L. Tauc

Tetanus toxin is a zinc protein and its inhibition of neurotransmitter release and protease activity depend on zinc.

Tetanus and botulinum neurotoxins are the most potent toxins known. They bind to nerve cells, penetrate the cytosol and block neurotransmitter release. Comparison of their predicted amino acid sequences reveals a highly conserved segment that contains the HexxH zinc binding motif of metalloendopeptidases. The metal content of tetanus toxin was then measured and it was found that one atom of zinc is bound to the light chain of tetanus toxin. Zinc could be reversibly removed by incubation with heavy metal chelators. Zn2+ is coordinated by two histidines with no involvement in cysteines, suggesting that it plays a catalytic rather than a structural role. Bound Zn2+ was found to be essential for the tetanus toxin inhibition of neurotransmitter release in Aplysia neurons injected with the light chain. The intracellular activity of the toxin was blocked by phosphoramidon, a very specific inhibitor of zinc endopeptidases. Purified preparations of light chain showed a highly specific proteolytic activity against synaptobrevin, an integral membrane protein of small synaptic vesicles. The present findings indicate that tetanus toxin, and possibly also the botulinum neurotoxins, are metalloproteases and that they block neurotransmitter release via this protease activity.

Cyclic ADP‐ribose and calcium‐induced calcium release regulate neurotransmitter release at a cholinergic synapse of Aplysia

1 Presynaptic injection of cyclic ADP‐ribose (cADPR), a modulator of the ryanodine receptor, increased the postsynaptic response evoked by a presynaptic spike at an identified cholinergic synapse in the buccal ganglion of Aplysia californica. 2 The statistical analysis of long duration postsynaptic responses evoked by square depolarizations of the voltage‐clamped presynaptic neurone showed that the number of evoked acetylcholine (ACh) quanta released was increased following cADPR injection. 3 Overloading the presynaptic neurone with cADPR led to a transient increase of ACh release followed by a depression. 4 cADPR injections did not modify the presynaptic Ca2+ current triggering ACh release. 5 Ca2+ imaging with the fluorescent dye rhod‐2 showed that cADPR injection rapidly increased the free intracellular Ca2+ concentration indicating that the effects of cADPR on ACh release might be related to Ca2+ release from intracellular stores. 6 Ryanodine and 8‐amino‐cADPR, a specific antagonist of cADPR, decreased ACh release. 7 ADP‐ribosyl cyclase, which cyclizes NAD+ into cADPR, was present in the presynaptic neurone as shown by reverse transcriptase‐polymerase chain reaction experiments. 8 Application of NAD+, the substrate of ADP‐ribosyl cyclase, increased ACh release and this effect was prevented by both ryanodine and 8‐amino‐cADPR. 9 These results support the view that Ca2+‐induced Ca2+ release might be involved in the build‐up of the Ca2+ concentration which triggers ACh release, and thus that cADPR might have a role in transmitter release modulation.

Nitric oxide transforms serotonin into an inactive form and this affects neuromodulation.

Nitric oxide is a highly reactive molecule, diffusible and therefore ubiquitous in the central nervous system. Consequently, nitric oxide or nitric oxide-derived nitrogen oxides must enter into contact with neuromodulators and they can modify these molecules, especially monoamines, and thus change their regulatory action on synaptic transmission. We tested this possibility on a well-known, identified cholinergic synapse of Aplysia buccal ganglion, in which we have found that evoked acetylcholine release was decreased by extracellularly applied serotonin. We show that this modulatory effect of serotonin was largely reduced not only in the presence of 3-morpholinosydnonimine, a nitric oxide donor, but also when endogenous nitric oxide synthase was activated. We have shown that this decrease in the serotonin effect is due to the formation of chemical derivatives of serotonin, mainly a symmetric serotonin dimer, 4-nitroso-serotonin and 4-nitro-serotonin, which are ineffective in reproducing the modulatory effect of serotonin. Serotonin is involved in the regulation of several central functions, such as sleep-wake activity or mood. The consequences of chemical modifications of serotonin by nitric oxide must be taken into account in physiological as well as pathological situations. In addition, our results highlight the importance of the physiological implications of interactions between free radicals and neuromediators in the nervous system.

Light chain of tetanus toxin intracellularly inhibits acetylcholine release at neuro‐neuronal synapses, and its internalization is mediated by heavy chain

The ability of the two‐chain form of tetanus toxin (TeTx), its constituent light (LC) or heavy (HC) chains, and papain fragment to block evoked acetylcholine (ACh) release in the buccal ganglia of Aplysia californica was studied electrophysiologically. Extracellularly applied, TeTx or its B fragment (consisting of LC and β2, the amino‐terminal portion of HC) blocked ACh release, whereas LC, HC, or the β2 fragment did not affect it. Toxicity was restored when LC was bath applied together with HC or the β2 fragment. When injected into the presynaptic neuron, TeTx, the B fragment or LC, but not HC, induced inhibition of ACh release. These results indicate that the blockade of ACh release by TeTx is mimicked by intracellular action of LC, the internalization of which is mediated by the HC via its amino‐terminal moiety.

Acetylcholine receptors: topographic distribution and pharmacological properties of two receptor types on a single molluscan neurone.

1. The iontophoretic application of acetylcholine (ACh) on to identified neurones in the buccal ganglion of the mollusc Navanax produced a biphasic or monophasic membrane potential change which was a function of the current intensity and site of ACh application.

Calcium transients and neurotransmitter release at an identified synapse

It is widely accepted that the modulation of the presynaptic Ca2+ influx is one of the main mechanisms by which neurotransmitter release can be controlled. The well-identified cholinergic synapse in the buccal ganglion of Aplysia has been used to study the modulations that affect presynaptic Ca2+ transients and to relate this to quantal evoked neurotransmitter release. Three types of Ca2+ channel (L, N and P) are present in the presynaptic neurone at this synapse. Influxes of Ca2+ through N- and P-type channels trigger the release of ACh with only N-type Ca2+ channels being regulated by presynaptic neuromodulator receptors. In addition, presynaptic Ca2+ stores, via complex mechanisms of Ca2+ uptake and Ca2+ release, control the Ca2+ concentration that triggers this evoked ACh release.

A nitric oxide synthase activity is involved in the modulation of acetylcholine release inAplysia ganglion neurons: A histological, voltammetric and electrophysiological study

Abstract The role of nitric oxide or related molecules as neuromodulators was investigated in the buccal and the abdominal ganglia of the mollusc Aplysia californica . In a first step we showed that reduced nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry and specific nitric oxide synthase immunohistochemistry labelled the same neurons and fibres in both ganglia, pointing to the presence of a neuronal nitric oxide synthase. In a second step, we performed voltammetric detection of nitric oxide-related molecules using a microcarbon electrode in a reduction mode. A peak identified as N -nitroso- l -arginine was detected at −1.66 V in both ganglia. The identification of this compound as a product of endogenous nitric oxide synthase activity was reinforced by the fact that its peak amplitude was decreased in the presence of N G -monomethyl- l -arginine, an inhibitor of nitric oxide synthase, and increased with its substrate, l -arginine. An additional proof of a nitric oxide synthase activity was the detection of nitrites and nitrates in high concentrations (millimolar range) by capillary electrophoresis. We also showed that these nitric oxide-related molecules modulated acetylcholine release at two identified synapses in these ganglia. l -Arginine decreased acetylcholine release it the inhibitory synapse (buccal ganglion), whereas it increased acetylcholine release at the excitatory synapse (abdominal ganglion). The nitric oxide synthase inhibitors, N ω -nitro- l -arginine and N G -monomethyl- l -arginine, had opposite effects. Moreover, the exogenous nitric oxide donor, 3-morpholinosydnonimine hydrochloride mimicked the effects of l -arginine on both inhibitory and excitatory cholinergic synapses. The identification of two cholinergic synapses where nitric oxide affects acetylcholine release in opposite ways provides a useful tool to study the cellular mechanisms through which nitric oxide-related molecules modulate transmitter release.

Transmitter release: ruthenium red used to demonstrate a possible role of sialic acid containing substrates.

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.

Antibodies Against Rat Brain Vesicle-Associated Membrane Protein (Synaptobrevin) Prevent Inhibition of Acetylcholine Release by Tetanus Toxin or Botulinum Neurotoxin Type B

Abstract: Tetanus and botulinum B neurotoxins are zinc endopeptidases that cleave vesicle‐associated membrane protein (VAMP or synaptobrevin) at a single peptide bond. To test the possibility that in vivo also the toxin‐induced blockade of neurotransmission is due to cleavage of VAMP, rat brain VAMP‐specific antibodies were raised in rabbits. IgGs purified from one antiserum, which bind specifically to rat brain VAMP, also specifically recognize proteins from Aplysia californica in immunoblotting. When injected into neurons in the buccal ganglion of Aplysia, these IgGs did not affect the release of acetylcholine but effectively prevented the inhibitory action of both toxins on neurotrans‐ mitter release, thus indicating that the block of neurotransmission by these neurotoxins is consequent to the cleavage of VAMP or specific interaction with VAMP.

Histamine and FLRFamide regulate acetylcholine release at an identified synapse in Aplysia in opposite ways.

1. The effects of histamine and FLRFamide (Phe‐Leu‐Arg‐Phe‐NH2) on acetylcholine (ACh) release were studied in the buccal ganglion of Aplysia californica on an identified synapse (buccal ganglion inhibitory synapse, BGIS) involved in a small neuronal circuit controlling the feeding behaviour. The inhibitory postsynaptic current (IPSC) evoked by a presynaptic spike in the voltage‐clamped postsynaptic neurone was decreased by histamine and increased by FLRFamide. 2. Histamine and FLRFamide modified the amplitude of the presynaptic spike. To test if these drugs acted directly on presynaptic calcium influx, we evoked transmitter release by 3 s depolarizations of the presynaptic neurone (to +10 mV) under voltage clamp to avoid modifications of presynaptic membrane polarization induced by changes in presynaptic voltage‐dependent K+ and/or Na+ conductances. 3. Statistical analysis of this evoked long‐duration (3 s) induced postsynaptic current (LDIPSC) allowed us to calculate the amplitude and the decay time of the miniature postsynaptic current and consequently the number of quanta released by the presynaptic terminal. 4. The amplitude of the LDIPSC decreased during the 3 s presynaptic depolarization. This was not due to a lack of available transmitter, since LDIPSC amplitude could be maintained constant by a ‘clamp of the release of ACh’ which adequately depolarized the presynaptic neurone, but rather to changes in the calcium influx into the presynaptic neurone. 5. FLRFamide increased more the initial portions of the LDIPSC than the final portions. This effect of FLRFamide was only reduced and delayed by atropine or curare, antagonists of muscarinic‐like and nicotinic‐like autoreceptors previously demonstrated to be present at the same terminal. Activation of the nicotinic‐like receptors, which also increased transmitter release, induced a modification of the shape of the LDIPSC which was completely different from that due to FLRFamide. 6. Histamine decreased the amplitude of the LDIPSC. This effect was more pronounced at the beginning of the response. The effects of histamine were insensitive to curare and atropine, but were completely blocked by cimetidine, a specific histamine receptor antagonist. 7. The modifications of the shape and of the amplitude of the LDIPSC by FLRFamide and histamine suggested that these molecules alter presynaptic influx of calcium. This was confirmed by the analysis of calcium current recorded from the presynaptic neurone: the calcium inward current in the presynaptic neurone was increased by FLRFamide and reduced by histamine, whereas the activation of autoreceptors had no measurable effect on calcium current.(ABSTRACT TRUNCATED AT 400 WORDS)