Currents In CA1 Pyramidal Cells Research Articles

Dysregulation of Neuropilin-2 Expression in Inhibitory Neurons Impairs Hippocampal Circuit Development and Enhances Risk for Autism-Related Behaviors and Seizures.

Dysregulation of development, migration, and function of interneurons, collectively termed interneuronopathies, have been proposed as a shared mechanism for autism spectrum disorders (ASDs) and childhood epilepsy. Neuropilin-2 (Nrp2), a candidate ASD gene, is a critical regulator of interneuron migration from the median ganglionic eminence (MGE) to the pallium, including the hippocampus. While clinical studies have identified Nrp2 polymorphisms in patients with ASD, whether selective dysregulation of Nrp2-dependent interneuron migration contributes to pathogenesis of ASD and enhances the risk for seizures has not been evaluated. We tested the hypothesis that the lack of Nrp2 in MGE-derived interneuron precursors disrupts the excitation/inhibition balance in hippocampal circuits, thus predisposing the network to seizures and behavioral patterns associated with ASD. Embryonic deletion of Nrp2 during the developmental period for migration of MGE derived interneuron precursors (iCKO) significantly reduced parvalbumin, neuropeptide Y, and somatostatin positive neurons in the hippocampal CA1. Consequently, when compared to controls, the frequency of inhibitory synaptic currents in CA1 pyramidal cells was reduced while frequency of excitatory synaptic currents was increased in iCKO mice. Although passive and active membrane properties of CA1 pyramidal cells were unchanged, iCKO mice showed enhanced susceptibility to chemically evoked seizures. Moreover, iCKO mice exhibited selective behavioral deficits in both preference for social novelty and goal-directed learning, which are consistent with ASD-like phenotype. Together, our findings show that disruption of developmental Nrp2 regulation of interneuron circuit establishment, produces ASD-like behaviors and enhanced risk for epilepsy. These results support the developmental interneuronopathy hypothesis of ASD epilepsy comorbidity.

Delaying the GABA Shift Indirectly Affects Membrane Properties in the Developing Hippocampus.

During the first two postnatal weeks, intraneuronal chloride concentrations in rodents gradually decrease, causing a shift from depolarizing to hyperpolarizing GABA responses. The postnatal GABA shift is delayed in rodent models for neurodevelopmental disorders and in human patients, but the impact of a delayed GABA shift on the developing brain remains obscure. Here we examine the direct and indirect consequences of a delayed postnatal GABA shift on network development in organotypic hippocampal cultures made from 6- to 7-d-old mice by treating the cultures for 1 week with VU0463271, a specific inhibitor of the chloride exporter KCC2. We verified that VU treatment delayed the GABA shift and kept GABA signaling depolarizing until DIV9. We found that the structural and functional development of excitatory and inhibitory synapses at DIV9 was not affected after VU treatment. In line with previous studies, we observed that GABA signaling was already inhibitory in control and VU-treated postnatal slices. Surprisingly, 14 d after the VU treatment had ended (DIV21), we observed an increased frequency of spontaneous inhibitory postsynaptic currents in CA1 pyramidal cells, while excitatory currents were not changed. Synapse numbers and release probability were unaffected. We found that dendrite-targeting interneurons in the stratum radiatum had an elevated resting membrane potential, while pyramidal cells were less excitable compared with control slices. Our results show that depolarizing GABA signaling does not promote synapse formation after P7, and suggest that postnatal intracellular chloride levels indirectly affect membrane properties in a cell-specific manner.SIGNIFICANCE STATEMENT During brain development, the action of neurotransmitter GABA shifts from depolarizing to hyperpolarizing. This shift is a thought to play a critical role in synapse formation. A delayed shift is common in rodent models for neurodevelopmental disorders and in human patients, but its consequences for synaptic development remain obscure. Here, we delayed the GABA shift by 1 week in organotypic hippocampal cultures and carefully examined the consequences for circuit development. We find that delaying the shift has no direct effects on synaptic development, but instead leads to indirect, cell type-specific changes in membrane properties. Our data call for careful assessment of alterations in cellular excitability in neurodevelopmental disorders.

Early adenosine release contributes to hypoxia-induced disruption of stimulus-induced sharp wave-ripple complexes in rat hippocampal area CA3.

We investigated the effects of hypoxia on sharp wave-ripple complex (SPW-R) activity and recurrent epileptiform discharges in rat hippocampal slices, and the mechanisms underlying block of this activity. Oxygen levels were measured using Clark-style oxygen sensor microelectrodes. In contrast to recurrent epileptiform discharges, oxygen consumption was negligible during SPW-R activity. These network activities were reversibly blocked when oxygen levels were reduced to 20% or less for 3min. The prolongation of hypoxic periods to 6min caused reversible block of SPW-Rs during 20% oxygen and irreversible block when 0% oxygen (anoxia) was applied. In contrast, recurrent epileptiform discharges were more resistant to prolonged anoxia and almost fully recovered after 6min of anoxia. SPW-Rs were unaffected by the application of 1-butyl-3-(4-methylphenylsulfonyl) urea, a blocker of KATP channels, but they were blocked by activation of adenosine A1 receptors. In support of a modulatory function of adenosine, the amplitude and incidence of SPW-Rs were increased during application of the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). Interestingly, hypoxia decreased the frequency of miniature excitatory post-synaptic currents in CA3 pyramidal cells, an effect that was converted into increased frequency by the adenosine A1 agonist DPCPX. In addition, DPCPX also delayed the onset of hypoxia-mediated block of SPW-Rs. Our data suggest that early adenosine release during hypoxia induces a decrease in pre-synaptic glutamate release and that both might contribute to transient block of SPW-Rs during hypoxia/anoxia in area CA3.

Direct excitation of parvalbumin-positive interneurons by M1 muscarinic acetylcholine receptors: roles in cellular excitability, inhibitory transmission and cognition.

Parvalbumin-containing (PV) neurons, a major class of GABAergic interneurons, are essential circuit elements of learning networks. As levels of acetylcholine rise during active learning tasks, PV neurons become increasingly engaged in network dynamics. Conversely, impairment of either cholinergic or PV interneuron function induces learning deficits. Here, we examined PV interneurons in hippocampus (HC) and prefrontal cortex (PFC) and their modulation by muscarinic acetylcholine receptors (mAChRs). HC PV cells, visualized by crossing PV-CRE mice with Rosa26YFP mice, were anatomically identified as basket cells and PV bistratified cells in the stratum pyramidale; in stratum oriens, HC PV cells were electrophysiologically distinct from somatostatin-containing cells. With glutamatergic transmission pharmacologically blocked, mAChR activation enhanced PV cell excitability in both CA1 HC and PFC; however, CA1 HC PV cells exhibited a stronger postsynaptic depolarization than PFC PV cells. To delete M1 mAChRs genetically from PV interneurons, we created PV-M1 knockout mice by crossing PV-CRE and floxed M1 mice. The elimination of M1 mAChRs from PV cells diminished M1 mAChR immunoreactivity and muscarinic excitation of HC PV cells. Selective cholinergic activation of HC PV interneurons using Designer Receptors Exclusively Activated by Designer Drugs technology enhanced the frequency and amplitude of inhibitory synaptic currents in CA1 pyramidal cells. Finally, relative to wild-type controls, PV-M1 knockout mice exhibited impaired novel object recognition and, to a lesser extent, impaired spatial working memory, but reference memory remained intact. Therefore, the direct activation of M1 mAChRs on PV cells contributes to some forms of learning and memory.

GDPbS Activates Excitatory Synapses in CA1 Pyramidal Cells by Disinhibiting the PKA Activating Pathway

Objective: During the early postnatal brain developmental stages, excitatory synapses mediated by AMPA receptors are weak or silent. Activity-dependent insertion of AMPA receptors into synapses depends on the activation of protein kinase A. In this work, we investigated the effect of Guanosine 5’-[βthiol] diphosphate (GDPsS) on excitatory and inhibitory synaptic currents in CA1 pyramidal cells at postnatal days 9-12. Methods: Whole-cell patch-clamp recordings from identified hippocampal CA1 pyramidal cells were used. GDPβS Was applied through the recording electrode. Results: GDPβS induces an increase in excitatory synaptic current amplitude, but not in the inhibitory synaptic current amplitude. An analysis of the change in excitatory synaptic current amplitude in the presence of GDPβS revealed a progressive increase, which is blocked by the protein kinase A inhibitor Rp-3’,5’-cyclic monophosphothioatetriethylamine (Rp-cAMP), suggesting that GDPβS inhibits G-protein with a tonic negative control on a protein kinase A activating pathway. In addition, GDPβS has no effect on paired-pulse facilitation, suggesting that the glutamate release machinery is not affected. Moreover, as GDPβS was applied to postsynaptic neurons, the increase in excitatory postsynaptic current amplitude is related to changes at the postsynaptic side. Conclusion: Those results suggest that in developing hippocampal CA1 pyramidal cells, the tonic inhibition of a protein kinase A activating pathway by a G-protein prevents the activation of excitatory synapses

The activity within the CA3 excitatory network during Theiler's virus encephalitis is distinct from that observed during chronic epilepsy

Viral infections of the central nervous system (CNS) are associated with an increased risk for seizures during the acute infection period and the subsequent development of chronic epilepsy that is often difficult to treat. In previous work, we have shown that mice of the C57BL/6 strain infected with Theiler's murine encephalomyelitis virus (TMEV) exhibit a similar sequence, thereby providing a potential useful model of virus-induced epilepsy. The present study examines spontaneous and miniature excitatory postsynaptic currents in CA3 pyramidal cells recorded from brain slices prepared during both the acute phase during encephalitis and 2 months following TMEV infection. Animals that develop chronic epilepsy following TMEV infection exhibit considerable hippocampal sclerosis, directly implicating this brain region in the process of epileptogenesis. There are significant increases in amplitude and frequency of spontaneous and miniature excitatory currents in CA3 cells recorded in brain slices prepared during the acute infection period and 2 months after infection. However, the patterns of changes observed are markedly different during these two periods, suggesting that there are underlying changes in the network over time. These differences have implications for the treatment used during the acute infection and after chronic seizures develop.

Sphingomyelinase selectively reduces M1 muscarinic receptors in rat hippocampal membranes

Although there is evidence that nicotinic acetylcholine (Ach) receptors are influenced by ceramides, we do not currently know whether or not these sphingolipids can also regulate the muscarinic subtypes of Ach receptors. Using the whole-cell patch technique, we demonstrated that the effectiveness of the muscarinic receptor agonist pilocarpine, in enhancing spontaneous inhibitory postsynaptic currents in CA1 pyramidal cells, was completely abolished in hippocampal slices pre-exposed to the ceramide-generating enzyme sphingomyelinase (SMase). Western blot experiments, performed with biotinylated hippocampal membranes, showed that this electrophysiological defect possibly relies on the loss of M1 muscarinic Ach receptors at the cell surface. However, the effect appears to be relatively specific as the cell-surface expression of M4 muscarinic receptors was not found to be impacted by SMase treatment. Interestingly, we observed that G protein-coupled receptor kinases 2 and β-arrestin1/2 interactions with M1-immunoprecipitated proteins were substantially augmented in SMase-treated slices and that the reduction of cell-surface M1 muscarinic receptor expression generated was completely suppressed by the muscarinic antagonist atropine. Collectively, our data suggest that selective internalization of M1 muscarinic receptors can be accentuated in neurons subjected to high ceramide levels. The potential physiopathological implications of this finding are presented.

Cannabinoids attenuate hippocampal γ oscillations by suppressing excitatory synaptic input onto CA3 pyramidal neurons and fast spiking basket cells.

CB(1) cannabinoid receptor (CB(1)R) activation by exogenous ligands can impair memory processes, which critically depend on synchronous neuronal activities that are temporarily structured by oscillations. In this study, we aimed to reveal the mechanisms underlying the cannabinoid-induced decrease in gamma oscillations. We first verified that cannabinoids (CP55,940 and WIN55,212-2) readily suppressed carbachol-induced gamma oscillations in the CA3 region of hippocampal slices via activation of CB(1)Rs. The cannabinoid-induced decrease in the peak power of oscillations was accompanied by reduced and less precise firing activity in CA3 pyramidal cells and fast spiking basket cells. By examining the cannabinoid sensitivity of synaptic inputs we found that the amplitude of evoked excitatory postsynaptic currents was significantly suppressed upon CB(1)R activation in both CA3 pyramidal cells and fast spiking basket cells. In contrast, evoked inhibitory postsynaptic currents in CA3 pyramidal cells were unaltered. Furthermore, we observed that a CB(1)R agonist-induced decrease in the oscillation power at the beginning of the drug application was accompanied primarily by the reduced discharge of fast spiking basket cells, while pyramidal cell firing was unaltered. This result implies that the dampening of cholinergically induced gamma oscillations in the hippocampus by cannabinoids can be explained by a reduced excitatory input predominantly onto fast spiking basket cells, which leads to a reduction in neuronal firing frequency and precision, and thus to smaller field potentials. In addition, we uncovered that the spontaneously occurring sharp wave-ripple activities in hippocampal slices could also be suppressed by CB(1)R activation suggesting that cannabinoids profoundly reduce the intrinsically generated oscillatory activities at distinct frequencies in CA3 networks by reducing synaptic neurotransmission.

ClC-2 contributes to tonic inhibition mediated by α5 subunit-containing GABAA receptor in experimental temporal lobe epilepsy

Temporal lobe epilepsy (TLE), characterized by spontaneous recurrent seizures, learning and memory impairments is associated with neurodegeneration, abnormal reorganization of the circuitry and loss of functional inhibition in hippocampus. In adult hippocampus, the GABAergic cells mediate the major inhibitory function of the principal neurons, promoting the Cl(-) entry through the GABA(A) receptor, whether through phasic (synaptic) or tonic (extrasynaptic) conductance. Aside from classical synaptic component, tonic GABAergic inhibition mediated by extrasynaptic GABA(A) receptor received increasing attention over the past years. There is growing evidence that tonic inhibition plays an important role in epilepsy, memory and cognition. Since GABA(A) receptor-mediated inhibition depends on the maintenance of intracellular Cl(-) concentration at low levels in mature neurons, a shift in E(cl) is likely to participate in the generation and not merely a consequence of TLE. As we know, chloride channel-2 (ClC-2) is a member of the supergene family of voltage-gated chloride channels, it constitutes part of the background conductance and is involved in chloride extrusion. Here we show that ClC-2 were upregulated functionally in CA1 pyramidal cells in pilocarpine-treated rats, and that an observed increase in ClC-2 currents in CA1 pyramidal cells was reversed by L655,708, a specific antagonist of α5 subunit-containing GABA(A) receptor.

ErbB4 in parvalbumin-positive interneurons is critical for neuregulin 1 regulation of long-term potentiation

Neuregulin 1 (NRG1) is a trophic factor that acts by stimulating ErbB receptor tyrosine kinases and has been implicated in neural development and synaptic plasticity. In this study, we investigated mechanisms of its suppression of long-term potentiation (LTP) in the hippocampus. We found that NRG1 did not alter glutamatergic transmission at SC-CA1 synapses but increased the GABA(A) receptor-mediated synaptic currents in CA1 pyramidal cells via a presynaptic mechanism. Inhibition of GABA(A) receptors blocked the suppressing effect of NRG1 on LTP and prevented ecto-ErbB4 from enhancing LTP, implicating a role of GABAergic transmission. To test this hypothesis further, we generated parvalbumin (PV)-Cre;ErbB4(-/-) mice in which ErbB4, an NRG1 receptor in the brain, is ablated specifically in PV-positive interneurons. NRG1 was no longer able to increase inhibitory postsynaptic currents and to suppress LTP in PV-Cre;ErbB4(-/-) hippocampus. Accordingly, contextual fear conditioning, a hippocampus-dependent test, was impaired in PV-Cre;ErbB4(-/-) mice. In contrast, ablation of ErbB4 in pyramidal neurons had no effect on NRG1 regulation of hippocampal LTP or contextual fear conditioning. These results demonstrate a critical role of ErbB4 in PV-positive interneurons but not in pyramidal neurons in synaptic plasticity and support a working model that NRG1 suppresses LTP by enhancing GABA release. Considering that NRG1 and ErbB4 are susceptibility genes of schizophrenia, these observations contribute to a better understanding of how abnormal NRG1/ErbB4 signaling may be involved in the pathogenesis of schizophrenia.

Plasticity of Neuron-Glial Interactions Mediated by Astrocytic EphARs

Ephrin (Eph) signaling via Eph receptors affects neuronal structure and function. We report here that exogenous ephrinAs (EphAs) induce outgrowth of filopodial processes from astrocytes within minutes in rat hippocampal slice cultures. Identical effects were induced by release of endogenous ephrinAs by cleavage of their glycosylphosphatidylinositol anchor. Reverse transcription-PCR and immunocytochemistry revealed the expression of multiple EphA receptors (EphARs) in astrocytes. Exogenous and endogenous ephrins did not induce process outgrowth from astrocytes transfected with a kinase-dead EphAR construct, indicating that the critical EphARs were located on glia. Concomitant with these morphological changes, ephrinA reduced the frequency of (S)-3,5-dihydroxyphenylglycine-evoked NMDA receptor-mediated inward currents in CA1 pyramidal cells, elicited by release of glutamate from glial cells. The sensitivity of CA1 cell synaptic or extrasynaptic NMDA receptors was unaffected by ephrinA, indicating that this effect was mediated by inhibition of glutamate release from glial cells. Finally, ephrinA application decreased the frequency and increased the duration of spontaneous oscillations of the intracellular [Ca2+] in astrocytes. We conclude that ephrinA-EphA signaling is a pluripotent regulator of neuron-astrocyte interactions mediating rapid structural and functional plasticity.

Postnatal aniracetam treatment improves prenatal ethanol induced attenuation of AMPA receptor-mediated synaptic transmission

Aniracetam is a nootropic compound and an allosteric modulator of AMPA receptors (AMPARs) which mediate synaptic mechanisms of learning and memory. Here we analyzed impairments in AMPAR-mediated synaptic transmission caused by moderate prenatal ethanol exposure and investigated the effects of postnatal aniracetam treatment on these abnormalities. Pregnant Sprague-Dawley rats were gavaged with ethanol or isocaloric sucrose throughout pregnancy, and subsequently the offspring were treated with aniracetam on postnatal days (PND) 18 to 27. Hippocampal slices prepared from these pups on PND 28 to 34 were used for the whole-cell patch-clamp recordings of AMPAR-mediated spontaneous and miniature excitatory postsynaptic currents in CA1 pyramidal cells. Our results indicate that moderate ethanol exposure during pregnancy results in impaired hippocampal AMPAR-mediated neurotransmission, and critically timed aniracetam treatment can abrogate this deficiency. These results highlight the possibility that aniracetam treatment can restore synaptic transmission and ameliorate cognitive deficits associated with the fetal alcohol syndrome.

Alcohol is a potent stimulant of immature neuronal networks: implications for fetal alcohol spectrum disorder

Ethanol consumption during development affects the maturation of hippocampal circuits by mechanisms that are not fully understood. Ethanol acts as a depressant in the mature CNS and it has been assumed that this also applies to immature neurons. We investigated whether ethanol targets the neuronal network activity that is involved in the refinement of developing hippocampal synapses. This activity appears during the growth spurt period in the form of giant depolarizing potentials (GDPs). GDPs are generated by the excitatory actions of GABA and glutamate via a positive feedback circuit involving pyramidal neurons and interneurons. We found that ethanol potently increases GDP frequency in the CA3 hippocampal region of slices from neonatal rats. It also increased the frequency of GDP-driven Ca2+ transients in pyramidal neurons and increased the frequency of GABA(A) receptor-mediated spontaneous postsynaptic currents in CA3 pyramidal cells and interneurons. The ethanol-induced potentiation of GABAergic activity is probably the result of increased quantal GABA release at interneuronal synapses but not enhanced neuronal excitability. These findings demonstrate that ethanol is a potent stimulant of developing neuronal circuits, which might contribute to the abnormal hippocampal development associated with fetal alcohol syndrome and alcohol-related neurodevelopmental disorders.

Comparison of midazolam effects on inhibitory postsynaptic currents in hippocampal CA3 with those in CA1

We compared the effects of midazolam, one of the popular benzodiazepines, on inhibitory postsynaptic currents in CA3 pyramidal cells with those in CA1 pyramidal cells in rat hippocampal slices. With all of the midazolam concentrations tested, the conductance of the evoked inhibitory postsynaptic current of the CA3 and CA1 pyramidal cells was significantly larger than the control, and increased in a dose-dependent manner. The normalized conductance of the inhibitory postsynaptic currents of the CA3 and CA1 pyramidal cells was not significantly different. However, we found that midazolam significantly prolonged the decay time of inhibitory postsynaptic currents of CA3 pyramidal cells. The results suggest that the differential effect of midazolam on the inhibitory postsynaptic currents could be due to the different decay kinetics, which depend on the gamma-aminobutyric acid type A receptor subunit compositions of the CA3 and CA1 pyramidal cells.

Differential effects of midazolam on inhibitory postsynaptic currents in CA1 pyramidal cells and dentate gyrus granule cells of rat hippocampal slices

To examine regional differences of synaptic transmission, the effects of midazolam were observed on inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal cells (CA1-PCs) and dentate gyrus granule cells (DG-GCs) in rat hippocampal slices. Midazolam is one of the most popular benzodiazepines. The monosynaptic IPSCs in the CA1-PCs and DG-GCs were evoked by electrical stimulation of GABAergic interneurons and recorded by whole cell patch-clamp techniques. The effects of specific concentrations of midazolam (0.3, 1, 10 and 75 μM) on the IPSCs in CA1-PCs and DG-GCs were examined at particular membrane potentials (20 mV steps, from −120 to +40 mV). In all midazolam concentrations tested, the conductance of the IPSCs was significantly larger than that in control and was increased by increasing the concentration of midazolam in CA1-PCs (normalized conductance, 0.3 μM, 121%; 1 μM, 125%; 10 μM, 147%; 75 μM, 147%). However, midazolam did not significantly change the conductance of the IPSCs in DG-GCs (normalized conductance, 0.3 μM, 92%; 1 μM, 92%; 10 μM, 91%; 75 μM, 115%). The normalized conductance was significantly different between the CA1-PCs and DG-GCs in 1 and 10 μM midazolam. The results strongly suggest that the differential effects of midazolam on IPSCs in CA1-PCs and DG-GCs could be, at least in part, due to the different sensitivity to midazolam of the GABA A receptor subtypes.

Inhibition of GABA release by presynaptic ionotropic GABA receptors in hippocampal CA3.

Vesicular transmitter release can be regulated by transmitter-gated ion channels at presynaptic axon terminals. The central inhibitory transmitter GABA acts on such presynaptic ionotropic receptors in various cells, including inhibitory interneurons. Here we report that GABA-mediated postsynaptic inhibitory currents in CA3 pyramidal cells of rat hippocampal slices are suppressed by agonists of GABAA receptors. The effect is present for both stimulus-induced and miniature IPSCs, indicating a reduction in the probability of vesicular release by presynaptic, action-potential-independent mechanisms. We conclude that the release of GABA from hippocampal CA3 interneurons is regulated by a negative feedback via presynaptic ionotropic GABA autoreceptors.

Partial hippocampal kindling increases GABA B receptor-mediated postsynaptic currents in CA1 pyramidal cells

In previous studies, we showed that partial hippocampal kindling decreased the efficacy of the presynaptic GABA B receptors on both GABAergic and glutamatergic terminals of CA1 neurons in hippocampal slices in vitro. In this study, GABA B receptor-mediated inhibitory postsynaptic currents (GABA B-IPSCs) were assessed by whole-cell recordings in CA1 pyramidal neurons in hippocampal slices of male Long–Evans rats. The peak GABA B-IPSC evoked by a brief train of supramaximal stratum radiatum stimuli (20 pulses of 300 Hz) in the presence of picrotoxin (0.1 mM) and kynurenic acid (1 mM) was larger in neurons of kindled (65.9±5.2 pA, N=42 cells) than control (45.8±4.8 pA, N=32 cells) rats ( P<0.01). Adding GABA uptake blocker nipecotic acid (1 mM) or GABA B receptor agonist baclofen (0.01 mM) in the perfusate induced outward currents that were blocked by GABA B receptor antagonist CGP 55845A (1 μM). The peak outward current induced by nipecotic acid was larger in neurons of the kindled (55.4±5.7 pA, N=30) than the control group (39.8±4.5 pA, N=28) ( P<0.05). However, the magnitude of the baclofen-induced current was not different between kindled (90.8±6.9 pA, N=29) and control (87.2±5.9 pA, N=21) groups ( P>0.05). We concluded that partial hippocampal kindling increased GABA B-IPSCs in hippocampal CA1 pyramidal cells via multiple presynaptic mechanisms.

N-desmethylclozapine, an allosteric agonist at muscarinic 1 receptor, potentiates N-methyl-D-aspartate receptor activity.

The molecular and neuronal substrates conferring on clozapine its unique and superior efficacy in the treatment of schizophrenia remain elusive. The interaction of clozapine with many G protein-coupled receptors is well documented but less is known about its biologically active metabolite, N-desmethylclozapine. Recent clinical and preclinical evidences of the antipsychotic activity of the muscarinic agonist xanomeline prompted us to investigate the effects of N-desmethylclozapine on cloned human M1-M5 muscarinic receptors. N-desmethylclozapine preferentially bound to M1 muscarinic receptors with an IC50 of 55 nM and was a more potent partial agonist (EC50, 115 nM and 50% of acetylcholine response) at this receptor than clozapine. Furthermore, pharmacological and site-directed mutagenesis studies suggested that N-desmethylclozapine preferentially activated M1 receptors by interacting with a site that does not fully overlap with the acetylcholine orthosteric site. As hypofunction of N-methyl-d-aspartate (NMDA) receptor-driven neuronal ensembles has been implicated in psychotic disorders, the neuronal activity of N-desmethylclozapine was electrophysiologically investigated in hippocampal rat brain slices. N-desmethylclozapine was shown to dose-dependently potentiate NMDA receptor currents in CA1 pyramidal cells by 53% at 100 nM, an effect largely mediated by activation of muscarinic receptors. Altogether, our observations provide direct evidence that the brain penetrant metabolite N-desmethylclozapine is a potent, allosteric agonist at human M1 receptors and is able to potentiate hippocampal NMDA receptor currents through M1 receptor activation. These observations raise the possibility that N-desmethylclozapine contributes to clozapine's clinical activity in schizophrenics through modulation of both muscarinic and glutamatergic neurotransmission.

Recognition molecule associated carbohydrate inhibits postsynaptic GABA B receptors: a mechanism for homeostatic regulation of GABA release in perisomatic synapses

Extracellular matrix molecules are important cues in the shaping of nervous system structure and function. Here, we describe a novel mechanism by which the HNK-1 carbohydrate carried by recognition molecules regulates perisomatic inhibition in the hippocampus. Neutralization of HNK-1 activity by an HNK-1 antibody results in GABA B receptor-mediated activation of K + currents in CA1 pyramidal cells, which elevates extracellular K + concentration and reduces evoked GABA release in perisomatic inhibitory synapses. This mechanism is supported by pharmacological analysis in hippocampal slices and data showing that the HNK-1 carbohydrate binds to GABA B receptors and inhibits GABA B receptor-activated K + currents in a heterologous expression system. We suggest that the HNK-1 carbohydrate is involved in homeostatic regulation of GABA A receptor-mediated perisomatic inhibition by suppression of postsynaptic GABA B receptor activity.

Maturation of kainate-induced epileptiform activities in interconnected intact neonatal limbic structures in vitro.

In vivo studies suggest that ontogenesis of limbic seizures is determined by the development of the limbic circuit. We have now used the newly-developed in vitro intact interconnected neonatal rat limbic structures preparation to determine the developmental profile of kainate-induced epileptiform activity in the hippocampus and its propagation to other limbic structures. We report gradual alterations in the effects of kainate during the first postnatal week on an almost daily basis; from no epileptiform activity at birth, through interictal seizures around postnatal day (P) 2 and ictal seizures by the end of the first week. The developmental profile of kainate-induced hippocampal seizures is paralleled by the expression of postsynaptic kainate receptor-mediated currents in CA3 pyramidal cells. Intralimbic propagation of the hippocampal seizures is also age-dependent: whereas seizures readily propagate to the septum and to the contralateral hippocampus via the commissures on P2, propagation to the entorhinal cortex only takes place from P4 onwards. Finally, repeated brief applications of kainate to the hippocampus induce recurrent spontaneous glutamatergic ictal and interictal discharges which persist for several hours after the kainate is washed away and which replace the physiological pattern of network activity. Paroxysmal activities are thus generated by kainate in the hippocampus at an early developmental stage and are initially restricted to this structure. Before the end of the first week of postnatal life, kainate generates the epileptiform activities that may perturb activity-dependent mechanisms that modulate neuronal development. Although at this stage neurons are relatively resistant to the pathological effects of kainate, the epileptiform activities that it generates will perturb activity-dependent mechanisms that modulate neuronal development.