Activation of protein kinase C by presynaptic FLRFamide receptors facilitates transmitter release at an aplysia cholinergic synapse.

Modulation of evoked quantal transmitter release by protein kinase C (PKC) was investigated at an identified cholinergic neuro-neuronal synapse of the Aplysia buccal ganglion. Evoked acetylcholine release was increased by a diacylglycerol analog that activates PKC and was decreased by H-7, a blocker of PKC. FLRFamide facilitated evoked quantal release by increasing presynaptic Ca2+ influx. The inhibition of PKC by H-7 prevented both the increase of presynaptic Ca2+ influx and the facilitation of evoked acetylcholine release induced by the activation of presynaptic FLRFamide receptors. These results provide evidence that the activation of PKC could be a step in the intracellular pathway by which FLRFamide receptors increase evoked quantal acetylcholine release.

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.

Electrical transmission among neurons in the buccal ganglion of a mollusc, Navanax inermis.

The opisthobranch mollusc, Navanax, is carnivorous and cannibalistic. Prey are swallowed whole by way of a sudden expansion of the pharynx. The buccal ganglion which controls this sucking action was isolated and bathed in seawater. Attention was focused upon 10 identifiable cells visible on the ganglion's rostral side. Two cells were observed simultaneously, and each was penetrated with two glass microelectrodes, one for polarizing the membrane and the other for recording membrane potential variations. The coupling coefficients for direct current flow and action potentials of several identified cells were tabulated. Attenuation was essentially independent of the direction of current flow, but depended upon the relative size of the directly and indirectly polarized cells. The attenuation of subthreshold sinusoidally varying voltages increased with frequency above about 1 Hz. The coupling coefficient for spikes was lower than for DC due to greater high frequency attenuation. There is considerable similarity in the spontaneous PSP's of all cells, which is not due to the electrical coupling but to input from a common source. The 10 cells were not chemically interconnected but some were electrically connected to interneurons which fed back chemically mediated PSP's. The feedback can be negative or positive depending upon the membrane potential of the postsynaptic cell. We conclude that electrical coupling among the 10 cells plays a minor role in sudden pharyngeal contractions but that the dual electrical-chemical coupling with interneurons may be important in this respect.

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 beta 2, the amino-terminal portion of HC) blocked ACh release, whereas LC, HC, or the beta 2 fragment did not affect it. Toxicity was restored when LC was bath applied together with HC or the beta 2 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.

Presynaptic mechanisms regulating Ca2+ concentration triggering acetylcholine release at an identified neuro-neuronal synapse of Aplysia.

We have used an identified cholinergic neuro-neuronal synapse in the buccal ganglion of Aplysia to determine which types of Ca2+ channels are involved in triggering transmitter release. omega-Conotoxin as well as funnel web spider toxin partially reduced acetylcholine release indicating that both N- and P-type Ca2+ channels are involved. Nifedipine-sensitive L-type Ca2+ channels are also present but they are not directly implicated in acetylcholine release. We have identified presynaptic receptors to two peptides. FMRFamide and buccalin and to the neurotransmitter histamine. FMRFamide facilitates acetylcholine release by increasing the presynaptic Ca2+ influx whereas buccalin and histamine have an opposite effect. These neuromodulators control only the influx of Ca2+ through N-type Ca2+ channels since their action on transmitter release can be prevented by omega-conotoxin but not by funnel web spider toxin. FMRFamide and histamine, respectively, increased and decreased Ca2+ influx by shifting in opposite ways the voltage sensitivity to activation of the channels. Buccalin reduced Ca2+ influx by decreasing the number of available channels. 2,5-Diterbutyl 1,4-benzohydroquinone, a blocker of the reticulum Ca2+ pump, increased evoked transmitter release by increasing the intracellular concentration of Ca2+ without affecting the presynaptic Ca2+ influx. It is suggested that a reticulum-like Ca2+ buffer, in close proximity to N- and P-type Ca2+ channels, controls the intracellular concentrations of Ca2+ actually triggering acetylcholine release.

Transmitter release and calcium currents at an Aplysia buccal ganglion synapse--II. Modulation by presynaptic receptors.

Changes in evoked acetylcholine quantal release induced by histamine, FLRFamide and buccalin were investigated at an identified neuro-neuronal synapse in the buccal ganglion of Aplysia californica. Regulation of acetylcholine release by these neuromodulators was correlated with their actions on the presynaptic Ca2+ current. We have previously reported that FLRFamide and histamine, respectively, increase and decrease acetylcholine release from buccal neurons B4/B5. Buccalin, a peptide specific to the buccal ganglion, lowered the number of acetylcholine quanta released. Consistent with the synaptic effects, the presynaptic nifedipine-resistant Ca2+ current that triggers the release of acetylcholine in B4/B5 neurons [Trudeau L.-E. et al. (1993) Neuroscience 53, 571-580] was lowered by buccalin or by histamine and enhanced by FLRFamide. The analysis of tail currents showed that histamine shifts the voltage dependence of the nifedipine-resistant Ca2+ channels towards more positive voltages, whereas FLRFamide has an opposite action. Buccalin did not affect the voltage dependence of the channels but depressed the amplitude of the Ca2+ current, an effect which could be due either to a reduction of the number of available Ca2+ channels, to a decrease of their unitary conductance or to a modification of their gating. Inactivation of presynaptic G proteins prevented the modulatory actions of FLRFamide and histamine on quantal acetylcholine release and also on the voltage dependence of the nifedipine-resistant Ca2+ channels. This procedure, however, failed to prevent the suppressive effects of buccalin. The possibility of relating the voltage dependence shifts of the Ca2+ current induced by FLRFamide and histamine to the phosphorylation state of the Ca2+ channels is discussed. It is concluded that three independent presynaptic pathways initiated by histamine, FLRFamide and buccalin control presynaptic Ca2+ influx, these modulations being apparent within the physiological range of voltages required to activate Ca2+ channels.

Involvement of Ca2+ uptake by a reticulum-like store in the control of transmitter release.

At an identified neuro-neuronal synapse of Aplysia, 2,5-diterbutyl 1,4-benzohydroquinone, a selective blocker of the reticulum Ca2+ pump, was found to potentiate evoked quantal release of acetylcholine through an increased accumulation of Ca2+ in the presynaptic neuron during depolarization without any accompanying changes in the presynaptic Ca2+ current. We conclude that a rapid Ca2+ buffering system, similar to that associated with the endoplasmic reticulum, must be present in the nerve terminal and play a role in the control of Ca2+ which reaches the release system.

Transmitter release and calcium currents at an Aplysia buccal ganglion synapse--I. Characterization.

The Ca2+ current recorded in the presynaptic neuron (B4/B5) of an identified Aplysia synapse was characterized in terms of its activation, voltage sensitivity, Ca2+ dependence of inactivation and pharmacology. It was compared to that recorded in left upper quadrant abdominal ganglion neurons which, unlike B4/B5, display Ca2+ action potentials. The two Ca2+ currents could not be distinguished in terms of their activation threshold or voltage sensitivity. The Ca2+ current recorded in left upper quadrant neurons, however, displayed more important Ca(2+)-dependent inactivation. The peak Ca2+ current in B4/B5 neurons was significantly reduced (30-40%) by the dihydropyridine Ca2+ channel antagonist, nifedipine, while it was increased (15-20%) by the dihydropyridine Ca2+ channel agonist, BAY K8644, although none of these agents had any effect on transmitter release from B4/B5. omega-Conotoxin similarly reduced the Ca2+ current by 30-40%, but unlike nifedipine, it also caused a 50-60% reduction in B4/B5 transmitter release. The pharmacological properties of the Ca2+ current present in left upper quadrant neurons were somewhat different, as this current was unaffected by either BAY K8644 or omega-conotoxin and moderately suppressed (20%) by nifedipine.

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

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 NG-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 at the inhibitory synapse (buccal ganglion), whereas it increased acetylcholine release at the excitatory synapse (abdominal ganglion). The nitric oxide synthase inhibitors, N omega-nitro-L-arginine and NG-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.

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.

Differences in the multiple step process of inhibition of neurotransmitter release induced by tetanus toxin and botulinum neurotoxins type A and B at Aplysia synapses.

In order to gain insights into the steps (binding, uptake, intracellular effect) which differ in the inhibitory actions of tetanus toxin and botulinum neurotoxins types A or B, their temperature dependencies were investigated at identified cholinergic and non-cholinergic synapses in Aplysia. Upon lowering the temperature from 22 degrees C to 10 degrees C, extracellularly applied botulinum neurotoxin type A and B appeared unable to inhibit transmitter release whilst tetanus toxin exhibited a residual activity. Binding of each toxin to the neuronal membrane appeared virtually unaltered following this temperature change. By contrast, the intracellular effects of botulinum neurotoxin type B and tetanus toxin were strongly attenuated by temperature reduction whereas the inhibitory action of botulinum neurotoxin type A was only moderately reduced. Importantly, this discrepancy relates to the known proteolytic cleavage of different synaptic proteins by these two toxin groups. Since both the binding and intracellular activity of botulinum neurotoxin type A are minimally affected at 10 degrees C, its inability to inhibit neurotransmission at this low temperature when applied extracellularly indicated attenuation of its uptake. Due to the strict temperature dependence of the intracellular action of tetanus toxin and botulinum neurotoxin type B, but not A, an examination of the effects of changes in temperature on the internalization step was facilitated by the use of heterologous mixtures of the toxins' heavy and light chains. At 10 degrees C, heavy chain from tetanus toxin but not from botulinum neurotoxin type B mediated uptake of botulinum neurotoxin type A light chain. Collectively, these results provide evidence that, at least in Aplysia, the uptake mechanism for botulinum neurotoxin types A and B differs from that of tetanus toxin.

Control of the calcium concentration involved in acetylcholine release and its facilitation: an additional role for synaptic vesicles?

2,5-Diterbutyl-1,4-benzohydroquinone, a specific blocker of Ca2+-ATPase pumps, increased acetylcholine release from an identified synapse of Aplysia, as well as from Torpedo and mouse caudate nucleus synaptosomes. Because 2,5-diterbutyl-1,4-benzohydroquinone does not change the presynaptic Ca2+ influx, the enhancement of acetylcholine release could be due to an accumulation of Ca2+ in the terminal. This possibility was further checked by studying the effects of 2,5-diterbutyl-1,4-benzohydroquinone on twin pulse facilitation, classically attributed to residual Ca2+. While preventing the fast sequestration of Ca2+ by presynaptic organelles, 2,5-diterbutyl-1,4-benzohydroquinone magnified both twin pulse facilitation observed under low extracellular Ca2+ concentration and twin pulse dysfacilitation observed under high extracellular Ca2+ concentration. Thus, it is concluded that 2,5-diterbutyl-1,4-benzohydroquinone, by preventing Ca2+ buffering near transmitter release sites, modulates acetylcholine release. As 2,5-diterbutyl-1,4-benzohydroquinone was also shown to decrease by 50% the uptake of 45Ca2+ by isolated synaptic vesicles, we propose that synaptic vesicles can control the presynaptic Ca2+ concentration triggering the release of neurotransmitter.

Role of different types of Ca2+ channels and a reticulum-like Ca2+ pump in neurotransmitter release.

The factors controlling the Ca2+ concentration directly responsible for triggering acetylcholine (ACh) release were investigated at an identified neuro-neuronal synapse of the Aplysia buccal ganglion. The types of presynaptic voltage-gated Ca2+ channels associated with transmitter release were determined by using selective blockers such as nifedipine, omega-conotoxin and a partially purified extract from the venom of a funnel web spider (FTx). L-type, N-type and P-type Ca2+ channels are present in the presynaptic neuron. The influx of Ca2+ through both N- and P-types induces the release of ACh whereas Ca2+ flowing through L-type channels modulates the duration of the presynaptic action potential by controlling the Ca(2+)-dependent K+ current. tBuBHQ, a blocker of the reticulum Ca2+ pump, induces a potentiation of evoked release without modifying the presynaptic Ca2+ influx. This seems to indicate that a part of the Ca2+ entering the presynaptic terminal through N- and P-type Ca2+ channels is sequestered in a presynaptic reticulum-like Ca2+ buffer preventing these ions from contributing to ACh release. To exert its control, this Ca2+ buffer must be located close to both the presynaptic Ca2+ channels and the transmitter release mechanism.

Presynaptic receptors for FMRFamide, histamine and buccalin regulate acetylcholine release at a neuro-neuronal synapse of Aplysia by modulating N-type Ca2+ channels.

At an identified neuro-neuronal synapse of the buccal ganglion of Aplysia, quantal release of acetylcholine (ACh) is increased by FMRFamide and decreased by histamine or buccalin. Activation of presynaptic receptors for these neuromodulators modifies a presynaptic Ca2+ current which is nifedipine-resistant and omega-conotoxin-sensitive. The voltage-sensitivity of these N-type Ca2+ channels is increased by FMRFamide and decreased by histamine through the intermediate of G proteins. Buccalin does not implicate G proteins and reduces the Ca2+ current without affecting the voltage-sensitivity of N-type Ca2+ channels. The possibility of relating the shifts in voltage-dependence of the Ca2+ current induced by FMRFamide and histamine to the phosphorylation state of the N-type Ca2+ channels is discussed. A scheme for the complex regulation of ACh release by presynaptic auto- and heteroreceptors is proposed.

G proteins are involved in the regulation of transmitter release at an Aplysia cholinergic synapse.

At an identified cholinergic synapse of the Aplysia buccal ganglion, presynaptic injections of guanosine 5'-O-3-thiotriphosphate (GTP-gamma-S) depressed the amplitude of evoked postsynaptic responses. This reduction of acetylcholine (ACh) release by GTP-gamma-S, prevented by pre-injection of guanosine 5'-O-2-thiodiphosphate (GDP-beta-S) in the presynaptic neuron, was due to a reduction of the number of ACh quanta released. The mean amplitude of the evoked miniature postsynaptic current (MPSC) was unchanged. The presynaptic Ca2+ influx was lowered.

Postsynaptic acetylcholine receptor efficacy is similarly increased by detergents and acetylcholinesterase inhibitors at an Aplysia synapse.

At Aplysia H- and D-type cholinergic neuro-neuronal synapses, application of high concentrations of detergents (Triton X-100 and sodium deoxycholate) depressed synaptic transmission and the postsynaptic response to ionophoretic application of acetylcholine (ACh) or carbachol. However, when very low concentrations of detergents (of the order of 10(-9) M for sodium deoxycholate) were used, the nerve-evoked response as well as the ACh and carbachol ionophoretic responses were facilitated (by at least 200%), but only in H-type cells. This facilitation was similar to that previously observed in the same receptor type when acetylcholinesterase (AChE) was inhibited by various organophosphate or carbamate acetylcholinesterase inhibitors (AChEIs)3. Indeed, the effects of AChEI and detergents were not cumulative. We propose that on H-type synapses detergents may perturb a hypothetical molecular interaction between AChE and the acetylcholine receptor (AChR) by which AChE modulates the ability of the AChR to be activated by ACh or carbachol.

Potassium channels in mouse neonate dorsal root ganglion cells: a patch-clamp study.

Isolated neurons from mouse neonate dorsal root ganglia were analyzed using both whole-cell clamp and single-channel recording techniques and presented a complex repertoire of potassium (K) channels. Different types of potassium channels have been found: calcium-activated K channel presenting a large unit conductance of 260 pS in symmetrical K; voltage-dependent K channels of 130 pS without calcium-dependence; two types of inward rectifying K channels (90 and 120 pS in symmetrical K); low probability K channels; delayed rectifier channels and non-selective cationic channels.

Hemicholinium-3 facilitates the release of acetylcholine by acting on presynaptic nicotinic receptors at a central synapse in Aplysia.

The effects of hemicholinium-3 (HC-3) on acetylcholine (ACh) release were studied on central inhibitory or excitatory synapses of Aplysia californica. HC-3 was used at concentrations below 10(-5) M, which did not affect choline uptake by this preparation. Statistical analysis of the synaptic noise evoked by sustained depolarization of the presynaptic neuron allowed us to calculate the amplitude and mean duration of the miniature postsynaptic responses at an inhibitory synapse in the buccal ganglion. Taking into account the modifications of miniature and evoked responses, it was concluded that HC-3 potentiates ACh release. A similar presynaptic effect was observed at an excitatory synapse in the abdominal ganglion. This facilitation of ACh release was prevented by tubocurarine or hexamethonium, pointing to an agonistic action of HC-3 on nicotinic presynaptic receptors implicated in a positive feedback on ACh release. The possible blockage of muscarinic presynaptic receptors by HC-3 was also considered. Hemicholinium-15 was without effect on ACh release but was nevertheless able to prevent the presynaptic action of HC-3.

Changes in serotonin concentration in a living neurone: a study by on-line intracellular voltammetry.

Intraneuronal concentration of serotonin (5-HT) and its changes were measured in live serotonergic metacerebral cells of Aplysia for several hours following neuronal stimulation, after intracellular injection of 5-HT or extracellular application of L-tryptophan, reserpine, or p-chlorophenylalanine. This was achieved by an on-line intracellular differential pulse voltammetric method using a new, needle-tipped and glass-insulated, platinum microelectrode sensitive to 5-HT.

Xanthine derivatives IBMX and S-9977-2 potentiate transmission at an Aplysia central cholinergic synapse.

In an attempt to investigate the role of cAMP-dependent phosphorylations on synaptic transmission at an Aplysia cholinergic buccal ganglion synapse, the effects of xanthine derivatives such as 3-isobutyl-1-methylxanthine (IBMX), which is well known to inhibit phosphodiesterase activity thereby promoting cAMP accumulation, and a novel xanthine derivative, S-9977-2 were evaluated. They were found to potentiate cholinergic transmission by significantly increasing the time constant of decay (Tc) of inhibitory postsynaptic currents (IPSCs). The postsynaptic origin of the phenomenon was supported by the observation that responses to the ionophoretic application of acetylcholine (ACh) were also potentiated in duration as well as in amplitude. No effects of S-9977-2 on the ACh-gated Cl- channel conductance or mean open time were observed. The finding that responses to the hydrolysis-resistant cholinergic analogue carbachol were unaffected by the two xanthines suggested that the observed effects were at least partly caused by an inhibition of acetylcholinesterase (AChE) activity. That these substances inhibit AChE activity was confirmed in vitro. Phosphorylation processes nonetheless appear to be partly involved in the synaptic effect of the xanthines as the kinase blocker H-8 blocked part of the IPSC Tc lengthening. Possible mechanisms are discussed.