-NE by field stimulation. These observations indicate that the long-term administration of desipramine and lithium produce a functional subsensitivity of the alpha/sub 2/ adrenoreceptor which regulates norepinephrine release in the rat brain.

  10. Acute Exercise Improves Prefrontal Cortex but not Hippocampal Function in Healthy Adults.

    PubMed

    Basso, Julia C; Shang, Andrea; Elman, Meredith; Karmouta, Ryan; Suzuki, Wendy A

    2015-11-01

    The effects of acute aerobic exercise on cognitive functions in humans have been the subject of much investigation; however, these studies are limited by several factors, including a lack of randomized controlled designs, focus on only a single cognitive function, and testing during or shortly after exercise. Using a randomized controlled design, the present study asked how a single bout of aerobic exercise affects a range of frontal- and medial temporal lobe-dependent cognitive functions and how long these effects last. We randomly assigned 85 subjects to either a vigorous intensity acute aerobic exercise group or a video watching control group. All subjects completed a battery of cognitive tasks both before and 30, 60, 90, or 120 min after the intervention. This battery included the Hopkins Verbal Learning Test-Revised, the Modified Benton Visual Retention Test, the Stroop Color and Word Test, the Symbol Digit Modalities Test, the Digit Span Test, the Trail Making Test, and the Controlled Oral Word Association Test. Based on these measures, composite scores were formed to independently assess prefrontal cortex- and hippocampal-dependent cognition. A three-way mixed Analysis of Variance was used to determine whether differences existed between groups in the change in cognitive function from pre- to post-intervention testing. Acute exercise improved prefrontal cortex- but not hippocampal-dependent functioning, with no differences found between delay groups. Vigorous acute aerobic exercise has beneficial effects on prefrontal cortex-dependent cognition and these effects can last for up to 2 hr after exercise.

  11. Slice orientation and muscarinic acetylcholine receptor activation determine the involvement of N-methyl D-aspartate receptor subunit GluN2B in hippocampal area CA1 long-term depression

    PubMed Central

    2011-01-01

    Background The contribution of different GluN2 subunits of the N-methyl D-aspartate (NMDA) receptor to the induction of bidirectional hippocampal synaptic plasticity is a controversial topic. As both supporting and refuting evidence for the hypothesis of subunit specialization in opposing directions of plasticity has accumulated since it was first proposed a few years ago, we hypothesize that differences in experimental conditions may have in part contributed to some of the inconsistent results from these studies. Here we investigate the controversial hypothesis that long-term depression (LTD) is preferentially induced by GluN2B-containing NMDA receptors in area CA1 of hippocampal slices. Results We find that brain slices from 2-3 week old rats prepared in the sagittal orientation have GluN2B-independent LTD whereas slices prepared in the coronal orientation have GluN2B-dependent LTD. There was no difference between the orientations in the fraction of the NMDAR EPSC sensitive to a GluN2B-selective antagonist, leading us to believe that the intracellular signaling properties of the NMDARs were different in the two preparations. Coronal slices had greater association of LTD-related intracellular signaling protein RasGRF1 with GluN2B relative to sagittal slices. Antagonism of muscarinic acetylcholine receptors (mAChRs) in the sagittal slices returned LTD to a GluN2B-dependent form and increased the association of GluN2B with RasGRF1. Conclusions These results suggest a novel form of NMDAR modulation by mAChRs and clarify some disagreement in the literature. PMID:22082088

  12. Motor skill learning and offline-changes in TGA patients with acute hippocampal CA1 lesions.

    PubMed

    Döhring, Juliane; Stoldt, Anne; Witt, Karsten; Schönfeld, Robby; Deuschl, Günther; Born, Jan; Bartsch, Thorsten

    2017-04-01

    Learning and the formation of memory are reflected in various memory systems in the human brain such as the hippocampus based declarative memory system and the striatum-cortex based system involved in motor sequence learning. It is a matter of debate how both memory systems interact in humans during learning and consolidation and how this interaction is influenced by sleep. We studied the effect of an acute dysfunction of hippocampal CA1 neurons on the acquisition (on-line condition) and off-line changes of a motor skill in patients with a transient global amnesia (TGA). Sixteen patients (68 ± 4.4 yrs) were studied in the acute phase and during follow-up using a declarative and procedural test, and were compared to controls. Acute TGA patients displayed profound deficits in all declarative memory functions. During the acute amnestic phase, patients were able to acquire the motor skill task reflected by increasing finger tapping speed across the on-line condition, albeit to a lesser degree than during follow-up or compared to controls. Retrieval two days later indicated a greater off-line gain in motor speed in patients than controls. Moreover, this gain in motor skill performance was negatively correlated to the declarative learning deficit. Our results suggest a differential interaction between procedural and declarative memory systems during acquisition and consolidation of motor sequences in older humans. During acquisition, hippocampal dysfunction attenuates fast learning and thus unmasks the slow and rigid learning curve of striatum-based procedural learning. The stronger gains in the post-consolidation condition in motor skill in CA1 lesioned patients indicate a facilitated consolidation process probably occurring during sleep, and suggest a competitive interaction between the memory systems. These findings might be a reflection of network reorganization and plasticity in older humans and in the presence of CA1 hippocampal pathology.

  13. Genome-wide alterations in hippocampal 5-hydroxymethylcytosine links plasticity genes to acute stress

    PubMed Central

    Li, Sisi; Papale, Ligia A.; Zhang, Qi; Madrid, Andy; Chen, Li; Chopra, Pankaj; Keleş, Sündüz; Jin, Peng; Alisch, Reid S.

    2015-01-01

    Environmental stress is among the most important contributors to increased susceptibility to develop psychiatric disorders, including anxiety and post-traumatic stress disorder. While even acute stress alters gene expression, the molecular mechanisms underlying these changes remain largely unknown. 5-hydroxymethylcytosine (5hmC) is a novel environmentally sensitive DNA modification that is highly enriched in post-mitotic neurons and is associated with active transcription of neuronal genes. Recently, we found a hippocampal increase of 5hmC in the glucocorticoid receptor gene (Nr3c1) following acute stress, warranting a deeper investigation of stress-related 5hmC levels. Here, we used an established chemical labeling and affinity purification method coupled with high-throughput sequencing technology to generate the first genome-wide profile of hippocampal 5hmC following exposure to acute restraint stress and a one-hour recovery. This approach found a genome-wide disruption in 5hmC associated with acute stress response, primarily in genic regions, and identified known and potentially novel stress-related targets that have a significant enrichment for neuronal ontological functions. Integration of these data with hippocampal gene expression data from these same mice found stress-related hydroxymethylation correlated to altered transcript levels and sequence motif predictions indicated that 5hmC may function by mediating transcription factor binding to these transcripts. Together, these data reveal an environmental impact on this newly discovered epigenetic mark in the brain and represent a critical step toward understanding stress-related epigenetic mechanisms that alter gene expression and can lead to the development of psychiatric disorders. PMID:26598390

  14. A comparison of distal and proximal dendritic synapses on CAi pyramids in guinea-pig hippocampal slices in vitro.

    PubMed

    Andersen, P; Silfvenius, H; Sundberg, S H; Sveen, O

    1980-10-01

    1. In vitro slices of guinea-pig hippocampus have been employed to compare excitatory synapses located distally and proximally on the dendritic tree of CA1 pyramidal cells.2. The main orientation of unmyelinated afferent fibres was found to be parallel to each other and perpendicular to the dendritic axis.3. The density of boutons ending on dendritic spines was roughly similar throughout the greater part of the dendritic tree with an average of 42 +/- 7.2 synapses per 100 mum(2). Their number did, however, decrease in the distal fifth of the apical and in the distal third of the basal dendritic region in parallel with an increase of boutons on the dendritic shafts.4. Negative synaptic field potentials (extracellular field e.p.s.p.s) had their maximum in the region where activated afferent fibres terminated and showed reversal when recorded from sufficiently displaced positions along the dendritic axis. The field e.p.s.p. was preceded by a diphasic presynaptic fibre volley. By cutting all but a narrow bundle of afferent fibres selective activation of a small group of dendritic synapses was possible. Stimulation of fibres crossing tissue bridges (35-100 mum wide) evoked field e.p.s.p.s comparable in amplitude to those seen in slices without lesions. The size of the field e.p.s.p.s evoked via distal and proximal bridges was remarkably similar and linearly related to the size of the appropriate stimulus current and presynaptic volley.5. Selective activation of a small group of afferent fibres gave rise to large amplitude population spikes. Proximal and distal bridges were largely equipotent when they were equally wide. Above the threshold amplitude, the evoked population spikes were linearly related to both the presynaptic volley and the stimulus current. Constant current stimulation of fibres at all apical dendritic levels was equally effective in evoking population spikes, with the exception of the outer fifth of the tree where stimulation was unsuccessful. Input

  15. Quantitative effects produced by modifications of neuronal activity on the size of GABAA receptor clusters in hippocampal slice cultures.

    PubMed

    Marty, Serge; Wehrlé, Rosine; Fritschy, Jean-Marc; Sotelo, Constantino

    2004-07-01

    The number and strength of GABAergic synapses needs to be precisely adjusted for adequate control of excitatory activity. We investigated to what extent the size of GABA(A) receptor clusters at inhibitory synapses is under the regulation of neuronal activity. Slices from P7 rat hippocampus were cultured for 13 days in the presence of bicuculline or 4-aminopyridine (4-AP) to increase neuronal activity, or DNQX to decrease activity. The changes provoked by these treatments on clusters immunoreactive for the alpha1 and alpha2 subunits of the GABA(A) receptor or gephyrin were quantitatively evaluated. While an increase in activity augmented the density of these clusters, a decrease in activity provoked, in contrast, a decrease in their density. An inverse regulation was observed for the size of individual clusters. Bicuculline and 4-AP decreased whilst DNQX increased the mean size of the clusters. When the pharmacological treatments were applied for 2 days instead of 2 weeks, no effects on the size of the clusters were observed. The variations in the mean size of individual clusters were mainly due to changes in the number of small clusters. Finally, a regulation of the size of GABA(A) receptor clusters occurred during development in vivo, with a decrease of the mean size of the clusters between P7 and P21. This physiological change was also the result of an increase in the number of small clusters. These results indicate that neuronal activity regulates the mean size of GABA(A) receptor- and gephyrin-immunoreactive clusters by modifying specifically the number of synapses with small clusters of receptors.

  16. Magnetic Resonance Imaging Findings of Mumps Meningoencephalitis with Bilateral Hippocampal Lesions without Preceding Acute Parotitis: A Case Report

    PubMed Central

    Woo, Ah Reum; Lim, Myung Kwan; Kang, Young Hye; Cho, Soon Gu; Choi, Seong Hye; Baek, Ji Hyeon

    2017-01-01

    Meningitis is a common central nervous system (CNS) complication of the mumps, a viral infection, but encephalitis and meningoencephalitis are less common in mumps. We describe magnetic resonance imaging findings of acute mumps meningoencephalitis in a 32-year-old male who showed bilateral hippocampal lesions without preceding parotitis. Although it is rare, hippocampal involvement should be considered a CNS complication of mumps infection. PMID:28246518

  17. Quantal analysis of inhibitory synaptic transmission in the dentate gyrus of rat hippocampal slices: a patch-clamp study.

    PubMed Central

    Edwards, F A; Konnerth, A; Sakmann, B

    1990-01-01

    1. Synaptically connected neurones were identified in the granule cell layer of slices of 17- to 21-day-old rat hippocampus. Whole-cell current recording using the patch-clamp technique revealed synaptic currents ranging from less than 10 to 200 pA in symmetrical Cl- conditions, at a holding potential of -50 mV. These currents were blocked by 2 microM-bicuculline, indicating that they result from the activation of postsynaptic gamma-aminobutyric acid receptor (GABAA-receptor) channels. 2. Addition of tetrodotoxin (TTX, 1 microM) resulted in the loss of most currents of more than 40 pA in amplitude. Currents which disappeared after TTX treatment were assumed to be the result of spontaneous presynaptic action potentials. The currents seen in the absence of TTX are referred to as spontaneously occurring inhibitory postsynaptic currents (IPSCs); those remaining in the presence of TTX were defined as miniature IPSCs. 3. Similar currents were observed when recording in the whole-cell configuration while extracellular stimulation was applied to a nearby neurone. These currents were also completely blocked by 2 microM-bicuculline and by 0.5 microM-TTX. They were thus defined as stimulus-evoked IPSCs. 4. The half rise time of both miniature and stimulus-evoked IPSCs was fast (less than 1 ms). The time course of decay of both miniature IPSCs and stimulus-evoked IPSCs could be well fitted with the sum of two exponentials. At a membrane potential of -50 mV, the mean decay time constants of the two components were 2.0 +/- 0.38 and 54.4 +/- 18 ms (mean +/- S.D.) for miniature IPSCs (six cells) and 2.2 +/- 1.3 and 66 +/- 20 ms (three cells) for stimulus-evoked IPSCs. 5. Stimulus-evoked IPSCs varied in amplitude from less than ten to hundreds of picoamperes. In eight of eleven cells histograms of IPSC amplitudes showed several clear peaks which, when fitted with the sum of Gaussian curves, were found to be equidistant. This is consistent with the view that stimulus-evoked IPSC

  18. Sensitivity of N-methyl-D-aspartate receptor-mediated excitatory postsynaptic potentials and synaptic plasticity to TCN 201 and TCN 213 in rat hippocampal slices.

    PubMed

    Izumi, Yukitoshi; Zorumski, Charles F

    2015-02-01

    Whereas ifenprodil has been used as a selective GluN1/GluN2B (NR1/NR2B, B-type) receptor antagonist to distinguish between GluN2B (NR2B) and GluN2A (NR2A)-containing N-methyl-d-aspartate receptors (NMDARs), TCN 201 (3-chloro-4-fluoro-N-[4-[[2-(phenylcarbonyl)hydrazino]carbonyl]benzyl]benzenesulphonamide) and TCN 213 [N-(cyclohexylmethyl)-2-[{5-[(phenylmethyl)amino]-1,3,4-thiadiazol-2-yl}thio]acetamide] have been found to be selective GluN1/GluN2A (NR1/NR2A, A-type) antagonists. Based on the premise that A- and B-types are major synaptic NMDARs, we examined whether inhibition of NMDAR excitatory postsynaptic potentials (EPSPs) by the TCN compounds and ifenprodil are complementary. Contrary to this prediction, inhibition of NMDAR EPSPs by the TCN compounds and ifenprodil were largely overlapping in the CA1 region of hippocampal slices from 30-day-old rats. After partial inhibition by ifenprodil, TCN compounds produced little further suppression of NMDAR EPSPs. Similarly, after partial inhibition by TCN compounds ifenprodil failed to further suppress NMDAR EPSPs. However, low micromolar d-2-amino-5-phosphonovalerate, a competitive NMDAR antagonist, which alone only partially inhibits NMDAR EPSPs, markedly suppresses residual NMDAR responses in the presence of ifenprodil or the TCNs, suggesting that low 2-amino-5-phosphonovalerate antagonizes both ifenprodil- and TCN-insensitive synaptic NMDARs. These observations can be most readily interpreted if ifenprodil and TCNs act on a similar population of synaptic NMDARs. Recent lines of evidence suggest that the majority of hippocampal synaptic NMDARs are triheteromers. If so, modulation of GluN2A, and not just GluN2B NMDARs, could dampen long-term depression (LTD). Indeed, both TCNs, like ifenprodil, blocked LTD, suggesting the involvement of ifenprodil- and TCN-sensitive NMDARs in LTD induction. However, the TCNs plus ifenprodil failed to inhibit long-term potentiation (LTP), suggesting that neither ifenprodil- nor TCN

  19. Acute inactivation of PSD-95 destabilizes AMPA receptors at hippocampal synapses.

    PubMed

    Yudowski, Guillermo A; Olsen, Olav; Adesnik, Hillel; Marek, Kurt W; Bredt, David S

    2013-01-01

    Postsynatptic density protein (PSD-95) is a 95 kDa scaffolding protein that assembles signaling complexes at synapses. Over-expression of PSD-95 in primary hippocampal neurons selectively increases synaptic localization of AMPA receptors; however, mice lacking PSD-95 display grossly normal glutamatergic transmission in hippocampus. To further study the scaffolding role of PSD-95 at excitatory synapses, we generated a recombinant PSD-95-4c containing a tetracysteine motif, which specifically binds a fluorescein derivative and allows for acute and permanent inactivation of PSD-95. Interestingly, acute inactivation of PSD-95 in rat hippocampal cultures rapidly reduced surface AMPA receptor immunostaining, but did not affected NMDA or transferrin receptor localization. Acute photoinactivation of PSD-95 in dissociated neurons causes ∼80% decrease in GluR2 surface staining observed by live-cell microscopy within 15 minutes of PSD-95-4c ablation. These results confirm that PSD-95 stabilizes AMPA receptors at postsynaptic sites and provides insight into the dynamic interplay between PSD-95 and AMPA receptors in live neurons.

  20. An in situ measurement of extracellular cysteamine, homocysteine, and cysteine concentrations in organotypic hippocampal slice cultures by integration of electroosmotic sampling and microfluidic analysis.

    PubMed

    Wu, Juanfang; Xu, Kerui; Landers, James P; Weber, Stephen G

    2013-03-19

    We demonstrate an all-electric sampling/derivatization/separation/detection system for the quantitation of thiols in tissue cultures. Extracellular fluid collected from rat organotypic hippocampal slice cultures (OHSCs) by electroosmotic flow through an 11 cm (length) × 50 μm (i.d.) sampling capillary is introduced to a simple microfluidic chip for derivatization, continuous flow-gated injection, separation, and detection. With the help of a fluorogenic, thiol-specific reagent, ThioGlo-1, we have successfully separated and detected the extracellular levels of free reduced cysteamine, homocysteine, and cysteine from OHSCs within 25 s in a 23 mm separation channel with a confocal laser-induced fluorescence (LIF) detector. Attention to the conductivities of the fluids being transported is required for successful flow-gated injections. When the sample conductivity is much higher than the run buffer conductivities, the electroosmotic velocities are such that there is less fluid coming by electroosmosis into the cross from the sample/reagent channel than is leaving by electroosmosis into the separation and waste channels. The resulting decrease in the internal fluid pressure in the injection cross pulls flow from the gated channel. This process may completely shut down the gated injection. Using a glycylglycine buffer with physiological osmolarity but only 62% of physiological conductivity and augmenting the conductivity of the run buffers solved this problem. Quantitation is by standard additions. Concentrations of cysteamine, homocysteine, and cysteine in the extracellular space of OHSCs are 10.6 ± 1.0 nM (n = 70), 0.18 ± 0.01 μM (n = 53), and 11.1 ± 1.2 μM (n = 70), respectively. This is the first in situ quantitative estimation of endogenous cysteamine in brain tissue. Extracellular levels of homocysteine and cysteine are comparable with other reported values.

  1. An in situ Measurement of Extracellular Cysteamine, Homocysteine and Cysteine Concentrations in Organotypic Hippocampal Slice Cultures by Integration of Electroosmotic Sampling and Microfluidic Analysis

    PubMed Central

    Wu, Juanfang; Xu, Kerui; Landers, James P.; Weber, Stephen G.

    2013-01-01

    We demonstrate an all-electric sampling/derivatization/separation/detection system for the quantitation of thiols in tissue cultures. Extracellular fluid collected from rat organotypic hippocampal slice cultures (OHSCs) by electroosmotic flow through an11 cm (length) × 50 μm (ID) sampling capillary is introduced to a simple microfluidic chip for derivatization, continuous flow-gated injection, separation and detection.With the help of a fluorogenic, thiol-specific reagent, ThioGlo-1, we have successfully separated and detected the extracellular levels of free reduced cysteamine, homocysteineand cysteinefrom OHSCs within 25 s in a 23 mm separation channel with a confocal laser induced fluorescence (LIF) detector. Attention to the conductivities of the fluids being transported is required for successful flow-gated injections.When the sample conductivity is much higher than the run buffer conductivities, the electroosmotic velocities are such that there is less fluid coming by electroosmosis into the cross from the sample/reagent channel than is leaving by electroosmosis into the separation and waste channels. The resulting decrease in the internal fluid pressure in the injection cross pulls flow from the gated channel. This process may completely shut down the gated injection. Using a glycylglycine buffer with physiological osmolarity but only 62% of physiological conductivity and augmenting the conductivity of the run buffers solved this problem. Quantitation is by standard additions. Concentrations of cysteamine, homocysteine and cysteine in the extracellular space of OHSCs are10.6±1.0 nM (n=70), 0.18±0.01 μM (n=53) and 11.1±1.2 μM (n=70), respectively. This is the first in situquantitative estimation of endogenous cysteamine in brain. Extracellular levels of homocysteine and cysteine are comparable with other reported values. PMID:23330713

  2. Loss of the Kv1.1 potassium channel promotes pathologic sharp waves and high frequency oscillations in in vitro hippocampal slices

    PubMed Central

    Simeone, Timothy A.; Simeone, Kristina A.; Samson, Kaeli K.; Kim, Do Young; Rho, Jong M.

    2013-01-01

    In human disease, channelopathies involving functional reduction of the delayed rectifier potassium channel α-subunit Kv1.1 – either by mutation or autoimmune inhibition – result in temporal lobe epilepsy. Kv1.1 is prominently expressed in the axons of the hippocampal tri-synaptic pathway, suggesting its absence will result in widespread effects on normal network oscillatory activity. Here, we performed in vitro extracellular recordings using a multielectrode array to determine the effects of loss of Kv1.1 on spontaneous sharp waves (SPWs) and high frequency oscillations (HFOs). We found that Kcna1-null hippocampi generate SPWs and ripples (80–200 Hz bandwidth) with a 50% increased rate of incidence and 50% longer duration, and that epilepsy-associated pathologic HFOs in the fast ripple bandwidth (200–600 Hz) are also present. Furthermore, Kcna1-null CA3 has enhanced coupling of excitatory inputs and population spike generation and CA3 principal cells have reduced spike timing reliability. Removing the influence of mossy fiber and perforant path inputs by microdissecting the Kcna1-null CA3 region mostly rescued the oscillatory behavior and improved spike timing. We found that Kcna1-null mossy fibers and medial perforant path axons are hyperexcitable and produce greater pre- and post-synaptic responses with reduced paired-pulse ratios suggesting increased neurotransmitter release at these terminals. These findings were recapitulated in wild-type slices exposed to the Kv1.1 inhibitor dendrotoxin-κ. Collectively, these data indicate that loss of Kv1.1 enhances synaptic release in the CA3 region, which reduces spike timing precision of individual neurons leading to disorganization of network oscillatory activity and promotes the emergence of fast ripples. PMID:23466697

  3. The protein kinase C inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) disinhibits CA1 pyramidal cells in rat hippocampal slices.

    PubMed Central

    Corradetti, R.; Pugliese, A. M.; Ropert, N.

    1989-01-01

    1. The effects of the protein kinase C (PKC) inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) on evoked synaptic potentials were investigated in the CA1 region of rat hippocampal slices by use of extracellular and intracellular recording techniques. 2. Extracellular recordings showed that superfusion with H-7 (10-100 microM) increased the amplitude of the population spike and the initial slope of the dendritic field e.p.s.p. H-7 also produced the appearance of multiple population spikes in the somatic region and in the dendritic field e.p.s.p. 3. H-7 (30 microM) induced the disappearance of intracellularly recorded inhibitory potentials elicited by orthodromic stimulation of CA1 pyramidal cells. At this concentration H-7 had no effect on resting membrane potential, input membrane resistance, and spike threshold. In voltage-clamped neurones H-7 blocked the antidromically evoked inhibitory currents and the spontaneous miniature inhibitory currents. 4. The hyperpolarizing effect of bath applied gamma-aminobutyric acid (GABA, 500 microM) or isoguvacine (30 microM) was not affected by 30 microM H-7. 5. Neither the PKC activity regulator sphingosine (10-40 microM) nor the H-7 analogue N-(2-guanidinoethyl)-5-isoquinolinesulphonamide (HA-1004, 20-50 microM) which is devoid of activity on PKC at these concentrations, affected the extracellularly recorded dendritic field e.p.s.p. or population spike. 6. It is concluded that the disinhibitory effect produced by H-7 is due to the block of a H-7-sensitive PKC which is involved in the spontaneous and evoked release of GABA. PMID:2611497

  4. Sr2+ supports depolarization-induced suppression of inhibition and provides new evidence for a presynaptic expression mechanism in rat hippocampal slices.

    PubMed Central

    Morishita, W; Alger, B E

    1997-01-01

    1. We studied the transient suppression of evoked GABAA ergic inhibitory postsynaptic currents (eIPSCs) that follows brief membrane depolarization in rat CA1 hippocampal pyramidal cells, a process called depolarization-induced suppression of inhibition (DSI). We used whole-cell patch electrodes filled with a CsCl-based solution to voltage clamp the currents. All experiments were done in the presence of 50 microM 2-amino-5-phosphonovaleric acid (APV) and 20 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to block ionotropic glutamate-induced currents and polysynaptic transmission in the slice preparation. 2. Substituting strontium (Sr2+) for extracellular calcium (Ca2+) led to the appearance of numerous 'asynchronous' small IPSCs following an eIPSC. These asynchronous IPSCs were indistinguishable from TTX-insensitive quantal IPSCs. 3. Although somewhat less effective than Ca2+, Sr2+ was capable of supporting DSI, and both asynchronous and synchronous IPSCs were blocked by the DSI process. 4. During DSI, quantal content of eIPSCs, but not quantal size, was significantly reduced. 5. Sr2+ converted paired-pulse depression (PPD) of eIPSCs to a paired-pulse facilitation (PPF), presumably by altering the probability of release at inhibitory nerve terminals. DSI had no effect on either PPD or PPF. 6. The results show that Sr2+ induces asynchronous release of GABA as it does of other neurotransmitters and changes the probability of release at GABAA ergic terminals as well. Most importantly, the results support the hypothesis that, despite being induced postsynaptically, DSI is expressed presynaptically as a decrease in GABA release, possibly by acting at a site other than the Ca(2+)-dependent release step. PMID:9423174

  5. Sr2+ supports depolarization-induced suppression of inhibition and provides new evidence for a presynaptic expression mechanism in rat hippocampal slices.

    PubMed

    Morishita, W; Alger, B E

    1997-12-01

    1. We studied the transient suppression of evoked GABAA ergic inhibitory postsynaptic currents (eIPSCs) that follows brief membrane depolarization in rat CA1 hippocampal pyramidal cells, a process called depolarization-induced suppression of inhibition (DSI). We used whole-cell patch electrodes filled with a CsCl-based solution to voltage clamp the currents. All experiments were done in the presence of 50 microM 2-amino-5-phosphonovaleric acid (APV) and 20 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to block ionotropic glutamate-induced currents and polysynaptic transmission in the slice preparation. 2. Substituting strontium (Sr2+) for extracellular calcium (Ca2+) led to the appearance of numerous 'asynchronous' small IPSCs following an eIPSC. These asynchronous IPSCs were indistinguishable from TTX-insensitive quantal IPSCs. 3. Although somewhat less effective than Ca2+, Sr2+ was capable of supporting DSI, and both asynchronous and synchronous IPSCs were blocked by the DSI process. 4. During DSI, quantal content of eIPSCs, but not quantal size, was significantly reduced. 5. Sr2+ converted paired-pulse depression (PPD) of eIPSCs to a paired-pulse facilitation (PPF), presumably by altering the probability of release at inhibitory nerve terminals. DSI had no effect on either PPD or PPF. 6. The results show that Sr2+ induces asynchronous release of GABA as it does of other neurotransmitters and changes the probability of release at GABAA ergic terminals as well. Most importantly, the results support the hypothesis that, despite being induced postsynaptically, DSI is expressed presynaptically as a decrease in GABA release, possibly by acting at a site other than the Ca(2+)-dependent release step.

  6. Kainic acid-induced neurodegeneration and activation of inflammatory processes in organotypic hippocampal slice cultures: treatment with cyclooxygenase-2 inhibitor does not prevent neuronal death.

    PubMed

    Järvelä, Juha T; Ruohonen, Saku; Kukko-Lukjanov, Tiina-Kaisa; Plysjuk, Anna; Lopez-Picon, Francisco R; Holopainen, Irma E

    2011-06-01

    In the postnatal rodent hippocampus status epilepticus (SE) leads to age- and region-specific excitotoxic neuronal damage, the precise mechanisms of which are still incompletely known. Recent studies suggest that the activation of inflammatory responses together with glial cell reactivity highly contribute to excitotoxic neuronal damage. However, pharmacological tools to attenuate their activation in the postnatal brain are still poorly elucidated. In this study, we investigated the role of inflammatory mediators in kainic acid (KA)-induced neuronal damage in organotypic hippocampal slice cultures (OHCs). A specific cyclooxygenase-2 (COX-2) inhibitor N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398) was used to study whether or not it could ameliorate neuronal death. Our results show that KA treatment (24 h) resulted in a dose-dependent degeneration of CA3a/b pyramidal neurons. Furthermore, COX-2 immunoreactivity was pronouncedly enhanced particularly in CA3c pyramidal neurons, microglial and astrocyte morphology changed from a resting to active appearance, the expression of the microglial specific protein, Iba1, increased, and prostaglandin E₂ (PGE₂) production increased. These indicated the activation of inflammatory processes. However, the expression of neither proinflammatory cytokines, i.e. tumour necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β), nor the anti-inflammatory cytokine IL-10 mRNA was significantly altered by KA treatment as studied by real-time PCR. Despite activation of an array of inflammatory processes, neuronal damage could not be rescued either with the combined pre- and co-treatment with a specific COX-2 inhibitor, NS-398. Our results suggest that KA induces activation of a repertoire of inflammatory processes in immature OHCs, and that the timing of anti-inflammatory treatment to achieve neuroprotection is a challenge due to developmental properties and the complexity of inflammatory processes activated by

  7. Clodronate inhibits the secretion of proinflammatory cytokines and NO by isolated microglial cells and reduces the number of proliferating glial cells in excitotoxically injured organotypic hippocampal slice cultures.

    PubMed

    Dehghani, Faramarz; Conrad, Ariane; Kohl, Angelika; Korf, Horst-Werner; Hailer, Nils P

    2004-10-01

    Treatment of excitotoxically injured organotypic hippocampal slice cultures (OHSC) with clodronate is known to result in the inhibition of microglial activation. We hypothesized that this is due to direct effects of clodronate on microglial cells, and investigated microglial proliferation in OHSC, and cytokine and NO secretion in isolated microglial cells. N-methyl-D-aspartate (NMDA) lesioning of OHSC resulted in a massive increase in the number of proliferating, bromo-desoxy-uridine (BrdU)-labeled cells that was reduced to control levels after treatment with clodronate (0.1, 1, 10 microg/ml). Triple-labeling revealed that clodronate abrogated the proliferation of both glial fibrillary acidic protein (GFAP)-labeled astrocytes and Griffonia simplicifolia isolectin B4 (IB4)-labeled microglial cells. Furthermore, isolated microglial cells were treated with clodronate after stimulation with lipopolysaccharide (LPS) or macrophage colony stimulating factor (M-CSF). Clodronate (0.01, 0.1, 1 microg/ml) significantly down-regulated the LPS-stimulated microglial secretion of tumor necrosis factor (TNF)-alpha, Interleukin (IL)-1beta and NO, but not of IL-6. In contrast, clodronate significantly reduced the microglial IL-6-release induced by M-CSF, indicating different intracellular pathways. The number and morphology of isolated microglial cells did not change significantly after treatment with clodronate. In summary, the number of proliferating microglial cells and astrocytes after excitotoxic injury is reduced to control levels after treatment with clodronate. Furthermore, clodronate inhibits microglial secretion of proinflammatory cytokines and NO. Clodronate could therefore prove to be a useful tool in the investigation of interactions between damaged neurons and microglial cells.

  8. Neuroprotection by JM-20 against oxygen-glucose deprivation in rat hippocampal slices: Involvement of the Akt/GSK-3β pathway.

    PubMed

    Ramírez-Sánchez, Jeney; Simões Pires, Elisa Nicoloso; Nuñez-Figueredo, Yanier; Pardo-Andreu, Gilberto L; Fonseca-Fonseca, Luis Arturo; Ruiz-Reyes, Alberto; Ochoa-Rodríguez, Estael; Verdecia-Reyes, Yamila; Delgado-Hernández, René; Souza, Diogo O; Salbego, Christianne

    2015-11-01

    Cerebral ischemia is the third most common cause of death and a major cause of disability worldwide. Beyond a shortage of essential metabolites, ischemia triggers many interconnected pathophysiological events, including excitotoxicity, oxidative stress, inflammation and apoptosis. Here, we investigated the neuroprotective mechanisms of JM-20, a novel synthetic molecule, focusing on the phosphoinositide-3-kinase (PI3K)/Akt survival pathway and glial cell response as potential targets of JM-20. For this purpose, we used organotypic hippocampal slice cultures exposed to oxygen-glucose deprivation (OGD) to achieve ischemic/reperfusion damage in vitro. Treatment with JM-20 at 0.1 and 10 μM reduced PI incorporation (indicative of cell death) after OGD. OGD decreased the phosphorylation of Akt (pro-survival) and GSK 3β (pro-apoptotic), resulting in respective inhibition and activation of these proteins. Treatment with JM20 prevented the reduced phosphorylation of these proteins after OGD, representing a shift from pro-apoptotic to pro-survival signaling. The OGD-induced activation of caspase-3 was also attenuated by JM-20 treatment at 10 μM. Moreover, in cultures treated with JM-20 and exposed to OGD conditioning, we observed a decrease in activated microglia, as well as a decrease in interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α release into the culture medium, while the level of the anti-inflammatory IL-10 increased. GFAP immunostaining and IB4 labeling showed that JM-20 treatment significantly augmented GFAP immunoreactivity after OGD, when compared with cultures exposed to OGD only, suggesting the activation of astroglial cells. Our results confirm that JM-20 has a strong neuroprotective effect against ischemic injury and suggest that the mechanisms involved in this effect may include the modulation of reactive astrogliosis, as well as neuroinflammation and the anti-apoptotic cell signaling pathway.

  9. BK channel activity determines the extent of cell degeneration after oxygen and glucose deprivation: a study in organotypical hippocampal slice cultures.

    PubMed

    Rundén-Pran, E; Haug, F M; Storm, J F; Ottersen, O P

    2002-01-01

    BK channels are voltage- and calcium-dependent potassium channels whose activation tends to reduce cellular excitability. In hippocampal pyramidal cells, BK channels repolarize somatic action potentials, and recent immunogold and electrophysiological analyses have revealed a presynaptic pool of BK channels that can regulate glutamate release. Agents that modulate BK channel activity would therefore be expected to affect cell excitability and neurotransmitter release also under pathological conditions. We have investigated the role of BK potassium channels in a model of ischemia-induced nerve cell degeneration. Organotypical slice cultures of rat hippocampus were exposed to oxygen and glucose deprivation (OGD), and cell death was assessed by the fluorescent dye propidium iodide. OGD induced cell death in the CA1 region and to a lesser extent in CA3. Treatment with the BK channel blockers, paxilline and iberiotoxin, during and after OGD induced increased cell death in CA1 and CA3. Both BK channel blockers also sensitized the relatively resistant granule cells in fascia dentata to OGD. The effect of paxilline and iberiotoxin was evident from 3 h after OGD, indicating a role of BK channels early in the post-ischemic phase or during OGD itself. The BK channel opener, NS1619, turned out to be gliotoxic, and this effect was not counteracted by paxilline and iberiotoxin. Our data show that blockade of BK channels aggravates OGD-induced cell damage and suggest that BK channels act as a kind of 'emergency brake' during and/or after ischemia. Accordingly, the BK channel is a potential molecular target for neuroprotective therapy in stroke.

  10. Brief anoxia preconditioning and HIF prolyl-hydroxylase inhibition enhances neuronal resistance in organotypic hippocampal slices on model of ischemic damage.

    PubMed

    Lushnikova, Iryna; Orlovsky, Maxim; Dosenko, Victor; Maistrenko, Anastasiia; Skibo, Galina

    2011-04-22

    It is well known that a brief anoxia or hypoxia episodes can render brain resistant to a subsequent ischemia. Recent investigations indicate that mechanisms of such stimulated endogenous neuroprotection are related to the family of hypoxia-inducible factors (HIF), however there are still little data available on the role of HIF family members in hippocampus-a brain structure, highly sensitive to oxygen deficiency. We have used the model of cultured hippocampal slices and single-cell quantitative RT-PCR to study HIF-1α and HIF-3α mRNA expression following triple 5-min mild anoxia, 30-min oxygen-glucose deprivation and their combination. We also tested the effects of HIF prolyl-hydroxylase inhibition with 2,4-pyridinedicarboxylic acid diethyl ester pre-treatment followed by a 30-min oxygen-glucose deprivation. It was found that neuronal damage induced by oxygen-glucose deprivation was accompanied by a significant decrease in both HIF-1α and HIF-3α mRNA levels in CA1 but not CA3 neurons. Anoxia preconditioning did not affect cell viability and HIF mRNA levels but applied before oxygen-glucose deprivation prevented neuronal damage and suppression of HIF-1α and HIF-3α mRNA expression. It was also found that effects of the prolyl-hydroxylase inhibitor were similar to anoxia preconditioning. These results suggest that anoxia preconditioning increases anti-ischemic neuronal resistance which to a certain extent correlates with the changes of HIF-1α and HIF-3α expression.

  11. GDNF pre-treatment aggravates neuronal cell loss in oxygen-glucose deprived hippocampal slice cultures: a possible effect of glutamate transporter up-regulation.

    PubMed

    Bonde, C; Sarup, A; Schousboe, A; Gegelashvili, G; Noraberg, J; Zimmer, J

    2003-01-01

    Besides its neurotrophic and neuroprotective effects on dopaminergic neurons and spinal motoneurons, glial cell line-derived neurotrophic factor (GDNF) has potent neuroprotective effects in cerebral ischemia. The protective effect has so far been related to reduced activation of N-methyl-D-aspartate receptors (NMDAr). This study tested the effects of GDNF on glutamate transporter expression, with the hypothesis that modulation of glutamate transporter activity would affect the outcome of cerebral ischemia. Organotypic hippocampal slice cultures, derived from 1-week-old rats, were treated with 100 ng/ml GDNF for either 2 or 5 days, followed by Western blot analysis of NMDAr subunit 1 (NR1) and two glutamate transporter subtypes, GLAST and GLT-1. After 5-day exposure to GDNF, expression of GLAST and GLT-1 was up-regulated to 169 and 181% of control values, respectively, whereas NR1 was down-regulated to 64% of control. However, despite these changes that potentially would support neuronal resistance to excitotoxicity, the long-term treatment with GDNF was found to aggravate the neuronal damage induced by oxygen-glucose deprivation (OGD). The increased cell death, assessed by propidium iodide (PI) uptake, occurred not only among the most susceptible CA1 pyramidal cells, but also in CA3 and fascia dentata. Given that glutamate transporters are able to release glutamate by reversed action during energy failure, it is suggested that the observed increase in OGD-induced cell death in the GDNF-pretreated cultures was caused by the build-up of excitotoxic concentrations of extracellular glutamate released through the glutamate transporters, which were up-regulated by GDNF. Although the extent and consequences of glutamate release via reversal of GLAST and GLT-1 transporters seem to vary in different energy failure models, the present findings should be taken into account in clinical trials of GDNF.

  12. The Effect of Acute and Chronic Social Stress on the Hippocampal Transcriptome in Mice

    PubMed Central

    Stankiewicz, Adrian M.; Goscik, Joanna; Majewska, Alicja; Swiergiel, Artur H.; Juszczak, Grzegorz R.

    2015-01-01

    Psychogenic stress contributes to the formation of brain pathology. Using gene expression microarrays, we analyzed the hippocampal transcriptome of mice subjected to acute and chronic social stress of different duration. The longest period of social stress altered the expression of the highest number of genes and most of the stress-induced changes in transcription were reversible after 5 days of rest. Chronic stress affected genes involved in the functioning of the vascular system (Alas2, Hbb-b1, Hba-a2, Hba-a1), injury response (Vwf, Mgp, Cfh, Fbln5, Col3a1, Ctgf) and inflammation (S100a8, S100a9, Ctla2a, Ctla2b, Lcn2, Lrg1, Rsad2, Isg20). The results suggest that stress may affect brain functions through the stress-induced dysfunction of the vascular system. An important issue raised in our work is also the risk of the contamination of brain tissue samples with choroid plexus. Such contamination would result in a consistent up- or down-regulation of genes, such as Ttr, Igf2, Igfbp2, Prlr, Enpp2, Sostdc1, 1500015O10RIK (Ecrg4), Kl, Clic6, Kcne2, F5, Slc4a5, and Aqp1. Our study suggests that some of the previously reported, supposedly specific changes in hippocampal gene expression, may be a result of the inclusion of choroid plexus in the hippocampal samples. PMID:26556046

  13. The Effect of Acute and Chronic Social Stress on the Hippocampal Transcriptome in Mice.

    PubMed

    Stankiewicz, Adrian M; Goscik, Joanna; Majewska, Alicja; Swiergiel, Artur H; Juszczak, Grzegorz R

    2015-01-01

    Psychogenic stress contributes to the formation of brain pathology. Using gene expression microarrays, we analyzed the hippocampal transcriptome of mice subjected to acute and chronic social stress of different duration. The longest period of social stress altered the expression of the highest number of genes and most of the stress-induced changes in transcription were reversible after 5 days of rest. Chronic stress affected genes involved in the functioning of the vascular system (Alas2, Hbb-b1, Hba-a2, Hba-a1), injury response (Vwf, Mgp, Cfh, Fbln5, Col3a1, Ctgf) and inflammation (S100a8, S100a9, Ctla2a, Ctla2b, Lcn2, Lrg1, Rsad2, Isg20). The results suggest that stress may affect brain functions through the stress-induced dysfunction of the vascular system. An important issue raised in our work is also the risk of the contamination of brain tissue samples with choroid plexus. Such contamination would result in a consistent up- or down-regulation of genes, such as Ttr, Igf2, Igfbp2, Prlr, Enpp2, Sostdc1, 1500015O10RIK (Ecrg4), Kl, Clic6, Kcne2, F5, Slc4a5, and Aqp1. Our study suggests that some of the previously reported, supposedly specific changes in hippocampal gene expression, may be a result of the inclusion of choroid plexus in the hippocampal samples.

  14. Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

    PubMed Central

    Qi, Guanxiao; Radnikow, Gabriele; Feldmeyer, Dirk

    2015-01-01

    The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain. PMID:25650985

  15. Correlated memory defects and hippocampal dendritic spine loss after acute stress involve corticotropin-releasing hormone signaling.

    PubMed

    Chen, Yuncai; Rex, Christopher S; Rice, Courtney J; Dubé, Céline M; Gall, Christine M; Lynch, Gary; Baram, Tallie Z

    2010-07-20

    Stress affects the hippocampus, a brain region crucial for memory. In rodents, acute stress may reduce density of dendritic spines, the location of postsynaptic elements of excitatory synapses, and impair long-term potentiation and memory. Steroid stress hormones and neurotransmitters have been implicated in the underlying mechanisms, but the role of corticotropin-releasing hormone (CRH), a hypothalamic hormone also released during stress within hippocampus, has not been elucidated. In addition, the causal relationship of spine loss and memory defects after acute stress is unclear. We used transgenic mice that expressed YFP in hippocampal neurons and found that a 5-h stress resulted in profound loss of learning and memory. This deficit was associated with selective disruption of long-term potentiation and of dendritic spine integrity in commissural/associational pathways of hippocampal area CA3. The degree of memory deficit in individual mice correlated significantly with the reduced density of area CA3 apical dendritic spines in the same mice. Moreover, administration of the CRH receptor type 1 (CRFR(1)) blocker NBI 30775 directly into the brain prevented the stress-induced spine loss and restored the stress-impaired cognitive functions. We conclude that acute, hours-long stress impairs learning and memory via mechanisms that disrupt the integrity of hippocampal dendritic spines. In addition, establishing the contribution of hippocampal CRH-CRFR(1) signaling to these processes highlights the complexity of the orchestrated mechanisms by which stress impacts hippocampal structure and function.

  16. Pine needle extract prevents hippocampal memory impairment in acute restraint stress mouse model.

    PubMed

    Lee, Jin-Seok; Kim, Hyeong-Geug; Lee, Hye-Won; Kim, Won-Yong; Ahn, Yo-Chan; Son, Chang-Gue

    2017-07-31

    The Pinus densiflora leaf has been traditionally used to treat mental health disorders as a traditional Chinese medicine. Here we examined the ethnopharmacological relevance of pine needle on memory impairment caused by stress. To elucidate the possible modulatory actions of 30% ethanolic pine needle extract (PNE) on stress-induced hippocampal excitotoxicity, we adopted an acute restraint stress mouse model. Mice were orally administered with PNE (25, 50, or 100mg/kg) or ascorbic acid (100mg/kg) for 9 days, and were then subjected to restraint stress (6h/day) for 3 days (from experimental day 7-9). To evaluate spatial cognitive and memory function, the Morris water maze was performed during experimental days 5-9. Restraint stress induced the memory impairment (the prolonged escape latency and cumulative path-length, and reduced time spent in the target quadrant), and these effects were significantly prevented by PNE treatment. The levels of corticosterone and its receptor in the sera/hippocampus were increased by restraint stress, which was normalized by PNE treatment. Restraint stress elicited the hippocampal excitotoxicity, the inflammatory response and oxidative injury as demonstrated by the increased glutamate levels, altered levels of tumor necrosis factor (TNF)-α and imbalanced oxidant-antioxidant balance biomarkers. Two immunohistochemistry activities against glial fibrillary acidic protein (GFAP)-positive astrocytes and neuronal nuclei (NeuN)-positive neurons supported the finding of excitotoxicity especially in the cornu ammonis (CA)3 region of the hippocampus. Those alterations were notably attenuated by administration of PNE. The above findings showed that PNE has pharmacological properties that modulate the hippocampal excitotoxicity-derived memory impairment under severe stress conditions. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  17. Effects of normobaric versus hyperbaric oxygen on cell injury induced by oxygen and glucose deprivation in acute brain slices

    PubMed Central

    Chazalviel, Laurent; Blatteau, Jean-Eric; Vallée, Nicolas; Risso, Jean-Jacques; Besnard, Stéphane; Abraini, Jacques H.

    2016-01-01

    Normobaric oxygen (NBO) and hyperbaric oxygen (HBO) are emerging as a possible co-treatment of acute ischemic stroke. Both have been shown to reduce infarct volume, to improve neurologic outcome, to promote endogenous tissue plasminogen activator-induced thrombolysis and cerebral blood flow, and to improve tissue oxygenation through oxygen diffusion in the ischemic areas, thereby questioning the interest of HBO compared to NBO. In the present study, in order to investigate and compare the oxygen diffusion effects of NBO and HBO on acute ischemic stroke independently of their effects at the vascular level, we used acute brain slices exposed to oxygen and glucose deprivation, an ex vivo model of brain ischemia that allows investigating the acute effects of NBO (partial pressure of oxygen (pO2) = 1 atmospheres absolute (ATA) = 0.1 MPa) and HBO (pO2 = 2.5 ATA = 0.25 MPa) through tissue oxygenation on ischemia-induced cell injury as measured by the release of lactate dehydrogenase. We found that HBO, but not NBO, reduced oxygen and glucose deprivation-induced cell injury, indicating that passive tissue oxygenation (i.e. without vascular support) of the brain parenchyma requires oxygen partial pressure higher than 1 ATA. PMID:27867486

  18. Effects of normobaric versus hyperbaric oxygen on cell injury induced by oxygen and glucose deprivation in acute brain slices.

    PubMed

    Chazalviel, Laurent; Blatteau, Jean-Eric; Vallée, Nicolas; Risso, Jean-Jacques; Besnard, Stéphane; Abraini, Jacques H

    2016-01-01

    Normobaric oxygen (NBO) and hyperbaric oxygen (HBO) are emerging as a possible co-treatment of acute ischemic stroke. Both have been shown to reduce infarct volume, to improve neurologic outcome, to promote endogenous tissue plasminogen activator-induced thrombolysis and cerebral blood flow, and to improve tissue oxygenation through oxygen diffusion in the ischemic areas, thereby questioning the interest of HBO compared to NBO. In the present study, in order to investigate and compare the oxygen diffusion effects of NBO and HBO on acute ischemic stroke independently of their effects at the vascular level, we used acute brain slices exposed to oxygen and glucose deprivation, an ex vivo model of brain ischemia that allows investigating the acute effects of NBO (partial pressure of oxygen (pO2) = 1 atmospheres absolute (ATA) = 0.1 MPa) and HBO (pO2 = 2.5 ATA = 0.25 MPa) through tissue oxygenation on ischemia-induced cell injury as measured by the release of lactate dehydrogenase. We found that HBO, but not NBO, reduced oxygen and glucose deprivation-induced cell injury, indicating that passive tissue oxygenation (i.e. without vascular support) of the brain parenchyma requires oxygen partial pressure higher than 1 ATA.

  19. Analyzing the relationship between decorrelation time and tissue thickness in acute rat brain slices using multispeckle diffusing wave spectroscopy

    PubMed Central

    Brake, Joshua; Jang, Mooseok; Yang, Changhuei

    2016-01-01

    Novel techniques in the field of wavefront shaping have enabled light to be focused deep inside or through scattering media such as biological tissue. However, most of these demonstrations have been limited to thin, static samples since these techniques are very sensitive to changes in the arrangement of the scatterers within. As the samples of interest get thicker, the influence of the dynamic nature of the sample becomes even more pronounced and the window of time in which the wavefront solutions remain valid shrinks further. In this paper, we examine the time scales upon which this decorrelation happens in acute rat brain slices via multispeckle diffusing wave spectroscopy and investigate the relationship between this decorrelation time and the thickness of the sample using diffusing wave spectroscopy theory and Monte Carlo photon transport simulation. PMID:26831778

  20. Acute Slices of Mice Testis Seminiferous Tubules Unveil Spontaneous and Synchronous Ca2+ Oscillations in Germ Cell Clusters1

    PubMed Central

    Sánchez-Cárdenas, Claudia; Guerrero, Adán; Treviño, Claudia Lydia; Hernández-Cruz, Arturo; Darszon, Alberto

    2012-01-01

    ABSTRACT Spermatogenic cell differentiation involves changes in the concentration of cytoplasmic Ca2+ ([Ca2+]i); however, very few studies exist on [Ca2+]i dynamics in these cells. Other tissues display Ca2+ oscillations involving multicellular functional arrangements. These phenomena have been studied in acute slice preparations that preserve tissue architecture and intercellular communications. Here we report the implementation of intracellular Ca2+ imaging in a sliced seminiferous tubule (SST) preparation to visualize [Ca2+]i changes of living germ cells in situ within the SST preparation. Ca2+ imaging revealed that a subpopulation of male germ cells display spontaneous [Ca2+]i fluctuations resulting from Ca2+ entry possibly throughout CaV3 channels. These [Ca2+]i fluctuation patterns are also present in single acutely dissociated germ cells, but they differ from those recorded from germ cells in the SST preparation. Often, spontaneous Ca2+ fluctuations of spermatogenic cells in the SST occur synchronously, so that clusters of cells can display Ca2+ oscillations for at least 10 min. Synchronous Ca2+ oscillations could be mediated by intercellular communication via gap junctions, although intercellular bridges could also be involved. We also observed an increase in [Ca2+]i after testosterone application, suggesting the presence of functional Sertoli cells in the SST. In summary, we believe that the SST preparation is suitable to explore the physiology of spermatogenic cells in their natural environment, within the seminiferous tubules, in particular Ca2+ signaling phenomena, functional cell-cell communication, and multicellular functional arrangements. PMID:22914313

  1. Pharmacological antagonism of the actions of group II and III mGluR agonists in the lateral perforant path of rat hippocampal slices.

    PubMed Central

    Bushell, T. J.; Jane, D. E.; Tse, H. W.; Watkins, J. C.; Garthwaite, J.; Collingridge, G. L.

    1996-01-01

    1. An understanding of the physiological and pathological roles of metabotropic glutamate receptors (mGluRs) is currently hampered by the lack of selective antagonists. Standard extracellular recording techniques were used to investigate the activity of recently reported mGluR antagonists on agonist-induced depressions of synaptic transmission in the lateral perforant path of hippocampal slices obtained from 12-16 day-old rats. 2. The group III specific mGluR agonist, (S)-2-amino-4-phosphonobutanoate (L-AP4) depressed basal synaptic transmission in a reversible and dose-dependent manner. The mean (+/-s.e. mean) depression obtained with 100 microM L-AP4 (the maximum concentration tested) was 74 +/- 3% and the IC50 value was 3 +/- 1 microM (n = 5). 3. The selective group II mGluR agonists, (1S,3S)-1-aminocyclopentane-1, 3-dicarboxylate ((1S,3s)-ACPD) and (2S, 1'R, 2'R, 3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) also depressed basal synaptic transmission in a reversible and dose-dependent manner. The mean depression obtained with 200 microM (1S,3S)-ACPD was 83 +/- 8% and the IC50 value was 12 +/- 3 microM (n = 5). The mean depression obtained with 1 microM DCG-IV was 73 +/- 7% and the IC50 value was 88 +/- 15 nM (n = 4). 4. Synaptic depressions induced by the actions of 20 microM (1S,3S)-ACPD and 10 microM L-AP4 were antagonized by the mGluR antagonists (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG), (S)-2-methyl-2-amino-4-phosphonobutanoate (MAP4), (2S,1'S,2'S)-2-methyl-2(2'-carboxycyclopropyl)glycine (MCCG), (RS)-alpha-methyl-4-tetrazolylphenylglycine (MTPG), (RS)-alpha-methyl-4-sulphonophenylglycine (MSPG) and (RS)-alpha-methyl-4-phosphonophenylglycine (MPPG) (all tested at 500 microM). 5. (+)-MCPG was a weak antagonist of both L-AP4 and (1S,3S)-ACPD-induced depressions. MCCG was selective towards (1S,3S)-ACPD, but analysis of its effects were complicated by apparent partial agonist activity. MAP4 showed good selectivity for L-AP4-induced effects. 6

  2. Pharmacological antagonism of the actions of group II and III mGluR agonists in the lateral perforant path of rat hippocampal slices.

    PubMed

    Bushell, T J; Jane, D E; Tse, H W; Watkins, J C; Garthwaite, J; Collingridge, G L

    1996-04-01

    1. An understanding of the physiological and pathological roles of metabotropic glutamate receptors (mGluRs) is currently hampered by the lack of selective antagonists. Standard extracellular recording techniques were used to investigate the activity of recently reported mGluR antagonists on agonist-induced depressions of synaptic transmission in the lateral perforant path of hippocampal slices obtained from 12-16 day-old rats. 2. The group III specific mGluR agonist, (S)-2-amino-4-phosphonobutanoate (L-AP4) depressed basal synaptic transmission in a reversible and dose-dependent manner. The mean (+/-s.e. mean) depression obtained with 100 microM L-AP4 (the maximum concentration tested) was 74 +/- 3% and the IC50 value was 3 +/- 1 microM (n = 5). 3. The selective group II mGluR agonists, (1S,3S)-1-aminocyclopentane-1, 3-dicarboxylate ((1S,3s)-ACPD) and (2S, 1'R, 2'R, 3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) also depressed basal synaptic transmission in a reversible and dose-dependent manner. The mean depression obtained with 200 microM (1S,3S)-ACPD was 83 +/- 8% and the IC50 value was 12 +/- 3 microM (n = 5). The mean depression obtained with 1 microM DCG-IV was 73 +/- 7% and the IC50 value was 88 +/- 15 nM (n = 4). 4. Synaptic depressions induced by the actions of 20 microM (1S,3S)-ACPD and 10 microM L-AP4 were antagonized by the mGluR antagonists (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG), (S)-2-methyl-2-amino-4-phosphonobutanoate (MAP4), (2S,1'S,2'S)-2-methyl-2(2'-carboxycyclopropyl)glycine (MCCG), (RS)-alpha-methyl-4-tetrazolylphenylglycine (MTPG), (RS)-alpha-methyl-4-sulphonophenylglycine (MSPG) and (RS)-alpha-methyl-4-phosphonophenylglycine (MPPG) (all tested at 500 microM). 5. (+)-MCPG was a weak antagonist of both L-AP4 and (1S,3S)-ACPD-induced depressions. MCCG was selective towards (1S,3S)-ACPD, but analysis of its effects were complicated by apparent partial agonist activity. MAP4 showed good selectivity for L-AP4-induced effects. 6

  3. Postsynaptic potentials mediated by excitatory and inhibitory amino acids in interneurons of stratum pyramidale of the CA1 region of rat hippocampal slices in vitro.

    PubMed

    Lacaille, J C

    1991-11-01

    1. Because interneurons of stratum pyramidale partly mediate the feed-forward inhibition of pyramidal cells, intracellular postsynaptic potentials (PSPs) evoked by activation of afferent fibers were examined in 32 nonpyramidal cells of stratum pyramidale of the CA1 region of rat hippocampal slices. 2. Electrical stimulation of stratum radiatum at the CA1-CA3 border elicited, in interneurons, PSPs that were composed of four components: a fast excitatory postsynaptic potential (EPSP), an early inhibitory postsynaptic potential (IPSPA), a late IPSPB, and in some cells a delayed, slower EPSP. These synaptic potentials summated and elicited single action potentials in 57% of cells (17/30) and burst of action potentials (2-10) in the remaining 43%. 3. The fast EPSP was observed in all cells, and the mean stimulation intensity at its threshold was 53.4 microA. Its amplitude increased with membrane hyperpolarization, and it was associated with a 45.4% decrease in cellular input resistance. The fast EPSP always elicited an action potential at short latencies (3.6-6.4 ms poststimulation). It was reversibly reduced by 6-cyano-7-nitroquinoxaline-2,3- dione (CNQX), a blocker of non-N-methyl-D-aspartate (non-NMDA) excitatory amino acid receptors. 4. The IPSPA was observed in 28/32 cells, and the mean intensity of stimulation was 57.6 microA at its threshold. The mean latency of its peak amplitude was 17.4 ms. The mean equilibrium potential (Erev) was -72.8 mV, and it was associated with a 38.9% decrease in cellular input resistance. IPSPA was blocked by the GABAA antagonist bicuculline. 5. The IPSPB was seen in 29/32 cells, and the mean intensity of stimulation at its threshold was 80.3 microA. Its latency to peak was 130.6 ms, its Erev was -107.6 mV, and it was associated with a small (7.6%) decrease in cellular input resistance. IPSPB was blocked by the GABAB antagonist phaclofen. 6. In 11/32 cells a slower EPSP was also observed. Its mean latency to peak was 53.3 ms, and the

  4. Modifications of excitatory and inhibitory transmission in rat hippocampal pyramidal neurons by acute lithium treatment.

    PubMed

    Wakita, Masahito; Nagami, Hideaki; Takase, Yuko; Nakanishi, Ryoji; Kotani, Naoki; Akaike, Norio

    2015-08-01

    The acute effects of high-dose Li(+) treatment on glutamatergic and GABAergic transmissions were studied in the "synaptic bouton" preparation of isolated rat hippocampal pyramidal neurons by using focal electrical stimulation. Both action potential-dependent glutamatergic excitatory and GABAergic inhibitory postsynaptic currents (eEPSC and eIPSC, respectively) were dose-dependently inhibited in the external media containing 30-150 mM Li(+), but the sensitivity for Li(+) was greater tendency for eEPSCs than for eIPSCs. When the effects of Li(+) on glutamate or GABAA receptor-mediated whole-cell responses (IGlu and IGABA) elicited by an exogenous application of glutamate or GABA were examined in the postsynaptic soma membrane of CA3 neurons, Li(+) slightly inhibited both IGlu and IGABA at the 150 mM Li(+) concentration. Present results suggest that acute treatment with high concentrations of Li(+) acts preferentially on presynaptic terminals, and that the Li(+)-induced inhibition may be greater for excitatory than for inhibitory transmission.

  5. Acute running stimulates hippocampal dopaminergic neurotransmission in rats, but has no influence on brain-derived neurotrophic factor.

    PubMed

    Goekint, Maaike; Bos, Inge; Heyman, Elsa; Meeusen, Romain; Michotte, Yvette; Sarre, Sophie

    2012-02-01

    Hippocampal brain-derived neurotrophic factor (BDNF) protein is increased with exercise in rats. Monoamines seem to play a role in the regulation of BDNF, and monoamine neurotransmission is known to increase with exercise. The purpose of this study was to examine the influence of acute exercise on monoaminergic neurotransmission and BDNF protein concentrations. Hippocampal microdialysis was performed in rats that were subjected to 60 min of treadmill running at 20 m/min or rest. Two hours postexercise, the rats were killed, and the hippocampus was dissected. In experiments without microdialysis, hippocampus and serum samples were collected immediately after exercise. Exercise induced a twofold increase in hippocampal dopamine release. Noradrenaline and serotonin release were not affected. Hippocampal BDNF levels were not influenced, whether they were measured immediately or 2 h after the exercise protocol. Serum BDNF levels did not change either, but serum BDNF was negatively correlated to peripheral corticosterone concentrations, indicating a possible inhibitory reaction to the stress of running. Sixty minutes of exercise enhances dopamine release in the hippocampus of the rat in vivo. However, this increase is not associated with changes in BDNF protein levels immediately nor 2 h after the acute exercise bout. An increased corticosterone level might be the contributing factor for the absence of changes in BDNF.

  6. Dibucaine Mitigates Spreading Depolarization in Human Neocortical Slices and Prevents Acute Dendritic Injury in the Ischemic Rodent Neocortex

    PubMed Central

    Risher, W. Christopher; Lee, Mark R.; Fomitcheva, Ioulia V.; Hess, David C.; Kirov, Sergei A.

    2011-01-01

    Background Spreading depolarizations that occur in patients with malignant stroke, subarachnoid/intracranial hemorrhage, and traumatic brain injury are known to facilitate neuronal damage in metabolically compromised brain tissue. The dramatic failure of brain ion homeostasis caused by propagating spreading depolarizations results in neuronal and astroglial swelling. In essence, swelling is the initial response and a sign of the acute neuronal injury that follows if energy deprivation is maintained. Choosing spreading depolarizations as a target for therapeutic intervention, we have used human brain slices and in vivo real-time two-photon laser scanning microscopy in the mouse neocortex to study potentially useful therapeutics against spreading depolarization-induced injury. Methodology/Principal Findings We have shown that anoxic or terminal depolarization, a spreading depolarization wave ignited in the ischemic core where neurons cannot repolarize, can be evoked in human slices from pediatric brains during simulated ischemia induced by oxygen/glucose deprivation or by exposure to ouabain. Changes in light transmittance (LT) tracked terminal depolarization in time and space. Though spreading depolarizations are notoriously difficult to block, terminal depolarization onset was delayed by dibucaine, a local amide anesthetic and sodium channel blocker. Remarkably, the occurrence of ouabain-induced terminal depolarization was delayed at a concentration of 1 µM that preserves synaptic function. Moreover, in vivo two-photon imaging in the penumbra revealed that, though spreading depolarizations did still occur, spreading depolarization-induced dendritic injury was inhibited by dibucaine administered intravenously at 2.5 mg/kg in a mouse stroke model. Conclusions/Significance Dibucaine mitigated the effects of spreading depolarization at a concentration that could be well-tolerated therapeutically. Hence, dibucaine is a promising candidate to protect the brain from

  7. Short-term memory deficits correlate with hippocampal-thalamic functional connectivity alterations following acute sleep restriction.

    PubMed

    Chengyang, Li; Daqing, Huang; Jianlin, Qi; Haisheng, Chang; Qingqing, Meng; Jin, Wang; Jiajia, Liu; Enmao, Ye; Yongcong, Shao; Xi, Zhang

    2016-07-21

    Acute sleep restriction heavily influences cognitive function, affecting executive processes such as attention, response inhibition, and memory. Previous neuroimaging studies have suggested a link between hippocampal activity and short-term memory function. However, the specific contribution of the hippocampus to the decline of short-term memory following sleep restriction has yet to be established. In the current study, we utilized resting-state functional magnetic resonance imaging (fMRI) to examine the association between hippocampal functional connectivity (FC) and the decline of short-term memory following total sleep deprivation (TSD). Twenty healthy adult males aged 20.9 ± 2.3 years (age range, 18-24 years) were enrolled in a within-subject crossover study. Short-term memory and FC were assessed using a Delay-matching short-term memory test and a resting-state fMRI scan before and after TSD. Seed-based correlation analysis was performed using fMRI data for the left and right hippocampus to identify differences in hippocampal FC following TSD. Subjects demonstrated reduced alertness and a decline in short-term memory performance following TSD. Moreover, fMRI analysis identified reduced hippocampal FC with the superior frontal gyrus (SFG), temporal regions, and supplementary motor area. In addition, an increase in FC between the hippocampus and bilateral thalamus was observed, the extent of which correlated with short-term memory performance following TSD. Our findings indicate that the disruption of hippocampal-cortical connectivity is linked to the decline in short-term memory observed after acute sleep restriction. Such results provide further evidence that support the cognitive impairment model of sleep deprivation.

  8. The trapping block of NMDA receptor channels in acutely isolated rat hippocampal neurones

    PubMed Central

    Sobolevsky, Alexander I; Yelshansky, Maria V

    2000-01-01

    N-methyl-d-aspartate (NMDA) receptor responses were recorded from acutely isolated rat hippocampal neurones using the whole-cell patch-clamp technique. A rapid perfusion system was used to study the voltage-dependent block of NMDA channels by Mg2+, amantadine (AM) and N-2-(adamantyl)-hexamethylenimine (A-7). Mg2+, AM and A-7-induced stationary blockade of NMDA channels increased with the blocker concentration but did not depend on the agonist (aspartate; Asp) concentration. Blockade by AM and A-7, but not Mg2+, was weakly use dependent. ‘Hooked’ tail currents were observed after coapplication of Asp and Mg2+, AM or A-7. The hooked tail current kinetics, amplitude and carried charge indicated that Mg2+, AM and A-7 did not prevent closure and desensitization of NMDA channels nor agonist dissociation. Tail currents following Asp application in the absence and continuous presence of Mg2+, AM or A-7 had similar kinetics. Application of multiple stationary and kinetic criteria to the Mg2+, AM and A-7 blockade led us to conclude that their effects on NMDA channels can be described in terms of a ‘trapping’ model, which is fully symmetrical with respect to the blocking transition. In general, the apparent blocking/recovery kinetics predicted by the fully symmetrical trapping model differ significantly from the microscopic kinetics and depend on the rate of binding and unbinding of the blocker, the NMDA channel open probability and the rate of solution exchange. PMID:10922002

  9. Acute postoperative seizures and long-term seizure outcome after surgery for hippocampal sclerosis.

    PubMed

    Di Gennaro, Giancarlo; Casciato, Sara; Quarato, Pier Paolo; Mascia, Addolorata; D'Aniello, Alfredo; Grammaldo, Liliana G; De Risi, Marco; Meldolesi, Giulio N; Romigi, Andrea; Esposito, Vincenzo; Picardi, Angelo

    2015-01-01

    To assess the incidence and the prognostic value of acute postoperative seizures (APOS) in patients surgically treated for drug-resistant temporal lobe epilepsy due to hippocampal sclerosis (TLE-HS). We studied 139 consecutive patients with TLE-HS who underwent epilepsy surgery and were followed up for at least 5 years (mean duration of follow-up 9.1 years, range 5-15). Medical charts were reviewed to identify APOS, defined as ictal events with the exception of auras occurring within the first 7 days after surgery. Seizure outcome was determined at annual intervals. Patients who were in Engel Class Ia at the last contact were classified as having a favorable outcome. Seizure outcome was favorable in 99 patients (71%). Six patients (4%) experienced APOS and in all cases their clinical manifestations were similar to the habitual preoperative seizures. All patients with APOS had unfavorable long-term outcome, as compared with 35 (26%) of 133 in whom APOS did not occur (p<0.001). Our study suggests that APOS, despite being relatively uncommon in patients undergoing resective surgery for TLE-HS, are associated with a worse long-term seizure outcome. Given some study limitations, our findings should be regarded as preliminary and need confirmation from future larger, prospective, multicenter studies. Copyright © 2014 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

  10. KCNQ/Kv7 channel activator flupirtine protects against acute stress-induced impairments of spatial memory retrieval and hippocampal LTP in rats.

    PubMed

    Li, C; Huang, P; Lu, Q; Zhou, M; Guo, L; Xu, X

    2014-11-07

    Spatial memory retrieval and hippocampal long-term potentiation (LTP) are impaired by stress. KCNQ/Kv7 channels are closely associated with memory and the KCNQ/Kv7 channel activator flupirtine represents neuroprotective effects. This study aims to test whether KCNQ/Kv7 channel activation prevents acute stress-induced impairments of spatial memory retrieval and hippocampal LTP. Rats were placed on an elevated platform in the middle of a bright room for 30 min to evoke acute stress. The expression of KCNQ/Kv7 subunits was analyzed at 1, 3 and 12 h after stress by Western blotting. Spatial memory was examined by the Morris water maze (MWM) and the field excitatory postsynaptic potential (fEPSP) in the hippocampal CA1 area was recorded in vivo. Acute stress transiently decreased the expression of KCNQ2 and KCNQ3 in the hippocampus. Acute stress impaired the spatial memory retrieval and hippocampal LTP, the KCNQ/Kv7 channel activator flupirtine prevented the impairments, and the protective effects of flupirtine were blocked by XE-991 (10,10-bis(4-Pyridinylmethyl)-9(10H)-anthracenone), a selective KCNQ channel blocker. Furthermore, acute stress decreased the phosphorylation of glycogen synthase kinase-3β (GSK-3β) at Ser9 in the hippocampus, and flupirtine inhibited the reduction. These results suggest that the KCNQ/Kv7 channels may be a potential target for protecting both hippocampal synaptic plasticity and spatial memory retrieval from acute stress influences.

  11. Neuroprotective effects of inhibiting N-methyl-D-aspartate receptors, P2X receptors and the mitogen-activated protein kinase cascade: a quantitative analysis in organotypical hippocampal slice cultures subjected to oxygen and glucose deprivation.

    PubMed

    Rundén-Pran, E; Tansø, R; Haug, F M; Ottersen, O P; Ring, A

    2005-01-01

    Cell death was assessed by quantitative analysis of propidium iodide uptake in rat hippocampal slice cultures transiently exposed to oxygen and glucose deprivation, an in vitro model of brain ischemia. The hippocampal subfields CA1 and CA3, and fascia dentata were analyzed at different stages from 0 to 48 h after the insult. Cell death appeared at 3 h and increased steeply toward 12 h. Only a slight additional increase in propidium iodide uptake was seen at later intervals. The mitogen-activated protein kinases extracellular signal-regulated kinase 1 and extracellular signal-regulated kinase 2 were activated immediately after oxygen and glucose deprivation both in CA1 and in CA3/fascia dentata. Inhibition of the specific mitogen-activated protein kinase activator mitogen-activated protein kinase kinase by PD98059 or U0126 offered partial protection against oxygen and glucose deprivation-induced cell damage. The non-selective P2X receptor antagonist suramin gave neuroprotection of the same magnitude as the N-methyl-D-aspartate channel blocker MK-801 (approximately 70%). Neuroprotection was also observed with the P2 receptor blocker PPADS. Immunogold data indicated that hippocampal slice cultures (like intact hippocampi) express several isoforms of P2X receptors at the synaptic level, consistent with the idea that the effects of suramin and PPADS are mediated by P2X receptors. Virtually complete neuroprotection was obtained by combined blockade of N-methyl-D-aspartate receptors, P2X receptors, and mitogen-activated protein kinase kinase. Both P2X receptors and N-methyl-D-aspartate receptors mediate influx of calcium. Our results suggest that inhibition of P2X receptors has a neuroprotective potential similar to that of inhibition of N-methyl-D-aspartate receptors. In contrast, our comparative analysis shows that only partial protection can be achieved by inhibiting the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase cascade, one of the

  12. Neuroprotection of rat hippocampal slices exposed to oxygen-glucose deprivation by enrichment with docosahexaenoic acid and by inhibition of hydrolysis of docosahexaenoic acid-containing phospholipids by calcium independent phospholipase A2.

    PubMed

    Strokin, M; Chechneva, O; Reymann, K G; Reiser, G

    2006-06-30

    Polyunsaturated fatty acids play an important role in the development of pathological states in brain after hypoxia/ischemia. Here, we investigated the role of docosahexaenoic acid (22:6n-3) in brain phospholipids for neuronal survival. We used organotypic cultures of rat brain hippocampal slices exposed to 40 min of oxygen-glucose deprivation, to study the consequences of experimental ischemia. In [14C]docosahexaenoic acid-labeled cultures, oxygen-glucose deprivation induced significant release of radioactive docosahexaenoic acid. This release could be blocked by the selective inhibitor of the Ca2+-independent phospholipase A2, 4-bromoenol lactone (10 microM), when it was added 30 min prior to oxygen-glucose deprivation. Addition of 4-bromoenol lactone at 30 min prior to oxygen-glucose deprivation markedly decreased the neuronal damage induced by oxygen-glucose deprivation. The protective effect was substantially higher in dentate gyrus than in CA1 and CA3 areas. Enrichment of the hippocampal tissue with docosahexaenoic acid by incubation with 10 microM docosahexaenoic acid for 24 h exerted the same neuroprotective effect, which was observed after treatment with 4-bromoenol lactone. In contrast to the 24 h-preincubation, simultaneous addition of docosahexaenoic acid with the onset of oxygen-glucose deprivation had no protective effect. This suggests that incorporation of docosahexaenoic acid into phospholipids is required for the protective effect observed. Then the possible involvement of arachidonic acid metabolism in docosahexaenoic acid-induced neuroprotection was tested. Inhibition of prostaglandin production by ibuprofen produced no change in neuroprotection after 24-h incubation of the hippocampal slices with docosahexaenoic acid. Simultaneous inhibition of Ca2+-independent and Ca2+-dependent phospholipases A2 by treatment with the general phospholipase A2 inhibitor methyl arachidonyl fluorophosphonate (3 microM, 30 min prior to oxygen-glucose deprivation

  13. Apoptosis of Hippocampal Pyramidal Neurons Is Virus Independent in a Mouse Model of Acute Neurovirulent Picornavirus Infection

    PubMed Central

    Buenz, Eric J.; Sauer, Brian M.; LaFrance-Corey, Reghann G.; Deb, Chandra; Denic, Aleksandar; German, Christopher L.; Howe, Charles L.

    2009-01-01

    Many viruses, including picornaviruses, have the potential to infect the central nervous system (CNS) and stimulate a neuroinflammatory immune response, especially in infants and young children. Cognitive deficits associated with CNS picornavirus infection result from injury and death of neurons that may occur due to direct viral infection or during the immune responses to virus in the brain. Previous studies have concluded that apoptosis of hippocampal neurons during picornavirus infection is a cell-autonomous event triggered by direct neuronal infection. However, these studies assessed neuron death at time points late in infection and during infections that lead to either death of the host or persistent viral infection. In contrast, many neurovirulent picornavirus infections are acute and transient, with rapid clearance of virus from the host. We provide evidence of hippocampal pathology in mice acutely infected with the Theiler’s murine encephalomyelitis picornavirus. We found that CA1 pyramidal neurons exhibited several hallmarks of apoptotic death, including caspase-3 activation, DNA fragmentation, and chromatin condensation within 72 hours of infection. Critically, we also found that many of the CA1 pyramidal neurons undergoing apoptosis were not infected with virus, indicating that neuronal cell death during acute picornavirus infection of the CNS occurs in a non–cell-autonomous manner. These observations suggest that therapeutic strategies other than antiviral interventions may be useful for neuroprotection during acute CNS picornavirus infection. PMID:19608874

  14. [Effects of coriaria lactone on the calcium currents of acutely isolated rat hippocampal neurons].

    PubMed

    Lai, Xiaohui; Zhang, Qin; Zhou, Dong

    2008-10-01

    The hippocampal neurons of Sprague-Dawley rat (post natal days 7 to 14) were acutely isolated using trypsin and mechanical dissociation. AgNO3 staining was performed to identify them. Voltage-dependent inward calcium currents were recorded by employing the patch-clamp technique in the whole-cell voltage-clamp mode. With different stimulation processes, we studied the changes of low voltage activated (LVA) calcium currents, the transient and sustained components of high voltage-activated calcium currents (HVA) in the hippocampal neuron membrane. The effects of CL on the peak currents in the neuron membrane were assessed and analyzed. The main sustained components ofHVA were L-type calcium currents. The HVA calcium currents were slowly inactivated in 300 ms. The changes of the current amplitude of the sustained components of HVA were insignificant with the holding potential of -100 mV and -50 mV which indicated that the inactivation could be removed with the potential of -50 mV. The HVA calcium currents were evoked by depolarizing voltage steps over the range of -70 mV to +50 mV for 300ms with the holding potential of -50 mV and the threshold potential of -40 mV or so. The HVA and LVA calcium currents were evoked by the same stimulation with the holding potential of -100 mV and the total calcium currents appeared at the potential of -60 mV. The amplitude of peak currents significantly increased (P<0.05). The transient LVA calcium currents as the peak calcium currents were evoked by depolarizing voltage steps over the range of -70 mV to -30 mV with the holding potential of -100 mV. Both HVA and LVA calcium currents appeared over the range of -30 mV to +10 mV. The HVA calcium currents mainly appeared above the membrane potential of +10 mV. There was no obvious borderline between these components. About 3 minutes after the application of CL, the peak density of LVA calcium currents in 20 microl/ml and 40 microl/ml CL group increased respectively (75.56% +/- 23.09% vs

  15. Acute restraint stress reduces hippocampal oxidative damage and behavior in rats: Effect of S-allyl cysteine.

    PubMed

    Colín-González, Ana Laura; Becerríl, Hugo; Flores-Reyes, Bianca Rubí; Torres, Ismael; Pinzón, Enrique; Santamaría-Del Angel, Daniel; Túnez, Isaac; Serratos, Iris; Pedraza-Chaverrí, José; Santamaría, Abel; Maldonado, Perla D

    2015-08-15

    This simple study was designed to investigate whether acute restraint stress can generate changes in behavioral tests and hippocampal endpoints of oxidative stress in rats, and if the antioxidant S-allyl cysteine (SAC) can prevent these alterations. We evaluated motor activity, forced swimming and anxiety behavior, as well as the hippocampal levels of lipid peroxidation and the activities of glutathione-related enzymes in animals submitted to mild immobilization. The effect of SAC (100 mg/kg, i.p.), given to rats every day 30 min before starting the immobilization session, was also investigated. Immobilization (restraint) stress was induced for a period of 6 h per day for five consecutive days. Our results indicate that, under the tested conditions, acute restraint stimulates compensatory behavioral tasks (motor activity, anxiety and forced swimming) to counteract the stressing conditions prevailing, and selectively increased the levels of lipid peroxidation and the enzyme activities of glutathione-S-transferase (GST) and glutathione peroxidase (GPx) in the hippocampus also as adaptive responses. SAC exhibited preventive effects in the stressed group as it improved behavior, reduced lipid peroxidation and prevented the increase of GST and GPx activities, suggesting that this antioxidant blunted primary pro-oxidative stimuli induced by restraint stress. Findings of this work also confirm that the use of antioxidants such as SAC can provide effective protection against the acute oxidative damage associated with anxiety produced by stressing conditions. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Multi-walled carbon nanotube increases the excitability of hippocampal CA1 neurons through inhibition of potassium channels in rat's brain slices.

    PubMed

    Chen, Ting; Yang, Jiajia; Ren, Guogang; Yang, Zhuo; Zhang, Tao

    2013-02-27

    This study was to investigate the neurotoxicity of multi-walled carbon nanotube (MWCNT) by measuring neuronal excitability in rat hippocampal neurons and exploring the underlying mechanism. Whole cell patch-clamp technique was used. Action potential properties and the pattern of repetitive firing rate were assessed. Our data showed that spike half-width and repetitive firing rate were significantly increased in a concentration-dependent manner. Furthermore, voltage-activated potassium currents were recorded. It was found that MWCNT produced a concentration-dependent inhibition in amplitudes of I(A) and I(K). In addition, MWCNT had effect on the activation kinetics of I(A) and I(K) with V(h) being shifted to the negative potential at high concentration, while I(A) inactivation curve was considerably shifted to the hyperpolarize potential with V(h) being increased. However, no effect was found on the recovery from inactivation of I(A). The results suggest that MWCNT increases the excitability of hippocampal CA1 neurons by inhibiting voltage-gated potassium current. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  17. Resveratrol suppresses calcium-mediated microglial activation and rescues hippocampal neurons of adult rats following acute bacterial meningitis.

    PubMed

    Sheu, Ji-Nan; Liao, Wen-Chieh; Wu, Un-In; Shyu, Ling-Yuh; Mai, Fu-Der; Chen, Li-You; Chen, Mei-Jung; Youn, Su-Chung; Chang, Hung-Ming

    2013-03-01

    Acute bacterial meningitis (ABM) is a serious disease with severe neurological sequelae. The intense calcium-mediated microglial activation and subsequently pro-inflammatory cytokine release plays an important role in eliciting ABM-related oxidative damage. Considering resveratrol possesses significant anti-inflammatory and anti-oxidative properties, the present study aims to determine whether resveratrol would exert beneficial effects on hippocampal neurons following ABM. ABM was induced by inoculating Klebsiella pneumoniae into adult rats intraventricularly. The time-of-flight secondary ion mass spectrometry (TOF-SIMS), Griffonia simplicifolia isolectin-B4 (GSA-IB4) and ionized calcium binding adaptor molecule 1 (Iba1) immunohistochemistry, enzyme-linked immunosorbent assay as well as malondialdehyde (MDA) measurement were used to examine the calcium expression, microglial activation, pro-inflammatory cytokine level, and extent of oxidative stress, respectively. In ABM rats, strong calcium signaling associated with enhanced microglial activation was observed in hippocampus. Increased microglial expression was coincided with intense production of pro-inflammatory cytokines and oxidative damage. However, in rats receiving resveratrol after ABM, the calcium intensity, microglial activation, pro-inflammatory cytokine and MDA levels were all significantly decreased. Quantitative data showed that much more hippocampal neurons were survived in resveratrol-treated rats following ABM. As resveratrol successfully rescues hippocampal neurons from ABM by suppressing the calcium-mediated microglial activation, therapeutic use of resveratrol may act as a promising strategy to counteract the ABM-induced neurological damage.

  18. Reduced Hippocampal Dendritic Spine Density and BDNF Expression following Acute Postnatal Exposure to Di(2-Ethylhexyl) Phthalate in Male Long Evans Rats

    PubMed Central

    Smith, Catherine A.; Holahan, Matthew R.

    2014-01-01

    Early developmental exposure to di(2-ethylhexyl) phthalate (DEHP) has been linked to a variety of neurodevelopmental changes, particularly in rodents. The primary goal of this work was to establish whether acute postnatal exposure to a low dose of DEHP would alter hippocampal dendritic morphology and BDNF and caspase-3 mRNA expression in male and female Long Evans rats. Treatment with DEHP in male rats led to a reduction in spine density on basal and apical dendrites of neurons in the CA3 dorsal hippocampal region compared to vehicle-treated male controls. Dorsal hippocampal BDNF mRNA expression was also down-regulated in male rats exposed to DEHP. No differences in hippocampal spine density or BDNF mRNA expression were observed in female rats treated with DEHP compared to controls. DEHP treatment did not affect hippocampal caspase-3 mRNA expression in male or female rats. These results suggest a gender-specific vulnerability to early developmental DEHP exposure in male rats whereby postnatal DEHP exposure may interfere with normal synaptogenesis and connectivity in the hippocampus. Decreased expression of BDNF mRNA may represent a molecular mechanism underlying the reduction in dendritic spine density observed in hippocampal CA3 neurons. These findings provide initial evidence for a link between developmental exposure to DEHP, reduced levels of BDNF and hippocampal atrophy in male rats. PMID:25295592

  19. Reduced hippocampal dendritic spine density and BDNF expression following acute postnatal exposure to di(2-ethylhexyl) phthalate in male Long Evans rats.

    PubMed

    Smith, Catherine A; Holahan, Matthew R

    2014-01-01

    Early developmental exposure to di(2-ethylhexyl) phthalate (DEHP) has been linked to a variety of neurodevelopmental changes, particularly in rodents. The primary goal of this work was to establish whether acute postnatal exposure to a low dose of DEHP would alter hippocampal dendritic morphology and BDNF and caspase-3 mRNA expression in male and female Long Evans rats. Treatment with DEHP in male rats led to a reduction in spine density on basal and apical dendrites of neurons in the CA3 dorsal hippocampal region compared to vehicle-treated male controls. Dorsal hippocampal BDNF mRNA expression was also down-regulated in male rats exposed to DEHP. No differences in hippocampal spine density or BDNF mRNA expression were observed in female rats treated with DEHP compared to controls. DEHP treatment did not affect hippocampal caspase-3 mRNA expression in male or female rats. These results suggest a gender-specific vulnerability to early developmental DEHP exposure in male rats whereby postnatal DEHP exposure may interfere with normal synaptogenesis and connectivity in the hippocampus. Decreased expression of BDNF mRNA may represent a molecular mechanism underlying the reduction in dendritic spine density observed in hippocampal CA3 neurons. These findings provide initial evidence for a link between developmental exposure to DEHP, reduced levels of BDNF and hippocampal atrophy in male rats.

  20. Precise spatial and temporal control of oxygen within in vitro brain slices via microfluidic gas channels.

    PubMed

    Mauleon, Gerardo; Fall, Christopher P; Eddington, David T

    2012-01-01

    The acute brain slice preparation is an excellent model for studying the details of how neurons and neuronal tissue respond to a variety of different physiological conditions. But open slice chambers ideal for electrophysiological and imaging access have not allowed the precise spatiotemporal control of oxygen in a way that might realistically model stroke conditions. To address this problem, we have developed a microfluidic add-on to a commercially available perfusion chamber that diffuses oxygen throughout a thin membrane and directly to the brain slice. A microchannel enables rapid and efficient control of oxygen and can be modified to allow different regions of the slice to experience different oxygen conditions. Using this novel device, we show that we can obtain a stable and homogeneous oxygen environment throughout the brain slice and rapidly alter the oxygen tension in a hippocampal slice. We also show that we can impose different oxygen tensions on different regions of the slice preparation and measure two independent responses, which is not easily obtainable with current techniques.

  1. Influences of nanoparticle zinc oxide on acutely isolated rat hippocampal CA3 pyramidal neurons.

    PubMed

    Zhao, Jingxia; Xu, Lanju; Zhang, Tao; Ren, Guogang; Yang, Zhuo

    2009-03-01

    The effects of zinc oxide nanoparticles (nano-ZnO) on the properties of voltage-dependent sodium, potassium currents and evoked action potentials were studied in acutely isolated rat hippocampal CA3 pyramidal neurons at postnatal ages of 10-14 days rats using the whole-cell patch-clamp technique. The results indicated that: (1) in the present of final concentration of 10(-4)g/ml nano-ZnO, the current-voltage curve of sodium current (I(Na)) was decreased, and the peak amplitudes of I(Na) were increased considerably from -50 to +20mV (p<0.05). Meanwhile, the inactivation and the recovery from inactivation of I(Na) were also promoted by the nano-ZnO solution (10(-4)g/ml) (p<0.01). However, the steady-state activation curve of I(Na) was not shifted by the nano-ZnO. (2) The amplitudes of transient outward potassium current (I(A)) were increased by the nano-ZnO solution (10(-4)g/ml), while the current-voltage curve of delayed rectifier potassium current (I(K)) was significantly increased from +20 to +90mV (p<0.05). However, it is apparent that the nano-ZnO solution did not shift the steady-state activation curve of I(A) and I(K), and neither had significant effects on the inactivation and the recovery from inactivation of I(A). (3) Peak amplitude and overshoot of the evoked single action potential were increased and half-width was diminished in the presence of the 10(-4)g/ml nano-ZnO solution (p<0.05). Simultaneously, a prolonged depolarizing current injection enhanced (p<0.05) repetitive firing evoked firing rate. These results suggested that 10(-4)g/ml nano-ZnO solution can lead to an enhancement in the current amplitudes of I(Na) and I(K) by increasing the opening number of sodium channels, delaying rectifier potassium channels, and enhancing the excitability of neurons, which lead to Na(+) influx and the accumulation of intracellular Na(+), as well as K(+) efflux plus the loss of cytoplasmic K(+). These may disturb the ionic homeostasis and the physiological

  2. Analysis of acute brain slices by electron microscopy: a correlative light-electron microscopy workflow based on Tokuyasu cryo-sectioning.

    PubMed

    Loussert Fonta, Celine; Leis, Andrew; Mathisen, Cliff; Bouvier, David S; Blanchard, Willy; Volterra, Andrea; Lich, Ben; Humbel, Bruno M

    2015-01-01

    Acute brain slices are slices of brain tissue that are kept vital in vitro for further recordings and analyses. This tool is of major importance in neurobiology and allows the study of brain cells such as microglia, astrocytes, neurons and their inter/intracellular communications via ion channels or transporters. In combination with light/fluorescence microscopies, acute brain slices enable the ex vivo analysis of specific cells or groups of cells inside the slice, e.g. astrocytes. To bridge ex vivo knowledge of a cell with its ultrastructure, we developed a correlative microscopy approach for acute brain slices. The workflow begins with sampling of the tissue and precise trimming of a region of interest, which contains GFP-tagged astrocytes that can be visualised by fluorescence microscopy of ultrathin sections. The astrocytes and their surroundings are then analysed by high resolution scanning transmission electron microscopy (STEM). An important aspect of this workflow is the modification of a commercial cryo-ultramicrotome to observe the fluorescent GFP signal during the trimming process. It ensured that sections contained at least one GFP astrocyte. After cryo-sectioning, a map of the GFP-expressing astrocytes is established and transferred to correlation software installed on a focused ion beam scanning electron microscope equipped with a STEM detector. Next, the areas displaying fluorescence are selected for high resolution STEM imaging. An overview area (e.g. a whole mesh of the grid) is imaged with an automated tiling and stitching process. In the final stitched image, the local organisation of the brain tissue can be surveyed or areas of interest can be magnified to observe fine details, e.g. vesicles or gold labels on specific proteins. The robustness of this workflow is contingent on the quality of sample preparation, based on Tokuyasu's protocol. This method results in a reasonable compromise between preservation of morphology and maintenance of

  3. Exogenous hydrogen sulfide eliminates spatial memory retrieval impairment and hippocampal CA1 LTD enhancement caused by acute stress via promoting glutamate uptake.

    PubMed

    He, Jin; Guo, Ruixian; Qiu, Pengxin; Su, Xingwen; Yan, Guangmei; Feng, Jianqiang

    2017-03-20

    Acute stress impairs the hippocampus-dependent spatial memory retrieval, and its synaptic mechanisms are associated with hippocampal CA1 long-term depression (LTD) enhancement in the adult rats. Endogenous hydrogen sulfide (H2S) is recognized as a novel gasotransmitter and has the neural protective roles. However, very little attention has been paid to understanding the effects of H2S on spatial memory retrieval impairment. We observed the protective effects of NaHS (a donor of H2S) against spatial memory retrieval impairment caused by acute stress and its synaptic mechanisms. Our results showed that NaHS abolished spatial memory retrieval impairment and hippocampal CA1 LTD enhancement caused by acute stress, but not by glutamate transporter inhibitor l-trans-pyrrolidine-2,4-dicarboxylic (tPDC), indicating that the activation of glutamate transporters is necessary for exogenous H2S to exert its roles. Moreover, NaHS restored the decreased glutamate uptake in the hippocampal CA1 synaptosomal fraction caused by acute stress. Dithiothreitol (DTT, a disulfide reducing agent) abolished a decrease in the glutamate uptake caused by acute stress, and NaHS eradicated the decreased glutamate uptake caused by 5,5'-dithio-bis(2-nitrobenzoic)acid (DTNB, a thiol oxidizing agent), collectively, revealing that exogenous H2S increases glutamate uptake by reducing disulfide bonds of the glutamate transporters. Additionally, NaHS inhibited the increased expression level of phosphorylated c-Jun-N-terminal kinase (JNK) in the hippocampal CA1 region caused by acute stress. The JNK inhibitor SP600125 eliminated spatial memory retrieval impairment, hippocampal CA1 LTD enhancement and the decreased glutamate uptake caused by acute stress, indicating that exogenous H2S exerts these roles by inhibiting the activation of JNK signaling pathway.

  4. Simultaneous monitoring of tissue P2 and NADH fluorescence during synaptic stimulation and spreading depression reveals a transient dissociation between oxygen utilization and mitochondrial redox state in rat hippocampal slices

    PubMed Central

    Galeffi, Francesca; Somjen, George G; Foster, Kelley A; Turner, Dennis A

    2011-01-01

    Nicotinamide adenine dinucleotide (NADH) imaging can be used to monitor neuronal activation and ascertain mitochondrial dysfunction, for example during hypoxia. During neuronal stimulation in vitro, NADH normally becomes more oxidized, indicating enhanced oxygen utilization. A subsequent NADH overshoot during activation or on recovery remains controversial and reflects either increased metabolic activity or limited oxygen availability. Tissue P2 measurements, obtained simultaneously with NADH imaging in area CA1 in hippocampal slices, reveal that during prolonged train stimulation (ST) in 95% O2, a persistent NADH oxidation is coupled with increased metabolic demand and oxygen utilization, for the duration of the stimulation. However, under conditions of either decreased oxygen supply (ST-50% O2) or enhanced metabolic demand (K+-induced spreading depression (K+-SD) 95% O2) the NADH oxidation is brief and the redox balance shifts early toward reduction, leading to a prolonged NADH overshoot. Yet, oxygen utilization remains elevated and is correlated with metabolic demand. Under these conditions, it appears that the rate of NAD+ reduction may transiently exceed oxidation, to maintain an adequate oxygen flux and ATP production. In contrast, during SD in 50% O2, the oxygen levels dropped to a point at which oxidative metabolism in the electron transport chain is limited and the rate of utilization declined. PMID:20736960

  5. Caffeine-dependent stimulus-triggered oscillations in the CA3 region of hippocampal slices from rats chronically exposed to lead.

    PubMed

    He, Shui-Jin; Xiao, Cheng; Wu, Zhi-Yuan; Ruan, Di-Yun

    2004-12-01

    Yoshimura et al. [Yoshimura, H., Sugai, T., Onoda, N., Segami, N., Kato, N., 2002. Age-dependent occurrence of synchronized population oscillation suggestive of a developing functional coupling between NMDA and ryanodine receptors in the neocortex. Dev. Brain Res., 136, 63-68.] have shown that caffeine can elicit synchronized oscillations (10-12 Hz) dependent on calcium-induced calcium release in rat neocortex neurons. In the present work, synchronized oscillations in the CA3 region of rat hippocampus were studied by recording field excitatory postsynaptic potentials (fEPSPs) in vitro. In the presence of 0.1 mM caffeine, in CA3 of 44 of 45 (97.8%) slices from chronic lead-exposed rats, single electrical stimuli triggered a burst of high-frequency oscillations (approximately 230 Hz), whereas in CA3 of caffeine-treated slices from control rats, such oscillations could be elicited in only 2 of 24 (8.3%) slices. The complete (but fully reversible) block of caffeine-dependent oscillations by 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX; 20 microM) indicates that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors are necessary for the high-frequency synchronized oscillations. 2-Amino-5-phosphonopentanoate (AP-5; 50 micoM) partially reduced the amplitude of caffeine-dependent oscillations without significantly altering their frequency. Caffeine-dependent oscillations could be abolished by application of AP-5 and 3 mM Mg2+ during the initial period of bursting, indicating that N-methyl-D-aspartate (NMDA) receptors play an important role in the generation of oscillations. The Ca2+ chelator ethylene glycol bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA; 5 mM) added in standard artificial cerebrospinal fluid (ACSF) containing 0.1 mM caffeine fully blocked the oscillations. Caffeine-dependent oscillations are insensitive to an antagonist of gamma-aminobutyric acid (GABAA) receptors (10 microM bicuculline), L-type Ca2+ channels (10 mu

  6. BONLAC: A Combinatorial Proteomic Technique to Measure Stimulus-induced Translational Profiles in Brain Slices

    PubMed Central

    Bowling, Heather; Bhattacharya, Aditi; Zhang, Guoan; Lebowitz, Joseph Z.; Alam, Danyal; Smith, Peter T.; Kirshenbaum, Kent; Neubert, Thomas A.; Vogel, Christine; Chao, Moses V.; Klann, Eric

    2015-01-01

    Stimulus-triggered protein synthesis is critical for brain health and function. However, due to technical hurdles, de novo neuronal translation is predominantly studied in cultured cells, whereas electrophysiological and circuit analyses often are performed in brain slices. The different properties of these two experimental systems create an information gap about stimulus-induced alterations in the expression of new proteins in mature circuits. To address this, we adapted two existing techniques, BONCAT and SILAC, to a combined proteomic technique, BONLAC, for use in acute adult hippocampal slices. Using BDNF-induced protein synthesis as a proof of concept, we found alterations in expression of proteins involved in neurotransmission, trafficking, and cation binding that differed from those found in a similar screen in cultured neurons. Our results indicate important differences between cultured neurons and slices, and suggest that BONLAC could be used to dissect proteomic changes underlying synaptic events in adult circuits. PMID:26205778

  7. OLM interneurons are transiently recruited into field gamma oscillations evoked by brief kainate pressure ejections onto area CA1 in mice hippocampal slices.

    PubMed

    Kipiani, E

    2009-02-01

    Oscillations (30-100 Hz) are correlated with the cognitive functions of the brain. In the hippocampus interactions between perisomatic and trilaminar interneurons with pyramidal cells are thought to underlie generation of field gamma oscillations. In area CA3 OLM interneurons receive synaptic input in gamma range but generate action potential (AP) output in theta band and are involved in theta oscillations synchronized along the longitudinal axis of the hippocampus. In slice preparations of CA3 area the spike timing of OLM cells could be modulated by carbachole induced gamma oscillations, although their firing rates are limited to theta frequency. Normally, OLM interneurons are somatostatin positive cells. In this study we tested whether parvalbumin (PV) containing OLM interneurons in area CA1 limit AP output during kainate pressure ejection also to theta frequency. We used focal short applications of kainate in area CA1 to induce filed gamma oscillations with an average frequency of about 44.7+/-4.4 Hz. The duration of field gamma was on average 8.9+/-3.5 s. During such oscillations CA1 PV positive OLM interneurons of mice hippocampus received excitatory synaptic input at gamma frequency. Moreover, their AP output was in gamma range as well. Thus, we show that beside the somatostatin containing OLM interneurons, which generate theta rhythm there are PV containing OLM cells, which could synchronize the distal dendrites of CA1 pyramidal cells to the field gamma oscillations.

  8. Oxygen glucose deprivation causes mitochondrial dysfunction in cultivated rat hippocampal slices: protective effects of CsA, its immunosuppressive congener [D-Ser](8)CsA, the novel non-immunosuppressive cyclosporin derivative Cs9, and the NMDA receptor antagonist MK 801.

    PubMed

    Trumbeckaite, Sonata; Gizatullina, Zemfira; Arandarcikaite, Odeta; Röhnert, Peter; Vielhaber, Stefan; Malesevic, Miroslav; Fischer, Gunter; Seppet, Enn; Striggow, Frank; Gellerich, Frank Norbert

    2013-09-01

    We have introduced a sensitive method for studying oxygen/glucose deprivation (OGD)-induced mitochondrial alterations in homogenates of organotypic hippocampal slice cultures (slices) by high-resolution respirometry. Using this approach, we tested the neuroprotective potential of the novel non-immunosuppressive cyclosporin (CsA) derivative Cs9 in comparison with CsA, the immunosuppressive CsA analog [D-Ser](8)CsA, and MK 801, a N-methyl-d-aspartate (NMDA) receptor antagonist. OGD/reperfusion reduced the glutamate/malate dependent (and protein-related) state 3 respiration to 30% of its value under control conditions. All of the above drugs reversed this effect, with an increase to >88% of the value for control slices not exposed to OGD. We conclude that Cs9, [D-Ser](8)CsA, and MK 801, despite their different modes of action, protect mitochondria from OGD-induced damage.

  9. Bicarbonate efflux via GABAA receptors depolarizes membrane potential and inhibits two-pore domain potassium channels of astrocytes in rat hippocampal slices

    PubMed Central

    Ma, Bao-Feng; Xie, Min-Jie; Zhou, Min

    2014-01-01

    Increasing evidence indicates the functional expression of ionotropic γ-aminobutyric acid receptor (GABAA-R) in astrocytes. However, it remains controversial in regard to the intracellular Cl− concentration ([Cl−]i) and the functional role of anion-selective GABAA-R in astrocytes. In gramicidin perforated-patch recordings from rat hippocampal CA1 astrocytes, GABA and GABAA-R specific agonist THIP depolarized astrocyte membrane potential (Vm), and the THIP induced currents reversed at the voltages between −75.3 to −78.3 mV, corresponding to a [Cl−]i of 3.1 – 3.9 mM that favors a passive distribution of Cl− anions across astrocyte membrane. Further analysis showed that GABAA-R induced Vm depolarization is ascribed to HCO3− efflux, while a passively distributed Cl− mediates no net flux or influx of Cl-that leads to an unchanged or hyperpolarized Vm. In addition to a rapidly activated GABAA-R current component, GABA and THIP also induced a delayed inward current (DIC) in 63% of astrocytes. The DIC became manifest after agonist withdrawal and enhanced in amplitude with increasing agonist application duration or concentrations. Astrocytic two-pore domain K+ channels (K2Ps), especially TWIK-1, appeared to underlie the DIC, because 1) acidic intracellular pH, as a result of HCO3− efflux, inhibited TWIK-1; 2) the DIC remained in the Cs+ recording solutions that inhibited conventional K+ channels and 3) the DIC was completely inhibited by 1 mM quinine but not by blockers for other cation/anion channels. Altogether, HCO3− efflux through activated GABAA-R depolarizes astrocyte Vm and induces a delayed inhibition of K2Ps K+ channels via intracellular acidification. PMID:22855415

  10. Neuroprotective Effect of Amantadine on Corticosterone-induced Abnormal Glutamatergic Synaptic Transmission of CA3-CA1 pathway in rat's hippocampal slices.

    PubMed

    Xiao, Xi; Zhang, Hui; Wang, Hui; Li, Qun; Zhang, Tao

    2017-09-13

    Depression is a psychiatric disorder and chronic stress, leading to altered glucocorticoid secretion patterns, is one of the factors that induce depression. Our previous study showed that amantadine significantly attenuated the impairments of synaptic plasticity and cognitive function a rat model of CUS. However, little is known regarding the underlying mechanism. In the present study, the whole-cell patch-clamp technique was applied to examine the protection effect of amantadine on the hippocampus CA3-CA1 pathway. Evoked excitatory postsynaptic currents (eEPSCs), miniature excitatory postsynaptic currents (mEPSCs), paired-pulse ratio (PPR) and the action potentials of CA3 neurons were recorded. Our data showed that corticosterone increased the amplitude of eEPSCs and decreased the value of paired-pulse ratio (PPR), but both of them were significantly reversed by amantadine. In addition, the frequency of mEPSC was considerably increased by corticosterone, but it was reduced by amantadine. Moreover, we used the Fluo-3/AM image to detect the Ca(2+) influx in primary cultured hippocampal neurons. The results showed that the intracellular calcium levels were significantly decreased by amantadine in the corticosterone treated neurons. Additionally, the superoxide dismutase (SOD) and catalase (CAT) activities were reduced by corticosterone, while they were enhanced by either amantadine or low-calcium artificial cerebral spinal fluid (ACSF). These results suggest that amantadine significantly improves corticosterone-induced abnormal glutamatergic synaptic transmission of CA3-CA1 synapses presynaptically and alleviates the activities of antioxidant enzymes via regulating the calcium influx. This article is protected by copyright. All rights reserved. © 2017 Wiley Periodicals, Inc.

  11. Role of P2 purinergic receptors in synaptic transmission under normoxic and ischaemic conditions in the CA1 region of rat hippocampal slices

    PubMed Central

    Coppi, Elisabetta; Pugliese, Anna Maria; Stephan, Holger; Müller, Christa E.

    2007-01-01

    The role of ATP and its stable analogue ATPγS [adenosine-5′-o-(3-thio)triphosphate] was studied in rat hippocampal neurotransmission under normoxic conditions and during oxygen and glucose deprivation (OGD). Field excitatory postsynaptic potentials (fEPSPs) from the dendritic layer or population spikes (PSs) from the soma were extracellularly recorded in the CA1 area of the rat hippocampus. Exogenous application of ATP or ATPγS reduced fEPSP and PS amplitudes. In both cases the inhibitory effect was blocked by the selective A1 adenosine receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine) and was potentiated by different ecto-ATPase inhibitors: ARL 67156 (6-N,N-diethyl-D-β,γ-dibromomethylene), BGO 136 (1-hydroxynaphthalene-3,6-disulfonate) and PV4 [hexapotassium dihydrogen monotitanoundecatungstocobaltate(II) tridecahydrate, K6H2[TiW11CoO40]·13H2O]. ATPγS-mediated inhibition was reduced by the P2 antagonist suramin [8-(3-benzamido-4-methylbenzamido)naphthalene-1,3,5-trisulfonate] at the somatic level and by other P2 blockers, PPADS (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonate) and MRS 2179 (2′-deoxy-N6-methyladenosine 3′,5′-bisphosphate), at the dendritic level. After removal of both P2 agonists, a persistent increase in evoked synaptic responses was recorded both at the dendritic and somatic levels. This effect was prevented in the presence of different P2 antagonists. A 7-min OGD induced tissue anoxic depolarization and was invariably followed by irreversible loss of fEPSP. PPADS, suramin, MRS2179 or BBG (brilliant blue G) significantly prevented the irreversible failure of neurotransmission induced by 7-min OGD. Furthermore, in the presence of these P2 antagonists, the development of anoxic depolarization was blocked or significantly delayed. Our results indicate that P2 receptors modulate CA1 synaptic transmission under normoxic conditions by eliciting both inhibitory and excitatory effects. In the same brain region, P2 receptor

  12. BDNF increases release probability and the size of a rapidly recycling vesicle pool within rat hippocampal excitatory synapses

    PubMed Central

    Tyler, William J; Zhang, Xiao-lei; Hartman, Kenichi; Winterer, Jochen; Muller, Wolfgang; Stanton, Patric K; Pozzo-Miller, Lucas

    2006-01-01

    Exerting its actions pre-, post- and peri-synaptically, brain-derived neurotrophic factor (BDNF) is one of the most potent modulators of hippocampal synaptic function. Here, we examined the effects of BDNF on a rapidly recycling pool (RRP) of vesicles within excitatory synapses. First, we estimated vesicular release in hippocampal cultures by performing FM4-64 imaging in terminals impinging on enhanced green fluorescent protein (eGFP)-labelled dendritic spines – a hallmark of excitatory synapses. Consistent with a modulation of the RRP, BDNF increased the evoked destaining rate of FM4-64 only during the initial phase of field stimulation. Multiphoton microscopy in acute hippocampal slices confirmed these observations by selectively imaging the RRP, which was loaded with FM1-43 by hyperosmotic shock. Slices exposed to BDNF showed an increase in the evoked and spontaneous rates of FM1-43 destaining from terminals in CA1 stratum radiatum, mostly representing excitatory terminals of Schaffer collaterals. Variance-mean analysis of evoked EPSCs in CA1 pyramidal neurons further confirmed that release probability is increased in BDNF-treated slices, without changes in the number of independent release sites or average postsynaptic quantal amplitude. Because BDNF was absent during dye loading, imaging, destaining and whole-cell recordings, these results demonstrate that BDNF induces a long-lasting enhancement in the probability of transmitter release at hippocampal excitatory synapses by modulating the RRP. Since the endogenous BDNF scavenger TrkB-IgG prevented the enhancement of FM1-43 destaining rate caused by induction of long-term potentiation in acute hippocampal slices, the modulation of a rapidly recycling vesicle pool may underlie the role of BDNF in hippocampal long-term synaptic plasticity. PMID:16709633

  13. Risperidone reverses the spatial object recognition impairment and hippocampal BDNF-TrkB signalling system alterations induced by acute MK-801 treatment

    PubMed Central

    Chen, Guangdong; Lin, Xiaodong; Li, Gongying; Jiang, Diego; Lib, Zhiruo; Jiang, Ronghuan; Zhuo, Chuanjun

    2017-01-01

    The aim of the present study was to investigate the effects of a commonly-used atypical antipsychotic, risperidone, on alterations in spatial learning and in the hippocampal brain-derived neurotrophic factor (BDNF)-tyrosine receptor kinase B (TrkB) signalling system caused by acute dizocilpine maleate (MK-801) treatment. In experiment 1, adult male Sprague-Dawley rats subjected to acute treatment of either low-dose MK801 (0.1 mg/kg) or normal saline (vehicle) were tested for spatial object recognition and hippocampal expression levels of BDNF, TrkB and the phophorylation of TrkB (p-TrkB). We found that compared to the vehicle, MK-801 treatment impaired spatial object recognition of animals and downregulated the expression levels of p-TrkB. In experiment 2, MK-801- or vehicle-treated animals were further injected with risperidone (0.1 mg/kg) or vehicle before behavioural testing and sacrifice. Of note, we found that risperidone successfully reversed the deleterious effects of MK-801 on spatial object recognition and upregulated the hippocampal BDNF-TrkB signalling system. Collectively, the findings suggest that cognitive deficits from acute N-methyl-D-aspartate receptor blockade may be associated with the hypofunction of hippocampal BDNF-TrkB signalling system and that risperidone was able to reverse these alterations. PMID:28451387

  14. Risperidone reverses the spatial object recognition impairment and hippocampal BDNF-TrkB signalling system alterations induced by acute MK-801 treatment.

    PubMed

    Chen, Guangdong; Lin, Xiaodong; Li, Gongying; Jiang, Diego; Lib, Zhiruo; Jiang, Ronghuan; Zhuo, Chuanjun

    2017-03-01

    The aim of the present study was to investigate the effects of a commonly-used atypical antipsychotic, risperidone, on alterations in spatial learning and in the hippocampal brain-derived neurotrophic factor (BDNF)-tyrosine receptor kinase B (TrkB) signalling system caused by acute dizocilpine maleate (MK-801) treatment. In experiment 1, adult male Sprague-Dawley rats subjected to acute treatment of either low-dose MK801 (0.1 mg/kg) or normal saline (vehicle) were tested for spatial object recognition and hippocampal expression levels of BDNF, TrkB and the phophorylation of TrkB (p-TrkB). We found that compared to the vehicle, MK-801 treatment impaired spatial object recognition of animals and downregulated the expression levels of p-TrkB. In experiment 2, MK-801- or vehicle-treated animals were further injected with risperidone (0.1 mg/kg) or vehicle before behavioural testing and sacrifice. Of note, we found that risperidone successfully reversed the deleterious effects of MK-801 on spatial object recognition and upregulated the hippocampal BDNF-TrkB signalling system. Collectively, the findings suggest that cognitive deficits from acute N-methyl-D-aspartate receptor blockade may be associated with the hypofunction of hippocampal BDNF-TrkB signalling system and that risperidone was able to reverse these alterations.

  15. Changes in C57BL6 Mouse Hippocampal Transcriptome Induced by Hypergravity Mimic Acute Corticosterone-Induced Stress

    PubMed Central

    Pulga, Alice; Porte, Yves; Morel, Jean-Luc

    2016-01-01

    Centrifugation is a widely used procedure to study the impact of altered gravity on Earth, as observed during spaceflights, allowing us to understand how a long-term physical constraint can condition the mammalian physiology. It is known that mice, placed in classical cages and maintained during 21 days in a centrifuge at 3G gravity level, undergo physiological adaptations due to hypergravity, and/or stress. Indeed, an increase of corticosterone levels has been previously measured in the plasma of 3G-exposed mice. Corticosterone is known to modify neuronal activity during memory processes. Although learning and memory performances cannot be assessed during the centrifugation, literature largely described a large panel of proteins (channels, second messengers, transcription factors, structural proteins) which expressions are modified during memory processing. Thus, we used the Illumina technology to compare the whole hippocampal transcriptome of three groups of C57Bl6/J mice, in order to gain insights into the effects of hypergravity on cerebral functions. Namely, a group of 21 days 3G-centrifuged mice was compared to (1) a group subjected to an acute corticosterone injection, (2) a group receiving a transdermal chronic administration of corticosterone during 21 days, and (3) aged mice because aging could be characterized by a decrease of hippocampus functions and memory impairment. Our results suggest that hypergravity stress induced by corticosterone administration and aging modulate the expression of genes in the hippocampus. However, the modulations of the transcriptome observed in these conditions are not identical. Hypergravity affects per-se the hippocampus transcriptome and probably modifies its activity. Hypergravity induced changes in hippocampal transcriptome were more similar to acute injection than chronic diffusion of corticosterone or aging. PMID:28082866

  16. Changes in C57BL6 Mouse Hippocampal Transcriptome Induced by Hypergravity Mimic Acute Corticosterone-Induced Stress.

    PubMed

    Pulga, Alice; Porte, Yves; Morel, Jean-Luc

    2016-01-01

    Centrifugation is a widely used procedure to study the impact of altered gravity on Earth, as observed during spaceflights, allowing us to understand how a long-term physical constraint can condition the mammalian physiology. It is known that mice, placed in classical cages and maintained during 21 days in a centrifuge at 3G gravity level, undergo physiological adaptations due to hypergravity, and/or stress. Indeed, an increase of corticosterone levels has been previously measured in the plasma of 3G-exposed mice. Corticosterone is known to modify neuronal activity during memory processes. Although learning and memory performances cannot be assessed during the centrifugation, literature largely described a large panel of proteins (channels, second messengers, transcription factors, structural proteins) which expressions are modified during memory processing. Thus, we used the Illumina technology to compare the whole hippocampal transcriptome of three groups of C57Bl6/J mice, in order to gain insights into the effects of hypergravity on cerebral functions. Namely, a group of 21 days 3G-centrifuged mice was compared to (1) a group subjected to an acute corticosterone injection, (2) a group receiving a transdermal chronic administration of corticosterone during 21 days, and (3) aged mice because aging could be characterized by a decrease of hippocampus functions and memory impairment. Our results suggest that hypergravity stress induced by corticosterone administration and aging modulate the expression of genes in the hippocampus. However, the modulations of the transcriptome observed in these conditions are not identical. Hypergravity affects per-se the hippocampus transcriptome and probably modifies its activity. Hypergravity induced changes in hippocampal transcriptome were more similar to acute injection than chronic diffusion of corticosterone or aging.

  17. Impaired Hippocampal Glutamate and Glutamine Metabolism in the db/db Mouse Model of Type 2 Diabetes Mellitus

    PubMed Central

    Andersen, Jens Velde; Nissen, Jakob Dahl; Christensen, Sofie Kjellerup; Markussen, Kia Hjulmand

    2017-01-01

    Type 2 diabetes mellitus (T2DM) is a risk factor for the development of Alzheimer's disease, and changes in brain energy metabolism have been suggested as a causative mechanism. The aim of this study was to investigate the cerebral metabolism of the important amino acids glutamate and glutamine in the db/db mouse model of T2DM. Glutamate and glutamine are both substrates for mitochondrial oxidation, and oxygen consumption was assessed in isolated brain mitochondria by Seahorse XFe96 analysis. In addition, acutely isolated cerebral cortical and hippocampal slices were incubated with [U-13C]glutamate and [U-13C]glutamine, and tissue extracts were analyzed by gas chromatography-mass spectrometry. The oxygen consumption rate using glutamate and glutamine as substrates was not different in isolated cerebral mitochondria of db/db mice compared to controls. Hippocampal slices of db/db mice exhibited significantly reduced 13C labeling in glutamate, glutamine, GABA, citrate, and aspartate from metabolism of [U-13C]glutamate. Additionally, reduced 13C labeling were observed in GABA, citrate, and aspartate from [U-13C]glutamine metabolism in hippocampal slices of db/db mice when compared to controls. None of these changes were observed in cerebral cortical slices. The results suggest specific hippocampal impairments in glutamate and glutamine metabolism, without affecting mitochondrial oxidation of these substrates, in the db/db mouse. PMID:28695014

  18. Traumatic alterations in GABA signaling disrupt hippocampal network activity in the developing brain

    PubMed Central

    Dzhala, Volodymyr; Valeeva, Guzel; Glykys, Joseph; Khazipov, Rustem; Staley, Kevin

    2012-01-01

    Severe head trauma causes widespread neuronal shear injuries and acute seizures. Shearing of neural processes might contribute to seizures by disrupting the transmembrane ion gradients that subserve normal synaptic signaling. To test this possibility, we investigated changes in intracellular chloride concentration ([Cl−]i) associated with the widespread neural shear injury induced during preparation of acute brain slices. In hippocampal slices and intact hippocampal preparations from immature CLM-1 mice, increases in [Cl−]i correlated with disruption of neural processes and biomarkers of cell injury. Traumatized neurons with higher [Cl−]i demonstrated excitatory GABA signaling, remained synaptically active, and facilitated network activity as assayed by the frequency of extracellular action potentials and spontaneous network-driven oscillations. These data support a more inhibitory role for GABA in the unperturbed immature brain, demonstrate the utility of the acute brain slice preparation for the study of the consequences of trauma, and provide potential mechanisms for both GABA-mediated excitatory network events in the slice preparation and early post-traumatic seizures. PMID:22442068

  19. Comparison of image characteristics of plaques in culprit coronary arteries by 64 slice CT and intravascular ultrasound in acute coronary syndromes.

    PubMed

    Takaoka, Hiroyuki; Ishibashi, Iwao; Uehara, Masae; Rubin, Geoffrey D; Komuro, Issei; Funabashi, Nobusada

    2012-10-04

    To evaluate plaque image characteristics in coronary artery culprit-lesions in subjects with acute coronary syndromes (ACS), we retrospectively compared coronary arterial images by 64-slice CT before conventional-coronary-angiogram with those by intravascular ultrasound (IVUS). Retrospective analysis of coronary arterial images from thirty-one subjects (26-males, mean age 59.3 ± 12.0 years) exhibiting acute symptoms with suspicion of ACS, where either (1) ECG was un-interpretable or (2) ECG was non diagnostic/cardiac biomarkers was equivocal; with significant stenosis on emergent 64 slice CT and subjects were finally diagnosed as having ACS confirmed by conventional-coronary-angiogram, followed by IVUS before coronary-intervention. After principal culprit-lesion components were classified into 1) thrombus, 2) soft plaques, and 3) fibrotic plaques by IVUS, corresponding culprit-lesion CT values were measured (two-observers). Nineteen and 12 of 31 subjects were finally diagnosed as unstable angina pectoris and non-ST elevation acute myocardial infarction respectively. Main culprit-lesion components of ACS were identified on MSCT in all subjects. Culprit-lesion CT values diagnosed as soft plaques by IVUS (n=6, 32.9 ± 8.7 HU) were not lower than those of thrombi (n=18, 43.2 ± 10.7 HU, p=0.268); both values were significantly lower than those of fibrotic plaques (n=7, 82.5 ± 22.6 HU) (both p<0.01). Calcification, spotty calcification, and positive arterial remodeling were observed in 67.7%, 61.3%, 58.1% (IVUS) and 58.1%, 51.6%, 74.2% (MSCT), respectively (all p=NS). CT value reproducibilities and culprit-lesion areas, were 0.87 and 0.86, respectively (two analyzers). 64-slice CT can non-invasively evaluate image characteristics in coronary artery culprit-lesions in ACS subjects accurately; this may help to differentiate soft plaques or thrombi generated by plaque rupture from fibrotic plaques. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  20. Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

    PubMed Central

    Mosa, Adam J.; Wang, Sabrina; Tan, Yao Fang; Wojtowicz, J. Martin

    2015-01-01

    Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research. PMID:25867138

  1. Organotypic slice cultures for studies of postnatal neurogenesis.

    PubMed

    Mosa, Adam J; Wang, Sabrina; Tan, Yao Fang; Wojtowicz, J Martin

    2015-03-04

    Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

  2. Basic presynaptic functions in hippocampal neurons are not affected by acute or chronic lithium treatment.

    PubMed

    Lueke, Katharina; Kaiser, Tobias; Svetlitchny, Alexei; Welzel, Oliver; Wenzel, Eva M; Tyagarajan, Shiva; Kornhuber, Johannes; Groemer, Teja W

    2014-02-01

    Lithium is an effective mood-stabilizer in the treatment of bipolar affective disorder. While glycogen synthase kinase 3-mediated and inositol depletion-dependent effects of lithium have been described extensively in literature, there is very little knowledge about the consequences of lithium treatment on vesicle recycling and neurotransmitter availability. In the present study we have examined acute and chronic effects of lithium on synaptic vesicle recycling using primary hippocampal neurons. We found that exocytosis of readily releasable pool vesicles as well as recycling pool vesicles was unaffected by acute and chronic treatment within the therapeutic range or at higher lithium concentrations. Consistent with this observation, we also noticed that the network activity and number of active synapses within the network were also not significantly altered after lithium treatment. Taken together, as lithium treatment does not affect synaptic vesicle release at even high concentrations, our data suggest that therapeutic effects of lithium in bipolar affective disorder are not directly related to presynaptic function.

  3. Gonadal Steroids: Effects on Excitability of Hippocampal Pyramidal Cells

    NASA Astrophysics Data System (ADS)

    Teyler, Timothy J.; Vardaris, Richard M.; Lewis, Deborah; Rawitch, Allen B.

    1980-08-01

    Electrophysiological field potentials from hippocampal slices of rat brain show sex-linked differences in response to 1 × 10-10M concentrations of estradiol and testosterone added to the incubation medium. Slices from male rats show increased excitability to estradiol and not to testosterone. Slices from female rats are not affected by estradiol, but slices from female rats in diestrus show increased excitability in response to testosterone whereas slices from females in proestrus show decreased excitability.

  4. Osteopontin Expression in Acute Immune Response Mediates Hippocampal Synaptogenesis and Adaptive Outcome Following Cortical Brain Injury

    PubMed Central

    Chan, Julie L.; Reeves, Thomas M.; Phillips, Linda L.

    2014-01-01

    Traumatic brain injury (TBI) produces axotomy, deafferentation and reactive synaptogenesis. Inflammation influences synaptic repair, and the novel brain cytokine osteopontin (OPN) has potential to support axon regeneration through exposure of its integrin receptor binding sites. This study explored whether OPN secretion and proteolysis by matrix metalloproteinases (MMPs) mediate the initial degenerative phase of synaptogenesis, targeting reactive neuroglia to affect successful repair. Adult rats received unilateral entorhinal cortex lesion (UEC) modeling adaptive synaptic plasticity. Over the first week postinjury, hippocampal OPN protein and mRNA were assayed and histology performed. At 1–2d, OPN protein increased up to 51 fold, and was localized within activated, mobilized glia. OPN transcript also increased over 50 fold, predominantly within reactive microglia. OPN fragments known to be derived from MMP proteolysis were elevated at 1d, consistent with prior reports of UEC glial activation and enzyme production. Postinjury minocycline immunosuppression attenuated MMP-9 gelatinase activity, which was correlated with reduction of neutrophil gelatinase-associated lipocalin (LCN2) expression, and reduced OPN fragment generation. The antibiotic also attenuated removal of synapsin-1 positive axons from the deafferented zone. OPN KO mice subjected to UEC had similar reduction of hippocampal MMP-9 activity, as well as lower synapsin-1 breakdown over the deafferented zone. MAP1B and N-cadherin, surrogates of cytoarchitecture and synaptic adhesion, were not affected. OPN KO mice with UEC exhibited time dependent cognitive deficits during the synaptogenic phase of recovery. This study demonstrates that OPN can mediate immune response during TBI synaptic repair, positively influencing synapse reorganization and functional recovery. PMID:25151457

  5. Ovarian hormone deficiency reduces intrinsic excitability and abolishes acute estrogen sensitivity in hippocampal CA1 pyramidal neurons

    PubMed Central

    Wu, Wendy W.; Adelman, John P.; Maylie, James

    2011-01-01

    Premature and uncompensated loss of ovarian hormones following ovariectomy (OVX) elevates the risks of cognitive impairment and dementia. These risks are prevented with estrogen (E2)-containing hormone replacement therapy initiated shortly following OVX but not after substantial delay. Currently the cellular bases underlying these clinical findings are unknown. At the cellular level, intrinsic membrane properties regulate the efficiency of synaptic inputs to initiate output action potentials (APs), thereby affecting neuronal communication hence cognitive processing. This study tested the hypothesis that in CA1 pyramidal neurons, intrinsic membrane properties and their acute regulation by E2 require ovarian hormones for maintenance. Whole-cell current clamp recordings were performed on neurons from ~7 months old OVX rats that experienced either short-term (10 days, control OVX) or long-term (5 months, OVXLT) ovarian hormone deficiency. The results reveal that long-term hormone deficiency reduced intrinsic membrane excitability (IE) as measured by the number of evoked action potentials (APs) and firing duration for a given current injection. This was accompanied by AP broadening, an increased slow afterhyperpolarization (sAHP), and faster accumulation of NaV channel inactivation during repetitive firing. In the control OVX neurons, E2 acutely increased IE and reduced the sAHP. In contrast, acute regulation of IE by E2 was absent in the OVXLT neurons. Since the degree of IE of hippocampal pyramidal neurons is positively related with hippocampus-dependent learning ability, and modulation of IE is observed following successful learning, these findings provide a framework for understanding hormone deficiency-related cognitive impairment and the critical window for therapy initiation. PMID:21325532

  6. Large-scale, high-resolution electrophysiological imaging of field potentials in brain slices with microelectronic multielectrode arrays

    PubMed Central

    Ferrea, E.; Maccione, A.; Medrihan, L.; Nieus, T.; Ghezzi, D.; Baldelli, P.; Benfenati, F.; Berdondini, L.

    2012-01-01

    Multielectrode arrays (MEAs) are extensively used for electrophysiological studies on brain slices, but the spatial resolution and field of recording of conventional arrays are limited by the low number of electrodes available. Here, we present a large-scale array recording simultaneously from 4096 electrodes used to study propagating spontaneous and evoked network activity in acute murine cortico-hippocampal brain slices at unprecedented spatial and temporal resolution. We demonstrate that multiple chemically induced epileptiform episodes in the mouse cortex and hippocampus can be classified according to their spatio-temporal dynamics. Additionally, the large-scale and high-density features of our recording system enable the topological localization and quantification of the effects of antiepileptic drugs in local neuronal microcircuits, based on the distinct field potential propagation patterns. This novel high-resolution approach paves the way to detailed electrophysiological studies in brain circuits spanning spatial scales from single neurons up to the entire slice network. PMID:23162432

  7. Large-scale, high-resolution electrophysiological imaging of field potentials in brain slices with microelectronic multielectrode arrays.

    PubMed

    Ferrea, E; Maccione, A; Medrihan, L; Nieus, T; Ghezzi, D; Baldelli, P; Benfenati, F; Berdondini, L

    2012-01-01

    Multielectrode arrays (MEAs) are extensively used for electrophysiological studies on brain slices, but the spatial resolution and field of recording of conventional arrays are limited by the low number of electrodes available. Here, we present a large-scale array recording simultaneously from 4096 electrodes used to study propagating spontaneous and evoked network activity in acute murine cortico-hippocampal brain slices at unprecedented spatial and temporal resolution. We demonstrate that multiple chemically induced epileptiform episodes in the mouse cortex and hippocampus can be classified according to their spatio-temporal dynamics. Additionally, the large-scale and high-density features of our recording system enable the topological localization and quantification of the effects of antiepileptic drugs in local neuronal microcircuits, based on the distinct field potential propagation patterns. This novel high-resolution approach paves the way to detailed electrophysiological studies in brain circuits spanning spatial scales from single neurons up to the entire slice network.

  8. Murine precision-cut lung slices exhibit acute responses following exposure to gasoline direct injection engine emissions.

    PubMed

    Maikawa, Caitlin L; Zimmerman, Naomi; Rais, Khaled; Shah, Mittal; Hawley, Brie; Pant, Pallavi; Jeong, Cheol-Heon; Delgado-Saborit, Juana Maria; Volckens, John; Evans, Greg; Wallace, James S; Godri Pollitt, Krystal J

    2016-10-15

    Gasoline direct injection (GDI) engines are increasingly prevalent in the global vehicle fleet. Particulate matter emissions from GDI engines are elevated compared to conventional gasoline engines. The pulmonary effects of these higher particulate emissions are unclear. This study investigated the pulmonary responses induced by GDI engine exhaust using an ex vivo model. The physiochemical properties of GDI engine exhaust were assessed. Precision cut lung slices were prepared using Balb/c mice to evaluate the pulmonary response induced by one-hour exposure to engine-out exhaust from a laboratory GDI engine operated at conditions equivalent to vehicle highway cruise conditions. Lung slices were exposed at an air-liquid interface using an electrostatic aerosol in vitro exposure system. Particulate and gaseous exhaust was fractionated to contrast mRNA production related to polycyclic aromatic hydrocarbon (PAH) metabolism and oxidative stress. Exposure to GDI engine exhaust upregulated genes involved in PAH metabolism, including Cyp1a1 (2.71, SE=0.22), and Cyp1b1 (3.24, SE=0.12) compared to HEPA filtered air (p<0.05). GDI engine exhaust further increased Cyp1b1 expression compared to filtered GDI engine exhaust (i.e., gas fraction only), suggesting this response was associated with the particulate fraction. Exhaust particulate was dominated by high molecular weight PAHs. Hmox1, an oxidative stress marker, exhibited increased expression after exposure to GDI (1.63, SE=0.03) and filtered GDI (1.55, SE=0.04) engine exhaust compared to HEPA filtered air (p<0.05), likely attributable to a combination of the gas and particulate fractions. Exposure to GDI engine exhaust contributes to upregulation of genes related to the metabolism of PAHs and oxidative stress.

  9. Acute deep brain stimulation in the thalamic reticular nucleus protects against acute stress and modulates initial events of adult hippocampal neurogenesis.

    PubMed

    Magdaleno-Madrigal, Víctor Manuel; Pantoja-Jiménez, Christopher Rodrigo; Bazaldúa, Adrián; Fernández-Mas, Rodrigo; Almazán-Alvarado, Salvador; Bolaños-Alejos, Fernanda; Ortíz-López, Leonardo; Ramírez-Rodriguez, Gerardo Bernabé

    2016-11-01

    Deep brain stimulation (DBS) is used as an alternative therapeutic procedure for pharmacoresistant psychiatric disorders. Recently the thalamic reticular nucleus (TRN) gained attention due to the description of a novel pathway from the amygdala to this nucleus suggesting that may be differentially disrupted in mood disorders. The limbic system is implicated in the regulation of these disorders that are accompanied by neuroplastic changes. The hippocampus is highly plastic and shows the generation of new neurons, process affected by stress but positively regulated by antidepressant drugs. We explored the impact of applying acute DBS to the TRN (DBS-TRN) in male Wistar rats exposed to acute stress caused by the forced-swim Porsolt's test (FST) and on initial events of hippocampal neurogenesis. After the first session of forced-swim, rats were randomly subdivided in a DBS-TRN and a Sham group. Stimulated rats received 10min of DBS, thus the depressant-like behavior reflected as immobility was evaluated in the second session of forced-swim. Locomotricity was evaluated in the open field test. Cell proliferation and doublecortin-associated cells were quantified in the hippocampus of other cohorts of rats. No effects of electrode implantation were found in locomotricity. Acute DBS-TRN reduced immobility in comparison to the Sham group (p<0.001). DBS-TRN increased cell proliferation (Ki67 or BrdU-positive cells; p=0.02, p=0.02) and the number of doublecortin-cells compared to the Sham group (p<0.02). Similar effects were found in rats previously exposed to the first session of forced-swim. Our data could suggest that TRN brain region may be a promising target for DBS to treat intractable depression.

  10. Neuroprotective effect of acute melatonin treatment on hippocampal neurons against irradiation by inhibition of caspase-3

    PubMed Central

    LI, JIANGUO; ZHANG, GUOWEI; MENG, ZHUANGZHI; WANG, LINGZHAN; LIU, HAIYING; LIU, QIANG; BUREN, BATU

    2016-01-01

    Neuronal cell apoptosis is associated with various factors that induce neurological damage, including radiation exposure. When administered prior to exposure to radiation, a protective agent may prevent cellular and molecular injury. The present study aimed to investigate whether melatonin exerts a neuroprotective effect by inhibiting the caspase cell death pathway. Male Sprague-Dawley rats were administered melatonin (100 mg/kg body weight) 30 min prior to radiation exposure in red light during the evening. In order to elucidate whether melatonin has a neuroprotective role, immunohistochemistry, terminal deoxynucleotidyl transferase dUTP nick-end labeling, Nissl staining, reverse transcription-quantitative polymerase chain reaction, reactive oxygen species analysis and western blotting were performed. At 24 h post-melatonin treatment, caspase-3 mRNA and protein expression levels were significantly decreased. These results demonstrated that melatonin may protect hippocampal neurons via the inhibition of caspase-3 when exposed to irradiation. Therefore, caspase-3 inhibition serves a neuroprotective and antioxidant role in the interventional treatment of melatonin. The results of the present study suggested that melatonin may have a potential therapeutic effect against irradiation; however, further studies are required in order to elucidate the underlying antioxidant mechanisms. PMID:27313671

  11. Fingolimod Limits Acute Aβ Neurotoxicity and Promotes Synaptic Versus Extrasynaptic NMDA Receptor Functionality in Hippocampal Neurons

    PubMed Central

    Joshi, Pooja; Gabrielli, Martina; Ponzoni, Luisa; Pelucchi, Silvia; Stravalaci, Matteo; Beeg, Marten; Mazzitelli, Sonia; Braida, Daniela; Sala, Mariaelvina; Boda, Enrica; Buffo, Annalisa; Gobbi, Marco; Gardoni, Fabrizio; Matteoli, Michela; Marcello, Elena; Verderio, Claudia

    2017-01-01

    Fingolimod, also known as FTY720, is an analogue of the sphingolipid sphingosine, which has been proved to be neuroprotective in rodent models of Alzheimer’s disease (AD). Several cellular and molecular targets underlying the neuroprotective effects of FTY720 have been recently identified. However, whether the drug directly protects neurons from toxicity of amyloid-beta (Aβ) still remains poorly defined. Using a combination of biochemical assays, live imaging and electrophysiology we demonstrate that FTY720 induces a rapid increase in GLUN2A-containing neuroprotective NMDARs on the surface of dendritic spines in cultured hippocampal neurons. In addition, the drug mobilizes extrasynaptic GLUN2B-containing NMDARs, which are coupled to cell death, to the synapses. Altered ratio of synaptic/extrasynaptic NMDARs decreases calcium responsiveness of neurons to neurotoxic soluble Aβ 1–42 and renders neurons resistant to early alteration of calcium homeostasis. The fast defensive response of FTY720 occurs through a Sphingosine-1-phosphate receptor (S1P-R) -dependent mechanism, as it is lost in the presence of S1P-R1 and S1P-R3 antagonists. We propose that rapid synaptic relocation of NMDARs might have direct impact on amelioration of cognitive performance in transgenic APPswe/PS1dE9 AD mice upon sub-chronic treatment with FTY720. PMID:28134307

  12. Acute low dose of MK-801 prevents memory deficits without altering hippocampal DARPP-32 expression and BDNF levels in sepsis survivor rats.

    PubMed

    Cassol-Jr, Omar J; Comim, Clarissa M; Constantino, Larissa S; Rosa, Daniela V F; Mango, Luiz Alexandre V; Stertz, Laura; Kapczinski, Flávio; Romano-Silva, Marco A; Quevedo, João; Dal-Pizzol, Felipe

    2011-01-01

    Sepsis is characterized by an intense inflammatory reaction with potential neurotoxic effects in the central nervous system and damage to memory and learning ability. We assessed the effects of acute low dose of MK-801 on the memory impairment, hippocampal BDNF levels and DARPP-32 expression ten days after sepsis. Under anesthesia, male Wistar rats underwent either cecal ligation and perforation (CLP) or sham. Then, the animals received either a single systemic injection of MK-801 (0.025 mg/kg) or saline solution. Ten days after CLP, the animals were submitted to the step-down inhibitory avoidance and object recognition tests. Also, the hippocampal BDNF protein levels and DARPP-32 expression were evaluated. MK-801 prevented cognitive impairment, but did not affect the hippocampal BDNF levels. DARPP-32 expression was significantly different only in the animals submitted to sepsis that received MK-801 treatment. Thus, we demonstrated that a single low dose of MK-801 prevented memory impairment without altering hippocampal DARPP-32 expression and BDNF levels.

  13. Patch-clamp recordings of rat neurons from acute brain slices of the somatosensory cortex during magnetic stimulation

    PubMed Central

    Pashut, Tamar; Magidov, Dafna; Ben-Porat, Hana; Wolfus, Shuki; Friedman, Alex; Perel, Eli; Lavidor, Michal; Bar-Gad, Izhar; Yeshurun, Yosef; Korngreen, Alon

    2014-01-01

    Although transcranial magnetic stimulation (TMS) is a popular tool for both basic research and clinical applications, its actions on nerve cells are only partially understood. We have previously predicted, using compartmental modeling, that magnetic stimulation of central nervous system neurons depolarized the soma followed by initiation of an action potential in the initial segment of the axon. The simulations also predict that neurons with low current threshold are more susceptible to magnetic stimulation. Here we tested these theoretical predictions by combining in vitro patch-clamp recordings from rat brain slices with magnetic stimulation and compartmental modeling. In agreement with the modeling, our recordings demonstrate the dependence of magnetic stimulation-triggered action potentials on the type and state of the neuron and its orientation within the magnetic field. Our results suggest that the observed effects of TMS are deeply rooted in the biophysical properties of single neurons in the central nervous system and provide a framework both for interpreting existing TMS data and developing new simulation-based tools and therapies. PMID:24917788

  14. New illumination technique for IR-video guided patch-clamp recording from neurons in slice cultures on biomembrane.

    PubMed

    Alix, Philippe; Winterer, Jochen; Müller, Wolfgang

    2003-09-30

    Slice cultures on biomembrane are the method of choice for studying Ca2+-dependent plastic changes occurring over several days to weeks. Using IR-differential interference contrast, good visualization of neurons in biomembrane slice cultures has been achieved despite a negative optical effect of the biomembrane, but epifluorescence imaging requires removal of a Wollaston prism and the analyzer. Here, we describe a novel illumination method to overcome this problem. Using optic fiber illumination at a shallow angle from the top of the slice culture, with or without additional illumination from the bottom, we obtained good cellular resolution of neurons in biomembrane slice cultures as well as in acute slices with an infrared-video camera. With this technique, we demonstrate visually guided whole-cell patch-clamp recording of Na+- and K+-currents as well as combination of whole-cell recording with fluorescence imaging of hippocampal and entorhinal cortex neurons in biomembrane slice cultures. Our inexpensive method should prove very useful for studying in vitro effects of long-term manipulations on membrane currents and intracellular Ca2+-signaling.

  15. Restraint stress increases hemichannel activity in hippocampal glial cells and neurons

    PubMed Central

    Orellana, Juan A.; Moraga-Amaro, Rodrigo; Díaz-Galarce, Raúl; Rojas, Sebastián; Maturana, Carola J.; Stehberg, Jimmy; Sáez, Juan C.

    2015-01-01

    Stress affects brain areas involved in learning and emotional responses, which may contribute in the development of cognitive deficits associated with major depression. These effects have been linked to glial cell activation, glutamate release and changes in neuronal plasticity and survival including atrophy of hippocampal apical dendrites, loss of synapses and neuronal death. Under neuro-inflammatory conditions, we recently unveiled a sequential activation of glial cells that release ATP and glutamate via hemichannels inducing neuronal death due to activation of neuronal NMDA/P2X7 receptors and pannexin1 hemichannels. In the present work, we studied if stress-induced glia activation is associated to changes in hemichannel activity. To this end, we compared hemichannel activity of brain cells after acute or chronic restraint stress in mice. Dye uptake experiments in hippocampal slices revealed that acute stress induces opening of both Cx43 and Panx1 hemichannels in astrocytes, which were further increased by chronic stress; whereas enhanced Panx1 hemichannel activity was detected in microglia and neurons after acute/chronic and chronic stress, respectively. Moreover, inhibition of NMDA/P2X7 receptors reduced the chronic stress-induced hemichannel opening, whereas blockade of Cx43 and Panx1 hemichannels fully reduced ATP and glutamate release in hippocampal slices from stressed mice. Thus, we propose that gliotransmitter release through hemichannels may participate in the pathogenesis of stress-associated psychiatric disorders and possibly depression. PMID:25883550

  16. Restraint stress increases hemichannel activity in hippocampal glial cells and neurons.

    PubMed

    Orellana, Juan A; Moraga-Amaro, Rodrigo; Díaz-Galarce, Raúl; Rojas, Sebastián; Maturana, Carola J; Stehberg, Jimmy; Sáez, Juan C

    2015-01-01

    Stress affects brain areas involved in learning and emotional responses, which may contribute in the development of cognitive deficits associated with major depression. These effects have been linked to glial cell activation, glutamate release and changes in neuronal plasticity and survival including atrophy of hippocampal apical dendrites, loss of synapses and neuronal death. Under neuro-inflammatory conditions, we recently unveiled a sequential activation of glial cells that release ATP and glutamate via hemichannels inducing neuronal death due to activation of neuronal NMDA/P2X7 receptors and pannexin1 hemichannels. In the present work, we studied if stress-induced glia activation is associated to changes in hemichannel activity. To this end, we compared hemichannel activity of brain cells after acute or chronic restraint stress in mice. Dye uptake experiments in hippocampal slices revealed that acute stress induces opening of both Cx43 and Panx1 hemichannels in astrocytes, which were further increased by chronic stress; whereas enhanced Panx1 hemichannel activity was detected in microglia and neurons after acute/chronic and chronic stress, respectively. Moreover, inhibition of NMDA/P2X7 receptors reduced the chronic stress-induced hemichannel opening, whereas blockade of Cx43 and Panx1 hemichannels fully reduced ATP and glutamate release in hippocampal slices from stressed mice. Thus, we propose that gliotransmitter release through hemichannels may participate in the pathogenesis of stress-associated psychiatric disorders and possibly depression.

  17. Acute and fractionated exposure to high-LET (56)Fe HZE-particle radiation both result in similar long-term deficits in adult hippocampal neurogenesis.

    PubMed

    Rivera, Phillip D; Shih, Hung-Ying; Leblanc, Junie A; Cole, Mara G; Amaral, Wellington Z; Mukherjee, Shibani; Zhang, Shichuan; Lucero, Melanie J; Decarolis, Nathan A; Chen, Benjamin P C; Eisch, Amelia J

    2013-12-01

    Astronauts on multi-year interplanetary missions will be exposed to a low, chronic dose of high-energy, high-charge particles. Studies in rodents show acute, nonfractionated exposure to these particles causes brain changes such as fewer adult-generated hippocampal neurons and stem cells that may be detrimental to cognition and mood regulation and thus compromise mission success. However, the influence of a low, chronic dose of these particles on neurogenesis and stem cells is unknown. To examine the influence of galactic cosmic radiation on neurogenesis, adult-generated stem and progenitor cells in Nestin-CreER(T2)/R26R-YFP transgenic mice were inducibly labeled to allow fate tracking. Mice were then sham exposed or given one acute 100 cGy (56)Fe-particle exposure or five fractionated 20 cGy (56)Fe-particle exposures. Adult-generated hippocampal neurons and stem cells were quantified 24 h or 3 months later. Both acute and fractionated exposure decreased the amount of proliferating cells and immature neurons relative to sham exposure. Unexpectedly, neither acute nor fractionated exposure decreased the number of adult neural stem cells relative to sham expsoure. Our findings show that single and fractionated exposures of (56)Fe-particle irradiation are similarly detrimental to adult-generated neurons. Implications for future missions and ground-based studies in space radiation are discussed.

  18. The differential effects of acute vs. chronic stress and their combination on hippocampal parvalbumin and inducible heat shock protein 70 expression.

    PubMed

    Filipović, D; Zlatković, J; Gass, P; Inta, D

    2013-04-16

    The hippocampus plays a central role in stress-related mood disorders. The effects of acute vs. chronic stress on the integrity of hippocampal circuitry in influencing the vulnerability to, or resiliency against, neuronal injury are poorly understood. Here we investigated whether acute vs. chronic psychosocial isolation stress or a combination of the two (chronic stress followed by acute stress) influences the expression of the interneuronal marker parvalbumin (PV) and the chaperone-inducible heat shock protein 70 (Hsp70i) in different subregions of the hippocampus. Low levels of the Ca(2+)-binding protein (PV) may increase the vulnerability to neuronal injury, and Hsp70i represents an indicator of intense excitation-induced neuronal stress. Adult male Wistar rats were exposed to 2h of immobilization (IM) or cold (4°C) (acute stressors), 21d of social isolation (chronic stress), or a combination of both acute and chronic stress. Both chronic isolation and the combined stressors strongly decreased the PV-immunoreactive cells in the CA1, CA3 and dentate gyrus (DG) region of the hippocampus, while acute stress did not affect PV expression. The combination of acute and chronic stress induced a dramatic increase in Hsp70i expression in the DG, but Hsp70i expression was unaffected in acute and chronic stress alone. We also monitored serum corticosterone (CORT) levels as a neuroendocrine marker of the stress response. Acute stress increased CORT levels, while chronic isolation stress compromised hypothalamic-pituitary-adrenocortical (HPA) axis activity such that the normal stress response was impaired following subsequent acute stress. These results indicate that in contrast to acute stress, chronic isolation compromises the HPA axis and generates a considerable reduction in PV expression, representing a decrease in the calcium-buffering capacity and a putatively higher vulnerability of specific hippocampal interneurons to excitotoxic injury. The induction of Hsp70i

  19. Distinct Trajectories of Cortisol Response to Prolonged Acute Stress Are Linked to Affective Responses and Hippocampal Gray Matter Volume in Healthy Females.

    PubMed

    Admon, Roee; Treadway, Michael T; Valeri, Linda; Mehta, Malavika; Douglas, Samuel; Pizzagalli, Diego A

    2017-08-16

    The development of robust laboratory procedures for acute stress induction over the last decades has greatly advanced our understanding of stress responses in humans and their underlying neurobiological mechanisms. Nevertheless, attempts to uncover linear relationships among endocrine, neural, and affective responses to stress have generally yielded inconsistent results. Here, 79 healthy females completed a well established laboratory procedure of acute stress induction that was modified to prolong its effect. Endocrinological and subjective affect assessments revealed stress-induced increases in cortisol release and negative affect that persisted 65 and 100 min after stress onset, respectively, confirming a relatively prolonged acute stress induction. Applying latent class linear mixed modeling on individuals' patterns of cortisol responses identified three distinct trajectories of cortisol response: the hyper-response (n = 10), moderate-response (n = 21), and mild-response (n = 48) groups. Notably, whereas all three groups exhibited a significant stress-induced increase in cortisol release and negative affect, the hyper-response and mild-response groups both reported more negative affect relative to the moderate-response group. Structural MRI revealed no group differences in hippocampal and amygdala volumes, yet a continuous measure of cortisol response (area under the curve) showed that high and low levels of stress-induced cortisol release were associated with less hippocampal gray matter volume compared with moderate cortisol release. Together, these results suggest that distinct trajectories of cortisol response to prolonged acute stress among healthy females may not be captured by conventional linear analyses; instead, quadratic relations may better describe links between cortisol response to stress and affective responses, as well as hippocampal structural variability.SIGNIFICANCE STATEMENT Despite substantial research, it is unclear whether and how

  20. Metabotropic glutamate response in acutely dissociated hippocampal CA1 pyramidal neurones of the rat.

    PubMed Central

    Shirasaki, T; Harata, N; Akaike, N

    1994-01-01

    1. The metabotropic glutamate (mGlu) response was investigated in dissociated rat hippocampal CA1 pyramidal neurones using conventional and nystatin-perforated whole-cell modes of the patch recording configuration. 2. In the perforated patch recording configuration, the application of glutamate (Glu), quisqualate (QA), aspartate (Asp) and N-methyl-D-aspartate (NMDA) induced a slow outward current superimposed on a fast ionotropic inward current, whereas alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate (KA) induced only an ionotropic inward current at a holding potential (VH) of -20 mV. A specific agonist of the mGlu receptor (mGluR), trans-1-aminocyclopentane-1,3-dicarboxylate (tACPD), induced an outward current in approximately 80% of the neurones tested. Asp- and NMDA-induced outward currents were antagonized by D-2-amino-5-phosphonopentanoate (D-AP5) whereas Glu-, QA- and tACPD-induced outward currents were not antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 6,7-dinitroquinoxaline-2,3-dione (DNQX) and D-AP5, indicating that the mGlu response is an outward current component. 3. L-2-Amino-3-phosphonopropionate (L-AP3) and DL-2-amino-4-phosphonobutyrate (AP4) did not block the mGlu response. 4. The relative potencies of mGlu agonists were QA > Glu > tACPD. The threshold and EC50 values of metabotropic outward currents were 10-100 times lower than those of the ionotropic inward current (iGlu response). 5. The reversal potential of the mGlu response (EmGlu) was close to EK (K+ equilibrium potential), and it shifted 59.5 mV for a tenfold change in extracellular K+ concentration. 6. In Ca(2+)-free external solution, the mGlu response was elicited by an initial application of Glu, but subsequent applications failed to induce the response. There was also an increase in the intracellular free Ca2+ concentration ([Ca2+]i) during the application of Glu and QA but not of AMPA, indicating Ca2+ release from an intracellular Ca2+ store. 7

  1. Suppression of acute seizures by theta burst electrical stimulation of the hippocampal commissure using a closed-loop system.

    PubMed

    Siah, Boon Hong; Chiang, Chia-Chu; Ju, Ming-Shaung; Lin, Chou-Ching K

    2014-12-17

    This study investigated the effects of electrical stimulation with theta burst stimulation (eTBS) on seizure suppression. Optimal parameters of eTBS were determined through open-loop stimulation experiments and then implemented in a close-loop seizure control system. For the experiments, 4-aminopyridine (4-AP) was injected into the right hippocampus of Sprague-Dawley rats to induce an acute seizure. eTBS was applied on the ventral hippocampal commissure and the effects of eTBS with different combinations of burst frequency and number of pulses per burst were analyzed in terms of seizure suppression. A closed-loop seizure control system was then implemented based on optimal eTBS parameters. The efficiency of the closed-loop eTBS was evaluated and compared to that of high frequency stimulation. The results show that eTBS induced global suppression in the hippocampus and this was sustained even after the application of eTBS. The optimal parameter of eTBS in the open-loop stimulation experiments was a burst frequency at 100Hz with nine pulses in a burst. The eTBS integrated with the on-off control law yielded less actions and cumulative delivered charge, but induced longer after-effects of seizure suppression compared to continuous high frequency stimulation (cHFS). To conclude, eTBS has suppressive effects on 4-AP induced seizure. A closed-loop eTBS system provides a more effective way of suppressing seizure and requires less effort compared to cHFS. eTBS may be a novel stimulation protocol for effective seizure control. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. The Relationship between Membrane Potential and Calcium Dynamics in Glucose-Stimulated Beta Cell Syncytium in Acute Mouse Pancreas Tissue Slices

    PubMed Central

    Miller, Evan W.; Slak Rupnik, Marjan

    2013-01-01

    Oscillatory electrical activity is regarded as a hallmark of the pancreatic beta cell glucose-dependent excitability pattern. Electrophysiologically recorded membrane potential oscillations in beta cells are associated with in-phase oscillatory cytosolic calcium activity ([Ca2+]i) measured with fluorescent probes. Recent high spatial and temporal resolution confocal imaging revealed that glucose stimulation of beta cells in intact islets within acute tissue slices produces a [Ca2+]i change with initial transient phase followed by a plateau phase with highly synchronized [Ca2+]i oscillations. Here, we aimed to correlate the plateau [Ca2+]i oscillations with the oscillations of membrane potential using patch-clamp and for the first time high resolution voltage-sensitive dye based confocal imaging. Our results demonstrated that the glucose-evoked membrane potential oscillations spread over the islet in a wave-like manner, their durations and wave velocities being comparable to the ones for [Ca2+]i oscillations and waves. High temporal resolution simultaneous records of membrane potential and [Ca2+]i confirmed tight but nevertheless limited coupling of the two processes, with membrane depolarization preceding the [Ca2+]i increase. The potassium channel blocker tetraethylammonium increased the velocity at which oscillations advanced over the islet by several-fold while, at the same time, emphasized differences in kinetics of the membrane potential and the [Ca2+]i. The combination of both imaging techniques provides a powerful tool that will help us attain deeper knowledge of the beta cell network. PMID:24324777

  3. Hippocampal stroke.

    PubMed

    Szabo, Kristina

    2014-01-01

    The first to link disturbance of memory and lesions of the medial temporal lobe was the Russian neurologist von Bechterew, who in 1989 presented the brain of a 60-year-old man who had suffered from severe amnesia. Autopsy showed bilateral damage of the medial temporal lobe. Several following postmortem case studies confirmed the association between permanent amnesia and bitemporal stroke. Reports of transient memory deficits in unilateral stroke in combination with other neurological and neuropsychological deficits followed. With the advent of brain imaging, persistent or transient amnesia as the sole or primary manifestation of acute - mostly left-sided - hippocampal stroke was described. With the use of modern MRI techniques the identification of typical ischemic stroke lesion patterns affecting the hippocampus has become possible. Although overt cognitive deficits in unilateral hippocampal stroke seem to be rare, a careful neuropsychological examination might be necessary to detect resulting neuropsychological deficits including disturbances of verbal and nonverbal episodic long-term memory and spatial orientation.

  4. Ketogenic diet sensitizes glucose control of hippocampal excitability1

    PubMed Central

    Kawamura, Masahito; Ruskin, David N.; Geiger, Jonathan D.; Boison, Detlev; Masino, Susan A.

    2014-01-01

    A high-fat low-carbohydrate ketogenic diet (KD) is an effective treatment for refractory epilepsy, yet myriad metabolic effects in vivo have not been reconciled clearly with neuronal effects. A KD limits blood glucose and produces ketone bodies from β-oxidation of lipids. Studies have explored changes in ketone bodies and/or glucose in the effects of the KD, and glucose is increasingly implicated in neurological conditions. To examine the interaction between altered glucose and the neural effects of a KD, we fed rats and mice a KD and restricted glucose in vitro while examining the seizure-prone CA3 region of acute hippocampal slices. Slices from KD-fed animals were sensitive to small physiological changes in glucose, and showed reduced excitability and seizure propensity. Similar to clinical observations, reduced excitability depended on maintaining reduced glucose. Enhanced glucose sensitivity and reduced excitability were absent in slices obtained from KD-fed mice lacking adenosine A1 receptors (A1Rs); in slices from normal animals effects of the KD could be reversed with blockers of pannexin-1 channels, A1Rs, or KATP channels. Overall, these studies reveal that a KD sensitizes glucose-based regulation of excitability via purinergic mechanisms in the hippocampus and thus link key metabolic and direct neural effects of the KD. PMID:25170119

  5. Modification of hippocampal markers of synaptic plasticity by memantine in animal models of acute and repeated restraint stress: implications for memory and behavior.

    PubMed

    Amin, Shaimaa Nasr; El-Aidi, Ahmed Amro; Ali, Mohamed Mostafa; Attia, Yasser Mahmoud; Rashed, Laila Ahmed

    2015-06-01

    Stress is any condition that impairs the balance of the organism physiologically or psychologically. The response to stress involves several neurohormonal consequences. Glutamate is the primary excitatory neurotransmitter in the central nervous system, and its release is increased by stress that predisposes to excitotoxicity in the brain. Memantine is an uncompetitive N-methyl D-aspartate glutamatergic receptors antagonist and has shown beneficial effect on cognitive function especially in Alzheimer's disease. The aim of the work was to investigate memantine effect on memory and behavior in animal models of acute and repeated restraint stress with the evaluation of serum markers of stress and the expression of hippocampal markers of synaptic plasticity. Forty-two male rats were divided into seven groups (six rats/group): control, acute restraint stress, acute restraint stress with Memantine, repeated restraint stress, repeated restraint stress with Memantine and Memantine groups (two subgroups as positive control). Spatial working memory and behavior were assessed by performance in Y-maze. We evaluated serum cortisol, tumor necrotic factor, interleukin-6 and hippocampal expression of brain-derived neurotrophic factor, synaptophysin and calcium-/calmodulin-dependent protein kinase II. Our results revealed that Memantine improved spatial working memory in repeated stress, decreased serum level of stress markers and modified the hippocampal synaptic plasticity markers in both patterns of stress exposure; in ARS, Memantine upregulated the expression of synaptophysin and brain-derived neurotrophic factor and downregulated the expression of calcium-/calmodulin-dependent protein kinase II, and in repeated restraint stress, it upregulated the expression of synaptophysin and downregulated calcium-/calmodulin-dependent protein kinase II expression.

  6. The Energy Demand of Fast Neuronal Network Oscillations: Insights from Brain Slice Preparations

    PubMed Central

    Kann, Oliver

    2012-01-01

    Fast neuronal network oscillations in the gamma range (30–100 Hz) in the cerebral cortex have been implicated in higher cognitive functions such as sensual perception, working memory, and, perhaps, consciousness. However, little is known about the energy demand of gamma oscillations. This is mainly caused by technical limitations that are associated with simultaneous recordings of neuronal activity and energy metabolism in small neuronal networks and at the level of mitochondria in vivo. Thus recent studies have focused on brain slice preparations to address the energy demand of gamma oscillations in vitro. Here, reports will be summarized and discussed that combined electrophysiological recordings, oxygen sensor microelectrodes, and live-cell fluorescence imaging in acutely prepared slices and organotypic slice cultures of the hippocampus from both, mouse and rat. These reports consistently show that gamma oscillations can be reliably induced in hippocampal slice preparations by different pharmacological tools. They suggest that gamma oscillations are associated with high energy demand, requiring both rapid adaptation of oxidative energy metabolism and sufficient supply with oxygen and nutrients. These findings might help to explain the exceptional vulnerability of higher cognitive functions during pathological processes of the brain, such as circulatory disturbances, genetic mitochondrial diseases, and neurodegeneration. PMID:22291647

  7. Bacopa monnieri extract enhances learning-dependent hippocampal long-term synaptic potentiation.

    PubMed

    Promsuban, Charkriya; Limsuvan, Suveerawan; Akarasereenont, Pravit; Tilokskulchai, Kanokwan; Tapechum, Sompol; Pakaprot, Narawut

    2017-11-08

    Bacopa monnieri has been used in Ayurvedic medicine as a memory enhancer for a long time; however, its direct effect on synaptic plasticity has not been investigated. To the best of our knowledge, this study is the first to report the effect of B. monnieri on long-term synaptic potentiation in acute hippocampal slices. Adult male Wistar rats were orally administered either sterile water or the ethanolic extract of B. monnieri for 60 days. The extracellular recording was performed to measure the field excitatory postsynaptic potential in the acute hippocampal slices of these rats. Our results showed that B. monnieri extract significantly increased long-term potentiation magnitude compared with the control group, whereas there was no change in basal synaptic transmission. The data support the beneficial mnemonic effect of B. monnieri, and suggest that this effect might be because of the increase of learning-associated synaptic machinery, resulting in the long-term potentiation enhancement and strengthening of hippocampal synapses, which plays a critical role in learning and memory formation.

  8. Mild hypothermia, but not propofol, is neuroprotective in organotypic hippocampal cultures.

    PubMed

    Feiner, John R; Bickler, Philip E; Estrada, Sergio; Donohoe, Paul H; Fahlman, Christian S; Schuyler, Jennifer A

    2005-01-01

    The neuroprotective potency of anesthetics such as propofol compared to mild hypothermia remains undefined. Therefore, we determined whether propofol at two clinically relevant concentrations is as effective as mild hypothermia in preventing delayed neuron death in hippocampal slice cultures (HSC). Survival of neurons was assessed 2 and 3 days after 1 h oxygen and glucose deprivation (OGD) either at 37 degrees C (with or without 10 or 100 microM propofol) or at an average temperature of 35 degrees C during OGD (mild hypothermia). Cell death in CA1, CA3, and dentate neurons in each slice was measured with propidium iodide fluorescence. Mild hypothermia eliminated death in CA1, CA3, and dentate neurons but propofol protected dentate neurons only at a concentration of 10 microM; the more ischemia vulnerable CA1 and CA3 neurons were not protected by either 10 microM or 100 microM propofol. In slice cultures, the toxicity of 100 muM N-methyl-D-aspartate (NMDA), 500 microM glutamate, and 20 microM alpha-amino-5-methyl-4-isoxazole propionic acid (AMPA) was not reduced by 100 microM propofol. Because propofol neuroprotection may involve gamma-aminobutyric acid (GABA)-mediated indirect inhibition of glutamate receptors (GluRs), the effects of propofol on GluR activity (calcium influx induced by GluR agonists) were studied in CA1 neurons in HSC, in isolated CA1 neurons, and in cortical brain slices. Propofol (100 and 200 microM, approximate burst suppression concentrations) decreased glutamate-mediated [Ca2+]i increases (Delta[Ca2+]i) responses by 25%-35% in isolated CA1 neurons and reduced glutamate and NMDA Delta[Ca2+]i in acute and cultured hippocampal slices by 35%-50%. In both CA1 neurons and cortical slices, blocking GABAA receptors with picrotoxin reduced the inhibition of GluRs substantially. We conclude that mild hypothermia, but not propofol, protects CA1 and CA3 neurons in hippocampal slice cultures subjected to oxygen and glucose deprivation. Propofol was not

  9. Brain Slices as Models for Neurodegenerative Disease and Screening Platforms to Identify Novel Therapeutics

    PubMed Central

    Cho, Seongeun; Wood, Andrew; Bowlby, Mark R

    2007-01-01

    Recent improvements in brain slice technology have made this biological preparation increasingly useful for examining pathophysiology of brain diseases in a tissue context. Brain slices maintain many aspects of in vivo biology, including functional local synaptic circuitry with preserved brain architecture, while allowing good experimental access and precise control of the extracellular environment, making them ideal platforms for dissection of molecular pathways underlying neuronal dysfunction. Importantly, these ex vivo systems permit direct treatment with pharmacological agents modulating these responses and thus provide surrogate therapeutic screening systems without recourse to whole animal studies. Virus or particle mediated transgenic expression can also be accomplished relatively easily to study the function of novel genes in a normal or injured brain tissue context. In this review we will discuss acute brain injury models in organotypic hippocampal and co-culture systems and the effects of pharmacological modulation on neurodegeneration. The review will also cover the evidence of developmental plasticity in these ex vivo models, demonstrating emergence of injury-stimulated neuronal progenitor cells, and neurite sprouting and axonal regeneration following pathway lesioning. Neuro-and axo-genesis are emerging as significant factors contributing to brain repair following many acute and chronic neurodegenerative disorders. Therefore brain slice models may provide a critical contextual experimental system to explore regenerative mechanisms in vitro. PMID:18615151

  10. Effects of extracellular delta-aminolaevulinic acid on sodium currents in acutely isolated rat hippocampal CA1 neurons.

    PubMed

    Wang, Lang; Yan, Dan; Gu, Yan; Sun, Li-Guang; Ruan, Di-Yun

    2005-12-01

    The effects of delta-aminolaevulinic acid (ALA) on voltage-gated sodium channel (VGSC) currents (I(Na)) in acutely isolated hippocampal CA1 neurons from 10- to 12-day-old Wistar rats were examined by using the whole-cell patch-clamp technique under voltage-clamp conditions. ALA from 0.01 microm to 20 microm was applied to the recorded neurons. Low concentrations of ALA (0.01-1.0 microM) increased I(Na) amplitude, whereas high concentrations of ALA (5.0-20.0 microM) decreased it. The average I(Na) amplitude reached a maximum of 117.4 +/- 3.9% (n = 9, P < 0.05) with 0.1 microM ALA, and decreased to 78.1 +/- 3.8% (n = 13, P < 0.05) with 10 microm ALA. ALA shifted the steady-state activation and inactivation curves of I(Na) in the hyperpolarizing direction with different V0.5, suggesting that ALA could depress the opening threshold of the voltage-gated sodium channel (VGSC) and thus increase the excitability of neurons through facilitating the opening of VGSC. The time course of recovery from inactivation was significantly prolonged at both low and high concentrations of ALA, whereas either low or high concentrations of ALA had no significant effect on the attenuation of I(Na) during stimulation at 5 Hz, indicating that the effect of ALA on VGSC is state-independent. Furthermore, we found that application of ascorbic acid, which blocks pro-oxidative effects in neurons, could prevent the increase of I(Na) amplitude at low concentrations of ALA. Baclofen, an agonist of GABAb receptors, induced some similar effects to ALA on VGSC, whereas bicuculline, an antagonist of GABAa receptors, could not prevent ALA-induced effects on VGSC. These results suggested that ALA regulated VGSC mainly through its pro-oxidative effects and GABAb receptor-mediated effects.

  11. Lamina-specific synaptic connections of hippocampal neurons in vitro.

    PubMed

    Frotscher, M; Heimrich, B

    1995-03-01

    By using slice cultures as a model, we demonstrate here that different target selectivities exist among the various afferent fibers to the hippocampus. As in intact animals, septohippocampal cholinergic fibers, provided by a slice culture of septum, innervate a co-cultured slice of hippocampus diffusely, that is, without forming distinct layers of termination. As in vivo, the septal cholinergic fibers establish synapses with a variety of target cells. Conversely, fibers from an entorhinal slice co-cultured to a hippocampal slice display their normal laminar specificity. They preferentially terminate in the outer molecular layer of the fascia dentata, thereby selectively contacting peripheral dendrites of the granule cells. This preferential termination on peripheral dendritic segments is remarkable, since these fibers do not have to compete with commissural fibers, hypothalamic fibers, and septal afferents for dendritic space under these culture conditions. Moreover, in triplet cultures in which first two hippocampal slices were co-cultured and then, with a delay of 5 days, an entorhinal slice was added, the fibers from the entorhinal slice and those from the hippocampal culture terminated in their appropriate layers in the hippocampal target culture. However, in this approach the normal sequence of ingrowth of these two afferents was reversed. In normal ontogenetic development, entorhinal afferents arrive in the hippocampus before the commissural fibers. The results show that there are different degrees of target selectivity of hippocampal afferents and that the characteristic lamination of certain afferent fibers in the hippocampus is not determined by their sequential ingrowth during development.

  12. In vitro detection of oxygen and glucose deprivation-induced neurodegeneration and pharmacological neuroprotection based on hippocampal stratum pyramidale width.

    PubMed

    Öz, Pınar; Saybaşılı, Hale

    2017-01-01

    Ischemia is one of the most prominent risk factors of neurodegenerative diseases such as Alzheimer's disease. The effects of oxygen and glucose depletion in hippocampal tissue due to ischemia can be mimicked in vitro using the oxygen and glucose deprivation (OGD) model. In this study, we applied OGD on acute rat hippocampal slices in order to design an elementary yet quantitative histological technique that compares the neuroprotective effects of (l)-carnitine to known neuroprotectors, such as the N-methyl-d-aspartate (NMDA) receptor antagonist memantine and the gamma-aminobutyric acid (GABA)-B receptor agonist baclofen. The level of neurodegeneration and the efficiency of pharmacological applications were estimated via stratum pyramidale width measurements in CA1 and CA3 regions of Nissl-stained 200-μm thick hippocampal slices. We demonstrated that (l)-carnitine is an effective pharmacological target against the neurodegeneration induced by in vitro ischemia in a narrow range of concentrations. Even though the effect of chemical neuroprotection was significant, full recovery was not achieved in the dose interval of 5-100μM. In addition to chemical applications, hypothermia was used as a physical neuroprotection against ischemia-related neurodegeneration. Our results showed that incubation of slices for 60min at 4°C provided the same level of neuroprotection as the most effective doses of memantine, baclofen, and (l)-carnitine.

  13. Acute p38-mediated inhibition of NMDA-induced outward currents in hippocampal CA1 neurons by interleukin-1beta.

    PubMed

    Zhang, Ruoyu; Sun, Li; Hayashi, Yoshinori; Liu, Xia; Koyama, Susumu; Wu, Zhou; Nakanishi, Hiroshi

    2010-04-01

    Interleukin-1beta (IL-1beta) is a potent pro-inflammatory cytokine that is primarily produced by microglia in the brain. IL-1beta inhibits N-methyl-d-aspartate (NMDA)-induced outward currents (I(NMDA-OUT)) through IL-1 type I receptor (IL-1RI) in hippocampal CA1 neurons (Zhang, R., Yamada, J., Hayashi, Y., Wu, Z, Koyama, S., Nakanishi, H., 2008. Inhibition of NMDA-induced outward currents by interleukin-1beta in hippocampal neurons, Biochem. Biophys. Res. Commun. 372, 816-820). Although IL-1RI is associated with mitogen-activated protein kinases, their involvement in the effect of IL-1beta on I(NMDA-OUT) remains unclear. In the present study, we demonstrate that IL-1beta caused activation of p38 mitogen-activated protein kinase and that the p38 inhibitor SB203580 significantly blocked the effect of IL-1beta on I(NMDA-OUT) in hippocampal CA1 neurons. Furthermore, the intracellular perfusion of active recombinant p38alpha significantly decreased the mean amplitude of I(NMDA-OUT). In neurons prepared from inflamed hippocampus, the mean amplitude of I(NMDA-OUT) was significantly reduced. In the inflamed hippocampus, IL-1beta and IL-1RI were expressed mainly in microglia and neurons, respectively. These results suggest that IL-1beta increases the excitability of hippocampal CA1 neurons in the p38-dependent inhibition of I(NMDA-OUT).

  14. Thick Slice and Thin Slice Teaching Evaluations

    ERIC Educational Resources Information Center

    Tom, Gail; Tong, Stephanie Tom; Hesse, Charles

    2010-01-01

    Student-based teaching evaluations are an integral component to institutions of higher education. Previous work on student-based teaching evaluations suggest that evaluations of instructors based upon "thin slice" 30-s video clips of them in the classroom correlate strongly with their end of the term "thick slice" student evaluations. This study's…

  15. Mechanical ventilation triggers hippocampal apoptosis by vagal and dopaminergic pathways.

    PubMed

    González-López, Adrián; López-Alonso, Inés; Aguirre, Alina; Amado-Rodríguez, Laura; Batalla-Solís, Estefanía; Astudillo, Aurora; Tomás-Zapico, Cristina; Fueyo, Antonio; dos Santos, Claudia C; Talbot, Konrad; Albaiceta, Guillermo M

    2013-09-15

    Critically ill patients frequently develop neuropsychological disturbances including acute delirium or memory impairment. The need for mechanical ventilation is a risk factor for these adverse events, but a mechanism that links lung stretch and brain injury has not been identified. To identify the mechanisms that lead to brain dysfunction during mechanical ventilation. Brains from mechanically ventilated mice were harvested, and signals of apoptosis and alterations in the Akt survival pathway were studied. These measurements were repeated in vagotomized or haloperidol-treated mice, and in animals intracerebroventricularly injected with selective dopamine-receptor blockers. Hippocampal slices were cultured and treated with micromolar concentrations of dopamine, with or without dopamine receptor blockers. Last, levels of dysbindin, a regulator of the membrane availability of dopamine receptors, were assessed in the experimental model and in brain samples from ventilated patients. Mechanical ventilation triggers hippocampal apoptosis as a result of type 2 dopamine receptor activation in response to vagal signaling. Activation of these receptors blocks the Akt/GSK3β prosurvival pathway and activates the apoptotic cascade, as demonstrated in vivo and in vitro. Vagotomy, systemic haloperidol, or intracerebroventricular raclopride (a type 2 dopamine receptor blocker) ameliorated this effect. Moreover, ventilation induced a concomitant change in the expression of dysbindin-1C. These results were confirmed in brain samples from ventilated patients. These results prove the existence of a pathogenic mechanism of lung stretch-induced hippocampal apoptosis that could explain the neurological changes in ventilated patients and may help to identify novel therapeutic approaches.

  16. Activation of Wnt signaling promotes hippocampal neurogenesis in experimental autoimmune encephalomyelitis.

    PubMed

    Schneider, Reiner; Koop, Barbara; Schröter, Friederike; Cline, Jason; Ingwersen, Jens; Berndt, Carsten; Hartung, Hans-Peter; Aktas, Orhan; Prozorovski, Tim

    2016-07-14

    Disease progression in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), as one of its animal models, is characterized by demyelination and neuronal damage in white and gray matter structures, including the hippocampus. It is thought that dysfunction of the hippocampus, a primary locus of learning and memory consolidation, may contribute to cognitive impairment in MS patients. Previously, we reported an increased generation of hippocampal neuronal progenitors in the acute stage of EAE, whereas the microenvironmental signals triggering this process remained uninvestigated. In the present study, we used the Wnt signaling reporter mouse Axin2(LacZ), to elucidate the molecular mechanisms underlying the activation of the hippocampal neurogenic niche upon autoimmune neuroinflammation. Histological and enzymatic examinations of β-gal during the disease course of EAE, allowed us to survey hippocampal Wnt/β-catenin activity, one of the key signaling pathways of adult neurogenesis. We found that Wnt signaling is transiently upregulated in the acute stage of disease, consistent with a timely induction of canonical Wnt ligands. The enhancement of signaling coincided with hippocampal neuronal damage and local expression of immune cytokines such as TNFα and IFNγ, implicating the role of the inflammatory milieu in activation of the Wnt/β-catenin pathway. Supporting this finding, we show that transient exposure to pro-inflammatory cytokine TNFα triggers Wnt signaling in hippocampal organotypic slice cultures. Importantly, inflammation-mediated activation of the Wnt/β-catenin pathway was associated with enhanced neurogenesis in vitro and in vivo, indicating its potential role in hippocampal tissue regeneration and repair. This study raises the possibility that enhancement of Wnt signaling may support neurogenic processes to cope with neuronal deficits upon immune-mediated neuroinflammation.

  17. Emergence of NMDAR-independent long-term potentiation at hippocampal CA1 synapses following early adolescent exposure to chronic intermittent ethanol: role for sigma-receptors.

    PubMed

    Sabeti, Jilla; Gruol, Donna L

    2008-01-01

    Adolescent humans who abuse alcohol are more vulnerable than adults to the development of memory impairments. Memory impairments often involve modifications in the ability of hippocampal neurons to establish long-term potentiation (LTP) of excitatory neurotransmission; however, few studies have examined how chronic ethanol exposure during adolescence affects LTP mechanisms in hippocampus. We investigated changes in LTP mechanisms in hippocamal slices from rats exposed to intoxicating concentrations of chronic intermittent ethanol (CIE) vapors in their period of early-adolescent (i.e., prepubescent) or late-adolescent (i.e., postpubescent) development. LTP was evaluated at excitatory CA1 synapses in hippocampal slices at 24 h after the cessation of air (control) or CIE vapor treatments. CA1 synapses in control slices showed steady LTP following induction by high-frequency stimulation, which was fully dependent on NMDAR function. By contrast, slices from early-adolescent CIE exposed animals showed a compound form of LTP consisting of an NMDAR-dependent component and a slow-developing component independent of NMDARs. These components summated to yield LTP of robust magnitude above LTP levels in age-matched control slices. Bath-application of the sigma-receptor antagonist BD1047 and the neuroactive steroid pregnenolone sulfate, but not acute ethanol application, blocked NMDAR-independent LTP, while leaving NMDAR-dependent LTP intact. Analysis of presynaptic function during NMDAR-independent LTP induction demonstrated increased presynaptic function via a sigma-receptor-dependent mechanism in slices from early-adolescent CIE-exposed animals. By contrast, CIE exposure after puberty onset in late-adolescent animals produced decrements in LTP levels. The identification of a role for sigma-receptors and neuroactive steroids in the development of NMDAR-independent LTP suggests an important pathway by which hippocampal synaptic plasticity, and perhaps memory, may be uniquely

  18. Repeated Acute Oral Exposure to Cannabis sativa Impaired Neurocognitive Behaviours and Cortico-hippocampal Architectonics in Wistar Rats.

    PubMed

    Imam, A; Ajao, M S; Akinola, O B; Ajibola, M I; Ibrahim, A; Amin, A; Abdulmajeed, W I; Lawal, Z A; Ali-Oluwafuyi, A

    2017-03-06

    The most abused illicit drug in both the developing and the developed world is Cannabis disposing users to varying forms of personality disorders. However, the effects of cannabis on cortico-hippocampal architecture and cognitive behaviours still remain elusive.  The present study investigated the neuro-cognitive implications of oral cannabis use in rats. Eighteen adult Wistar rats were randomly grouped to three. Saline was administered to the control rats, cannabis (20 mg/kg) to the experimental group I, while Scopolamine (1 mg/kg. ip) was administered to the last group as a standard measure for the cannabis induced cognitive impairment. All treatments lasted for seven consecutive days. Open Field Test (OFT) was used to assess locomotor activities, Elevated Plus Maze (EPM) for anxiety-like behaviour, and Y maze paradigm for spatial memory and data subjected to ANOVA and T test respectively. Thereafter, rats were sacrificed and brains removed for histopathological studies. Cannabis significantly reduced rearing frequencies in the OFT and EPM, and increased freezing period in the OFT. It also reduced percentage alternation similar to scopolamine in the Y maze, and these effects were coupled with alterations in the cortico-hippocampal neuronal architectures. These results point to the detrimental impacts of cannabis on cortico-hippocampal neuronal architecture and morphology, and consequently cognitive deficits.

  19. Preserving GABAergic interneurons in acute brain slices of mice using the N-methyl-D-glucamine-based artificial cerebrospinal fluid method.

    PubMed

    Pan, Geng; Li, Yue; Geng, Hong-Yan; Yang, Jian-Ming; Li, Ke-Xin; Li, Xiao-Ming

    2015-04-01

    Defects in the function and development of GABAergic interneurons have been linked to psychiatric disorders, so preservation of these interneurons in brain slices is important for successful electrophysiological recording in various ex vivo methods. However, it is difficult to maintain the activity and morphology of neurons in slices from mice of >30 days old. Here we evaluated the N-methyl-D-glucamine (NMDG)-based artificial cerebrospinal fluid (aCSF) method for the preservation of interneurons in slices from mice of up to ∼6 months old and discussed the steps that may affect their quality during slicing. We found that the NMDG-aCSF method rescued more cells than sucrose-aCSF and successfully preserved different types of interneurons including parvalbumin- and somatostatin-positive interneurons. In addition, both the chemical and electrical synaptic signaling of interneurons were maintained. These results demonstrate that the NMDG-aCSF method is suitable for the preservation of interneurons, especially in studies of gap junctions.

  20. Thrombin induces ischemic LTP (iLTP): implications for synaptic plasticity in the acute phase of ischemic stroke

    PubMed Central

    Stein, Efrat Shavit; Itsekson-Hayosh, Zeev; Aronovich, Anna; Reisner, Yair; Bushi, Doron; Pick, Chaim G.; Tanne, David; Chapman, Joab; Vlachos, Andreas; Maggio, Nicola

    2015-01-01

    Acute brain ischemia modifies synaptic plasticity by inducing ischemic long-term potentiation (iLTP) of synaptic transmission through the activation of N-Methyl-D-aspartate receptors (NMDAR). Thrombin, a blood coagulation factor, affects synaptic plasticity in an NMDAR dependent manner. Since its activity and concentration is increased in brain tissue upon acute stroke, we sought to clarify whether thrombin could mediate iLTP through the activation of its receptor Protease-Activated receptor 1 (PAR1). Extracellular recordings were obtained in CA1 region of hippocampal slices from C57BL/6 mice. In vitro ischemia was induced by acute (3 minutes) oxygen and glucose deprivation (OGD). A specific ex vivo enzymatic assay was employed to assess thrombin activity in hippocampal slices, while OGD-induced changes in prothrombin mRNA levels were assessed by (RT)qPCR. Upon OGD, thrombin activity increased in hippocampal slices. A robust potentiation of excitatory synaptic strength was detected, which occluded the ability to induce further LTP. Inhibition of either thrombin or its receptor PAR1 blocked iLTP and restored the physiological, stimulus induced LTP. Our study provides important insights on the early changes occurring at excitatory synapses after ischemia and indicates the thrombin/PAR1 pathway as a novel target for developing therapeutic strategies to restore synaptic function in the acute phase of ischemic stroke. PMID:25604482

  1. Transient impairment of hippocampus-dependent learning and memory in relatively low-dose of acute radiation syndrome is associated with inhibition of hippocampal neurogenesis.

    PubMed

    Kim, Joong-Sun; Lee, Hae-June; Kim, Jong Choon; Kang, Seong Soo; Bae, Chun-Sik; Shin, Taekyun; Jin, Jae-Kwang; Kim, Sung Ho; Wang, Hongbing; Moon, Changjong

    2008-09-01

    Neurogenesis in the adult hippocampus, which occurs constitutively, is vulnerable to ionizing radiation. In the relatively low-dose exposure of acute radiation syndrome (ARS), the change in the adult hippocampal function is poorly understood. This study analyzed the changes in apoptotic cell death and neurogenesis in the DGs of hippocampi from adult ICR mice with single whole-body gamma-irradiation using the TUNEL method and immunohistochemical markers of neurogenesis, Ki-67 and doublecortin (DCX). In addition, the hippocampus-dependent learning and memory tasks after single whole-body gamma-irradiation were examined in order to evaluate the hippocampus-related behavioral dysfunction in the relatively low-dose exposure of ARS. The number of TUNEL-positive apoptotic nuclei in the dentate gyrus (DG) was increased 6-12 h after acute gamma-irradiation (a single dose of 0.5 to 4 Gy). In contrast, the number of Ki-67- and DCX-positive cells began to decrease significantly 6 h postirradiation, reaching its lowest level 24 h after irradiation. The level of Ki-67 and DCX immunoreactivity decreased in a dose-dependent manner within the range of irradiation applied (0-4 Gy). In passive avoidance and object recognition memory test, the mice trained 1 day after acute irradiation (2 Gy) showed significant memory deficits, compared with the sham controls. In conclusion, the pattern of the hippocampus-dependent memory dysfunction is consistent with the change in neurogenesis after acute irradiation. It is suggested that a relatively low dose of ARS in adult ICR mice is sufficiently detrimental to interrupt the functioning of the hippocampus, including learning and memory, possibly through the inhibition of neurogenesis.

  2. The Relationship Between Acute Glucocorticoid Levels and Hippocampal Function Depends Upon Task Aversiveness and Memory Processing Stage

    PubMed Central

    Conrad, Cheryl D.

    2005-01-01

    This review evaluates the effects of glucocorticoids (GCs), the adrenal steroids released in response to stress, on memory functions requiring the hippocampus in animals and humans. The data support the hypothesis that the learning function between GCs and hippocampal-dependent memory is modulated by 1) the aversive nature of the learning paradigm and 2) stage of memory processing (acquisition, consolidation, retrieval). When tasks are minimally aversive, the glucocorticoid receptor (GR) mediates an inverted U-shaped relationship between GC levels and hippocampal function, while the mineralocorticoid receptor (MR) mediates attentional processes and/or reaction to novelty. This inverted U-shaped relationship during minimally aversive training paradigms describes GC-mediated memory processing at both acquisition and consolidation. In contrast, highly aversive paradigms activate the amygdala and elevate GCs as part of the training procedure, revealing a nonlinear inverted U-shaped relationship during acquisition and a positive linear function during consolidation. Thus, highly aversive tasks that activate the amygdala shift the memory function from an inverted U-shaped curve to a linear representation between GC levels and memory consolidation. PMID:16601824

  3. Neuregulin directly decreases voltage-gated sodium current in hippocampal ErbB4-expressing interneurons.

    PubMed

    Janssen, Megan J; Leiva-Salcedo, Elias; Buonanno, Andres

    2012-10-03

    The Neuregulin 1 (NRG1)/ErbB4 signaling pathway has been genetically and functionally implicated in the etiology underlying schizophrenia, and in the regulation of glutamatergic pyramidal neuron function and plasticity. However, ErbB4 receptors are expressed in subpopulations of GABAergic interneurons, but not in hippocampal or cortical pyramidal neurons, indicating that NRG1 effects on principal neurons are indirect. Consistent with these findings, NRG1 effects on hippocampal long-term potentiation at CA1 pyramidal neuron synapses in slices are mediated indirectly by dopamine. Here we studied whether NRG/ErbB signaling directly regulates interneuron intrinsic excitability by pharmacologically isolating ErbB4-expressing neurons in rat dissociated hippocampal cultures, which lack dopaminergic innervation. We found that NRG1 acutely attenuates ErbB4-expressing interneuron excitability by depolarizing the firing threshold; neurons treated with the pan-ErbB inhibitor PD158780 or negative for ErbB4 were unaffected. These effects of NRG1 are primarily attributable to decreased voltage-gated sodium channel activity, as current density was attenuated by ∼60%. In stark contrast, NRG1 had minor effects on whole-cell potassium currents. Our data reveal the direct actions of NRG1 signaling in ErbB4-expressing interneurons, and offer novel insight into how NRG1/ErbB4 signaling can impact hippocampal activity.

  4. Anti-inflammatory efficacy of dexamethasone and Nrf2 activators in the CNS using brain slices as a model of acute injury.

    PubMed

    Graber, David J; Hickey, William F; Stommel, Elijah W; Harris, Brent T

    2012-03-01

    Limiting excessive production of inflammatory mediators is an effective therapeutic strategy for many diseases. It's also a promising remedy for neurodegenerative diseases and central nervous system (CNS) injuries. Glucocorticoids are valuable anti-inflammatory agents, but their use is constrained by adverse side-effects. Activators of NF-E2-related factor-2 (Nrf2) signaling represent an attractive anti-inflammatory alternative. In this study, dexamethasone, a synthetic glucocorticoid, and several molecular activators of Nrf2 were evaluated for efficacy in slices of cerebral cortex derived from adult SJL/J mice. Cortical explants increased expression of IL-1β and TNF-α mRNAs in culture within 5 h of sectioning. This expression was inhibited with dexamethasone in the explant medium or injected systemically in mice before sectioning. Semi-synthetic triterpenoid (SST) derivatives, potent activators of the Nrf2 pathway, demonstrated fast-acting anti-inflammatory activity in microglia cultures, but not in the cortical slice system. Quercetin, luteolin, and dimethyl fumarate were also evaluated as molecular activators of Nrf2. While expression of inflammatory mediators in microglia cultures was inhibited, these compounds did not demonstrate anti-inflammatory efficacy in cortical slices. In conclusion, brain slices were amenable to pharmacological modification as demonstrated by anti-inflammatory activity with dexamethasone. The utilization of Nrf2 activators to limit inflammatory mediators within the CNS requires further investigation. Inactivity in CNS tissue, however, suggests their safe use without neurological side-effects in treating non-CNS disorders. Short-term CNS explants may provide a more accurate model of in vivo conditions than microglia cultures since the complex tissue microenvironment is maintained.

  5. Acute chest pain in emergency room. Preliminary findings with 40-64-slice CT ECG-gated of the whole chest.

    PubMed

    Coche, E

    2007-01-01

    ECG-gated MDCT of the entire chest represents the latest technical advance in the diagnostic work-up of atypical chest pain. The authors report their preliminary experience with the use of 40 and 64-slice CT in the emergency room and recommend to study only patients with moderate likelihood of coronary artery disease. ECG-gated MDCT of the entire chest will be preferentially performed on 64-slice MDCT rather than 40-slice MDCT because it enable to reduce the scan time (18 seconds versus 28 seconds acquisition time), the volume of contrast medium (82 mL + 15 mL versus 97 mL + 15 mL of highly concentrated contrast agent for a patient of 70 kgs) and radiation exposure (17 mSv versus 19 mSv). Approximately 1500 to 2000 of images are produced and need to be analysed on a dedicated workstation by a radiologist expert in cardiac and thoracic disorders. At the present time, only a few studies exist in the literature showing some promising results but further large clinical studies are needed before to implement such sophisticated protocol in emergency room.

  6. Parametric Trace Slicing

    NASA Technical Reports Server (NTRS)

    Rosu, Grigore (Inventor); Chen, Feng (Inventor); Chen, Guo-fang; Wu, Yamei; Meredith, Patrick O. (Inventor)

    2014-01-01

    A program trace is obtained and events of the program trace are traversed. For each event identified in traversing the program trace, a trace slice of which the identified event is a part is identified based on the parameter instance of the identified event. For each trace slice of which the identified event is a part, the identified event is added to an end of a record of the trace slice. These parametric trace slices can be used in a variety of different manners, such as for monitoring, mining, and predicting.

  7. Silicon slicing by fixed abrasive slicing technique

    NASA Technical Reports Server (NTRS)

    Schmid, F.; Khattak, C. P.

    1982-01-01

    One of the major cost factors in silicon ingot technology adaptation for terrestrial photovoltaic application is in slicing boules into wafers. The most developed industrial practice is the Internal Diameter (ID) slicing. This method utilizes diamond cutting. The diamond stands up for long periods, hence, the cost of expendable materials is low. However, the ID technology as practiced today has poor material utilization. The Multiblade Slurry (MBS) method has low equipment and labor costs but its expendable material costs are high. Recently Multiwire Slurry (MWS) technology has shown very good material utilization, but its expendable material costs are even higher than MBS. The multiwire Fixed Abrasive Slicing Technique (FAST) still in advanced development stage, combines the low expendable material costs of ID method, the low labor and equipment costs of MBS and high material utilization of MWS.

  8. Single fluoxetine treatment before but not after stress prevents stress-induced hippocampal long-term depression and spatial memory retrieval impairment in rats.

    PubMed

    Han, Huili; Dai, Chunfang; Dong, Zhifang

    2015-07-28

    A growing body of evidence has shown that chronic treatment with fluoxetine, a widely prescribed medication for treatment of depression, can affect synaptic plasticity in the adult central nervous system. However, it is not well understood whether acute fluoxetine influences synaptic plasticity, especially on hippocampal CA1 long-term depression (LTD), and if so, whether it subsequently impacts hippocampal-dependent spatial memory. Here, we reported that LTD facilitated by elevated-platform stress in hippocampal slices was completely prevented by fluoxetine administration (10 mg/kg, i.p.) 30 min before stress. The LTD was not, however, significantly inhibited by fluoxetine administration immediately after stress. Similarly, fluoxetine incubation (10 μM) during electrophysiological recordings also displayed no influence on the stress-facilitated LTD. In addition, behavioral results showed that a single fluoxetine treatment 30 min before but not after acute stress fully reversed the impairment of spatial memory retrieval in the Morris water maze paradigm. Taken together, these results suggest that acute fluoxetine treatment only before, but not after stress, can prevent hippocampal CA1 LTD and spatial memory retrieval impairment caused by behavioral stress in adult animals.

  9. Automated Computer-Assisted Diagnosis of Obstructive Coronary Artery Disease in Emergency Department Patients Undergoing 256-Slice Coronary Computed Tomography Angiography for Acute Chest Pain.

    PubMed

    Hashoul, Sharbell; Gaspar, Tamar; Halon, David A; Lewis, Basil S; Shenkar, Yuval; Jaffe, Ronen; Peled, Nathan; Rubinshtein, Ronen

    2015-10-01

    A 256-slice coronary computed tomography angiography (CCTA) is an accurate method for detection and exclusion of obstructive coronary artery disease (OBS-CAD). However, accurate image interpretation requires expertise and may not be available at all hours. The purpose of this study was to evaluate the usefulness of a fully automated computer-assisted diagnosis (COMP-DIAG) tool for exclusion of OBS-CAD in patients in the emergency department (ED) presenting with chest pain. Three hundred sixty-nine patients in ED without known coronary disease underwent 256-slice CCTA as part of the assessment of chest pain of uncertain origin. COMP-DIAG (CorAnalyzer II) automatically reported presence or exclusion of OBS-CAD (>50% stenosis, ≥1 vessel). Performance characteristics of COMP-DIAG for exclusion and detection of OBS-CAD were determined using expert reading as the reference standard. Seventeen (5%) studies were unassessable by COMP-DIAG software, and 352 patients (1,056 vessels) were therefore available for analysis. COMP-DIAG identified 33% of assessable studies as having OBS-CAD, but the prevalence of OBS-CAD on CCTA was only 18% (66 of 352 patients) by standard expert reading. However, COMP-DIAG correctly identified 61 of the 66 patients (93%) with OBS-CAD with 21 vessels (2%) with OBS-CAD misclassified as negative. In conclusion, compared to expert reading, automated computer-assisted diagnosis using the CorAnalyzer showed high sensitivity but only moderate specificity for detection of obstructive coronary disease in patients in ED who underwent 256-slice CCTA. The high negative predictive value of this computer-assisted algorithm may be useful in the ED setting.

  10. Abnormal endogenous amino acid release in brain slices from vitamin B-6 restricted neonatal rats.

    PubMed

    Guilarte, T R

    1991-01-02

    The basal and potassium-evoked efflux of glutamate, glycine, taurine, and gamma-aminobutyric acid (GABA) was measured in brain slices from vitamin B-6 restricted and sufficient 14-day-old rats. The results indicate a reduced level of basal glutamate, taurine, and GABA efflux in hippocampal slices and taurine and GABA in cortical slices from vitamin B-6 restricted animals. In the presence of depolarizing potassium concentrations, there was a reduced level of GABA efflux in hippocampal and cortical slices, and a marked reduction in the release of glutamate in cortical slices from B-6 restricted rats. The abnormalities in the secretion process of these neuroactive amino acids may be related to the neurological sequelae associated with neonatal vitamin B-6 restriction.

  11. The TNFα-Transgenic Rat: Hippocampal Synaptic Integrity, Cognition, Function, and Post-Ischemic Cell Loss

    PubMed Central

    Pettigrew, L. Creed; Kryscio, Richard J.; Norris, Christopher M.

    2016-01-01

    The cytokine, tumor necrosis factor α (TNFα), is a key regulator of neuroinflammation linked to numerous neurodegenerative conditions and diseases. The present study used transgenic rats that overexpress a murine TNFα gene, under the control of its own promoter, to investigate the impact of chronically elevated TNFα on hippocampal synaptic function. Neuronal viability and cognitive recovery in TNFα Tg rats were also determined following an ischemic insult arising from reversible middle cerebral artery occlusion (MCAO). Basal CA3-CA1 synaptic strength, recorded in acute brain slices, was not significantly different between eight-week-old TNFα Tg rats and non-Tg rats. In contrast, slices from TNFα Tg rats showed significantly greater levels of long-term potentiation (LTP) in response to 100 Hz stimulation, suggesting that synaptic networks may be hyperexcitable in the context of elevated TNFα. Cognitive and motor deficits (assessed on the Morris Water Maze and Rotarod task, respectively) were present in TNFα Tg rats in the absence of significant differences in the loss of cortical and hippocampal neurons. TNF overexpression exacerbated MCAO-dependent deficits on the rotarod, but ameliorated cortical neuron loss in response to MCAO. PMID:27144978

  12. Clinically relevant concentration of pregabalin has no acute inhibitory effect on excitation of dorsal horn neurons under normal or neuropathic pain conditions: An intracellular calcium-imaging study in spinal cord slices from adult rats.

    PubMed

    Baba, Hiroshi; Petrenko, Andrey B; Fujiwara, Naoshi

    2016-10-01

    Pregabalin is thought to exert its therapeutic effect in neuropathic pain via binding to α2δ-1 subunits of voltage-gated calcium (Ca(2+)) channels. However, the exact analgesic mechanism after its binding to α2δ-1 subunits remains largely unknown. Whether a clinical concentration of pregabalin (≈10μM) can cause acute inhibition of dorsal horn neurons in the spinal cord is controversial. To address this issue, we undertook intracellular Ca(2+)-imaging studies using spinal cord slices with an intact attached L5 dorsal root, and examined if pregabalin acutely inhibits the primary afferent stimulation-evoked excitation of dorsal horn neurons in normal rats and in rats with streptozotocin-induced painful diabetic neuropathy. Under normal conditions, stimulation of a dorsal root evoked Ca(2+) signals predominantly in the superficial dorsal horn. Clinically relevant (10μM) and a very high concentration of pregabalin (100μM) did not affect the intensity or spread of dorsal root stimulation-evoked Ca(2+) signals, whereas an extremely high dose of pregabalin (300μM) slightly but significantly attenuated Ca(2+) signals in normal rats and in diabetic neuropathic (DN) rats. There was no difference between normal rats and DN rats with regard to the extent of signal attenuation at all concentrations tested. These results suggest that the activity of dorsal horn neurons in the spinal cord is not inhibited acutely by clinical doses of pregabalin under normal or DN conditions. It is very unlikely that an acute inhibitory action in the dorsal horn is the main analgesic mechanism of pregabalin in neuropathic pain states. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Chronic treatment with ginsenoside Rg1 promotes memory and hippocampal long-term potentiation in middle-aged mice.

    PubMed

    Zhu, G; Wang, Y; Li, J; Wang, J

    2015-04-30

    Ginseng serves as a potential candidate for the treatment of aging-related memory decline or memory loss. However, the related mechanism is not fully understood. In this study, we applied an intraperitoneal injection of ginsenoside Rg1, an active compound from ginseng in middle-aged mice and detected memory improvement and the underlying mechanisms. Our results showed that a period of 30-day administration of ginsenoside Rg1 enhanced long-term memory in the middle-aged animals. Consistent with the memory improvement, ginsenoside Rg1 administration facilitated weak theta-burst stimulation (TBS)-induced long-term potentiation (LTP) in acute hippocampal slices from middle-aged animals. Ginsenoside Rg1 administration increased the dendritic apical spine numbers and area in the CA1 region. In addition, ginsenoside Rg1 administration up-regulated the expression of hippocampal p-AKT, brain-derived neurotrophic factor (BDNF), proBDNF and glutamate receptor 1 (GluR1), but not p-ERK. Interestingly, the phosphatase and tensin homolog deleted on chromosome ten (PTEN) inhibitor (bpV) mimicked the ginsenoside Rg1 effects, including increasing p-AKT expression, promoting hippocampal basal synaptic transmission, LTP and memory. Taken together, our data suggest that ginsenoside Rg1 treatment improves memory in middle-aged mice possibly through regulating the PI3K/AKT pathway, altering apical spines and facilitating hippocampal LTP. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  14. Physical exercise and acute restraint stress differentially modulate hippocampal brain-derived neurotrophic factor transcripts and epigenetic mechanisms in mice.

    PubMed

    Ieraci, Alessandro; Mallei, Alessandra; Musazzi, Laura; Popoli, Maurizio

    2015-11-01

    Physical exercise and stressful experiences have been shown to exert opposite effects on behavioral functions and brain plasticity, partly by involving the action of brain-derived neurotrophic factor (BDNF). Although epigenetic modifications are known to play a pivotal role in the regulation of the different BDNF transcripts, it is poorly understood whether epigenetic mechanisms are also implied in the BDNF modulation induced by physical exercise and stress. Here, we show that total BDNF mRNA levels and BDNF transcripts 1, 2, 3, 4, 6, and 7 were reduced immediately after acute restraint stress (RS) in the hippocampus of mice, and returned to control levels 24 h after the stress session. On the contrary, exercise increased BDNF mRNA expression and counteracted the stress-induced decrease of BDNF transcripts. Physical exercise-induced up-regulation of BDNF transcripts was accounted for by increase in histone H3 acetylated levels at specific BDNF promoters, whereas the histone H3 trimethylated lysine 27 and dimethylated lysine 9 levels were unaffected. Acute RS did not change the levels of acetylated and methylated histone H3 at the BDNF promoters. Furthermore, we found that physical exercise and RS were able to differentially modulate the histone deacetylases mRNA levels. Finally, we report that a single treatment with histone deacetylase inhibitors, prior to acute stress exposure, prevented the down-regulation of total BDNF and BDNF transcripts 1, 2, 3, and 6, partially reproducing the effect of physical exercise. Overall, these results suggest that physical exercise and stress are able to differentially modulate the expression of BDNF transcripts by possible different epigenetic mechanisms.

  15. Compartmental distribution of GABAB receptor-mediated currents along the somatodendritic axis of hippocampal principal cells.

    PubMed

    Degro, Claudius E; Kulik, Akos; Booker, Sam A; Vida, Imre

    2015-01-01

    Activity of cortical principal cells is controlled by the GABAergic system providing inhibition in a compartmentalized manner along their somatodendritic axis. While GABAAR-mediated inhibitory synaptic transmission has been extensively characterized in hippocampal principal cells, little is known about the distribution of postsynaptic effects of GABABRs. In the present study, we have investigated the functional localization of GABABRs and their effector inwardly rectifying potassium (Kir3) channels by combining electrophysiological recordings in acute rat hippocampal slices, high-resolution immunoelectron microscopic analysis and single cell simulations. Pharmacologically isolated slow inhibitory postsynaptic currents were elicited in the three major hippocampal principal cell types by endogenous GABA released by electrical stimulation, photolysis of caged-GABA, as well as the canonical agonist baclofen, with the highest amplitudes observed in the CA3. Spatially restricted currents were assessed along the axis of principal cells by uncaging GABA in the different hippocampal layers. GABABR-mediated currents were present along the entire somatodendritic axis of principal cells, but non-uniformly distributed: largest currents and the highest conductance densities determined in the simulations were consistently found on the distal apical dendrites. Finally, immunocytochemical localization of GABABRs and Kir3 channels showed that distributions overlap but their densities diverge, particularly on the basal dendrites of pyramidal cells. GABABRs current amplitudes and the conductance densities correlated better with Kir3 density, suggesting a bottlenecking effect defined by the effector channel. These data demonstrate a compartmentalized distribution of the GABABR-Kir3 signaling cascade and suggest differential control of synaptic transmission, dendritic integration and synaptic plasticity at afferent pathways onto hippocampal principal cells.

  16. Compartmental distribution of GABAB receptor-mediated currents along the somatodendritic axis of hippocampal principal cells

    PubMed Central

    Degro, Claudius E.; Kulik, Akos; Booker, Sam A.; Vida, Imre

    2015-01-01

    Activity of cortical principal cells is controlled by the GABAergic system providing inhibition in a compartmentalized manner along their somatodendritic axis. While GABAAR-mediated inhibitory synaptic transmission has been extensively characterized in hippocampal principal cells, little is known about the distribution of postsynaptic effects of GABABRs. In the present study, we have investigated the functional localization of GABABRs and their effector inwardly rectifying potassium (Kir3) channels by combining electrophysiological recordings in acute rat hippocampal slices, high-resolution immunoelectron microscopic analysis and single cell simulations. Pharmacologically isolated slow inhibitory postsynaptic currents were elicited in the three major hippocampal principal cell types by endogenous GABA released by electrical stimulation, photolysis of caged-GABA, as well as the canonical agonist baclofen, with the highest amplitudes observed in the CA3. Spatially restricted currents were assessed along the axis of principal cells by uncaging GABA in the different hippocampal layers. GABABR-mediated currents were present along the entire somatodendritic axis of principal cells, but non-uniformly distributed: largest currents and the highest conductance densities determined in the simulations were consistently found on the distal apical dendrites. Finally, immunocytochemical localization of GABABRs and Kir3 channels showed that distributions overlap but their densities diverge, particularly on the basal dendrites of pyramidal cells. GABABRs current amplitudes and the conductance densities correlated better with Kir3 density, suggesting a bottlenecking effect defined by the effector channel. These data demonstrate a compartmentalized distribution of the GABABR-Kir3 signaling cascade and suggest differential control of synaptic transmission, dendritic integration and synaptic plasticity at afferent pathways onto hippocampal principal cells. PMID:25852540

  17. Acute toxicity of CCl4 but not of paracetamol induces a transcriptomic signature of fibrosis in precision-cut liver slices.

    PubMed

    Vatakuti, Suresh; Schoonen, Willem G E J; Elferink, Marieke L G; Groothuis, Geny M M; Olinga, Peter

    2015-08-01

    In rat in vivo, both paracetamol (APAP) and carbon tetrachloride (CCl4) induce liver necrosis, but long-term treatment with CCl4, in contrast to paracetamol, causes liver fibrosis. The aim of this study was to perform transcriptomic analysis to compare the early changes in mRNA expression profiles induced by APAP and CCl4 in the rat precision-cut liver slice model (PCLS) and to identify early markers that could predict fibrosis-inducing potential. Microarray data of rat PCLS exposed to APAP andCCl4was generated using a toxic dose based on decrease in ATP levels. Toxicity pathway analysis using a custom made fibrosis-related gene list showed fibrosis as one of the predominant toxic endpoints in CCl4-treated, but not in APAP-treated PCLS. Moreover, genes which have a role in fibrosis such as alpha-B crystallin, jun proto-oncogene, mitogen-activated protein kinase 6, serpin peptidase inhibitor and also the transcription factor Kruppel-like-factor-6 were up-regulated by CCl4, but not by APAP. Predicted activation or inhibition of several upstream regulators due to CCl4 is in accordance with their role in fibrosis. In conclusion, transcriptomic analysis of PCLS successfully identified the fibrotic potential of CCl4 as opposed to APAP. The application of PCLS as an ex vivo model to identify early biomarkers to predict the fibrogenic potential of toxic compounds should be further explored. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Regulating hippocampal hyperexcitability through GABAB Receptors

    PubMed Central

    Lang, Min; Moradi‐Chameh, Homeira; Zahid, Tariq; Gane, Jonathan; Wu, Chiping; Valiante, Taufik; Zhang, Liang

    2014-01-01

    Abstract Disturbances of GABAergic inhibition are a major cause of epileptic seizures. GABA exerts its actions via ionotropic GABAA receptors and metabotropic G protein‐coupled GABAB receptors. Malfunction of GABAA inhibition has long been recognized in seizure genesis but the role of GABAB receptors in controlling seizure activity is still not well understood. Here, we examined the anticonvulsive, or inhibitory effects, of GABAB receptors in a mouse model of hippocampal kindling as well as mouse hippocampal slices through the use of GS 39783, a positive allosteric GABAB receptor modulator, and CGP 55845, a selective GABAB receptor antagonist. When administered via intraperitoneal injections in kindled mice, GS 39783 (5 mg/kg) did not attenuate hippocampal EEG discharges, but did reduce aberrant hippocampal spikes, whereas CGP 55845 (10 mg/kg) prolonged hippocampal discharges and increased spike incidences. When examined in hippocampal slices, neither GS 39783 at 5 μmol/L nor the GABAB receptor agonist baclofen at 0.1 μmol/L alone significantly altered repetitive excitatory field potentials, but GS 39783 and baclofen together reversibly abolished these field potentials. In contrast, CGP 55845 at 1 μmol/L facilitated induction and incidence of these field potentials. In addition, CGP 55845 attenuated the paired pulse depression of CA3 population spikes and increased the frequency of EPSCs in individual CA3 pyramidal neurons. Collectively, these data suggest that GABABB receptors regulate hippocampal hyperexcitability by inhibiting CA3 glutamatergic synapses. We postulate that positive allosteric modulation of GABAB receptors may be effective in reducing seizure‐related hyperexcitability. PMID:24771688

  19. Selective Dysregulation of Hippocampal Inhibition in the Mouse Lacking Autism Candidate Gene CNTNAP2.

    PubMed

    Jurgensen, Sofia; Castillo, Pablo E

    2015-10-28

    Mutations in the human gene encoding contactin-associated protein-like 2 (CNTNAP2) have been strongly associated with autism spectrum disorders (ASDs). Cntnap2(-/-) mice recapitulate major features of ASD, including social impairment, reduced vocalizations, and repetitive behavior. In addition, Cntnap2(-/-) mice show reduced cortical neuronal synchrony and develop spontaneous seizures throughout adulthood. As suggested for other forms of ASDs, this phenotype could reflect some form of synaptic dysregulation. However, the impact of lifelong deletion of CNTNAP2 on synaptic function in the brain remains unknown. To address this issue, we have assessed excitatory and inhibitory synaptic transmission in acute hippocampal slices of Cntnap2(-/-) mice. We found that although excitatory transmission was mostly normal, inhibition onto CA1 pyramidal cells was altered in Cntnap2(-/-) mice. Specifically, putative perisomatic, but not dendritic, evoked IPSCs were significantly reduced in these mice. Whereas both inhibitory short-term plasticity and miniature IPSC frequency and amplitude were normal in Cntnap2(-/-) mice, we found an unexpected increase in the frequency of spontaneous, action potential-driven IPSCs. Altered hippocampal inhibition could account for the behavioral phenotype Cntnap2(-/-) mice present later in life. Overall, our findings that Cntnap2 deletion selectively impairs perisomatic hippocampal inhibition while sparing excitation provide additional support for synaptic dysfunction as a common mechanism underlying ASDs.

  20. Dysfunctional hippocampal inhibition in the Ts65Dn mouse model of Down syndrome

    PubMed Central

    Best, Tyler K.; Cramer, Nathan P.; Chakrabarti, Lina; Haydar, Tarik F.; Galdzicki, Zygmunt

    2013-01-01

    GABAergic dysfunction is implicated in hippocampal deficits of the Ts65Dn mouse model of Down syndrome (DS). Since Ts65Dn mice overexpress G-protein coupled inward-rectifying potassium (GIRK2) containing channels, we sought to evaluate whether increased GABAergic function disrupts the functioning of hippocampal circuitry. After confirming that GABAB/GIRK current density is significantly elevated in Ts65Dn CA1 pyramidal neurons, we compared monosynaptic inhibitory inputs in CA1 pyramidal neurons in response to proximal (stratum radiatum; SR) and distal (stratum lacunosum moleculare; SLM) stimulation of diploid and Ts65Dn acute hippocampal slices. Synaptic GABAB and GABAA mediated currents evoked by SR stimulation were generally unaffected in Ts65Dn CA1 neurons. However, the GABAB/GABAA ratios evoked by stimulation within the SLM of Ts65Dn hippocampus were significantly larger in magnitude, consistent with increased GABAB/GIRK currents after SLM stimulation. These results indicate that GIRK overexpression in Ts65Dn has functional consequences which affect the balance between GABAB and GABAA inhibition of CA1 pyramidal neurons, most likely in a pathway specific manner, and may contribute to cognitive deficits reported in these mice. PMID:22178330

  1. Evaluation of biventricular ejection fraction with ECG-gated 16-slice CT: preliminary findings in acute pulmonary embolism in comparison with radionuclide ventriculography.

    PubMed

    Coche, Emmanuel; Vlassenbroek, Alain; Roelants, Véronique; D'Hoore, William; Verschuren, Franck; Goncette, Louis; Maldague, Baudouin

    2005-07-01

    This study aimed to assess the feasibility of cardiac global function evaluation during a whole-chest multi-slice CT (MSCT) acquisition in patients referred for suspicion of pulmonary embolism (PE), and to compare the results with planar equilibrium radionuclide ventriculography (ERNA). Ten consecutive haemodynamically stable patients (six female, four male; mean age 69.7 years; heart rate 65-99 bpm) with suspicion of PE underwent an MSCT and ERNA within a 6 h period. CT acquisition was performed after contrast medium injection by using 16x1.5 mm collimation and retrospective ECG gating. Left ventricular (LVEF) and right ventricular (RVEF) ejection fractions were calculated using dedicated three-dimensional software. Relationships between measurements obtained with MSCT and ERNA were assessed using linear regression analysis and reliability of MSCT was assessed with intra-class correlation coefficient. Bland-Altman analysis was performed to calculate limits of agreement between MSCT and ERNA. MSCT was performed successfully in ten patients with a mean acquisition time of 16.5+/-2.8 s. Functional cardiac evaluation was possible on CT for all patients except for one due to poor opacification of right ventricle. Linear regression analysis showed a good correlation between MSCT and ERNA for the LVEF (R=0.91) and the RVEF (R=0.89) measurements. Intra-class correlation was superior for LVEF (0.92) than for the RVEF (0.68). Bland-Altman plots demonstrated that MSCT substantially overestimated the ERNA RVEF. Morphological CT data demonstrated PE in four of ten of patients and alternative diagnoses in five of ten patients. Our study reveals that MSCT with retrospective ECG gating may provide in one modality a morphological and a functional cardiopulmonary evaluation. Comparison with ERNA demonstrated a good correlation for both ventricular ejection fractions.

  2. Corticosterone enhances the potency of ethanol against hippocampal long-term potentiation via local neurosteroid synthesis.

    PubMed

    Izumi, Yukitoshi; O'Dell, Kazuko A; Zorumski, Charles F

    2015-01-01

    Corticosterone is known to accumulate in brain after various stressors including alcohol intoxication. Just as severe alcohol intoxication is typically required to impair memory formation only high concentrations of ethanol (60 mM) acutely inhibit long-term potentiation (LTP), a cellular memory mechanism, in naïve hippocampal slices. This LTP inhibition involves synthesis of neurosteroids, including allopregnanolone, and appears to involve a form of cellular stress. In the CA1 region of rat hippocampal slices, we examined whether a lower concentration of ethanol (20 mM) inhibits LTP in the presence of corticosterone, a stress-related modulator, and whether corticosterone stimulates local neurosteroid synthesis. Although low micromolar corticosterone alone did not inhibit LTP induction, we found that 20 mM ethanol inhibited LTP in the presence of corticosterone. At 20 mM, ethanol alone did not stimulate neurosteroid synthesis or inhibit LTP. LTP inhibition by corticosterone plus ethanol was blocked by finasteride, an inhibitor of 5α-reductase, suggesting a role for neurosteroid synthesis. We also found that corticosterone alone enhanced neurosteroid immunostaining in CA1 pyramidal neurons and that this immunostaining was further augmented by 20 mM ethanol. The enhanced neurosteroid staining was blocked by finasteride and the N-methyl-D-aspartate antagonist, 2-amino-5-phosphonovalerate (APV). These results indicate that corticosterone promotes neurosteroid synthesis in hippocampal pyramidal neurons and can participate in ethanol-mediated synaptic dysfunction even at moderate ethanol levels. These effects may contribute to the influence of stress on alcohol-induced cognitive impairment.

  3. Corticosterone enhances the potency of ethanol against hippocampal long-term potentiation via local neurosteroid synthesis

    PubMed Central

    Izumi, Yukitoshi; O’Dell, Kazuko A.; Zorumski, Charles F.

    2015-01-01

    Corticosterone is known to accumulate in brain after various stressors including alcohol intoxication. Just as severe alcohol intoxication is typically required to impair memory formation only high concentrations of ethanol (60 mM) acutely inhibit long-term potentiation (LTP), a cellular memory mechanism, in naïve hippocampal slices. This LTP inhibition involves synthesis of neurosteroids, including allopregnanolone, and appears to involve a form of cellular stress. In the CA1 region of rat hippocampal slices, we examined whether a lower concentration of ethanol (20 mM) inhibits LTP in the presence of corticosterone, a stress-related modulator, and whether corticosterone stimulates local neurosteroid synthesis. Although low micromolar corticosterone alone did not inhibit LTP induction, we found that 20 mM ethanol inhibited LTP in the presence of corticosterone. At 20 mM, ethanol alone did not stimulate neurosteroid synthesis or inhibit LTP. LTP inhibition by corticosterone plus ethanol was blocked by finasteride, an inhibitor of 5α-reductase, suggesting a role for neurosteroid synthesis. We also found that corticosterone alone enhanced neurosteroid immunostaining in CA1 pyramidal neurons and that this immunostaining was further augmented by 20 mM ethanol. The enhanced neurosteroid staining was blocked by finasteride and the N-methyl-D-aspartate antagonist, 2-amino-5-phosphonovalerate (APV). These results indicate that corticosterone promotes neurosteroid synthesis in hippocampal pyramidal neurons and can participate in ethanol-mediated synaptic dysfunction even at moderate ethanol levels. These effects may contribute to the influence of stress on alcohol-induced cognitive impairment. PMID:26190975

  4. Metaplastic Effects of Subanesthetic Ketamine on CA1 Hippocampal Function

    PubMed Central

    Izumi, Yukitoshi; Zorumski, Charles F.

    2014-01-01

    Ketamine is a non-competitive N-methyl-D-aspartate receptor (NMDAR) antagonist of interest in neuropsychiatry. In the present studies, we examined the effects of subanesthetic, low micromolar ketamine on excitatory postsynaptic potentials (EPSPs), population spikes (PSs) and synaptic plasticity in the CA1 region of rat hippocampal slices. Ketamine acutely inhibited NMDAR-mediated synaptic responses with half-maximal effects near 10 µM. When administered for 15–30 min at 1–10 µM, ketamine had no effect on baseline dendritic AMPA receptor-mediated EPSPs, but persistently enhanced somatic EPSPs in the pyramidal cell body layer and augmented PS firing. Acute low micromolar ketamine also had no effect on the induction of long-term potentiation (LTP) but blocked long-term depression (LTD). Following 30 min administration of 1–10 µM ketamine, however, a slowly developing and persistent form of LTP inhibition was observed that took two hours following ketamine washout to become manifest. This LTP inhibition did not result from prolonged or enhanced NMDAR inhibition during drug washout. Effects of low ketamine on somatic EPSPs and LTP were not mimicked by a high ketamine concentration that completely inhibited NMDARs, and both of these effects were blocked by co-administration of low ketamine with a low concentration of the competitive NMDAR antagonist, 2-amino-5-phosphonovalerate or inhibitors of nitric oxide synthase. These results indicate that concentrations of ketamine relevant to psychotropic and psychotomimetic effects have complex metaplastic effects on hippocampal function that involve activation of unblocked NMDARs during ketamine exposure. PMID:25128848

  5. Gonadal Hormones Rapidly Enhance Spatial Memory and Increase Hippocampal Spine Density in Male Rats

    PubMed Central

    Jacome, Luis F.; Barateli, Ketti; Buitrago, Dina; Lema, Franklin; Frankfurt, Maya

    2016-01-01

    17β-estradiol (E2) rapidly, within minutes, activates behaviors and cognition by binding to membrane estrogen receptors, activating cell signaling cascades and increasing dendritic spines. In female rodents, E2 enhances spatial memory within 2–4 hours, and spine density is increased in the CA1 area of the hippocampus within 30–60 minutes. Although chronic gonadal hormone treatments in male rats alter cognition and spines/spine synapses and acute hormone effects occur in hippocampal slices, effects of acute, in vivo hormone administration in males are unknown. Therefore, we assessed rapid effects of E2 (20 μg/kg) and testosterone (T) (750 μg/kg) on spatial memory using the object placement task and on hippocampal spine density using Golgi impregnation. Orchidectomized rats received hormones immediately after the training trial and were tested for retention 2 hours later. Vehicle-injected orchidectomized males spent equal time exploring objects in the old and new locations, but E2- or T-treated subjects spent more time exploring objects at the new location, suggesting enhanced memory. Both hormones also increased spine density in CA1, but not the dentate gyrus, by 20%–40% at 30 minutes and 2 hours after injections. This report is the first, to our knowledge, to show E2 and T enhancements of memory and spine density within such a short time frame in male rats. PMID:26844375

  6. Exercise Prevents Amyloid-β-Induced Hippocampal Network Disruption by Inhibiting GSK3β Activation.

    PubMed

    Isla, Arturo G; Vázquez-Cuevas, Francisco Gabriel; Peña-Ortega, Fernando

    2016-03-16

    Exercise is becoming a promising therapeutic approach to prevent alterations both in Alzheimer's disease (AD) patients and in transgenic models of AD. This neuroprotection has been associated with changes in hippocampal structure and function, as well as with the reduction of amyloid-β (Aβ) production and accumulation. However, whether exercise produces lasting changes in hippocampal population activity and renders it resistant to Aβ-induced network dysfunction is still unknown. Thus, we tested whether voluntary exercise changes hippocampal population activity and prevents its alteration in the presence of Aβ, which has been associated to glycogen synthase kinase-3β (GSK3β) activation. We found that the hippocampal population activity recorded in slices obtained from mice that exercised voluntarily (with free access to a running wheel for 21 days) exhibits higher power and faster frequency composition than slices obtained from sedentary animals. Moreover, the hippocampal network of mice that exercised becomes insensitive to Aβ-induced inhibition of spontaneous population activity. This protective effect correlates with the inability of Aβ to activate GSK3β, is mimicked by GSK3β inhibition with SB126763 (in slices obtained from sedentary mice), and is abolished by the inhibition of PI3K with LY294002 (in slices obtained from mice that exercised). We conclude that voluntary exercise produces a lasting protective state in the hippocampus, maintained in hippocampal slices by a PI3K-dependent mechanism that precludes its functional disruption in the presence of Aβ by avoiding GSK3β activation.

  7. Slice profile distortions in single slice continuously moving table MRI

    NASA Astrophysics Data System (ADS)

    Sengupta, Saikat; Smith, David S.; Welch, E. B.

    2015-03-01

    Continuously Moving Table (CMT) MRI is a rapid imaging technique that allows scanning of extended fields of view (FOVs) such as the whole-body in a single continuous scan.1 A highly efficient approach to CMT MRI is single slice imaging, where data are continuously acquired from a single axial slice at isocenter with concurrent movement of the patient table.2 However, the continuous motion of the scanner table and supply of fresh magnetization into the excited slice can introduce deviations in the slice magnetization profile. The goal of this work is to investigate and quantify the distortion in the slice profile in CMT MRI. CMT MRI with a table speed of 20 mm/s was implemented on a 3 Tesla whole-body MRI scanner, with continuous radial data acquisition. Simulations were performed to characterize the transient and steady state slice profiles and magnetization effects. Simulated slice profiles were compared to actual slice profile measurements performed in the scanner. Both simulations and experiments revealed an asymmetric slice profile characterized by a skew towards the lagging edge of the moving table, in contrast to the nominal profiles associated with scanning a stationary object. The true excited slice width (FWHM) and pitch of the acquisition was observed to be dependent on table velocity, with larger table speeds resulting in larger slice profile deviations from the nominal shape.

  8. Oxidative Damage in the Guinea Pig Hippocampal Slice

    DTIC Science & Technology

    1989-01-01

    oxidation to the observed damage. the actions of the oxidants, chloramine -T and N-chlorosuccinimide (NCS). were studied on electrophysiological...hippocampus evoked a population postsynaptic potential (population PSP) in the dendritic layer and a population spike in the cell body layer. Chloramine -T (25...produced by free radicals but can rot account for the postsynaptic effects. Keywords- Chloramine -T. N-chlorosuccinimide. Oxidation. Free radical

  9. Fluoxetine impairs GABAergic signaling in hippocampal slices from neonatal rats

    PubMed Central

    Caiati, Maddalena D.; Cherubini, Enrico

    2013-01-01

    Fluoxetine (Prozac), an antidepressant known to selectively inhibit serotonin reuptake, is widely used to treat mood disorders in women suffering from depression during pregnancy and postpartum period. Several lines of evidence suggest that this drug, which crosses the human placenta and is secreted into milk during lactation, exerts its action not only by interfering with serotoninergic but also with GABAergic transmission. GABA is known to play a crucial role in the construction of neuronal circuits early in postnatal development. The immature hippocampus is characterized by an early type of network activity, the so-called Giant Depolarizing Potentials (GDPs), generated by the synergistic action of glutamate and GABA, both depolarizing and excitatory. Here we tested the hypothesis that fluoxetine may interfere with GABAergic signaling during the first postnatal week, thus producing harmful effects on brain development. At micromolar concentrations fluoxetine severely depressed GDPs frequency (IC50 22 μM) in a reversible manner and independently of its action on serotonin reuptake. This effect was dependent on a reduced GABAergic (but not glutamatergic) drive to principal cells most probably from parvalbumin-positive fast spiking neurons. Cholecystokinin-positive GABAergic interneurons were not involved since the effects of the drug persisted when cannabinoid receptors were occluded with WIN55,212-2, a CB1/CB2 receptor agonist. Fluoxetine effects on GABAergic transmission were associated with a reduced firing rate of both principal cells and interneurons further suggesting that changes in network excitability account for GDPs disruption. This may have critical consequences on the functional organization and stabilization of neuronal circuits early in postnatal development. PMID:23641199

  10. Presynaptic Modulation of the Hippocampal Mossy Fiber Synapse

    DTIC Science & Technology

    1991-10-07

    al., 1987). In addition, the nootropic (cognitive enhancing) drug bifemaline has been shown to increase the magnitude of MF LTP (Satoh et al., 1988...Different susceptibilities of long-term potentiations in CA3 and CAl regions of guinea pig hippocampal slices to nootropic drugs. Neurosci. Lett., 1988; 93

  11. Silhouette-Slice Theorems

    DTIC Science & Technology

    1987-03-20

    with standard expressions of spherical trigonometry is sinr)0 = cos0 sini//0 (4.37) which is consistent with the results obtained previously with...theorems for discrete transforms. However, sampling questions inlroduce difficult obstacles in the develop- ment of a discrete theory. First, sampling...additional obstacle to discrete represen- tations of the CT. An example of qualitative predication of the shape of silhouettes with the Silhouette-Slice

  12. Synaptic Function of Rab11Fip5: Selective Requirement for Hippocampal Long-Term Depression

    PubMed Central

    Ahmad, Mohiuddin; Jurado, Sandra; Malenka, Robert C.

    2015-01-01

    Postsynaptic AMPA-type glutamate receptors (AMPARs) are among the major determinants of synaptic strength and can be trafficked into and out of synapses. Neuronal activity regulates AMPAR trafficking during synaptic plasticity to induce long-term changes in synaptic strength, including long-term potentiation (LTP) and long-term depression (LTD). Rab family GTPases regulate most membrane trafficking in eukaryotic cells; particularly, Rab11 and its effectors are implicated in mediating postsynaptic AMPAR insertion during LTP. To explore the synaptic function of Rab11Fip5, a neuronal Rab11 effector and a candidate autism-spectrum disorder gene, we performed shRNA-mediated knock-down and genetic knock-out (KO) studies. Surprisingly, we observed robust shRNA-induced synaptic phenotypes that were rescued by a Rab11Fip5 cDNA but that were nevertheless not observed in conditional KO neurons. Both in cultured neurons and acute slices, KO of Rab11Fip5 had no significant effect on basic parameters of synaptic transmission, indicating that Rab11Fip5 is not required for fundamental synaptic operations, such as neurotransmitter release or postsynaptic AMPAR insertion. KO of Rab11Fip5 did, however, abolish hippocampal LTD as measured both in acute slices or using a chemical LTD protocol in cultured neurons but did not affect hippocampal LTP. The Rab11Fip5 KO mice performed normally in several behavioral tasks, including fear conditioning, but showed enhanced contextual fear extinction. These are the first findings to suggest a requirement for Rab11Fip5, and presumably Rab11, during LTD. PMID:25972173

  13. Chelation of hippocampal zinc enhances long-term potentiation and synaptic tagging/capture in CA1 pyramidal neurons of aged rats: implications to aging and memory.

    PubMed

    Shetty, Mahesh Shivarama; Sharma, Mahima; Sajikumar, Sreedharan

    2017-02-01

    Aging is associated with decline in cognitive functions, prominently in the memory consolidation and association capabilities. Hippocampus plays a crucial role in the formation and maintenance of long-term associative memories, and a significant body of evidence shows that impairments in hippocampal function correlate with aging-related memory loss. A number of studies have implicated alterations in hippocampal synaptic plasticity, such as long-term potentiation (LTP), in age-related cognitive decline although exact mechanisms underlying are not completely clear. Zinc deficiency and the resultant adverse effects on cognition have been well studied. However, the role of excess of zinc in synaptic plasticity, especially in aging, is not addressed well. Here, we have investigated the hippocampal zinc levels and the impairments in synaptic plasticity, such as LTP and synaptic tagging and capture (STC), in the CA1 region of acute hippocampal slices from 82- to 84-week-old male Wistar rats. We report increased zinc levels in the hippocampus of aged rats and also deficits in the tetani-induced and dopaminergic agonist-induced late-LTP and STC. The observed deficits in synaptic plasticity were restored upon chelation of zinc using a cell-permeable chelator. These data suggest that functional plasticity and associativity can be successfully established in aged neural networks by chelating zinc with cell-permeable chelating agents.

  14. Nitric oxide deficit in chronic intermittent hypoxia impairs large conductance calcium-activated potassium channel activity in rat hippocampal neurons.

    PubMed

    Tjong, Yung-Wui; Li, Meifang; Hung, Ming-Wai; Wang, Kun; Fung, Man-Lung

    2008-02-15

    Sleep apnea associated with chronic intermittent hypoxia (IH) impairs hippocampal functions but the pathogenic mechanisms involving dysfunction of nitric oxide (NO) and ionic channels remain unclear. We examined the hypothesis that hippocampal NO deficit impairs the activity of large conductance calcium-activated potassium (BK) channels in rats with chronic IH, mimicking conditions in patients with sleep apnea. A patch-clamp study was performed on hippocampal CA1 neurons acutely dissociated from IH and control rats. The levels of endogenous NO and intracellular calcium in the CA1 region of the hippocampal slices were measured respectively by electrochemical microsensors and spectrofluorometry. We found that the open probability of BK channels remarkably decreased in the CA1 pyramidal neurons in a time-dependent manner with the IH treatment, without changes in the unitary conductance and reversal potential. NO donors, SNP or DETA/NO, significantly restored the activity of BK channels in the IH neurons, which was prevented by blockade of S-nitrosylation with NEM or MTSES but not by inhibition of the cGMP pathway with ODQ or 8-bromo-cGMP. Endogenous NO levels were substantially lowered in the IH hippocampus during resting and hypoxia. Also, the level of protein expression of neuronal NO synthase was markedly lessened in the IH neurons with decreased intracellular calcium response to hypoxia. Collectively, the results suggest that the IH-induced NO deficit mediated by a down-regulation of the expression of neuronal NO synthase plays a causative role in the impaired activity of BK channels, which could account for the hippocampal injury in patients with sleep apnea.

  15. A multifunctional pipette for localized drug administration to brain slices.

    PubMed

    Ahemaiti, Aikeremu; Ainla, Alar; Jeffries, Gavin D M; Wigström, Holger; Orwar, Owe; Jesorka, Aldo; Jardemark, Kent

    2013-10-15

    We have developed a superfusion method utilizing an open-volume microfluidic device for administration of pharmacologically active substances to selected areas in brain slices with high spatio-temporal resolution. The method consists of a hydrodynamically confined flow of the active chemical compound, which locally stimulates neurons in brain slices, applied in conjunction with electrophysiological recording techniques to analyze the response. The microfluidic device, which is a novel free-standing multifunctional pipette, allows diverse superfusion experiments, such as testing the effects of different concentrations of drugs or drug candidates on neurons in different cell layers with high positional accuracy, affecting only a small number of cells. We demonstrate herein the use of the method with electrophysiological recordings of pyramidal cells in hippocampal and prefrontal cortex brain slices from rats, determine the dependence of electric responses on the distance of the superfusion device from the recording site, document a multifold gain in solution exchange time as compared to whole slice perfusion, and show that the device is able to store and deliver up to four solutions in a series. Localized solution delivery by means of open-volume microfluidic technology also reduces reagent consumption and tissue culture expenses significantly, while allowing more data to be collected from a single tissue slice, thus reducing the number of laboratory animals to be sacrificed for a study.

  16. A multifunctional pipette for localized drug administration to brain slices

    PubMed Central

    Ahemaiti, Aikeremu; Ainla, Alar; Jeffries, Gavin D. M.; Wigström, Holger; Orwar, Owe; Jesorka, Aldo; Jardemark, Kent

    2013-01-01

    We have developed a superfusion method utilizing an open-volume microfluidic device for administration of pharmacologically active substances to selected areas in brain slices with high spatio-temporal resolution. The method consists of a hydrodynamically confined flow of the active chemical compound, which locally stimulates neurons in brain slices, applied in conjunction with electrophysiological recording techniques to analyze the response. The microfluidic device, which is a novel free-standing multifunctional pipette, allows diverse superfusion experiments, such as testing the effects of different concentrations of drugs or drug candidates on neurons in different cell layers with high positional accuracy, affecting only a small number of cells. We demonstrate herein the use of the method with electrophysiological recordings of pyramidal cells in hippocampal and prefrontal cortex brain slices from rats, determine the dependence of electric responses on the distance of the superfusion device from the recording site, document a multifold gain in solution exchange time as compared to whole slice perfusion, and show that the device is able to store and deliver up to four solutions in a series. Localized solution delivery by means of open-volume microfluidic technology also reduces reagent consumption and tissue culture expenses significantly, while allowing more data to be collected from a single tissue slice, thus reducing the number of laboratory animals to be sacrificed for a study. PMID:23969260

  17. Physiological Effects of Enriched Environment Exposure and LTP Induction in the Hippocampus In Vivo Do Not Transfer Faithfully to In Vitro Slices

    ERIC Educational Resources Information Center

    Eckert, Michael J.; Abraham, Wickliffe C.

    2010-01-01

    A number of experimental paradigms use in vitro brain slices to test for changes in synaptic transmission and plasticity following a behavioral manipulation. For example, a number of previous studies have reported a variety of effects of environmental enrichment (EE) exposure on field potential responses in hippocampal slices, but in no study was…

  18. Physiological Effects of Enriched Environment Exposure and LTP Induction in the Hippocampus In Vivo Do Not Transfer Faithfully to In Vitro Slices

    ERIC Educational Resources Information Center

    Eckert, Michael J.; Abraham, Wickliffe C.

    2010-01-01

    A number of experimental paradigms use in vitro brain slices to test for changes in synaptic transmission and plasticity following a behavioral manipulation. For example, a number of previous studies have reported a variety of effects of environmental enrichment (EE) exposure on field potential responses in hippocampal slices, but in no study was…

  19. Transgenic Mice with Increased Astrocyte Expression of IL-6 Show Altered Effects of Acute Ethanol on Synaptic Function

    PubMed Central

    Hernandez, Ruben V.; Puro, Alana C.; Manos, Jessica C.; Huitron-Resendiz, Salvador; Reyes, Kenneth C.; Liu, Kevin; Vo, Khanh; Roberts, Amanda J.; Gruol, Donna L.

    2015-01-01

    A growing body of evidence has revealed that resident cells of the central nervous system (CNS), and particularly the glial cells, comprise a neuroimmune system that serves a number of functions in the normal CNS and during adverse conditions. Cells of the neuroimmune system regulate CNS functions through the production of signaling factors, referred to as neuroimmune factors. Recent studies show that ethanol can activate cells of the neuroimmune system, resulting in the elevated production of neuroimmune factors, including the cytokine interleukin-6 (IL-6). Here we analyzed the consequences of this CNS action of ethanol using transgenic mice that express elevated levels of IL-6 through increased astrocyte expression (IL-6-tg) to model the increased IL-6 expression that occurs with ethanol use. Results show that increased IL-6 expression induces neuroadaptive changes that alter the effects of ethanol. In hippocampal slices from non-transgenic (non-tg) littermate control mice, synaptically evoked dendritic field excitatory postsynaptic potential (fEPSP) and somatic population spike (PS) at the Schaffer collateral to CA1 pyramidal neuron synapse were reduced by acute ethanol (20 or 60 mM). In contrast, acute ethanol enhanced the fEPSP and PS in hippocampal slices from IL-6 tg mice. Long-term synaptic plasticity of the fEPSP (i.e., LTP) showed the expected dose-dependent reduction by acute ethanol in non-tg hippocampal slices, whereas LTP in the IL-6 tg hippocampal slices was resistant to this depressive effect of acute ethanol. Consistent with altered effects of acute ethanol on synaptic function in the IL-6 tg mice, EEG recordings showed a higher level of CNS activity in the IL-6 tg mice than in the non-tg mice during the period of withdrawal from an acute high dose of ethanol. These results suggest a potential role for neuroadaptive effects of ethanol-induced astrocyte production of IL-6 as a mediator or modulator of the actions of ethanol on the CNS, including

  20. Transgenic mice with increased astrocyte expression of IL-6 show altered effects of acute ethanol on synaptic function.

    PubMed

    Hernandez, Ruben V; Puro, Alana C; Manos, Jessica C; Huitron-Resendiz, Salvador; Reyes, Kenneth C; Liu, Kevin; Vo, Khanh; Roberts, Amanda J; Gruol, Donna L

    2016-04-01

    A growing body of evidence has revealed that resident cells of the central nervous system (CNS), and particularly the glial cells, comprise a neuroimmune system that serves a number of functions in the normal CNS and during adverse conditions. Cells of the neuroimmune system regulate CNS functions through the production of signaling factors, referred to as neuroimmune factors. Recent studies show that ethanol can activate cells of the neuroimmune system, resulting in the elevated production of neuroimmune factors, including the cytokine interleukin-6 (IL-6). Here we analyzed the consequences of this CNS action of ethanol using transgenic mice that express elevated levels of IL-6 through increased astrocyte expression (IL-6-tg) to model the increased IL-6 expression that occurs with ethanol use. Results show that increased IL-6 expression induces neuroadaptive changes that alter the effects of ethanol. In hippocampal slices from non-transgenic (non-tg) littermate control mice, synaptically evoked dendritic field excitatory postsynaptic potential (fEPSP) and somatic population spike (PS) at the Schaffer collateral to CA1 pyramidal neuron synapse were reduced by acute ethanol (20 or 60 mM). In contrast, acute ethanol enhanced the fEPSP and PS in hippocampal slices from IL-6 tg mice. Long-term synaptic plasticity of the fEPSP (i.e., LTP) showed the expected dose-dependent reduction by acute ethanol in non-tg hippocampal slices, whereas LTP in the IL-6 tg hippocampal slices was resistant to this depressive effect of acute ethanol. Consistent with altered effects of acute ethanol on synaptic function in the IL-6 tg mice, EEG recordings showed a higher level of CNS activity in the IL-6 tg mice than in the non-tg mice during the period of withdrawal from an acute high dose of ethanol. These results suggest a potential role for neuroadaptive effects of ethanol-induced astrocyte production of IL-6 as a mediator or modulator of the actions of ethanol on the CNS, including

  1. The roles of hippocampal microRNAs in response to acute postnatal exposure to di(2-ethylhexyl) phthalate in female and male rats.

    PubMed

    Luu, Bryan E; Green, Stuart R; Childers, Christine L; Holahan, Matthew R; Storey, Kenneth B

    2017-02-10

    Previous studies have shown that di(2-ethylhexyl) phthalate (DEHP) exposure impairs the normal development of pre- and post-synaptic elements of the male, but not female, rat hippocampus. While males seem to be vulnerable to the neurodevelopmental deficits resulting from DEHP exposure, females appear to show a protective response. The purpose of the present study was to characterize hippocampal microRNAs in female and male rats exposed to DEHP to assess whether any patterns emerged that would be consistent with vulnerability in males and resilience in females. Male and female rats were treated with 0, 1, 10, or 20mg/kg of DEHP by intraperitoneal injections from postnatal day 16 (PND16) - PND22 and brains were removed and flash frozen on PND78. A group of 85 microRNAs which have been previously shown to play a role in the development and maintenance of hippocampal neurons was assessed with RT-qPCR. In response to DEHP exposure, there were 19 microRNAs that increased in females and 52 that decreased in males. The strongest microRNA response in females occurred in conjunction with the 10mg/kg of DEHP dose, whereas suppression of microRNAs in males appeared to be dose-dependent. Select hippocampal microRNAs (such as miR-132-3p and miR-191-5p), previously shown to regulate dendrite morphology, were modulated by DEHP exposure in this study. The results suggest that DEHP exposure has the potential to regulate microRNAs in a sex-specific manner which may interfere with proper hippocampal development in males and preserve hippocampal development in females.

  2. System for slicing wafers

    NASA Technical Reports Server (NTRS)

    1982-01-01

    A newly patented process for slicing silicon wafers that has distinct advantages over methods now widely used is described. The primary advantage of the new system is that it allows the efficient slicing of a number of ingots simultaneously at high speed. The cutting action is performed mechanically, most often with diamond particles that are transported to the cutting zone by a fluid vehicle or have been made an integral part of the blade by plating or impregnation. The new system uses a multiple or ganged band saw, arranged and spaced so that each side, or length, segment of a blade element, or loop, provides a cutting function. Each blade is maintained precisely in position by guides as it enters and leaves each ingot. The cutting action is performed with a conventional abrasive slurry composed of diamond grit suspended in an oil- or water-based vehicle. The distribution system draws the slurry from the supply reservoir and pumps it to the injection tubes to supply it to each side of each ingot. A flush system is provided at the outer end of the work-station zone. In order to reduce potential damage, a pneumatically driven flushing fluid is provided.

  3. The theory of interface slicing

    NASA Technical Reports Server (NTRS)

    Beck, Jon

    1993-01-01

    Interface slicing is a new tool which was developed to facilitate reuse-based software engineering, by addressing the following problems, needs, and issues: (1) size of systems incorporating reused modules; (2) knowledge requirements for program modification; (3) program understanding for reverse engineering; (4) module granularity and domain management; and (5) time and space complexity of conventional slicing. The definition of a form of static program analysis called interface slicing is addressed.

  4. Serotonin dependent masking of hippocampal sharp wave ripples.

    PubMed

    ul Haq, Rizwan; Anderson, Marlene L; Hollnagel, Jan-Oliver; Worschech, Franziska; Sherkheli, Muhammad Azahr; Behrens, Christoph J; Heinemann, Uwe

    2016-02-01

    Sharp wave ripples (SPW-Rs) are thought to play an important role in memory consolidation. By rapid replay of previously stored information during slow wave sleep and consummatory behavior, they result from the formation of neural ensembles during a learning period. Serotonin (5-HT), suggested to be able to modify SPW-Rs, can affect many neurons simultaneously by volume transmission and alter network functions in an orchestrated fashion. In acute slices from dorsal hippocampus, SPW-Rs can be induced by repeated high frequency stimulation that induces long-lasting LTP. We used this model to study SPW-R appearance and modulation by 5-HT. Although stimulation in presence of 5-HT permitted LTP induction, SPW-Rs were "masked"--but appeared after 5-HT wash-out. This SPW-R masking was dose dependent with 100 nM 5-HT being sufficient--if the 5-HT re-uptake inhibitor citalopram was present. Fenfluramine, a serotonin releaser, could also mask SPW-Rs. Masking was due to 5-HT1A and 5-HT2A/C receptor activation. Neither membrane potential nor membrane conductance changes in pyramidal cells caused SPW-R blockade since both remained unaffected by combining 5-HT and citalopram. Moreover, 10 and 30 μM 5-HT mediated SPW-R masking preceded neuronal hyperpolarization and involved reduced presynaptic transmitter release. 5-HT, as well as a 5-HT1A agonist, augmented paired pulse facilitation and affected the coefficient of variance. Spontaneous SPW-Rs in mice hippocampal slices were also masked by 5-HT and fenfluramine. While neuronal ensembles can acquire long lasting LTP during higher 5-HT levels, lower 5-HT levels enable neural ensembles to replay previously stored information and thereby permit memory consolidation memory.

  5. Slice Segal-Bargmann transform

    NASA Astrophysics Data System (ADS)

    Cnudde, L.; De Bie, H.

    2017-06-01

    The Segal-Bargmann transform is a unitary map between the Schrödinger and Fock space, which is used, for example, to show the integrability of quantum Rabi models. Slice monogenic functions provide the framework in which functional calculus for quaternionic quantum mechanics can be developed. In this paper, a generalisation of the Segal-Bargmann transform, to the context of slice monogenic functions, is constructed and studied in detail. It is shown to interact appropriately with the recently constructed slice Fourier transform. This leads furthermore to a construction of a slice Fock space, which is shown to be a reproducing kernel space.

  6. Selective vulnerability of hippocampal sub-fields to oxygen-glucose deprivation is a function of animal age.

    PubMed

    Lalonde, Crystal C; Mielke, John G

    2014-01-16

    For more than a century, the hippocampal sub-fields have been recognized as being differentially vulnerable to injury. While the cause remains unknown, the explanations generally considered have involved either vascular differences, or innate variability among cells. To examine the latter possibility, we prepared acute hippocampal slices from Sprague-Dawley rats, applied a brief period of oxygen-glucose deprivation (OGD; an in vitro model of ischemia), and assessed the viability of dissected sub-fields (CA1, CA3, dentate gyrus) by measuring mitochondrial 2,3,5-triphenyltetrazolium chloride (TTC) metabolism. In slices from young animals (15 weeks of age), post-OGD TTC metabolism was significantly reduced in the CA sub-fields relative to the dentate gyrus. Since previous studies found increasing age may worsen ischemic injury, we completed the same experiment using tissue from animals at 52 weeks of age, and found no differences in TTC metabolism across sub-fields. Given the established role of glutamate receptors in ischemic cell death, we examined two key subunit proteins (GluN1, found in all NMDA receptors, and GluA2, found in most AMPA receptors) across sub-fields and age to determine whether their expression complemented our viability data. We found that, relative to the CA1, the DG displayed greater GluN1 expression and lower GluA2 expression in both young and old animals. Our results confirm that regional vulnerability can be shown in a slice model, that the property is not intransigent, and that these features are likely not attributable to the expression pattern of key glutamate receptor subunits, but another molecular variable that changes over the lifespan. © 2013 Published by Elsevier B.V.

  7. Ethanol exposure in early adolescence inhibits intrinsic neuronal plasticity via sigma-1 receptor activation in hippocampal CA1 neurons

    PubMed Central

    Sabeti, Jilla

    2011-01-01

    Background We demonstrated previously that rats exposed to chronic intermittent ethanol (CIE) vapors in early adolescence show increased magnitudes of long-term potentiation (LTP) of excitatory transmission when recorded at dendritic synapses in hippocampus. Large amplitude LTP following CIE exposure is mediated by sigma-1 receptors; however, not yet addressed is the role of sigma-1 receptors in modulating the intrinsic properties of neurons to alter their action potential firing during LTP. Methods Activity-induced plasticity of spike firing was investigated using rat hippocampal slice recordings to measure changes in both field excitatory postsynaptic potentials (fEPSPs) and population spikes (pop. spikes) concomitantly at dendritic inputs and soma of CA1 pyramidal neurons, respectively. Results We observed unique modifications in plasticity of action potential firing in hippocampal slices from CIE exposed adolescent rats, where the induction of large amplitude LTP by 100 Hz stimulations was accompanied by reduced CA1 neuronal excitability—reflected as decreased pop. spike efficacy and impaired activity-induced fEPSP-to-spike (E-S) potentiation. By contrast, LTP induction in ethanol-naïve control slices resulted in increased spike efficacy and robust E-S potentiation. E-S potentiation impairments emerged at 24 hr after CIE treatment cessation, but not before the alcohol withdrawal period, and were restored with bath-application of the sigma-1 receptor selective antagonist BD1047, but not the NMDA receptor antagonist D-AP5. Further evidence revealed a significantly shortened somatic fEPSP time course in adolescent CIE-withdrawn hippocampal slices during LTP; however, paired-pulse data show no apparent correspondence between E-S dissociation and altered recurrent feedback inhibition. Conclusions Results here suggest that acute withdrawal from adolescent CIE exposure triggers sigma-1 receptors that act to depress the efficacy of excitatory inputs in triggering

  8. Ethanol exposure in early adolescence inhibits intrinsic neuronal plasticity via sigma-1 receptor activation in hippocampal CA1 neurons.

    PubMed

    Sabeti, Jilla

    2011-05-01

    We demonstrated previously that rats exposed to chronic intermittent ethanol (CIE) vapors in early adolescence show increased magnitudes of long-term potentiation (LTP) of excitatory transmission when recorded at dendritic synapses in hippocampus. Large amplitude LTP following CIE exposure is mediated by sigma-1 receptors; however, not yet addressed is the role of sigma-1 receptors in modulating the intrinsic properties of neurons to alter their action potential firing during LTP. Activity-induced plasticity of spike firing was investigated using rat hippocampal slice recordings to measure changes in both field excitatory postsynaptic potentials (fEPSPs) and population spikes (pop. spikes) concomitantly at dendritic inputs and soma of CA1 pyramidal neurons, respectively. We observed unique modifications in plasticity of action potential firing in hippocampal slices from CIE exposed adolescent rats, where the induction of large amplitude LTP by 100 Hz stimulations was accompanied by reduced CA1 neuronal excitability--reflected as decreased pop. spike efficacy and impaired activity-induced fEPSP-to-spike (E-S) potentiation. In contrast, LTP induction in ethanol-naïve control slices resulted in increased spike efficacy and robust E-S potentiation. E-S potentiation impairments emerged at 24 hours after CIE treatment cessation, but not before the alcohol withdrawal period, and were restored with bath-application of the sigma-1 receptor selective antagonist BD1047, but not the NMDA receptor antagonist d-AP5. Further evidence revealed a significantly shortened somatic fEPSP time course in adolescent CIE-withdrawn hippocampal slices during LTP; however, paired-pulse data show no apparent correspondence between E-S dissociation and altered recurrent feedback inhibition. Results here suggest that acute withdrawal from adolescent CIE exposure triggers sigma-1 receptors that act to depress the efficacy of excitatory inputs in triggering action potentials during LTP. Such

  9. Mouse δ opioid receptors are located on presynaptic afferents to hippocampal pyramidal cells.

    PubMed

    Rezaï, Xavier; Faget, Lauren; Bednarek, Ewa; Schwab, Yannick; Kieffer, Brigitte L; Massotte, Dominique

    2012-05-01

    Delta opioid receptors participate in the control of chronic pain and emotional responses. Recent data have also identified their implication in drug-context associations pointing to a modulatory role on hippocampal activity. We used fluorescent knock-in mice that express a functional delta opioid receptor fused at its carboxy terminus with the green fluorescent protein in place of the native receptor to investigate the receptor neuroanatomical distribution in this structure. Fine mapping of the pyramidal layer was performed in hippocampal acute brain slices and organotypic cultures using fluorescence confocal imaging, co-localization with pre- and postsynaptic markers and correlative light-electron microscopy. The different approaches concurred to identify delta opioid receptors on presynaptic afferents to glutamatergic principal cells. In the latter, only scarce receptors were detected that were confined within the Golgi or vesicular intracellular compartments with no receptor present at the cell surface. In the mouse hippocampus, expression of functional delta opioid receptors is therefore mostly associated with interneurons emphasizing a presynaptic modulatory effect on the pyramidal cell firing rate.

  10. Pyrethroid insecticides evoke neurotransmitter release from rabbit striatal slices

    SciTech Connect

    Eells, J.T.; Dubocovich, M.L.

    1988-08-01

    The effects of the synthetic pyrethroid insecticide fenvalerate ((R,S)-alpha-cyano-3-phenoxybenzyl(R,S)-2-(4-chlorophenyl)-3- methylbutyrate) on neurotransmitter release in rabbit brain slices were investigated. Fenvalerate evoked a calcium-dependent release of (/sup 3/H)dopamine and (/sup 3/H)acetylcholine from rabbit striatal slices that was concentration-dependent and specific for the toxic stereoisomer of the insecticide. The release of (/sup 3/H)dopamine and (/sup 3/H)acetylcholine by fenvalerate was modulated by D2 dopamine receptor activation and antagonized completely by the sodium channel blocker, tetrodotoxin. These findings are consistent with an action of fenvalerate on the voltage-dependent sodium channels of the presynaptic membrane resulting in membrane depolarization, and the release of dopamine and acetylcholine by a calcium-dependent exocytotic process. In contrast to results obtained in striatal slices, fenvalerate did not elicit the release of (/sup 3/H)norepinephrine or (/sup 3/H)acetylcholine from rabbit hippocampal slices indicative of regional differences in sensitivity to type II pyrethroid actions.

  11. Hippocampal culture stimulus with 4-megahertz ultrasound

    NASA Astrophysics Data System (ADS)

    Muratore, Robert; LaManna, Justine K.; Lamprecht, Michael R.; Morrison, Barclay, III

    2012-10-01

    Among current modalities, ultrasound uniquely offers both millisecond and millimeter accuracy in noninvasively stimulating brain tissue. In addition, by sweeping the ultrasound beam within the refractory period of the neuronal tissue, ultrasonic neuromodulation can be adapted to target extended or multiply connected regions with quasi-simultaneity. Towards the development of this safe brain stimulus technique, the response of rat hippocampal cultures to ultrasound was investigated. Hippocampal slices, 0.4-mm thick, were obtained from 8-day old Sprague Dawley rats and cultured for 6 days. The in vitro cultures were exposed to multiple 100-ms 4.04-MHz ultrasound pulses from a 42-mm diameter, 90-mm spherical cap transducer. Peak pressure ranged from 0 through about 77 kPa. Responses in the form of electrical potentials from a sixty channel electrode array were digitized and recorded. The DG and CA1 regions of the hippocampus exhibited similar ultrasonically-evoked field potentials.

  12. Inhibition of local estrogen synthesis in the hippocampus impairs hippocampal memory consolidation in ovariectomized female mice

    PubMed Central

    Tuscher, Jennifer J.; Szinte, Julia S.; Starrett, Joseph R.; Krentzel, Amanda A.; Fortress, Ashley M.; Remage-Healey, Luke; Frick, Karyn M.

    2016-01-01

    The potent estrogen 17β-Estradiol (E2) plays a critical role in mediating hippocampal function, yet the precise mechanisms through which E2 enhances hippocampal memory remain unclear. In young adult female rodents, the beneficial effects of E2 on memory are generally attributed to ovarian-synthesized E2. However, E2 is also synthesized in the adult brain in numerous species, where it regulates synaptic plasticity and is synthesized in response to experiences such as exposure to females or conspecific song. Although de novo E2 synthesis has been demonstrated in rodent hippocampal cultures, little is known about the functional role of local E2 synthesis in mediating hippocampal memory function. Therefore, the present study examined the role of hippocampal E2 synthesis in hippocampal memory consolidation. Using bilateral dorsal hippocampal infusions of the aromatase inhibitor letrozole, we first found that blockade of dorsal hippocampal E2 synthesis impaired hippocampal memory consolidation. We next found that elevated levels of E2 in dorsal hippocampus observed 30 min after object training were blocked by dorsal hippocampal infusion of letrozole, suggesting that behavioral experience increases acute and local E2 synthesis. Finally, aromatase inhibition did not prevent exogenous E2 from enhancing hippocampal memory consolidation, indicating that hippocampal E2 synthesis is not necessary for exogenous E2 to enhance hippocampal memory. Combined, these data are consistent with the hypothesis that hippocampally-synthesized E2 is necessary for hippocampus-dependent memory consolidation in rodents. PMID:27178577

  13. Improvement of ischemic damage in gerbil hippocampal neurons by procaine.

    PubMed

    Chen, J; Adachi, N; Liu, K; Nagaro, T; Arai, T

    1998-05-04

    Acute cerebral ischemia induces membrane depolarization in the neuron, thereby incurring the simultaneous influx of various ions such as Na+ and Ca2+. Since procaine possesses the ability to inhibit the release of Ca2+ from intracellular Ca2+ stores to the cytosol as well as the ability to block Na+ channels, the effects of procaine on ischemia were investigated in the present study in gerbils both in vivo and in vitro. The histologic outcome was evaluated 7 days after 3 min of transient forebrain ischemia by assessing delayed neuronal death in hippocampal CA1 pyramidal cells in animals administered procaine (0.2, 0.4, or 2 micromol) intracerebroventricularly 10 min before ischemia and in animals given saline. The changes in the direct-current potential shift in the hippocampal CA1 area were measured using an identical animal model. A hypoxia-induced intracellular Ca2+ increase was evaluated by in vitro microfluorometry in gerbil hippocampal slices, and the effects of procaine (10, 50, and 100 micromol/l) on the Ca2+ accumulation were examined. Additionally, the effect of procaine (100 micromol/l) in a Ca2+-free condition was investigated. The histologic outcome was improved and the onset of the ischemia-induced membrane depolarization was prolonged by the preischemic administration of procaine. The increase in the intracellular concentration of Ca2+ induced by the in vitro hypoxia was suppressed by the perfusion of procaine-containing mediums (50 and 100 micromol/l), regarding both the initiation and the extent of the increase. A hypoxia-induced intracellular Ca2+ elevation in the Ca2+-free condition was observed, and the perfusion with procaine (100 micromol/l) inhibited this elevation. Procaine helps protect neurons from ischemia by suppressing the direct-current potential shift and by inhibiting the release of Ca2+ from the intracellular Ca2+ stores, as well as by inhibiting the influx of Ca2+ from the extracellular space. Copyright 1998 Elsevier Science B.V.

  14. Intranasal Oxytocin following Uncontrollable Stress Blocks Impairments in Hippocampal Plasticity and Recognition Memory in Stressed Rats.

    PubMed

    Park, Seong-Hae; Kim, Yoon-Jung; Park, Jung-Cheol; Han, Jung-Soo; Choi, Se-Young

    2017-10-01

    Nasal pretreatment with the neuropeptide oxytocin has been reported to prevent stress-induced impairments in hippocampal synaptic plasticity and spatial memory in rats. However, no study has asked if oxytocin application following a stress experience is effective in rescuing stress-induced impairments. Synaptic plasticity was measured in hippocampal Schaffer collateral-CA1 synapses of rats subjected to uncontrollable stress; their cognitive function was examined using an object recognition task. Impaired induction of long-lasting, long-term potentiation by uncontrollable stress was rescued, as demonstrated both in rats and hippocampal slices. Intranasal oxytocin after experiencing uncontrollable stress blocked cognitive impairments in stressed rats and in stressed hippocampal slices treated with a perfused bath solution containing oxytocin. These results indicated that posttreatment with oxytocin after experiencing a stressful event can keep synaptic plasticity and cognition function intact, indicating the therapeutic potential of oxytocin for stress-related disorders, including posttraumatic stress disorder.

  15. Intraneuronal Aβ accumulation induces hippocampal neuron hyperexcitability through A-type K(+) current inhibition mediated by activation of caspases and GSK-3.

    PubMed

    Scala, Federico; Fusco, Salvatore; Ripoli, Cristian; Piacentini, Roberto; Li Puma, Domenica Donatella; Spinelli, Matteo; Laezza, Fernanda; Grassi, Claudio; D'Ascenzo, Marcello

    2015-02-01

    Amyloid β-protein (Aβ) pathologies have been linked to dysfunction of excitability in neurons of the hippocampal circuit, but the molecular mechanisms underlying this process are still poorly understood. Here, we applied whole-cell patch-clamp electrophysiology to primary hippocampal neurons and show that intracellular Aβ42 delivery leads to increased spike discharge and action potential broadening through downregulation of A-type K(+) currents. Pharmacologic studies showed that caspases and glycogen synthase kinase 3 (GSK-3) activation are required for these Aβ42-induced effects. Extracellular perfusion and subsequent internalization of Aβ42 increase spike discharge and promote GSK-3-dependent phosphorylation of the Kv4.2 α-subunit, a molecular determinant of A-type K(+) currents, at Ser-616. In acute hippocampal slices derived from an adult triple-transgenic Alzheimer's mouse model, characterized by endogenous intracellular accumulation of Aβ42, CA1 pyramidal neurons exhibit hyperexcitability accompanied by increased phosphorylation of Kv4.2 at Ser-616. Collectively, these data suggest that intraneuronal Aβ42 accumulation leads to an intracellular cascade culminating into caspases activation and GSK-3-dependent phosphorylation of Kv4.2 channels. These findings provide new insights into the toxic mechanisms triggered by intracellular Aβ42 and offer potentially new therapeutic targets for Alzheimer's disease treatment.

  16. Intraneuronal Aβ accumulation induces hippocampal neuron hyperexcitability through A-type K+ current inhibition mediated by activation of caspases and GSK-3

    PubMed Central

    Scala, Federico; Fusco, Salvatore; Ripoli, Cristian; Piacentini, Roberto; Li Puma, Domenica Donatella; Spinelli, Matteo; Laezza, Fernanda; Grassi, Claudio; D’Ascenzo, Marcello

    2016-01-01

    Amyloid β-protein (Aβ) pathologies have been linked to dysfunction of excitability in neurons of the hippocampal circuit, but the molecular mechanisms underlying this process are still poorly understood. Here, we applied whole-cell patch-clamp electrophysiology to primary hippocampal neurons and show that intracellular Aβ42 delivery leads to increased spike discharge and action potential broadening through downregulation of A-type K+ currents. Pharmacologic studies showed that caspases and glycogen synthase kinase 3 (GSK-3) activation are required for these Aβ42-induced effects. Extracellular perfusion and subsequent internalization of Aβ42 increase spike discharge and promote GSK-3-dependent phosphorylation of the Kv4.2 α-subunit, a molecular determinant of A-type K+ currents, at Ser-616. In acute hippocampal slices derived from an adult triple-transgenic Alzheimer’s mouse model, characterized by endogenous intracellular accumulation of Aβ42, CA1 pyramidal neurons exhibit hyperexcitability accompanied by increased phosphorylation of Kv4.2 at Ser-616. Collectively, these data suggest that intraneuronal Aβ42 accumulation leads to an intracellular cascade culminating into caspases activation and GSK-3-dependent phosphorylation of Kv4.2 channels. These findings provide new insights into the toxic mechanisms triggered by intracellular Aβ42 and offer potentially new therapeutic targets for Alzheimer’s disease treatment. PMID:25541422

  17. Neurogliaform Cells in the Molecular Layer of the Dentate Gyrus as Feed-Forward γ-Aminobutyric Acidergic Modulators of Entorhinal–Hippocampal Interplay

    PubMed Central

    Armstrong, Caren; Szabadics, János; Tamás, Gábor; Soltesz, Ivan

    2014-01-01

    Feed-forward inhibition from molecular layer interneurons onto granule cells (GCs) in the dentate gyrus is thought to have major effects regulating entorhinal–hippocampal interactions, but the precise identity, properties, and functional connectivity of the GABAergic cells in the molecular layer are not well understood. We used single and paired intracellular patch clamp recordings from post-hoc-identified cells in acute rat hippocampal slices and identified a subpopulation of molecular layer interneurons that expressed immunocytochemical markers present in members of the neurogliaform cell (NGFC) class. Single NGFCs displayed small dendritic trees, and their characteristically dense axonal arborizations covered significant portions of the outer and middle one-thirds of the molecular layer, with frequent axonal projections across the fissure into the CA1 and subicular regions. Typical NGFCs exhibited a late firing pattern with a ramp in membrane potential prior to firing action potentials, and single spikes in NGFCs evoked biphasic, prolonged GABAA and GABAB postsynaptic responses in GCs. In addition to providing dendritic GABAergic inputs to GCs, NGFCs also formed chemical synapses and gap junctions with various molecular layer interneurons, including other NGFCs. NGFCs received low-frequency spontaneous synaptic events, and stimulation of perforant path fibers revealed direct, facilitating synaptic inputs from the entorhinal cortex. Taken together, these results indicate that NGFCs form an integral part of the local molecular layer microcircuitry generating feed-forward inhibition and provide a direct GABAergic pathway linking the dentate gyrus to the CA1 and subicular regions through the hippocampal fissure PMID:21452204

  18. In vivo quantification of hippocampal subfields using 4.7 T fast spin echo imaging.

    PubMed

    Malykhin, N V; Lebel, R M; Coupland, N J; Wilman, A H; Carter, R

    2010-01-15

    Several neuropsychiatric disorders involving hippocampal structural changes have been studied extensively using volumetric magnetic resonance imaging (MRI). These studies have mostly measured total hippocampal volume while the present study aimed to delineate and measure hippocampal subfields within the whole hippocampus and subdivisions along its longitudinal axis. Images were acquired at 4.7 T in 11 healthy subjects (5 males and 6 females, aged 23-56 years), using a fast spin echo (FSE) sequence with 0.52 x 0.68 x 1.0 mm(3) native resolution, collecting 90 contiguous coronal slices. Subiculum, cornu ammonis (CA1-3), and dentate gyrus were traced manually within the hippocampal head, body, and tail. We reported volumes for the subfields and demonstrated differences in the distribution within the hippocampus and its parts. The biggest part of the dentate gyrus was located in the hippocampal body, following the hippocampal head and tail. In contrast, the hippocampal head had the largest part of CA1-3, following the hippocampal body and tail. The hippocampal tail had the smallest portion of the subiculum compared to hippocampal head and tail. Subfield volumes were consistent between hemispheres and showed distributions within the longitudinal subdivisions that were consistent with histological data. Direct measurements of subfield distribution along the longitudinal axis of the hippocampus may be more sensitive to detecting disease effects than total volume measures and the differential distribution of subfield volumes may aid in the interpretation of measurements obtained at lower field strength and spatial resolution.

  19. Flat slices in Minkowski space

    NASA Astrophysics Data System (ADS)

    Murchadha, Niall Ó.; Xie, Naqing

    2015-03-01

    Minkowski space, flat spacetime, with a distance measure in natural units of d{{s}2}=-d{{t}2}+d{{x}2}+d{{y}2}+d{{z}2}, or equivalently, with spacetime metric diag(-1, +1, +1, +1), is recognized as a fundamental arena for physics. The Poincaré group, the set of all rigid spacetime rotations and translations, is the symmetry group of Minkowski space. The action of this group preserves the form of the spacetime metric. Each t = constant slice of each preferred coordinate system is flat. We show that there are also nontrivial non-singular representations of Minkowski space with complete flat slices. If the embedding of the flat slices decays appropriately at infinity, the only flat slices are the standard ones. However, if we remove the decay condition, we find non-trivial flat slices with non-vanishing extrinsic curvature. We write out explicitly the coordinate transformation to a frame with such slices.

  20. Benzodiazepines do not potentiate GABA responses in neonatal hippocampal neurons.

    PubMed

    Rovira, C; Ben-Ari, Y

    1991-09-16

    Benzodiazepines (midazolam; flunitrazepam) and pentobarbital increase the response to exogenous gamma-aminobutyric acid (GABA) in adult hippocampal cells. We report in this paper that in contrast pentobarbital but not benzodiazepine potentiate the effects of exogenous (GABA) in neurons recorded from slices of less than two weeks old. This finding suggests that the functional association of benzodiazepine and GABAA receptors is changed during early postnatal life.

  1. Cytotoxicity of gamma-ray in rat immature hippocampal neurons

    PubMed Central

    Yang, Miyoung; Song, Myoung-Sub; Kim, Sung-Ho; Kim, Jong-Choon; Kim, Joong-Sun; Shin, Taekyun

    2011-01-01

    This in vitro study evaluated the detrimental effect of acute gamma (γ)-irradiation on rat immature hippocampal neurons. Rat immature hippocampal neurons (0.5 day in vitro) were irradiated with 0~4 Gy γ-rays. Cytotoxicity was analyzed using a lactate dehydrogenase release assay at 24 h after γ-irradiation. Radiation-induced cytotoxicity in immature hippocampal neurons increased in a dose-dependent manner. Pre-treatments of pro-apoptotic caspase inhibitors and anti-oxidative substances significantly blocked γ-irradiation-induced cytotoxicity in immature hippocampal neurons. The results suggest that the caspase-dependent cytotoxicity of γ-rays in immature hippocampal cultured neurons may be caused by oxidative stress. PMID:21897091

  2. Matrix Metalloprotease 3 Activity Supports Hippocampal EPSP-to-Spike Plasticity Following Patterned Neuronal Activity via the Regulation of NMDAR Function and Calcium Flux.

    PubMed

    Brzdąk, Patrycja; Włodarczyk, Jakub; Mozrzymas, Jerzy W; Wójtowicz, Tomasz

    2017-01-01

    Matrix metalloproteases (MMPs) comprise a family of endopeptidases that are involved in remodeling the extracellular matrix and play a critical role in learning and memory. At least 24 different MMP subtypes have been identified in the human brain, but less is known about the subtype-specific actions of MMP on neuronal plasticity. The long-term potentiation (LTP) of excitatory synaptic transmission and scaling of dendritic and somatic neuronal excitability are considered substrates of memory storage. We previously found that MMP-3 and MMP-2/9 may be differentially involved in shaping the induction and expression of excitatory postsynaptic potential (EPSP)-to-spike (E-S) potentiation in hippocampal brain slices. MMP-3 and MMP-2/9 proteolysis was previously shown to affect the integrity or mobility of synaptic N-methyl-D-aspartate receptors (NMDARs) in vitro. However, the functional outcome of such MMP-NMDAR interactions remains largely unknown. The present study investigated the role of these MMP subtypes in E-S plasticity and NMDAR function in mouse hippocampal acute brain slices. The temporal requirement for MMP-3/NMDAR activity in E-S potentiation within the CA1 field largely overlapped, and MMP-3 but not MMP-2/9 activity was crucial for the gain-of-function of NMDARs following LTP induction. Functional changes in E-S plasticity following MMP-3 inhibition largely correlated with the expression of cFos protein, a marker of activity-related gene transcription. Recombinant MMP-3 promoted a gain in NMDAR-mediated field potentials and somatodendritic Ca(2+) waves. These results suggest that long-term hippocampal E-S potentiation requires transient MMP-3 activity that promotes NMDAR-mediated postsynaptic Ca(2+) entry that is vital for the activation of downstream signaling cascades and gene transcription.