Neurons In Hippocampal Slices Research Articles

Seizures exacerbate excitatory: inhibitory imbalance in Alzheimer's disease and 5XFAD mice.

Approximately 22% of Alzheimer's disease (AD) patients suffer from seizures, and the co-occurrence of seizures and epileptiform activity exacerbates AD pathology and related cognitive deficits, suggesting that seizures may be a targetable component of AD progression. Given that alterations in neuronal excitatory:inhibitory (E:I) balance occur in epilepsy, we hypothesized that decreased markers of inhibition relative to those of excitation would be present in AD patients. We similarly hypothesized that in 5XFAD mice, the E:I imbalance would progress from an early stage (prodromal) to later symptomatic stages and be further exacerbated by pentylenetetrazol (PTZ) kindling. Post-mortem AD temporal cortical tissues from patients with or without seizure history were examined for changes in several markers of E:I balance, including levels of the inhibitory GABAA receptor, the sodium potassium chloride cotransporter 1 (NKCC1) and potassium chloride cotransporter 2 (KCC2) and the excitatory NMDA and AMPA type glutamate receptors. We performed patch-clamp electrophysiological recordings from CA1 neurons in hippocampal slices and examined the same markers of E:I balance in prodromal 5XFAD mice. We next examined 5XFAD mice at chronic stages, after PTZ or control protocols, and in response to chronic mTORC1 inhibitor rapamycin, administered following kindled seizures, for markers of E:I balance. We found that AD patients with comorbid seizures had worsened cognitive and functional scores and decreased GABAA receptor subunit expression, as well as increased NKCC1/KCC2 ratios, indicative of depolarizing GABA responses. Patch clamp recordings of prodromal 5XFAD CA1 neurons showed increased intrinsic excitability, along with decreased GABAergic inhibitory transmission and altered glutamatergic neurotransmission, indicating that E:I imbalance may occur in early disease stages. Furthermore, seizure induction in prodromal 5XFAD mice led to later dysregulation of NKCC1/KCC2 and a reduction in GluA2 AMPA glutamate receptor subunit expression, indicative of depolarizing GABA receptors and calcium permeable AMPA receptors. Finally, we found that chronic treatment with the mTORC1 inhibitor, rapamycin, at doses we have previously shown to attenuate seizure-induced amyloid-β pathology and cognitive deficits, could also reverse elevations of the NKCC1/KCC2 ratio in these mice. Our data demonstrate novel mechanisms of interaction between AD and epilepsy and indicate that targeting E:I balance, potentially with US Food and Drug Administration-approved mTOR inhibitors, hold therapeutic promise for AD patients with a seizure history.

Increases in anterograde axoplasmic transport in neurons of the hyper-glutamatergic, glutamate dehydrogenase 1 (Glud1) transgenic mouse: Effects of glutamate receptors on transport.

The excitatory neurotransmitter glutamate has a role in neuronal migration and process elongation in the central nervous system (CNS). The effects of chronic glutamate hyperactivity on vesicular and protein transport within CNS neurons, that is, processes necessary for neurite growth, have not been examined previously. In this study, we measured the effects of lifelong hyperactivity of glutamate neurotransmission on axoplasmic transport in CNS neurons. We compared wild-type (wt) to transgenic (Tg) mice over-expressing the glutamate dehydrogenase gene Glud1 in CNS neurons and exhibiting increases in glutamate transmitter formation, release, and synaptic activation in brain throughout the lifespan. We found that Glud1 Tg as compared with wt mice exhibited increases in the rate of anterograde axoplasmic transport in neurons of the hippocampus measured in brain slices exvivo, and in olfactory neurons measured in vivo. We also showed that the in vitro pharmacologic activation of glutamate synapses in wt mice led to moderate increases in axoplasmic transport, while exposure to selective inhibitors of ion channel forming glutamate receptors very significantly suppressed anterograde transport, suggesting a link between synaptic glutamate receptor activation and axoplasmic transport. Finally, axoplasmic transport in olfactory neurons of Tg mice in vivo was partially inhibited following 14-day intake of ethanol, a known suppressor of axoplasmic transport and of glutamate neurotransmission. The same was true for transport in hippocampal neurons in slices from Glud1 Tg mice exposed to ethanol for 2 h exvivo. In conclusion, endogenous activity at glutamate synapses regulates and glutamate synaptic hyperactivity increases intraneuronal transport rates in CNS neurons.

Sodium channel endocytosis drives axon initial segment plasticity.

Activity-dependent plasticity of the axon initial segment (AIS) endows neurons with the ability to adapt action potential output to changes in network activity. Action potential initiation at the AIS highly depends on the clustering of voltage-gated sodium channels, but the molecular mechanisms regulating their plasticity remain largely unknown. Here, we developed genetic tools to label endogenous sodium channels and their scaffolding protein, to reveal their nanoscale organization and longitudinally image AIS plasticity in hippocampal neurons in slices and primary cultures. We find that N-methyl-d-aspartate receptor activation causes both long-term synaptic depression and rapid internalization of AIS sodium channels within minutes. The clathrin-mediated endocytosis of sodium channels at the distal AIS increases the threshold for action potential generation. These data reveal a fundamental mechanism for rapid activity-dependent AIS reorganization and suggests that plasticity of intrinsic excitability shares conserved features with synaptic plasticity.

Cell‐type Specific Effects of AD Mutations on Hippocampal Intrinsic Firing Properties in Genetically Diverse Mice

AbstractBackgroundCognitive resilience to Alzheimer’s disease (AD) is a phenomenon whereby individuals with known AD mutations or high levels of pathology exhibit better than predicted cognitive performance. Identification of the factors governing resilience may lead to novel therapeutic interventions to treat AD. Analysis of hippocampal transcriptional profiles from susceptible and resilient AD‐BXD strains indicates that transcriptomic changes in excitatory neurons in CA1 and dentate gyrus (DG) are associated with resilience and we now seek to examine physiological changes.MethodWe adapted the Patch‐Seq method (Cadwell et al., 2017) to investigate cell type‐specific transcriptional and electrophysiological changes associated with AD using the AD‐BXD genetic reference panel (Neuner et al., 2019). Whole‐cell patch clamp was used to evaluate intrinsic excitability of neurons within the DG and CA1 of 14m old mice with cognitive deficits compared to age matched controls; their cellular contents, including the nucleus, were collected after each recording to characterize the transcriptome.ResultComparison of the intrinsic properties of DG and CA1 neurons of hippocampal slices from 5XFAD mice and their non‐transgenic (Ntg) counterparts revealed that DG granule cells from 5XFAD mice have a lower input resistance and an increased sag ratio in response to hyperpolarizing current injections. Pyramidal neurons from the CA1 of 5xFAD mice exhibit significantly more depolarized action potential thresholds, a significant increase in the medium afterhyperpolarization (mAHP) and sag ratio compared to Ntg controls. While these intrinsic properties are proxy measures for neuronal excitability, we did not observe a significant effect of genotype on firing rate in response to 1s and 15s depolarizing current steps.ConclusionOur findings newly demonstrate disease related changes to intrinsic firing properties of DG granule cells at 14mo indicating that these neurons may be hypoexcitable. Also found, CA1 neurons from AD mice exhibit an increased mAHP and changes to sag ratio and AP threshold, indicating they too become less excitable. Future work will add additional AD‐BXD strains characterized as resilient or susceptible to the AD transgene to discover changes in intrinsic properties that could underlie resilience. We will also use an integrated analysis of transcriptomic and electrophysiological data to identify relevant functional targets for validation.

Removal of KCNQ2 from parvalbumin-expressing interneurons improves anti-seizure efficacy of retigabine

Anti-seizure drug (ASD) targets are widely expressed in both excitatory and inhibitory neurons. It remains unknown if the action of an ASD upon inhibitory neurons could counteract its beneficial effects on excitatory neurons (or vice versa), thereby reducing the efficacy of the ASD. Here, we examine whether the efficacy of the ASD retigabine (RTG) is altered after removal of the Kv7 potassium channel subunit KCNQ2, one of its drug targets, from parvalbumin-expressing interneurons (PV-INs). Parvalbumin-Cre (PV-Cre) mice were crossed with Kcnq2-floxed (Kcnq2fl/fl) mice to conditionally delete Kcnq2 from PV-INs. In these conditional knockout mice (cKO, PV-Kcnq2fl/fl), RTG (10 mg/kg, i.p.) significantly delayed the onset of either picrotoxin (PTX, 10 mg/kg, i.p)- or kainic acid (KA, 30 mg/kg, i.p.)-induced convulsive seizures compared to vehicle, while RTG was not effective in wild-type littermates (WT). Immunostaining for KCNQ2 and KCNQ3 revealed that both subunits were enriched at axon initial segments (AISs) of hippocampal CA1 PV-INs, and their specific expression was selectively abolished in cKO mice. Accordingly, the M-currents recorded from CA1 PV-INs and their sensitivity to RTG were significantly reduced in cKO mice. While the ability of RTG to suppress CA1 excitatory neurons in hippocampal slices was unchanged in cKO mice, its suppressive effect on the spike activity of CA1 PV-INs was significantly reduced compared with WT mice. In addition, the RTG-induced suppression on intrinsic membrane excitability of PV-INs in WT mice was significantly reduced in cKO mice. These findings suggest that preventing RTG from suppressing PV-INs improves its anticonvulsant effect.

A high-fat diet changes astrocytic metabolism to promote synaptic plasticity and behavior.

A high-fat diet (HFD) is generally considered to negatively influence the body, the brain, and cognition. Nonetheless, fat and fatty acids are essential for nourishing and constructing brain tissue. Astrocytes are central for lipolysis and fatty acids metabolism. We tested how HFD affects astrocyte metabolism, morphology, and physiology. We used Raman microspectroscopy to assess the redox state of mitochondria and lipid content in astrocytes and neurons in hippocampal slices of mice subjected to HFD. Astrocytes were loaded with fluorescent dye through patch pipette for morphological analysis. Whole-cell voltage-clamp recordings were performed to measure transporter and potassium currents. Western blot analysis quantified the expression of astrocyte-specific proteins. Field potential recordings measured the magnitude of long-term potentiation (LTP). Open filed test was performed to evaluate the effect of HFD on animal behavior. We found that exposure of young mice to 1 month of HFD increases lipid content and relative amount of reduced cytochromes in astrocytes but not in neurons. Metabolic changes were paralleled with an enlargement of astrocytic territorial domains due to an increased outgrowth of branches and leaflets. Astrocyte remodeling was associated with an increase in expression of ezrin and with no changes in glial fibrillary acidic protein (GFAP), glutamate transporter-1 (GLT-1), and glutamine synthetase (GS). Such physiological (non-reactive) enlargement of astrocytes in the brain active milieu promoted glutamate clearance and LTP and translated into behavioral changes. Dietary fat intake is not invariably harmful and might exert beneficial effects depending on the biological context.

A GLP-1/GIP/Gcg receptor triagonist improves memory behavior, as well as synaptic transmission, neuronal excitability and Ca2+ homeostasis in 3xTg-AD mice

Alzheimer's disease (AD) is a progressively neurodegenerative disorder, which seriously affects human health and cannot be stopped by current treatments. Type 2 diabetes mellitus (T2DM) is a risk factor for AD. Our recent studies reported the neuroprotective effects of a GLP-1/GIP/Glucagon receptor triagonist (Triagonist), a novel unimolecular anti-diabetic drug, in cognitive and pathological improvements of 3xTg-AD mice. However, the detailed electrophysiological and molecular mechanisms underlying neuroprotection remain unexplored. The present study investigated the underlying electrophysiological and molecular mechanisms further by using whole-cell patch clamp techniques. Our results revealed that chronic Triagonist treatment effectively reduced working memory and reference memory errors of 3xTg-AD mice in a radial maze test. In addition, the Triagonist increased spontaneous excitatory synaptic activities, differentially modulated voltage- and chemically-gated Ca2+ flux, and reduced the over-excitation of pyramidal neurons in hippocampal slices of 3xTg-AD mice. In addition, chronic Triagonist treatment also up-regulated the expression levels of synaptophysin and PSD-95 in the hippocampus of 3xTg-AD mice. These results indicate that the Triagonist could improve memory formation, as well as synaptic transmission, Ca2+ balance, and neuronal excitability in 3xTg-AD mice. These neuroprotective effects of Triagonist may be involved in the up-regulation of synaptophysin and PSD-95. Therefore, the study suggests that multi-receptor agonists might be a novel therapeutic strategy for the treatment of AD.

Zn2+ entry through the mitochondrial calcium uniporter is a critical contributor to mitochondrial dysfunction and neurodegeneration

Excitotoxic Ca2+ accumulation contributes to ischemic neurodegeneration, and Ca2+ can enter the mitochondria through the mitochondrial calcium uniporter (MCU) to promote mitochondrial dysfunction. Yet, Ca2+-targeted therapies have met limited success. A growing body of evidence has highlighted the underappreciated importance of Zn2+, which also accumulates in neurons after ischemia and can induce mitochondrial dysfunction and cell death. While studies have indicated that Zn2+ can also enter the mitochondria through the MCU, the specificity of the pore's role in Zn2+-triggered injury is still debated. Present studies use recently available MCU knockout mice to examine how the deletion of this channel impacts deleterious effects of cytosolic Zn2+ loading. In cultured cortical neurons from MCU knockout mice, we find significantly reduced mitochondrial Zn2+ accumulation. Correspondingly, these neurons were protected from both acute and delayed Zn2+-triggered mitochondrial dysfunction, including mitochondrial reactive oxygen species generation, depolarization, swelling and inhibition of respiration. Furthermore, when toxic extramitochondrial effects of Ca2+ entry were moderated, both cultured neurons (exposed to Zn2+) and CA1 neurons of hippocampal slices (subjected to prolonged oxygen glucose deprivation to model ischemia) from MCU knockout mice displayed decreased neurodegeneration. Finally, to examine the therapeutic applicability of these findings, we added an MCU blocker after toxic Zn2+ exposure in wildtype neurons (to induce post-insult MCU blockade). This significantly attenuated the delayed evolution of both mitochondrial dysfunction and neurotoxicity. These data—combining both genetic and pharmacologic tools—support the hypothesis that Zn2+ entry through the MCU is a critical contributor to ischemic neurodegeneration that could be targeted for neuroprotection.

Deep brain stimulation restores the glutamatergic and GABAergic synaptic transmission and plasticity to normal levels in kindled rats.

BackgroundThe precise effect of low frequency stimulation (LFS) as a newly postulated, anticonvulsant therapeutic approach on seizure-induced changes in synaptic transmission has not been completely determined.HypothesisIn this study, the LFS effect on impaired, synaptic plasticity in kindled rats was investigated.MethodsHippocampal kindled rats received LFS (4 trials consisting of one train of 200 monophasic square waves, 0.1 ms pulse duration, 1 Hz) on four occasions. LTP induction was evaluated using whole-cell recordings of evoked excitatory and inhibitory post-synaptic potentials (EPSPs and IPSPs respectively) in CA1 neurons in hippocampal slices. In addition, the hippocampal excitatory and inhibitory post-synaptic currents (EPSCs and IPSCs), and the gene expression of NR2A, GluR2 and γ2 were evaluated.ResultsLTP induction was attenuated in excitatory and inhibitory synapses in hippocampal slices of kindled rats. When LFS was applied in kindled animals, LTP was induced in EPSPs and IPSPs. Moreover, LFS increased and decreased the threshold intensities of EPSCs and IPSCs respectively. In kindled animals, NR2A gene expression increased, while γ2 gene expression decreased. GluR2 gene expression did not significantly change. Applying LFS in kindled animals mitigated these changes: No significant differences were observed in NR2A, γ2 and GluR2 gene expression in the kindled+LFS and control groups.ConclusionThe application of LFS in kindled animals restored LTP induction in both EPSPs and IPSPs, and returned the threshold intensity for induction of EPSCs, IPSCs and gene expression to similar levels as controls.

Obituary: Dr Carlos Barajas López (1959‐2018)

Obituary: Dr Carlos Barajas López (1959‐2018)

A mouse model of seizures in anti-N-methyl-d-aspartate receptor encephalitis.

Seizures develop in 80% of patients with anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis, and these represent a major cause of morbidity and mortality. Anti-NMDAR antibodies have been linked to memory loss in encephalitis; however, their role in seizures has not been established. We determined whether anti-NMDAR antibodies from autoimmune encephalitis patients are pathogenic for seizures. We performed continuous intracerebroventricular infusion of cerebrospinal fluid (CSF) or purified immunoglobulin (IgG) from the CSF of patients with anti-NMDAR encephalitis or polyclonal rabbit anti-NMDAR IgG, in male C57BL/6 mice. Seizure status during a 2-week treatment was assessed with video-electroencephalography. We assessed memory, anxiety-related behavior, and motor function at the end of treatment and assessed the extent of neuronal damage and gliosis in the CA1 region of hippocampus. We also performed whole-cell patch recordings from the CA1 pyramidal neurons in hippocampal slices of mice with seizures. Prolonged exposure to rabbit anti-NMDAR IgG, patient CSF, or human IgG purified from the CSF of patients with encephalitis induced seizures in 33 of 36 mice. The median number of seizures recorded in 2weeks was 13, 39, and 35 per mouse in these groups, respectively. We observed only 18 brief nonconvulsive seizures in 11 of 29 control mice (median seizure count of 0) infused with vehicle (n=4), normal CSF obtained from patients with noninflammatory central nervous system (CNS) conditions (n=12), polyclonal rabbit IgG (n=7), albumin (n=3), and normal human IgG (n=3). We did not observe memory deficits, anxiety-related behavior, or motor impairment measured at 2weeks in animals treated with CSF from affected patients or rabbit IgG. Furthermore, there was no evidence of hippocampal cell loss or astrocyte proliferation in the same mice. Our findings indicate that autoantibodies can induce seizures in anti-NMDAR encephalitis and offer a model for testing novel therapies for refractory autoimmune seizures.

Direct interaction with 14\u20133-3\u03b3 promotes surface expression of Best1 channel in astrocyte

BackgroundBestrophin-1 (Best1) is a calcium-activated anion channel (CAAC) that is expressed broadly in mammalian tissues including the brain. We have previously reported that Best1 is expressed in hippocampal astrocytes at the distal peri-synaptic regions, called microdomains, right next to synaptic junctions, and that it disappears from the microdomains in Alzheimer’s disease mouse model. Although Best1 appears to be dynamically regulated, the mechanism of its regulation and modulation is poorly understood. It has been reported that a regulatory protein, 14-3-3 affects the surface expression of numerous membrane proteins in mammalian cells.MethodsThe protein-protein interaction between Best1 and 14-3-3γ was confirmed by yeast-two hybrid assay and BiFC method. The effect of 14-3-3γ on Best1-mediated current was measured by whole-cell patch clamp technique.ResultsWe identified 14-3-3γ as novel binding partner of Best1 in astrocytes: among 7 isoforms of 14-3-3 protein, only 14-3-3γ was found to bind specifically. We determined a binding domain on the C-terminus of Best1 which is critical for an interaction with 14-3-3γ. We also revealed that interaction between Best1 and 14-3-3γ was mediated by phosphorylation of S358 in the C-terminus of Best1. We confirmed that surface expression of Best1 and Best1-mediated whole-cell current were significantly decreased after a gene-silencingof 14-3-3γ without a significant change in total Best1 expression in cultured astrocytes. Furthermore, we discovered that 14-3-3γ-shRNA reduced Best1-mediated glutamate release from hippocampal astrocyte by recording a PAR1 receptor-induced NMDA receptor-mediated current from CA1 pyramidal neurons in hippocampal slices injected with adenovirus carrying 14-3-3γ-shRNA. Finally, through a structural modeling, we found critical amino acid residues containing S358 of Best1 exhibiting binding affinities to 14-3-3γ.Conclusions14-3-3γ promotes surface expression of Best1 channel in astrocytes through direct interaction.

Rapamycin-Sensitive Late-LTP is Enhanced in the Hippocampus of IL-6 Transgenic Mice

The neuroimmune factor IL-6 has been shown to regulate hippocampal long-term potentiation (LTP), an activity-dependent enhancement of synaptic transmission that plays a central role in memory and learning. This IL-6 action was demonstrated with relatively short IL-6 exposure, and may reflect physiological actions of IL-6. IL-6 is also expressed chronically at elevated levels in the central nervous system (CNS) under pathological conditions such as neurological disorders. Little is known about the effects IL-6 on LTP under such conditions, an issue that we are addressing by electrophysiological recordings from CA1 pyramidal neurons of hippocampal slices from transgenic mice that persistently express elevated levels of IL-6 in the CNS (IL-6 tg). The current studies examined the long-lasting phase of LTP (late LTP; L-LTP) and the potential involvement mammalian target of rapamycin (mTOR), a known regulator of L-LTP and a downstream partner of IL-6 signal transduction pathways. Results show that basal synaptic transmission and L-LTP were increased in hippocampal slices from IL-6 tg mice compared to slices from non-transgenic (non-tg) control mice. An inhibitor of mTOR, rapamycin, reduced L-LTP in slices from both genotypes, and eliminated the difference in magnitude of L-LTP between IL-6 and non-tg hippocampus. There were no genotypic effect of rapamycin on basal synaptic transmission, but synaptic responses during the LTP induction protocol were reduced in IL-6 tg slices, an effect that could contribute to the reduction of L-LTP in the IL-6 tg slices. These results indicate that persistently increased levels of IL-6 can lead to alterations in mTOR regulation of L-LTP, possibly affecting learning and memory.

Epileptic pilocarpine-treated rats exhibit aberrant hippocampal EPSP-spike potentiation but retain long-term potentiation.

Hippocampal neuron plasticity is strongly associated with learning, memory, and cognition. In addition to modification of synaptic function and connectivity, the capacity of hippocampal neurons to undergo plasticity involves the ability to change nonsynaptic excitability. This includes altering the probability that EPSPs will generate action potentials (E‐S plasticity). Epilepsy is a prevalent neurological disorder commonly associated with neuronal hyperexcitability and cognitive dysfunction. We examined E‐S plasticity in chronically epileptic Sprague–Dawley rats 3–10 weeks after pilocarpine‐induced status epilepticus. CA1 neurons in hippocampal slices were assayed by whole‐cell current clamp to measure EPSPs evoked by Schaffer collateral stimulation. Using a weak spike‐timing‐dependent protocol to induce plasticity, we found robust E‐S potentiation in conjunction with weak long‐term potentiation (LTP) in saline‐treated rats. In pilocarpine‐treated rats, a similar degree of LTP was found, but E‐S potentiation was reduced. Additionally, the degree of E‐S potentiation was not correlated with the degree of LTP for either group, suggesting that they independently contribute to neuronal plasticity. E‐S potentiation also differed from LTP in that E‐S plasticity could be induced solely from action potentials generated by postsynaptic current injection. The calcium chelating agent BAPTA in the intracellular solution blocked LTP and E‐S potentiation, revealing the calcium dependence of both processes. These findings suggest that LTP and E‐S potentiation have overlapping but nonidentical mechanisms of inducing neuronal plasticity that may independently contribute to cognitive disruptions observed in the chronic epileptic state.

3-Iodothyroacetic acid (TA1), a by-product of thyroid hormone metabolism, reduces the hypnotic effect of ethanol without interacting at GABA-A receptors

3-iodothyroacetic acid (TA1) is among the by-products of thyroid hormone metabolism suspected to mediate the non-genomic effects of the hormone (T3).We aim to investigate whether TA1 systemically administered to mice stimulated mice wakefulness, an effect already described for T3 and for another T3 metabolite (i.e. 3-iodothryonamine; T1AM), and whether TA1 interacted at GABA-A receptors (GABA-AR).Mice were pre-treated with either saline (vehicle) or TA1 (1.32, 4 and 11 μg/kg) and, after 10 min, they received ethanol (3.5 g/kg, i.p.). In another set of experiments, TA1 was administered 5 min after ethanol. The latency of sleep onset and the time of sleep duration were recorded. Voltage-clamp experiments to evaluate the effect of 1 μM TA1 on bicuculline-sensitive currents in acute rat hippocampal slice neurons and binding experiments evaluating the capacity of 1, 10, 100 μM TA1 to displace [3H]flumazenil from mice brain membranes were also performed.4 μg/kg TA1 increases the latency of onset and at 1.32 and 4 μg/kg it reduces the duration of ethanol-induced sleep only if administered before ethanol. TA1 does not functionally interact at GABA-AR.Overall these results indicate a further similarity between the pharmacological profile of TA1 and that of T1AM.

Effects of copper on viability and functional properties of hippocampal neurons in vitro

Copper (Cu2+) is an essential metal presented in the mammalian brain and released from synaptic vesicles following neuronal depolarization. However, the disturbance of Cu2+ homeostasis results in neurotoxicity. In our study we performed for the first time a combined functional investigation of cultured hippocampal neurons under Cu2+ exposure, its effect on spontaneous spike activity of hippocampal neuronal network cultured on multielectrode array (MEA), and development of long-term potentiation (LTP) in acute hippocampal slices in the presence of Cu2+. Application of 0.2mM CuCl2 for 24h reduced viability of cultured neurons to 40±6%, whereas 0.01mM CuCl2 did not influence significantly on the neuronal survival. However, exposure to the action of 0.01mM Cu2+ resulted in pronounced reduction of network spike activity and abolished LTP induced by high-frequency stimulation of Schaffer's collaterals in CA1 pyramidal neurons of hippocampal slices. Antioxidant Trolox, the hydrosoluble vitamin E analogue, prevented neurotoxic effect and alterations of network activity under Cu2+ exposure, but didn't change the impairment of LTP in Cu2+-exposured hippocampal slices. We hypothesized that spontaneous network neuronal activity probably is one of the potential targets of Cu2+-induced neurotoxicity, in which free radicals can be involved. At the same time, it may be suggested that Cu2+-induced alterations of long-lasting trace processes (like LTP) are not mediated by oxidative damage.

Quantitative Imaging of Ca2+ by 3D-FLIM in Live Tissues.

The calcium concentration within living cells is highly dynamic and, for many cell types, a reliable indicator of the functional state of the cells-both of isolated cells, but even, more important, of cells in tissue. In order to dynamically quantify intracellular calcium levels, various genetically encoded calcium sensors have been developed-the best of which are those based on Förster resonant energy transfer (FRET). Here we present a fluorescence lifetime imaging (FLIM) method to measure FRET in such a calcium sensor (TN L15) in neurons of hippocampal slices and of the brain stem of anesthetized mice. The method gives the unique opportunity to determine absolute neuronal calcium concentrations in the living organism.

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

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.

Prior activation of inositol 1,4,5-trisphosphate receptors suppresses the subsequent induction of long-term potentiation in hippocampal CA1 neurons.

We investigated the role of inositol 1,4,5-trisphosphate receptors (IP3Rs) activated by preconditioning low-frequency afferent stimulation (LFS) in the subsequent induction of long-term potentiation (LTP) in CA1 neurons in hippocampal slices from mature guinea pigs. Induction of LTP in the field excitatory postsynaptic potential or the population spike by the delivery of high-frequency stimulation (HFS, a tetanus of 100 pulses at 100 Hz) to the Schaffer collateral–commissural pathway to CA1 neuron synapses was suppressed when group I metabotropic glutamate receptors (mGluRs) were activated prior to the delivery of HFS. LTP induction was also suppressed when CA1 synapses were preconditioned 60 min before HFS by LFS of 1000 pulses at 1 Hz and this effect was inhibited when the test stimulation delivered at 0.05 Hz was either halted or applied in the presence of an antagonist of N-methyl-d-aspartate receptors, group I mGluRs, or IP3Rs during a 20-min period from 20 to 40 min after the end of LFS. Furthermore, blockade of group I mGluRs or IP3Rs immediately before the delivery of HFS overcame the effects of the preconditioning LFS on LTP induction. These results suggest that, in CA1 neurons, after a preconditioning LFS, activation of group I mGluRs caused by the test stimulation results in IP3Rs activation that leads to a failure of LTP induction.

Role of BKCa Potassium Channels in the Mechanisms of Modulatory Effects of IL-10 on Hypoxia-Induced Changes in Activity of Hippocampal Neurons

We studied the contribution of large conductance Ca(2+)-activated potassium channels (BKCa) in the mechanisms of neuromodulatory effects of anti-inflammatory cytokine IL-10 on hypoxiainduced changes in activity of CA1 pyramidal neurons in rat hippocampus. We used the method of registration of population spikes from CA1 pyramidal neurons in hippocampal slices before, during, and after exposure to short-term episodes of hypoxia. Selective blocker (iberiotoxin) and selective activator of BKCa (BMS-191011) were used to evaluate the contribution of these channels in the mechanisms of suppressive effects of IL-10 on changes in neuronal activity during hypoxia and development of post-hypoxic hyperexcitability. It was shown that BKCa are involved in the modulatory effects of IL-10 on hypoxia-induced suppression of activity of CA1 pyramidal neurons in the hippocampus and development of post-hypoxic hyperexcitability in these neurons.