Su-Jane Wang

Mechanisms underlying the riluzole inhibition of glutamate release from rat cerebral cortex nerve terminals (synaptosomes)

We have examined the effect of riluzole, a neuroprotective agent with anticonvulsant properties, on the release of endogenous glutamate from rat cerebrocortical synaptosomes using an on-line enzyme-coupled fluorometric assay. Riluzole inhibited the calcium-dependent release of glutamate that was evoked by exposing cerebrocortical synaptosomes to the potassium channel blocker 4-aminopyridine, and this presynaptic inhibition was concentration-dependent. Riluzole did not alter either 4-aminopyridine-evoked depolarization of the synaptosomal membrane potential or ionomycin-mediated glutamate release, indicating that riluzole-mediated inhibition of glutamate release is not due to a decrease in synaptosomal excitability or a direct effect on the exocytotic machinery. Examination of the effect of riluzole on Ca2+ influx revealed that the diminution of glutamate release could be attributed to a reduction in cytosolic calcium. A possible effect of riluzole on synaptosomal calcium channels was confirmed in experiments where synaptosomes pretreated with P/Q-type calcium channel blocker omega-agatoxin IVA, which abolished the riluzole-mediated inhibition of glutamate release. In addition, pretreatment of synaptosomes with either the Gi/Go protein inhibitor pertussis toxin or the GABAB receptor agonist baclofen, completely prevented the inhibitory effect of riluzole on 4-aminopyridine-evoked glutamate release. It is concluded that riluzole exerts their presynaptic inhibition, likely through a reduction in the calcium influx mediated by P/Q-type calcium channels, and thereby inhibits the release of glutamate from rat cerebrocortical nerve terminals. This release inhibition may involve a pertussis toxin-sensitive G protein signalling pathway. This finding provides further support that presynaptic calcium channel blockade concomitant with inhibition of glutamate release could be an important mechanism underlying the therapeutic actions of this drug.

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 ω-cono-toxin-GVIA (1 μM). These results suggest that LAG may exert its anticonvulsant effect through inhibition of presynaptic N-type Ca2+ channels, thereby reducing glutamate release.

Serotonin depresses excitatory synaptic transmission and depolarization-evoked Ca2+ influx in rat basolateral amygdala via 5-HT1A receptors.

The actions of serotonin on rat basolateral amygdala neurons were studied with conventional intracellular recording techniques and fura‐2 fluorimetric recordings. Bath application of 5‐hydroxytryptamine (5‐HT or serotonin) reversibly suppressed the excitatory postsynaptic potential in a concentration‐dependent manner without affecting the resting membrane potential and neuronal input resistance. Extracellular Ba2+ or pertussis toxin pretreatment did not affect the depressing effect of 5‐HT suggesting that it is not mediated through activation of Gi/o protein‐coupled K+ conductance. The sensitivity of postsynaptic neurons to glutamate receptor agonist was unaltered by the 5‐HT pretreatment. In addition, the magnitude of paired‐pulse facilitation was increased in the presence of 5‐HT indicating a presynaptic mode of action. The effect of 5‐HT was mimicked by the selective 5‐HT1A agonist 8‐hydroxy‐dipropylaminotetralin (8‐OH‐DPAT) and was blocked by the selective 5‐HT1A antagonist 1‐(2‐methoxyphenyl)‐4[4‐(2‐phthalimido)butyl]piperazine oxadiazol‐3‐yl]methyl]phenyl]methanesulphonamide. In contrast, the selective 5‐HT2 receptor antagonist ketanserin failed to affect the action of 5‐HT. The effects of 5‐HT and 8‐OH‐DPAT on the high K+‐induced increase in [Ca2+]i were studied in acutely dissociated basolateral amygdala neurons. High K+‐induced increase in [Ca2+]i was blocked by Ca2+‐free solution and Cd2+ suggesting that Ca2+ entry responsible for the depolarizaton‐evoked increase in [Ca2+]i occurred through voltage‐dependent Ca2+ channels. Application of 5‐HT and 8‐OH‐DPAT reduced the K+‐induced Ca2+ influx in a concentration‐dependent manner. The effect of 5‐HT was completely abolished in slices pretreated with Rp‐cyclic adenosine 3′,5′‐monophosphothioate (Rp‐cAMP), a regulatory site antagonist of protein kinase A, suggesting that 5‐HT may act through a cAMP‐dependent mechanism. Taken together, these results suggest that functional 5‐HT1A receptors are present in the excitatory terminals and mediate the 5‐HT inhibition of synaptic transmission in the amygdala.

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.

Presynaptic inhibition of excitatory neurotransmission by lamotrigine in the rat amygdalar neurons

Lamotrigine (LAG) is a new antiepileptic drug which is licensed as adjunctive therapy for partial and secondary generalized seizures. In the present study, the mechanisms responsible for its antiepileptic effect were studied in rat amygdaloid slices using intracellular recording and whole‐cell patch clamp techniques. Bath application of LAG (50 μM) reversibly suppressed the excitatory postsynaptic potentials (EPSPs) and currents (EPSCs) evoked by stimulating ventral endopyriform nucleus. Synaptic response mediated by the N‐methyl‐D‐aspartate (NMDA) receptor (EPSP NMDA) was isolated pharmacologically by application of a solution containing non‐NMDA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX,10 μM) and γ‐aminobutyric acidA receptor antagonist bicuculline (20 μM). LAG produced a parallel inhibition of EPSP NMDA. Postsynaptic depolarization induced by α‐amino‐5‐methyl‐4‐isoxazole propionate (AMPA) was not altered by LAG. In addition, LAG increased the ratio of the second pulse response to the first pulse response (P2/P1), which is consistent with a presynaptic mode of action.

Lamotrigine inhibits depolarization-evoked Ca++ influx in dissociated amygdala neurons

Spectrophotometry with the Ca++‐sensitive dye fura‐2 was used to study the effect of lamotrigine (LAG) on the depolarization‐evoked Ca++ influx in the acutely isolated basolateral amygdala neurons. Depolarization of the neurons with high K+ resulted in the elevation of intracellular Ca++ concentration [Ca++]i in a concentration‐dependent manner. The K+‐induced Ca++ influx was completely blocked in the Ca++‐free solution or by Cd++, indicating that depolarization‐induced increases in [Ca++]i were triggered largely, if not at all, by Ca++ entry from extracellular space and Ca++ entry occurred through voltage‐dependent Ca++ channels. Application of LAG reduced the depolarization‐evoked Ca++ influx in a concentration‐dependent manner. The effect of LAG was markedly reduced in the presence of N‐type Ca++ channel blocker ω‐conotoxin‐GVIA (ω‐CgTX). These results suggest that the action of LAG is mediated, at least in part, by the modulation of N‐type Ca++ channels. Synapse 29:355–362, 1998.

Activation of neuropeptide Y Y1 receptors inhibits glutamate release through reduction of voltage-dependent Ca2+ entry in the rat cerebral cortex nerve terminals: Suppression of this inhibitory effect by the protein kinase C-dependent facilitatory pathway

Neuropeptide Y (NPY) is known to regulate the presynaptic glutamate release and neuronal responses to excitatory neurotransmission. The aim of this study was to investigate the effect of NPY on the release of endogenous glutamate from rat cerebrocortical nerve terminals (synaptosomes). NPY inhibited the Ca2+-dependent glutamate release evoked by 4-aminopyridine, and this inhibitory effect was mediated via NPY Y1 receptors, because it was mimicked by the specific NPY Y1 receptor agonist [Leu31 Pro34] NPY and blocked by the NPY Y1 receptor antagonist GR 231118. The inhibitory action of NPY was not due to it decreasing synaptosomal excitability or directly interfering with the release process at some point subsequent to Ca2+ influx, because NPY did not alter the 4-aminopyridine-evoked depolarization of the synaptosomal plasma membrane potential or ionomycin and hypertonic solution-induced glutamate release. Examination of the effect of NPY on the cytosolic [Ca2+] revealed that the inhibition of glutamate release could be attributed to a reduction in voltage-dependent Ca2+ influx. Consistent with this, the NPY-mediated inhibition of glutamate release was completely abolished in synaptosomes pretreated with N- and P/Q-type Ca2+ channel blocker, omega-conotoxin MVIIC. Moreover, NPY-mediated inhibition of 4-aminopyridine-evoked glutamate release was insensitive to KT 5720 and Ro32-0432 but was suppressed when protein kinase C was stimulated with phorbol ester. Together, these results suggest that NPY acting predominantly on NPY Y1 receptors inhibits glutamate release from rat cerebrocortical synaptosomes, likely by a mechanism involving direct coupling of receptors to N- and P/Q-type Ca2+ channels, and this coupling is subject to regulation by protein kinase C-dependent pathway. This implies that selective ligand for NPY receptors may be of value for treatment of conditions characterized by excessive glutamate release in the cerebral cortex.

Unexpected inhibitory regulation of glutamate release from rat cerebrocortical nerve terminals by presynaptic 5‐hydroxytryptamine‐2A receptors

Presynaptic 5‐HT2A receptor modulation of glutamate release from rat cerebrocortical nerve terminals (synaptosomes) was investigated by using the 5‐HT2A/2C receptor agonist (±)‐1‐[2,5‐dimethoxy‐4‐iodophenyl]‐2‐aminopropane (DOI). DOI potently inhibited 4‐aminopyridine (4AP)‐evoked glutamate release. Involvement of presynaptic 5‐HT2A receptors in this modulation of 4AP‐evoked release was confirmed by blockade of the DOI‐mediated inhibition by the 5‐HT2A receptor antagonist ketanserin but not by the 5‐HT2C receptor antagonist RS102221. Inhibition of glutamate release by DOI was associated with a reduction of 4AP‐evoked depolarization and downstream elevation of cytoplasmic free calcium concentration ([Ca2+]C) mediated via P/Q‐ and N‐type voltage‐dependent Ca2+ channels (VDCCs). In contrast to the DOI effect on 4AP‐evoked release, the agonist had no effect on high external [K+] (30 mM)‐induced (KCl) stimulation of VDCCs or glutamate release. Likewise, release mediated by direct Ca2+ entry with Ca2+ ionophore (ionomycin) or by hypertonic sucrose was unaffected by DOI. Mechanistically, DOI modulation of 4AP‐evoked glutamate release appeared to involve a phospholipase C/protein kinase C signaling cascade, insofar as pretreatment of synaptosomes with the phospholipase C inhibitor U73122 or protein kinase C inhibitors Ro320432 or GF109203X all effectively occluded the inhibitory effect of the agonist. Together, these results suggest that presynaptic 5‐HT2A receptors present on glutamatergic terminals effect an unexpected depression of glutamate release by negatively modulating nerve terminal excitability and downstream VDCC activation through a signaling cascade involving phospholipase C/protein kinase C. These observations invoke presynaptic inhibitory 5‐HT2A receptor function as a potential target for drugs to mitigate the effects of excessive glutamatergic transmission.

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 lamotrigines 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.

Blockade of isoproterenol-induced synaptic potentiation by tetra-9-aminoacridine in the rat amygdala ☆

The effects of tetrahydro-9-aminoacridine (THA) on beta-adrenoceptor activation-induced synaptic potentiation were studied in brain slices of the rat amygdala using intracellular recording techniques. To exclude the involvement of N-methyl-D-aspartate (NMDA) receptors, all the experiments were performed in the presence of NMDA receptor antagonist, D-APV (50 microM). Bath application of isoproterenol (Iso; 15 microM) results in a long-lasting enhancement of the amplitude of excitatory postsynaptic potentials (EPSPs) to 200 +/- 6% of baseline. Forskolin, which directly activates adenyl cyclase, produces a similar effect suggesting that Iso may act through a cyclic AMP-dependent mechanism. Pretreatment of the slices with THA (300 microM) completely abolishes the Iso- and forskolin-induced synaptic potentiation. We hypothesize that the locus of THA/beta-adrenoceptor interaction is presynaptic; the underlying mechanism is likely due to THAs depression of transmitter release via a presynaptic blockade of voltage-dependent Ca2+ channels.