| Summary: | 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.
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