A role for the PI-3 kinase signaling pathway in fear conditioning and synaptic plasticity in the amygdala.

Western blot analysis of neuronal tissues taken from fear-conditioned rats showed a selective activation of phosphatidylinositol 3-kinase (PI-3 kinase) in the amygdala. PI-3 kinase was also activated in response to long-term potentiation (LTP)-inducing tetanic stimulation. PI-3 kinase inhibitors blocked tetanus-induced LTP as well as PI-3 kinase activation. In parallel, these inhibitors interfered with long-term fear memory while leaving short-term memory intact. Tetanus and forskolin-induced activation of mitogen-activated protein kinase (MAPK) was blocked by PI-3 kinase inhibitors, which also inhibited cAMP response element binding protein (CREB) phosphorylation. These results provide novel evidence of a requirement of PI-3 kinase activation in the amygdala for synaptic plasticity and memory consolidation, and this activation may occur at a point upstream of MAPK activation.

Valproic acid and other histone deacetylase inhibitors induce microglial apoptosis and attenuate lipopolysaccharide-induced dopaminergic neurotoxicity.

Valproic acid (VPA), a widely prescribed drug for seizures and bipolar disorder, has been shown to be an inhibitor of histone deacetylase (HDAC). Our previous study has demonstrated that VPA pretreatment reduces lipopolysaccharide (LPS)-induced dopaminergic (DA) neurotoxicity through the inhibition of microglia over-activation. The aim of this study was to determine the mechanism underlying VPA-induced attenuation of microglia over-activation using rodent primary neuron/glia or enriched glia cultures. Other histone deacetylase inhibitors (HDACIs) were compared with VPA for their effects on microglial activity. We found that VPA induced apoptosis of microglia cells in a time- and concentration-dependent manner. VPA-treated microglial cells showed typical apoptotic hallmarks including phosphatidylserine externalization, chromatin condensation and DNA fragmentation. Further studies revealed that trichostatin A (TSA) and sodium butyrate (SB), two structurally dissimilar HDACIs, also induced microglial apoptosis. The apoptosis of microglia was accompanied by the disruption of mitochondrial membrane potential and the enhancement of acetylation levels of the histone H3 protein. Moreover, pretreatment with SB or TSA caused a robust decrease in LPS-induced pro-inflammatory responses and protected DA neurons from damage in mesencephalic neuron-glia cultures. Taken together, our results shed light on a novel mechanism whereby HDACIs induce neuroprotection and underscore the potential utility of HDACIs in preventing inflammation-related neurodegenerative disorders such as Parkinson's disease.

Long-term depression of excitatory synaptic transmission in the rat amygdala.

In view of the fact that both kindling and fear-potentiated startle are expressed by long-term enhancement of synaptic transmission in the amygdala, synaptic plasticity in this area of the brain is of particular importance. Here, we show for the first time that low-frequency stimulation of the lateral nucleus at 1 Hz for 15 min elicited a long-term depression (LTD) in the basolateral amygdala (BLA) neurons. LTD is expressed specifically at the lateral-BLA synapses but not at ventral endopyriform nucleus-BLA synapses. The induction of LTD requires activation of both NMDA and metabotropic glutamate receptors. Loading cells with a Ca(2+) chelator BAPTA or extracellular superfusion with protein phosphatase inhibitors prevents LTD, suggesting that LTD may result from dephosphorylation of AMPA receptors. The same stimulating protocol could not elicit LTD in neurons from kindled animals, whereas neurons from sham-operated or age-matched control rats were able to exhibit LTD. Together, this study characterizes the properties of LTD in the naïve amygdala slices for the first time and demonstrates that epileptogenesis in vivo induces disruption of LTD in the in vitro preparation.

Activation of group II metabotropic glutamate receptors induces long-term depression of synaptic transmission in the rat amygdala.

An animal model most sensitive for measuring anticipatory anxiety is fear conditioning, which is expressed by an enduring increase in synaptic strength in the amygdala. A converse view predicts that agents that induce long-term depression (LTD) of synaptic efficacy in the amygdala may be useful in the amelioration of stress disorders. In the present study, we show that activation of group II metabotropic glutamate receptor (mGluR II) by (2S,3S, 4S)-2-(carboxycyclopropyl) glycine (l-ccg) induces an LTD in the basolateral amygdala neurons. The effect was concentration-dependent with a maximal inhibition of approximately 30%. The induction of l-CCG LTD required concurrent synaptic activity, required presynaptic but not postsynaptic Ca(2+) increases, and was independent of NMDA receptors. l-CCG LTD was associated with an increase in the ratio of paired-pulse facilitation and was not occluded by low-frequency stimulation-induced LTD, suggesting that these two forms of LTD did not share a common underlying mechanism. After eliciting LTD with l-CCG, application of isoproterenol increased the synaptic responses back to its original baseline, demonstrating that chemically depressed synapses could be potentiated by another chemical. A selective PKA inhibitor, KT 5720, by its own caused a depression of synaptic transmission and blocked l-CCG LTD, presumably by mimicking and thereby occluding any further depression. Together, these results suggest that l-CCG LTD is induced by presynaptically mGluR II-mediated inhibition of Ca(2+)-sensitive adenylyl cyclase, resulting in a decrease in cAMP formation and PKA activation, which leads to a long-lasting decrease in transmitter release.

Bursting discharges in disinhibited amygdala slices: the role of excitatory amino acid receptors.

The involvement of N-methyl-D-aspartate (NMDA) and non-NMDA receptors in the epileptiform activity, induced by bicuculline, was studied in slices of amygdala in the rat, using intracellular recording techniques. Stimulation of the ventral endopyriform nucleus evoked an excitatory postsynaptic potential (EPSP). After exposure to bicuculline (20 microM), the same stimulus evoked burst firing. Occasionally, spontaneous bursts similar in waveform to synaptically triggered bursts also occurred in disinhibited slices. Superfusion of DL-2-amino-5-phosphonovalerate (DL-APV, 50 microM) or 3-((+)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acids (CPP, 10 microM), rapidly blocked the late component of the paroxysmal depolarizing shift. The spontaneous and evoked bursts were never completely abolished in the presence of DL-APV or CPP. These results suggest that NMDA receptors may contribute to but are not required for the generation of these bursts. In contrast, application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) largely abolished the bursts, indicating that activation of non-NMDA receptors is of primary importance in this model of epilepsy.

Epileptiform activity induced by magnesium-free solution in slices of rat amygdala: antagonism by N-methyl-D-aspartate receptor antagonists.

The effects of the N-methyl-D-aspartate (NMDA) receptor antagonists, D-2-amino-5-phosphonovalerate (D-APV) and phencyclidine (PCP), were studied in vitro on epileptiform activity induced in magnesium-free solution in neurons of the basolateral amygdala of the rat, using intracellular recording techniques. Twenty to 30 min after switching to magnesium-free medium, spontaneous interictal-like events were observed in 33 out of 37 amygdala slices. The spontaneous interictal-like events consisted of an initial burst followed by a number of afterdischarges. Superfusion with D-APV, a competitive NMDA receptor antagonist, reversibly reduced the duration of the events was also reduced. The IC50, estimated from the graph of the concentration-response relationship, was approximately 10 microM which is close to the IC50 for the binding of D-AVP to the NMDA receptor in other regions of the brain. The effect of phencyclidine, a noncompetitive NMDA receptor antagonist, was similar to that of D-APV. These results suggest that activation of NMDA receptors plays an intrinsic role in the induction or propagation of epileptiform activity seen in magnesium-free solution in the neurons of the amygdala.

Endogenous serotonin inhibits epileptiform activity in rat hippocampal CA1 neurons via 5-hydroxytryptamine1A receptor activation.

The modulatory effects of endogenous serotonin on the synaptic transmission and epileptiform activity were studied in the rat hippocampus with the use of extracellular and intracellular recording techniques. Field excitatory postsynaptic potential was reversibly depressed by serotonin in a concentration-dependent manner. Intracellular recordings revealed that serotonin-mediated synaptic depression was unaffected by extracellular Ba2+ or intracellular application of Cs+ while the postsynaptic hyperpolarizing effect was completely blocked. Epileptiform activity induced by picrotoxin (50 microM), a GABA(A) receptor antagonist, was also dose-dependently suppressed by serotonin. The antiepileptic effect was mimicked by 5-hydroxytryptamine1A agonist and was blocked by 5-hydroxytryptamine1A antagonists. 5-Hydroxytryptamine2 antagonist had no effect on the modulation. Similarly, fluoxetine, a selective serotonin re-uptake blocker, potently inhibited the epileptiform activity and this effect was blocked by 5-hydroxytryptamine1A receptor antagonist. Depletion of endogenous serotonin by pretreating the slices with p-chloroamphetamine completely prevented the antiepileptic action of fluoxetine, without modifying the action of serotonin in the same cells. These results suggest that the antiepileptic action of fluoxetine is due to an enhancement of endogenous serotonin which in turn is mediated by 5-hydroxytryptamine1A receptor. Endogenous serotonin transmission in the hippocampus is therefore capable of limiting the development and propagation of seizure activity.

Masking of forskolin-induced long-term potentiation by adenosine accumulation in area CA1 of the rat hippocampus.

At hippocampal Schaffer collateral-CA1 synapses, activation of beta-adrenergic receptors and adenylyl cyclase increases transmitter release. However, this effect is transient, which is in contrast to that seen at mossy fiber-CA3 synapses, where activation of cyclic-AMP-dependent protein kinase results in long-lasting facilitation of transmitter release, a phenomenon known as a presynaptic form of long-term potentiation. The present study was aimed at investigating whether forskolin, an adenylyl cyclase activator, could produce long-term effects at the Schaffer collateral-CA1 synapses using extracellular recording techniques. As has been reported previously, forskolin persistently increased the amplitude of evoked population spikes without having a long-term effect on the field excitatory postsynaptic potentials. However, under the conditions where adenosine A1 receptors are inhibited, cyclic-AMP metabolism is disrupted or the transport of cyclic-AMP is blocked, forskolin induces long-term potentiation. Forskolin-induced potentiation is associated with a decrease in paired-pulse facilitation and is blocked by the cyclic-AMP-dependent protein kinase inhibitor Rp-adenosine-3',5'-cyclic monophosphorothioate. Activation of N-methyl-D-aspartate receptors is not required for forskolin-induced long-term potentiation, because pretreatment of slices with the N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonovalerate did not prevent forskolin-induced potentiation. These results suggest that blockade of adenosine A1 receptors unmasks forskolin-induced long-term potentiation, and activation of cyclic-AMP-dependent protein kinase induces a form of long-term potentiation which is different from that induced by tetanic stimulation.

Lamotrigine inhibits tetraethylammonium-induced synaptic plasticity in the rat amygdala.

Although long-term potentiation was generally initiated by a brief tetanus, in the hippocampus, it could also be evoked by application of the K+ channel blocker tetraethylammonium. The present study was aimed at investigating the effect of lamotrigine, a new anticonvulsant, on the tetraethylammonium-induced potentiation in brain slices of the rat amygdala using intracellular recording techniques. Bath application of tetraethylammonium (20 mM) for 10 min resulted in a long-lasting enhancement of the amplitude of excitatory postsynaptic potentials to 235 +/- 12% of control (n = 6, P < 0.001). Pretreatment of the slices with nifedipine (10 microM) abolished the potentiation, suggesting that tetraethylammonium long-term potentiation in the amygdala is due to Ca2+ influx through voltage-dependent Ca2+ channels. By contrast, N-methyl-D-aspartate receptor activation was not required because D-2-amino-5-phosphonovalerate (50 microM) did not prevent the tetraehylammonium long-term potentiation. Superfusion of lamotrigine (50 microM) depressed the excitatory postsynaptic potential to 53.8 +/- 3.9% of control. Tetraethylammonium was subsequently added in the presence of lamotrigine but failed to enhance the excitatory postsynaptic potential. Bursts of Ca2+ spikes evoked by a depolarizing pulse or by synaptic stimulation under tetraethylammonium were depressed by lamotrigine. It is concluded that lamotrigine is capable of inhibiting tetraethylammonium-induced synaptic plasticity. The underlying mechanism is likely due to lamotrigine's inhibition of postsynaptic voltage-dependent Ca2+ channels. Considering that tetraethylammonium is a convulsant agent and brief seizure episodes induced long-term potentiation, fibre sprouting and the development of aberrant synaptic contacts, lamotrigine could be a potential neuroprotective agent, especially in pathological situations where excessive glutamate release occurs.

Investigations on the mechanism of tetrahydro-9-aminoacridine-induced presynaptic inhibition in the rat amygdala.

Tetrahydro-9-aminoacridine, a centrally acting anticholinesterase, has been reported to improve clinical conditions of certain patients with Alzheimer's disease. A previous study from our laboratory suggested that tetrahydro-9-aminoacridine presynaptically inhibited synaptic transmission. In the present study, the mechanism responsible for presynaptic inhibition mediated by tetrahydro-9-aminoacridine was studied in the rat amygdalar slice preparation using intracellular recording techniques. Bath application of tetrahydro-9-aminoacridine reversibly suppressed the excitatory postsynaptic potential. Tetrahydro-9-aminoacridine's inhibitory action was unaffected by the pretreatment of slices with baclofen (5 microM), suggesting that it did not act by eliciting the release of GABA, which binds presynaptic GABAB receptors to inhibit glutamate release. The synaptic depressant effect of tetrahydro-9-aminoacridine was blocked in the presence of 4-aminopyridine. The action of 4-aminopyridine could be reversed by reducing extracellular Ca2+ concentrations from a control level of 2.5 to 0.5 mM, suggesting that tetrahydro-9-aminoacridine inhibits excitatory postsynaptic potentials by acting directly at the terminals to decrease a Ca2+ influx. The L-type Ca2+ channel blocker nifedipine (50 microM) had no effect on tetrahydro-9-aminoacridine-induced presynaptic inhibition. However, the depressant effect of tetrahydro-9-aminoacridine was partially occluded in slices pretreated with the N-type Ca2+ channel blocker omega-conotoxin GVIA (1 microM). It is concluded that a reduction in omega-conotoxin GVIA-sensitive Ca2+ currents contributes to tetrahydro-9-aminoacridine-mediated presynaptic inhibition. After exposure to bicuculline, a GABAA receptor antagonist, afferent stimulation evoked epileptiform bursts. Occasionally, spontaneous bursts similar in waveform to synaptically triggered bursts also occurred in disinhibited slices. Application of tetrahydro-9-aminoacridine reversibly reduced the burst duration in a concentration-dependent manner. These results suggest that tetrahydro-9-aminoacridine possesses anticonvulsant activity against disinhibited bursts.

Carbachol induces inward current in neostriatal neurons through M1-like muscarinic receptors.

The effects of carbachol on rat neostriatal neurons were examined in the slice and the freshly dissociated neuron preparations using intracellular and whole-cell voltage-clamp recording methods. Superfusion of carbachol (30 microM) produced a depolarization concomitant with an increase in the rate of spontaneous action potentials. This depolarization was associated with an increase in the input resistance. The carbachol-induced membrane depolarization was blocked by pirenzepine (1 microM), a selective M1 muscarinic receptor antagonist. In other experiments, we observed that carbachol induced a transient inward current on the freshly dissociated neostriatal neuron at a holding potential of -60 mV in a concentration-dependent manner underlying the whole-cell voltage-clamp mode. The inward current caused by carbachol was not reduced by tetrodotoxin (1 microM), calcium-free recording solution or Cd2+ (100 microM). However, it was blocked by Ba2+ (100 microM). In addition, the carbachol-induced inward current reversed polarity at about the potassium equilibrium potential. The whole-cell membrane inward current in response to voltage-clamp step from -90 to -140 mV was reduced by 30 microM carbachol. With stronger hyperpolarization beyond the potassium equilibrium potential, carbachol produced a progressively greater reduction in membrane current. This inhibitory effect was also abolished by Ba2+ (100 microM). A concentration of 30 microM carbachol-induced inward current could be reversibly antagonized by the M1 muscarinic receptor antagonist pirenzepine (0.1-1 microM), with an estimated IC50 of 0.3 microM. However, other muscarinic receptor subtype (M2 or M3) antagonists could also block the carbachol-induced inward current. The rank order of antagonist potency was: pirenzepine (M1 antagonist) > 4-diphenylacetoxy-N,N-methyl-piperidine methiodide (M3/M1 antagonist) > gallamine (M2 antagonist). Based on these pharmacological data, we concluded that carbachol can act at M1-like muscarinic receptors to reduce the membrane K+ conductances and excite the neostriatal neurons.

Paired-pulse depression of the N-methyl-D-aspartate receptor-mediated synaptic potentials in the amygdala.

1. An in vitro slice preparation of rat amygdala was used to study the paired-pulse depression of the N-methyl-D-aspartate (NMDA) receptor-mediated synaptic potential e.p.s.p.NMDA. 2. The e.p.s.p.NMDA was isolated pharmacologically by applying a solution containing the non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the gamma-aminobutyric acidA (GABAA) blocker picrotoxin and increasing the stimulus intensity. 3. When two stimuli of identical strength were applied in close succession, the second e.p.s.p.NMDA was depressed. This paired-pulse depression was seen with interstimulus intervals of between 100 ms and 2000 ms; the maximal depression was observed at interval of 200 ms. 4. Superfusion of phaclofen or 2-hydroxy-saclofen inhibited the paired-pulse depression indicating the involvement of GABAB receptors. 5. Bath applications of Ba2+ or intracellular injection of Cs+ to block post- but not presynaptic GABAB receptors failed to inhibit the paired-pulse depression (PPD). 6. Incubation of slices with pertussis toxin prevented the postsynaptic hyperpolarization induced by baclofen. The PPD of e.p.s.p.NMDA, however, was not affected by pertussis toxin treatment. 7. These results suggest that GABA released by the first stimulus acts on GABAB receptors to suppress the second e.p.s.p.NMDA via mechanisms other than activation of a postsynaptic GABAB receptor-coupled K+ conductance.

Inhibition of synaptic transmission and epileptiform activity in central neurones by fluspirilene.

1. Recent studies have shown that fluspirilene, a dopamine D2 receptor antagonist which is a long-acting neuroleptic useful in the maintenance therapy of schizophrenic patients, also displays Ca2+ channel blocking activity. In the present study, we have investigated the effect of fluspirilene on synaptic transmission and epileptiform activity induced in slices of hippocampus and amygdala. 2. Fluspirilene reversibly suppressed the field excitatory postsynaptic potential (f-e.p.s.p) in a concentration-dependent manner in the area CA1 of the hippocampus without affecting the size and shape of fibre volley. Fluspirilene also inhibited the intracellularly recorded e.p.s.p. in amygdala neurones without affecting the resting membrane potential or neuronal input resistance. 3. Fluspirilene increased the ratio of paired-pulse facilitation suggesting a presynaptic mode of action. 4. Epileptiform activity induced in the disinhibited slices was suppessed by fluspirilene in a concentration-dependent manner. This antiepileptic effect was occluded in slices pretreated with the adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA). 5. It is concluded that fluspirilene-induced synaptic inhibition is probably due to a reduction in presynaptic Ca2+ currents. In clinical trials, the low incidence of seizures provoked by fluspirilene might be related to its intrinsic ability to inhibit synaptic transmission and epileptiform activity.

Frequency-dependent inhibition of neuronal activity by topiramate in rat hippocampal slices.

1. Topiramate is a structurally novel anticonvulsant which was recently approved for adjunctive therapy in partial and secondarily generalized seizures. The present study was aimed at elucidating the mechanisms underlying the anticonvulsant efficacy of topiramate using intra- and extracellular recording techniques in the in vitro hippocampal slices. 2. When stimuli were delivered every 20 s, topiramate had no measurable effect on both field excitatory postsynaptic potentials (fEPSPs) and population spikes (PSs). However, increasing the stimulation frequency from 0.05-0.2 Hz, topiramate significantly decreased the slope of fEPSP and the amplitude of PS in a concentration-dependent manner. The amplitude of presynaptic fiber volley was also reduced. 3. Topiramate did not affect the magnitude of paired-pulse inhibition and monosynaptically evoked inhibitory postsynaptic potentials (IPSPs). 4. Sustained repetitive firing was elicited by injection of long duration (500 ms) depolarizing current pulses (500-800 pA). Superfusion with topiramate significantly reduced the number of action potentials evoked by a given current pulse. 5. After blockade of GABA receptors by bicuculline, burst firing which consisted of a train of several spikes riding on a large depolarizing wave termed paroxysmal depolarizing shift (PDS) was recorded. Application of topiramate reduced the duration of PDS and later spikes with less effect on the initial action potential. 6. These results suggest that frequency-dependent inhibition of neuronal activity due to blockade of Na+ channels may account largely for the anticonvulsant efficacy of topiramate.

Activation of Metabotropic Glutamate Receptors in Conjunction with Postsynaptic Depolarization Triggers a Long-Term Depression of the N-Methyl-D-Aspartate Receptor-Mediated Synaptic Potential in the Rat Hippocampus.

The mechanism responsible for long-term depression (LTD) of pharmacologically isolated N-methyl-D-aspartate (NMDA) receptor-mediated excitatory postsynaptic potential (EPSP(NMDA)) was studied. Intracellular recordings were made from CA1 cells of rat hippocampal slices in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (10 &mgr;M) and picrotoxin (50 &mgr;M), which block non-NMDA and GABA(A) receptors, respectively. Intracellular injections of depolarizing pulses (500 ms, 0.3-0.7 nA) at 1 Hz for 5 min in the absence of synaptic stimulation caused a persistent increase in the amplitude of EPSP(NMDA). However, coupling postsynaptic depolarization with synaptic activity induced LTD. The EPSP(NMDA) LTD could be blocked by L-2-amino-3-phosphonopropionic acid (50 &mgr;M) or (RS)-alpha-methyl-4-carboxyphenylglycine (200 &mgr;M), specific antagonists for metabotropic glutamate receptors (mGluR). Furthermore, application of trans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD, 50 &mgr;M), a specific mGluR agonist, in conjunction with postsynaptic depolarizing elicited LTD. In contrast, the mGluR agonists quisqualate or t-ACPD when given alone produced a sustained enhancement of EPSP(NMDA). Finally, coupled depolarization did not evoke LTD in slices pretreated with the protein kinase C (PKC) inhibitor calphostin c (60 nM). The present results demonstrate that activation of mGluR is necessary for the induction of LTD of EPSP(NMDA) and suggest that NMDA receptors are subject to bidirectional regulation by mGluR. Furthermore, the induction of LTD is likely to involve the stimulation of PKC. Copyright 1995 S. Karger AG, Basel

Lamotrigine inhibition of glutamate release from isolated cerebrocortical nerve terminals (synaptosomes) by suppression of voltage-activated calcium channel activity.

Lamotrigine (LAG) is an antiepileptic drug which is believed to suppress seizures by inhibiting the release of excitatory neurotransmitters. The present study was aimed at investigating the effect of LAG on the 4-aminopyridine (4AP)-evoked glutamate release in cerebrocortical nerve terminals (synaptosomes). LAG inhibited the release of glutamate evoked by 4AP in a concentration-dependent manner. This inhibitory effect was associated with a reduction in the depolarization-evoked increase in the cytoplasmic free Ca2+ concentration ([Ca2+]C). In addition, LAG did not alter the resting synaptosomal membrane potential or 4AP-evoked depolarization. Furthermore, ionomycin-evoked glutamate release was not affected by LAG. Based on these results, we suggest that presynaptic calcium influx blockade and inhibition of glutamate release may underlie the mechanism of action of LAG. These action may also contribute to their neuroprotective properties in excitotoxic injury.

Inhibition of N-type calcium currents by lamotrigine in rat amygdalar neurones.

Lamotrigine (LAG) is a new anticonvulsant drug for the treatment of partial and secondarily generalized seizures. The present study was aimed at elucidating the possible involvement of Ca2+ channels in the action of LAG using whole-cell patch clamp recordings in acutely dissociated amygdalar neurones. Whole-cell Ca2+ currents (ICa) were elicited by 200 ms step commands from -70 mV to -10 mV. Application of LAG reduced the ICa by an average of 40.3 +/- 3.2%. The inhibition of ICa by LAG was markedly reduced or eliminated in the presence of the N-type Ca2+ channel blocker omega-conotoxin-GVIA (1 microM). These results suggest that LAG may exert its anticonvulsant effect through inhibition of presynaptic N-type Ca2+ channels, thereby reducing glutamate release.

The epileptiform activity induced by 4-aminopyridine in rat amygdala slices: antagonism by non-N-methyl-D-aspartate receptor antagonists.

The effects of excitatory amino acid receptor antagonists kynuretic acid and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) on epileptiform activity induced by 4-aminopyridine (4-AP) were studied in rat amygdala slices using intracellular recording techniques. Five to 10 min after switching to 4-AP-containing solution, spontaneous epileptiform bursts were observed in 35 out of 45 slices studied. The spontaneous epileptiform events consisted of an initial burst followed by a number of afterdischarges. Superfusion with kynuretic acid, a broad-spectrum excitatory amino acid receptor antagonist, reversibly reduced the duration of the spontaneous bursting discharges in a dose-dependent manner. The frequency of spontaneous bursting was also decreased. The IC50, estimated from the graph of the concentration-response relationship, was approximately 130 microM. In addition, CNQX which is a specific non-N-methyl-D-aspartate (NMDA) receptor antagonist blocked the spontaneous and evoked epileptiform bursting. In 11 out of 15 neurons tested, there was a residual depolarizing component remained. This depolarizing component was reversibly blocked by specific NMDA receptor antagonist, D,L-2-amino-5-phosphonovaleate (D,L-APV). Relative to the CNQX-sensitive component, the D,L-APV-sensitive component is much smaller in amplitude and shorter in duration indicating that NMDA receptor plays only a minor role in this process. These data suggest that the generation or propagation of spontaneous epileptiform events induced by 4-AP in the neurons of basolateral amygdala is mediated by excitatory amino acids and that activation of non-NMDA receptors is of primary importance.

Suppression of 4-aminopyridine-induced paroxysmal depolarizing shift in rat amygdaloid neurons by diltiazem.

The effects of organic Ca2+ channel blocker, diltiazem, on the epileptiform activity induced by 4-aminopyridine (4-AP) were studied in rat amygdaloid slices using intracellular recording techniques. Application of 4-AP (0.5 mM) resulted in spontaneous and evoked epileptiform activity which consisted of an initial burst followed by a number of afterdischarges. The initial burst began with rapidly rising action potentials superimposed on a large depolarizing wave termed paroxysmal depolarizing shift (PDS). Diltiazem reversibly suppressed the amplitude and duration of PDS in a concentration-dependent manner. The IC50, estimated from the graph of the concentration-response relationship, was approximately 60 microM. These results demonstrate that a calcium current sensitive to diltiazem is involved in the generation of PDS and suggest that PDS is based on giant synaptic conductance as well as endogenous calcium currents.

The transient potassium current, the A-current, is involved in spike frequency adaptation in rat amygdala neurons.

The possible functional roles of the transient K+ current, IA, in basolateral amygdala (BLA) neurons were studied using a rat brain slice preparation and conventional intracellular recording techniques. Conditioning depolarization, which inactivates IA, slowed the action potential repolarization while conditioning hyperpolarization accelerated the action potential repolarization. 4-Aminopyridine (4-AP, 100 microM), a specific IA antagonist, also caused a clear delay in spike repolarization similar to the effect of conditioning depolarization suggesting that IA is involved in the action potential repolarization. When BLA neurons were excited by injecting long depolarizing current pulses (500 ms), they responded with an initial rapid discharge of action potentials which slowed or accommodated; an afterhyperpolarization (AHP) followed the depolarizing current pulses. Superfusion of 4-AP (100 microM) blocked accommodation resulting in an increase in action potential discharge in 74% (32 out of 43) neurons tested. The remaining 11 cells responded with an increased frequency of discharge of the first few action potentials. Unlike the effect of cadmium (Cd2+, 100 microM), a calcium channel blocker, 4-AP did not reduce the AHP. In the presence of norepinephrine (NE, 10 microM), a neurotransmitter which has been shown to block calcium-activated potassium conductance, 4-AP caused a further increase in the number and frequency of action potential discharge. In addition, in BLA neurons, spontaneous interictal and ictal-like events were observed at low and high concentrations of 4-AP, respectively. We conclude that IA is involved in the action potential repolarization as well as spike frequency adaptation in BLA neurons and that these actions may contribute to the convulsant effect of 4-AP.