CA1 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.

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.

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.

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.

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.

Activation of inositol 1,4,5-trisphosphate receptors during preconditioning low-frequency stimulation suppresses subsequent induction of long-term potentiation in hippocampal CA1 neurons

We investigated the role of inositol 1,4,5-trisphosphate receptors (IP3Rs) activated during preconditioning low-frequency stimulation (LFS) in the subsequent high-frequency stimulation (HFS)-induced 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 (EPSP) or the population spike (PS) by delivery of HFS (a tetanus of 100 pulses at 100Hz) to the Schaffer collateral–commissural pathway to CA1 neuron synapses was suppressed when the CA1 synapses were preconditioned by LFS of 1000 pulses at 1Hz. This effect was inhibited when the preconditioning LFS was applied in the presence of an N-methyl-d-aspartate receptors (NMDARs) antagonist, a metabotropic glutamate receptor (mGluR) antagonist, IP3R antagonist, a calmodulin-dependent kinase II inhibitor or a calcineurin inhibitor. Furthermore, blockade of group I mGluRs immediately before the delivery of HFS blocked the inhibitory effect of the preconditioning LFS on subsequent induction of LTP by HFS. These results suggest that, in hippocampal CA1 neuron synapses, co-activation of NMDARs and IP3Rs during a preconditioning LFS results in both phosphorylation and dephosphorylation events that lead to prolonged activation of group I mGluRs that is responsible for the failure of LTP induction.

Social isolation-induced increase in NMDA receptors in the hippocampus exacerbates emotional dysregulation in mice.

Epidemiological studies have shown that early life adverse events have long-term effects on the susceptibility to subsequent stress exposure in adolescence, but the precise mechanism is unclear. In the present study, mice on postnatal day 21-28 were randomly assigned to either a group or isolated cages for 8 weeks. The socially isolated (SI) mice exhibited a higher level of spontaneous locomotor activity, a longer duration of immobility in the forced swimming test (FST), significantly less prepulse inhibition (PPI) and an increase in aggressive (but not attack) behavior. However, acute stress markedly exacerbated the attack counts of the SI mice but did not affect the group housing (GH) mice. SI mice exhibited higher synaptosomal NR2A and NR2B levels in the hippocampus as compared to the GH mice. Whole-cell patch clamp recordings of CA1 neurons in hippocampal slices showed that the SI mice exhibited a higher input-output relationship of NMDAR-EPSCs as compared to the GH mice. Application of the NR2B -specific antagonist ifenprodil produced a greater attenuating effect on NMDAR-EPSCs in slices from the SI mice. NMDAR EPSCs recorded from the SI mice had a slower deactivation kinetic. MK-801, CPP and ifenprodil, the NMDA antagonists, reversed acute stress-induced exaggeration of aggressive and depressive behaviors. Furthermore, acute stress-induced exacerbation of attack behavior in the SI mice was abolished after the knockdown of NR2B expression. These results suggest that social isolation-induced increased expression of NMDA receptors in the hippocampus involves stress exacerbation of aggressive behaviors. Amelioration of aggressive behaviors by NMDA antagonists may open a new avenue for the treatment of psychopathologies that involve outbursts of emotional aggression in neglected children.

The effects of eslicarbazepine on persistent Na+ current and the role of the Na+ channel β subunits

Eslicarbazepine is the major active metabolite of eslicarbazepine acetate, a once-daily antiepileptic drug approved in Europe as adjunctive therapy for refractory partial-onset seizures in adults. This study was aimed to determine the effects of eslicarbazepine on persistent Na(+) currents (INaP) and the role of β subunits in modulating these effects. To study the role of β subunits of the Na(+) channel we used a mouse line genetically lacking either the β1 or β2 subunit, encoded by the SCN1B or SCN2B gene, respectively. Whole cell patch-clamp recordings were performed on CA1 neurons in hippocampal slices under control conditions and application of 300 μM eslicarbazepine. We examined INaP in acutely isolated CA1 neurons and repetitive firing in hippocampal slices of mice lacking β subunits and corresponding wild-type littermates. We found that eslicarbazepine caused a significant reduction of maximal INaP conductance and an efficient reduction of the firing rate in wild-type mice. We have shown previously a paradoxical increase of conductance of INaP caused by carbamazepine in mice lacking β1 subunits in the subthreshold range, leading to a failure in affecting neuronal firing (Uebachs et al., 2010). In contrast, eslicarbazepine did not cause this paradoxical effect on INaP in SCN1B null mice. Consequently, the effects of eslicarbazepine on repetitive firing were maintained in these animals. These results indicate that eslicarbazepine exerts effects on INaP similar to those known for carbamazepine. However, in animals lacking the β1 Na(+) channel subunit these effects are maintained. Therefore, eslicarbazepine potentially overcomes a previously described putative mechanism of resistance to established Na(+) channel acting antiepileptic drugs.

Suppressive effect of preconditioning low-frequency stimulation on subsequent induction of long-term potentiation by high frequency stimulation in hippocampal CA3 neurons

We investigated the role of inositol 1, 4, 5-trisphosphate receptors (IP3Rs), activated during preconditioning low-frequency afferent stimulation (LFS), in the subsequent induction of long-term potentiation (LTP) in CA3 neurons in hippocampal slices from mature guinea pigs. Induction of LTP in the field excitatory postsynaptic potential (EPSP) by the delivery of high-frequency stimulation (HFS, a tetanus of two trains of 100 pulses at 100Hz with a 10s interval) to mossy fiber-CA3 neuron synapses was suppressed when CA3 synapses were preconditioned by the LFS of 1000 pulses at 2Hz and this effect was inhibited when the LFS preconditioning was performed in the presence of an IP3R antagonist or a protein phosphatase inhibitor. Furthermore, activation of group 1 metabotropic glutamate receptors (mGluRs) during HFS canceled the effects of an IP3R antagonist given during preconditioning LFS on the subsequent LTP induction at mossy fiber-CA3 synapses. These results suggest that, in hippocampal mossy fiber-CA3 neuron synapses, activation of IP3Rs during a preconditioning LFS results in dephosphorylation events that lead to failure of the HFS to induce subsequent LTP.

Activation of inositol 1, 4, 5-trisphosphate receptors during preconditioning low-frequency stimulation leads to reversal of long-term potentiation in hippocampal CA1 neurons

We investigated the role of inositol 1, 4, 5-trisphosphate receptors (IP3Rs) that were activated during preconditioning low-frequency afferent stimulation (LFS) in the subsequent induction of synaptic plasticity in CA1 neurons in hippocampal slices from mature guinea pigs. In standard perfusate, long-term potentiation (LTP) was induced in the field excitatory postsynaptic potential (EPSP) by the delivery of LFS (80 pulses at 1 Hz), and was reversed by an identical LFS applied 20 min later. However, when CA1 synapses were preconditioned in the presence of an IP3R antagonist and stimulated by the second LFS in the absence of the antagonist, LTP was not reversed, but was increased, by the second LFS. In addition, when CA1 synapses were preconditioned in standard solution, but stimulated by the second LFS in the presence of an N-methyl-d-aspartate receptor (NMDAR) antagonist, LTP was again not reversed, but increased. The excitatory postsynaptic current (EPSC) through NMDARs recorded from CA1 pyramidal neurons increased significantly 20 min after a single LFS and this increase was inhibited when the LFS was delivered in the presence of an IP3R antagonist or a Ca2+/calmodulin-dependent protein kinase II inhibitor. These results suggest that activation of IP3Rs by a preconditioning LFS results in postsynaptic protein phosphorylation and/or enhancement of NMDAR activation during a subsequent LFS, leading to reversal of LTP in the field EPSP in hippocampal CA1 neurons.

Down-Regulation of Synaptic GluN2B Subunit-Containing N-methyl-d-aspartate Receptors: A Physiological Brake on CA1 Neuron α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Hyperexcitability during Benzodiazepine Withdrawal

A significant link was previously established between benzodiazepine withdrawal anxiety and a progressive increase in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) potentiation in hippocampal CA1 neurons from rats withdrawn up to 2 days from 1-week oral administration of the benzodiazepine flurazepam (FZP). Despite AMPAR current potentiation, withdrawal anxiety was masked by a 2-fold reduction in CA1 neuron N-methyl-D-aspartate receptor (NMDAR) currents since preinjection of an NMDA antagonist restored NMDAR currents and unmasked anxiety in 2-day FZP-withdrawn rats. In the current study, GluN subunit levels in postsynaptic density (PSD)-enriched subfractions of CA1 minislices were compared with GluN2B-mediated whole-cell currents evoked in CA1 neurons in hippocampal slices from 1- and 2-day FZP-withdrawn rats. GluN1 and GluN2B, although not the phosphoSer1303-GluN2B ratio or GluN2A subunit levels, were decreased in PSD subfractions from 2-day, but not 1-day, FZP-withdrawn rats. Consistent with immunoblot analyses, GluN2B-mediated NMDAR currents evoked in slices from 2-day FZP-withdrawn rats were decreased in the absence, but not the presence, of the GluN2B subunit-selective antagonist ifenprodil. In contrast, ifenprodil-sensitive NMDAR currents were unchanged in slices from 1-day withdrawn rats. Because AMPA (1 μM) preincubation of slices from 1-day FZP-withdrawn rats induced depression of GluN2B subunit-mediated currents, depression of NMDAR currents was probably secondary to AMPAR potentiation. CA1 neuron NMDAR currents were depressed ∼50% after 2-day withdrawal and offset potentiation of AMPAR-mediated currents, leaving total charge transfer unchanged between groups. Collectively, these findings suggest that a reduction of GluN2B-containing NMDAR may serve as a homeostatic feedback mechanism to modulate glutamatergic synaptic strength during FZP withdrawal to alleviate benzodiazepine withdrawal symptoms.

Properties of subsequent induction of long-term potentiation and/or depression in one synaptic input in apical dendrites of hippocampal CA1 neurons in vitro

The hippocampus is a prominent structure to study mechanisms of learning and memory at the cellular level. Long-term potentiation (LTP) as well as long-term depression (LTD) are the major cellular models which could underlie learning and memory formation. LTP and LTD consist of at least two phases, an early protein synthesis-independent transient stage (<4 h; E-LTP, E-LTD) as well as a prolonged phase (>4 h; L-LTP, L-LTD) requiring the synthesis of new proteins. It is known that during E-LTP the further induction of longer lasting LTP is precluded. However, if E-LTP is transformed into L-LTP, the same synapses now allow the induction of LTP again. We reproduced the LTP-results first and then investigated whether hippocampal LTP or LTD also prevents the establishment of subsequent LTD-induction in the same synaptic input. We show that the prior induction of LTP or LTD does not prevent a short-term depression (STD) but occludes LTD in apical dendrites of CA1 neurons in hippocampal slices in vitro during the early phase of LTP or LTD. However, LTD can again be induced in addition to STD after the establishment of L-LTP or L-LTD, that is about 4 h after the induction of the first event in the same synaptic input. We suggest that the neuronal input preserves the capacity for STD immediately after an initial potentiation or depression, but for the onset of additional longer lasting LTD in the same synaptic input, the establishment of the late plasticity form of the preceding event is critical.

Oxygen‐induced superoxide production in solitary complex neurons in rat medullary slices

We previously reported that the rate of superoxide (•O2−) production in CA1 neurons in hippocampal slices decreases as O2 is decreased from 95% to 60, 40 or 20% (J. Neurophysiol. 98:1030‐1041, 2007). Here we report the effects of continuous exposure to O2 (95, 40 &amp; 20%) on •O2− production in solitary complex (SC) neurons in medullary slices (400μm, weaned rats). The rate of intracellular •O2− production was measured using the fluorogenic probe, DHE (2.5μM), added to the superfusate (35–37°C). Fluorescence imaging initially classified cells at the start of the experiment as dim versus bright. Dim SC neurons incubated in 95%O2 had a similar rate of •O2− production (increasing fluorescence) over time as that reported previously for CA1 hippocampal neurons. Continuous exposure of dim cells to 20 or 40%O2, however, revealed slightly lower but similar rates of •O2− production that were significantly greater than that reported for CA1 neurons. SC neurons initially identified as bright cells increased their rate of fluorescence during the first 60–90 minutes but thereafter fluorescence reached a plateau indicative of a decreased rate of •O2− production. In bright SC cells, 95% and 20%O2 caused the lowest and highest rate of •O2− production, respectively. We conclude the following: 1) dim SC neurons have a higher capacity for •O2− production during exposure to 20–40%O2 than CA1 hippocampal neurons. 2) Neurons with initially bright DHE fluorescence may be damaged from slicing and lose their •O2− generating mechanisms rapidly, especially in the presence of 95%O2 (ONR N000140110179, NIH R01HL56683).

Synaptic NR2A- but not NR2B-containing NMDA receptors increase with blockade of ionotropic glutamate receptors

NMDA receptors (NMDAR) are key molecules involved in physiological and pathophysiological brain processes such as plasticity and excitotoxicity. Neuronal activity regulates NMDA receptor levels in the cell membrane. However, little is known on which time scale this regulation occurs and whether the two main diheteromeric NMDA receptor subtypes in forebrain, NR1/NR2A and NR1/NR2B, are regulated in a similar fashion. As these differ considerably in their electrophysiological properties, the NR2A/NR2B ratio affects the neurons’ reaction to NMDA receptor activation. Here we provide evidence that the basal turnover rate in the cell membrane of NR2A- and NR2B-containing receptors is comparable. However, the level of the NR2A subtype in the cell membrane is highly regulated by NMDA receptor activity, resulting in a several-fold increased insertion of new receptors after blocking NMDAR for 8 h. Blocking AMPA receptors also increases the delivery of NR2A-containing receptors to the cell membrane. In contrast, the amount of NR2B-containing receptors in the cell membrane is not affected by ionotropic glutamate receptor block. Moreover, electrophysiological analysis of synaptic currents in hippocampal cultures and CA1 neurons of hippocampal slices revealed that after 8 h of NMDA receptor blockade the NMDA EPSCs increase as a result of augmented NMDA receptor-mediated currents. In conclusion, synaptic NR2A- but not NR2B-containing receptors are dynamically regulated, enabling neurons to change their NR2A/NR2B ratio within a time scale of hours.

Synaptic Metaplasticity through NMDA Receptor Lateral Diffusion

Lateral diffusion of glutamate receptors was proposed as a mechanism for regulating receptor numbers at synapses and affecting synaptic functions, especially the efficiency of synaptic transmission. However, a direct link between receptor lateral diffusion and change in synaptic function has not yet been established. In the present study, we demonstrated NMDA receptor (NMDAR) lateral diffusion in CA1 neurons in hippocampal slices by detecting considerable recovery of spontaneous or evoked EPSCs from the block of (+)-MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate], an irreversible NMDAR open-channel blocker. We observed changes on both the number and the composition of synaptic NMDAR on recovery. More importantly, after the recovery, long-term potentiation (LTP)-producing protocol induced only LTD (long-term depression) instead of LTP. In contrast, a complete recovery from competitive NMDAR blocker D,L-AP-5 was observed without subsequent changes on synaptic plasticity. Our data suggest a revised model of NMDAR trafficking wherein extrasynaptic NMDARs, mostly NR1/NR2B receptors, move laterally into synaptic sites, resulting in altered rule of synaptic modification. Thus, CA1 synapses exhibit a novel form of metaplasticity in which the direction of synaptic modification can be reverted through subtype-specific lateral diffusion of NMDA receptors.

Modulation of Network-Driven, GABA-Mediated Giant Depolarizing Potentials by SDF-1α in the Developing Hippocampus

Chemokine stromal cell-derived factor-1 (SDF-1, or CXCL12) plays an important role in brain development and functioning. Whole-cell patch clamp recordings were conducted on CA3 neurons in hippocampal slices prepared from neonatal rats between postnatal days 2 and 6 to study the modulatory effects of SDF-1α on network-driven, γ-aminobutyric-acid-mediated giant depolarizing potentials (GDPs), a hallmark of the developing hippocampus. We found that SDF-1α, the only natural ligand for chemokine CXC motif receptor 4 (CXCR4), decreased GDP firing without significant effects on neuronal passive membrane properties in neonatal hippocampal neurons. The SDF-1α-mediated decrease in GDP firing was blocked by T140, a CXCR4 receptor antagonist, suggesting that SDF-1α modulates GDP firing via CXCR4. We also showed that endogenous SDF-1 exerts a tonic inhibitory action on GDPs in the developing hippocampus. As SDF-1/CXCR4 are highly expressed in the developing brain and GDPs are involved in activity-dependent synapse formation and functioning, the inhibitory action of SDF-1α on GDPs may reflect a potential mechanism for chemokine regulation of neural development in early neonatal life.

Synaptic Sumoylation

Sumoylation--the posttranslational modification of proteins with the small ubiquitin-like modifier protein (SUMO)--is most familiar in the context of transcriptional regulation and nuclear transport; increasingly, however, roles for extranuclear sumoylation have begun to surface. Martin et al . found that the SUMO-conjugating enzyme Ubc9 and the SUMO ligase PIAS3 interacted with the GluR6 subunit of the kainate type of glutamate receptor (KAR) in a yeast two-hybrid screen of rat brain cDNA and coimmunoprecipitated with GluR6 from cultured hippocampal neuronal extracts. Truncation analysis indicated that interaction depended on a region of GluR6 containing the consensus sumoylation motif. Immunocytochemical analysis indicated that there was limited colocalization of GluR6 and SUMO-1 in cultured hippocampal neurons and that most of the sumoylated GluR6 was intracellular. Kainate, which stimulates KAR internalization and degradation, increased GluR6 sumoylation (as did glutamate), whereas desumoylation with the SUMO-specific isopeptidase SENP-1 inhibited kainate-dependent GluR6 endocytosis. Moreover, unlike fluorescently labeled wild-type GluR6a, a fluorescently labeled non-sumoylatable GluR6a point mutant expressed in COS-7 cells failed to undergo kainate-dependent endocytosis. Infusion of SUMO-1 into CA3 neurons in hippocampal slices decreased the amplitude of KAR-mediated excitatory postsynaptic currents (KAR-EPSCs) without affecting AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptor-mediated EPSCs, whereas infusion of SENP-1 increased the amplitude of KAR-EPSCs. Thus, the authors conclude that, under certain conditions, sumoylation may modify the postsynaptic response to glutamate and propose that sumoylation may regulate the function of many other synaptic proteins as well. Coussen and Choquet discuss some of the implications of this research. S. Martin, A. Nishimune, J. R. Mellor, J. M. Henley, SUMOylation regulates kainate-receptor-mediated synaptic transmission. Nature 447 , 321-325 (2007). [PubMed]

Inhibitory Gating Modulation of Small Conductance Ca2+-Activated K+Channels by the Synthetic Compound (R)-N-(Benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphtylamine (NS8593) Reduces Afterhyperpolarizing Current in Hippocampal CA1 Neurons

SK channels are small conductance Ca(2+)-activated K(+) channels important for the control of neuronal excitability, the fine tuning of firing patterns, and the regulation of synaptic mechanisms. The classic SK channel pharmacology has largely focused on the peptide apamin, which acts extracellularly by a pore-blocking mechanism. 1-Ethyl-2-benzimidazolinone (1-EBIO) and 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309) have been identified as positive gating modulators that increase the apparent Ca(2+) sensitivity of SK channels. In the present study, we describe inhibitory gating modulation as a novel principle for selective inhibition of SK channels. In whole-cell patch-clamp experiments, the compound (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphtylamine (NS8593) reversibly inhibited recombinant SK3-mediated currents (human SK3 and rat SK3) with potencies around 100 nM. However, in contrast to known pore blockers, NS8593 did not inhibit (125)I-apamin binding. Using excised patches, it was demonstrated that NS8593 decreased the Ca(2+) sensitivity by shifting the activation curve for Ca(2+) to the right, only slightly affecting the maximal Ca(2+)-activated SK current. NS8593 inhibited all the SK1-3 subtypes Ca(2+)-dependently (K(d) = 0.42, 0.60, and 0.73 microM, respectively, at 0.5 microM Ca(2+)), whereas the compound did not affect the Ca(2+)-activated K(+) channels of intermediate and large conductance (hIK and hBK channels, respectively). The site of action was accessible from both sides of the membrane, and the NS8593-mediated inhibition was prevented in the presence of a high concentration of the positive modulator NS309. NS8593 was further tested on mouse CA1 neurons in hippocampal slices and shown to inhibit the apaminand tubocurarine-sensitive SK-mediated afterhyperpolarizing current, at a concentration of 3 microM.

Calcium influx through presynaptic 5-HT 3 receptors facilitates GABA release in the hippocampus: in vitro slice and synaptosome studies

Serotonin 5-hydroxytryptamine type 3 receptors (5HT 3R) are Ca 2+-permeant, non-selective cation channels that have been localized to presynaptic terminals and demonstrated to modulate neurotransmitter release. In the present study the effect of 5-HT on GABA release in the hippocampus was characterized using both electrophysiological and biochemical techniques. 5-HT elicited a burst-like, 6- to 10-fold increase in the frequency of GABA A receptor-mediated inhibitory postsynaptic currents (IPSCs) measured with whole-cell voltage-clamp recordings of CA1 neurons in hippocampal slices. When tetrodotoxin was used to block action potential propagation, the 5-HT-induced burst of IPSCs was still observed. Stimulation of hippocampal synaptosomes with 5-HT resulted in a significant increase in the amount of [ 3H]GABA released by hyperosmotic saline. In both preparations, the 5-HT effect was shown to be mediated by 5HT 3Rs, as it was mimicked by the selective 5HT 3R agonist m-chlorophenyl biguanide and blocked by the selective 5HT 3R antagonist 3-tropanylindole-3-carboxylate hydrochloride. The 5HT 3R-mediated increase in GABA release was blocked by 100 μM cadmium or by omitting Ca 2+ in external solutions, indicating the Ca 2+-dependence of the effect. The high voltage-activated Ca 2+ channel blockers ω-conotoxin GVIA and ω-conotoxin MVIIC and 10 μM cadmium had no significant effect on the 5-HT 3R-mediated enhancement of GABA release, indicating that Ca 2+ influx through the 5-HT 3R facilitates GABA release. Taken together, these data provide direct evidence that Ca 2+ entry via presynaptic 5HT 3Rs facilitates the release of GABA from hippocampal interneurons.