Studies of the actions of LAG and THA on the rat amygdala neurons.

• Main Authors: Wang, Su-Jane, 王素珍
• Other Authors: Gean, Po-Wu
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/26053318924772952205

博士 === 國立成功大學 === 基礎醫學研究所 === 86 === There are two main research subjects in the present dissertation: The first subject is directed to studying the mechanism of action of lamotrigine (LAG) in rat amygdaloid slices using intracellular recording and whole-cell patch clamp techniques. Lamotrigine is a new antiepileptic drug which is licensed as adjunctive therapy for partial and secondary generalized seizures. It is generally accepted that the reduction in glutamate release previously demonstrated for LAG plays a part in its effects, but the underlying mechanism is not clear. Previous neurochemical studies have shown that LAG is capable of inhibiting glutamate release evoked by the Na+ channel activator veratrine. Electrophysiologically, LAG blocks high-frequency sustained firing of Na+-dependent action potentials in cultured mouse spinal cord neurones. It is suggested that LAG acts at voltage-dependent Na+ channels to stabilize the neuronal membrane and inhibit transmitter release. Bath application of LAG (50mM) 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 (EPSPNMDA) was isolated pharmacologically by application of a solution containing non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10mM) and g-aminob-utyric acidA receptor antagonist bicuculline (20mM). LAG produced a parallel inhibition of EPSPNMDA. Postsynaptic depolarization induced by a-amino-5-methyl-4-isoxazlole 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. The L-type Ca2+ channel blocker nifedipine (20mM) had no effect on LAG-induced presynaptic inhibition. However, the depressant effect of LAG was markedly reduced in slices pretreated with N-type Ca2+ channel blocker w-conotoxin-GVIA (w-CgTx-GVIA) or a broad spectrum Ca2+ channel blocker w-conotoxin-MVIIC (w-CgTx-MVIIC). These results suggest that antagonism of presynaptic N-type Ca2+ channels may contribute to LAG-induced presynaptic inhibition. To test this hypothesis directly, we used whole-cell patch clamp recording techniques to examine the effect of LAG on the Ca2+ currents in acutely dissociated amygdaloid neurones. Whole-cell Ca2+ currents (ICa) were elicited by 200 ms step commands from -70mV to -10mV. 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 w-conotoxin-GVIA. These results suggest that LAG may exert its anticonvulsant effect through inhibition of presynaptic N-type Ca2+ channels, thereby reducing glutamate release. Microfluorimetry with the Ca2+-sensitive dye fura-2 was used to study the effect of LAG on the depolarization-evoked Ca2+ influx in the acutely isolated basolateral amygdala neurons. Depolarization of the neurons with high K+ resulted in the elevation of intracellular Ca2+ concentration [Ca2+]i depending on the concentrations of K+ applied. The K+-induced Ca2+ influx was completely blocked in the Ca2+-free solution or by Cd2+ indicating that depolarization-induced increases in [Ca2+]i was triggered largely, if not at all, by Ca2+ entry from extacellular space and Ca2+ entry occurred through voltage-dependent Ca2+ channels. Application of LAG reduced the depolarization-evoked Ca2+ influx in a concentration-dependent manner. The effect of LAG was markedly reduced in the presence of N-type Ca2+ channel blocker w-CgTx-GVIA. These results suggest that the action of LAG is mediated, at least in part, by the modulation of N-type Ca2+ channels. 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 (TEA). The effect of LAG on the TEA-induced potentiation was investigated in rat amygdalar neurons using intracellular recording technique. Bath application of TEA (20mM) for 10 min resulted in a long-lasting enhancement of the amplitude of excitatory postsynaptic potentials to 235±12% of control. Pretreatment of the slices with nifedipine (10mM) abolished the potentiation, suggesting that TEA long-term potentiation in the amygdala is due to Ca2+ influx through voltage-dependent Ca2+ channels. By contrast, NMDA receptor activation was not required because D-APV (50mM) did not prevent the TEA long-term potentiation. Superfusion of LAG (50mM) depressed the excitatory postsynaptic potential to 53.8±3.9% of control. TEA was subsequently added in the presence of LAG but failed to enhance the excitatory postsynaptic potential. Burst of Ca2+ spikes evoked by a depolarizing pulse or by synaptic stimulation under TEA were depressed by LAG. These results suggest that LAG is capable of inhibiting TEA-induced synaptic plasticity. The underlying mechanism is likely due to LAG''s inhibition of postsynaptic voltage-dependent Ca2+ channels. Considering that TEA is a convulsant agent and brief seizure episodes induced long-term potentiation, fibre sprouting and the development of aberrant synaptic contacts, LAG could be a potential neuroprotective agent, especially in pathological situations where excessive glutamate release occurs. The second subject aims to studying the effect of tetrahydro-9- aminoacridine (THA) on synaptic transmission in rat amygdala neurons. A 60-90% loss of choline acetyltransferase, a enzyme catalyzes the synthesis of acetylcholine and is a specific marker for cholinergic neurons, has been discovered in patient with Alzheimer''s disease. The basolateral nuclens of the amygdala receives one of the densest cholinergic innervations, and it has been amply demonstrated that the major part of the innervation comes from neurons in the basal forebrain. Recently, it has been reported that long-term treatment with THA, a potent centrally acting anticholinesterase, produces improvement of cognitive functions in Alzheimer''s patients, although its mechanism of actionis not determined yet. THA inbihits excitatory postsynaptic potential (EPSP) without decreasing the postsynaptic depolarization induced by glutamate agonist suggesting a presynaptic mechanism. Pretreatment the slices with atropine did not affect THA''s effect, indicating that the presynaptic muscarinic receptors are not involved THA''s inhibitory action was unaffected by the pretreatment of slices with baclofen, 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 THA was blocked in the presence of 4-AP. The action of 4-AP could be reversed by reducing extracellular Ca2+ concentrations from a control level of 2.5 to 0.5 mM, suggesting that THA inhibits EPSP by acting directly at the terminals to decrease a Ca2+ influx. The L-type Ca2+ channel blocker nifedipine had no effect on THA-induced presynaptic inhibition. However, the depressant effect of THA was partially occluded in slices pretreated with the N-type Ca2+ channel blocker w-conotoxin GVIA. It is concluded that a reduction in w- conotoxin GVIA-sensitive Ca2+ currents contributes to THA-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 THA reversibly reduced the burst duration in a concentration- dependent manner. These results suggest that THA possesses anticonvulsant activity against disinhibited bursts. On the other hand, the effect of THA on b-adrenoceptor activation- induced synaptic potentiation were studied in brain slices of the rat amygdala using intracellalar recording technques. Bath application of ISO resulting a long-term enhancement of the amplitude of excitatory postsynaptic potential 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 (300mM) completely abolishes the ISO- and forskolin-induced synaptic potentiation. We hypothesize that the locus of THA/b-adrenoceptor interaction is presynaptic; the underlying mechanism is likely due to THA''s depression of neurotransmitter release via a presynaptic blockade of voltage-dependent Ca2+ channels.