The effects of nitric oxide on evoked acetylcholine (ACh) release were studied at two identified cholinergic neuro-neuronal synapses of the nervous system of the mollusc Aplysia californica. The NO-donor, 3-morpholinosydnonimine (SIN-1), decreased the amplitude of evoked inhibitory postsynaptic currents (buccal ganglion) and potentiated that of evoked excitatory postsynaptic currents (abdominal ganglion). SIN-1 acted by modulating the number of ACh quanta released. 8Br-cGMP mimicked the effects of NO on ACh release in both types of synapses thus pointing to the involvement of a NO-sensitive guanylate cyclase. Presynaptic voltage-dependent Ca2+ and K+ (IA and late outward rectifier) currents were not modified by SIN-1 suggesting another final target for NO/cGMP. The labelling of a NO-synthase by immunostaining in several neurones as well as the modulation of ACh release by L-arginine indicate that an endogenous NO-synthase is involved in the modulation of synaptic efficacy in both buccal and abdominal ganglia.
Nitric oxide (NO) produced opposite effects on acetylcholine (ACh) release in identified neuroneuronal Aplysia synapses depending on the excitatory or the inhibitory nature of the synapse. Extracellular application of the NO donor, SIN-1, depressed the inhibitory postsynaptic currents (IPSCs) and enhanced the excitatory postsynaptic currents (EPSCs) evoked by presynaptic action potentials (1/60 Hz). Application of a membrane-permeant cGMP analog mimicked the effect of SIN-1 suggesting the participation of guanylate cyclase in the NO pathway. The guanylate cyclase inhibitor, methylene blue, blocked the NO-induced enhancement of EPSCs but only reduced the inhibition of IPSCs indicating that an additional mechanism participates to the depression of synaptic transmission by NO. Using nicotinamide, an inhibitor of ADP-ribosylation, we found that the NO-induced depression of ACh release on the inhibitory synapse also involves ADP-ribosylation mechanism(s). Furthermore, application of SIN-1 paired with cGMP-dependent protein kinase (cGMP-PK) inhibitors showed that cGMP-PK could play a role in the potentiating but not in the depressing effect of NO on ACh release. Increasing the frequency of stimulation of the presynaptic neuron from 1/60 Hz to 0.25 or 1 Hz potentiated the EPSCs and reduced the IPSCs. In these conditions, the potentiating effect of NO on the excitatory synapse was reduced, whereas its depressing effect on the inhibitory synapse was unaffected. Moreover the frequency-dependent enhancement of ACh release in the excitatory synapse was greatly reduced by the inhibition of NO synthase. Our results indicate that NO may be involved in different ways of modulation of synaptic transmission depending on the type of the synapse including synaptic plasticity.
1. In a cholinergic synaptic couple in the buccal ganglion of Aplysia california, where the synaptic areas are situated close to the somata (500 micron), we were able to control transmitter release by stimulating the cell body of the presynaptic neurone with long depolarizing pulses in the presence of tetrodotoxin (TTX). 2. Statistical analysis of noise occurring at the peak of the long‐depolarization‐induced post‐synaptic current (p.s.c.) responses allowed us to calculate the amplitude and the decay time of the miniature post‐synaptic currents (m.p.s.c.s). These data were used to calculate the quantal content of the responses. 3. Bath‐applied tubocurarine reduced the amplitude of the long‐depolarization‐induced p.s.c. more than that of the m.p.s.c.s, indicating that tubocurarine exerts a depressive presynaptic action on the quantal content of the post‐synaptic responses. 4. Tubocurarine injected into the presynaptic neurone blocked synaptic transmission without decreasing the size of the m.p.s.c.s probably by acting on the mechanism of transmitter release. 5. Bath‐applied atropine (10(‐6) and 10(‐5) M) caused a slight decrease of the m.p.s.c.s but the long‐depolarization‐induced p.s.c.s increased, as did the quantal content. Higher concentrations of atropine depressed strongly both the m.p.s.c. and the quantal content. 6. Injection of atropine into the presynaptic neurone had the same effect as its bath application, probably due to the leakage of the drug into the synaptic cleft; the effect depended on the concentration reached in the cleft, i.e. on the quantity of injected drug. The synapses of the neighbouring cholinergic neurone were also affected by this leak of atropine. 7. The presence of nicotinic presynaptic receptors blocked by tubocurarine, and muscarinic presynaptic receptors blocked by atropine, which regulate synaptic transmission by facilitating and depressing the ACh release respectively, is discussed.
1. The possible function of sialic acid-containing substrates (SACS) in synaptic terminals of Aplysia was studied by intracellular injection of ruthenium red and of neuraminidase. 2. Ruthenium red, a dye known to have sialic acid as a molecular target, blocked transmission irreversibly in both cholinergic (buccal ganglion) and non-cholinergic (cerebral ganglion) synapses. 3. An intracellular site of action is likely because much less ruthenium red was necessary to block transmission when it was injected intracellularly than when it was presented by bath perfusion. 4. Ca2+ spikes recorded in the presence of tetrodotoxin or in Na+-free solution were not modified by ruthenium red or neuraminidase injections or perfusions. It is therefore improbable that these substances blocked transmission by blocking voltage-dependent Ca2+ influx. 5. Strong electrotonic depolarization of a pre-synaptic interneurone in the presence of 10(-4) M-tetrodotoxin caused a sustained post-synaptic response, which was abolished by ruthenium red. This result eliminates axonal conduction block as the principal mechanism of ruthenium red action. 6. Post-synaptic responses to ionophoretically applied acetylcholine (ACh) were not modified by bath perfusion of 2 x 10(-2) M-ruthenium red. 7. Biochemical analysis of pools of [3H]ACh was performed after injection of a precursor, [3H]acetate, into an identified interneurone. Ruthenium red appeared to increase significantly the 'free' (cytoplasmic) ACh pool without any change of 'bound' (vesicular) [3H]ACh-pool. 8. A model is proposed in which SACS act as intracellular Ca2+ receptors involved in transmitter release.
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