Biomedical subjects

L Tauc

Publications and source records attributed to L Tauc.

At least 55 records · Page 3Linked to original sources

PubMed1989

Multiple domains of botulinum neurotoxin contribute to its inhibition of transmitter release in Aplysia neurons.

The binding, internalization, and inhibition of transmitter release by botulinum neurotoxin (BoNT) was investigated using the intact toxin, its heavy (HC) or light (LC) chains, and a proteolytic fragment thereof. In Aplysia neurons, blockade of acetylcholine release upon external application of BoNT types A or E was prevented by reducing the temperature to 10 degrees C, due to arresting intoxication at the membrane binding step. At this low temperature, type A HC, H2 (comprised of the N-terminal of HC), or H2L (H2 disulfide-linked to LC) antagonized the neuroparalytic action of BoNT A or E, indicating that the latter bind saturably to common ecto-acceptor via the H2 region. In contrast, H2L was unable to counteract BoNT-induced paralysis at the murine neuromuscular junction. In accordance with this species difference, unlike native BoNT, saturable binding of 125I-labeled H2L could not be detected in mammalian peripheral or central nerve terminals. Possibly, more stringent structural requirements form the basis of the toxin's greater effectiveness in inhibiting neurotransmission at mouse nerve muscle synapses than Aplysia nerve terminals. In further identification of functional domains in the toxin, an unprocessed single-chain form of BoNT type E was found to be ineffective when applied extra- or intracellularly to Aplysia neurons. Notably, bath application of the latter to a neuron preinjected with HC, but not H2L or LC, resulted in a blockade of release. This shows that the single-chain species can become internalized and requires, not only LC, but also processed HC for its inhibitory action; consistently, the proteolyzed form of BoNT E was active.

Acetylcholine↗

PubMed1989

Inhibition of transmitter release by botulinum neurotoxin A. Contribution of various fragments to the intoxication process.

1. The contribution of a proteolytic fragment (H2L) of botulinum neurotoxin type A (comprised of the aminoterminal region of the heavy-chain disulphide-linked to the light chain) to inhibition of neurotransmitter release was investigated, using central cholinergic synapses of Aplysia, rodent nerve-diaphragm preparations and cerebrocortical synaptosomes. 2. No reduction in neurotransmitter release was observed following external application to these preparations of highly purified H2L or after intracellular injection into Aplysia neurons. 3. The lack of activity was not the result of alteration in the light chain of H2L during preparation of the latter because (a) renaturation of this light chain with intact heavy chain produced a toxic di-chain form and (b) simultaneous application of heavy chain and light chain from H2L inhibited transmitter release in Aplysia. 4. Bath application of H2L and heavy chain together inhibited release of transmitter; however, at the neuromuscular junction the potency of this mixture was much lower than that of native toxin. A similar blockade resulted when heavy chain was applied intracellularly and H2L added to the bath, demonstrating that H2L is taken up into cholinergic neurons of Aplysia. This uptake is shown to be mediated by the amino-terminal moiety of heavy chain (H2), because bath application of light chain plus H2 led to a decrease in acetylcholine release from a neuron that had been injected with heavy chain. 5. A role within the neuron is implicated for a carboxy-terminal portion of heavy chain (H1) since intracellular injection of light chain and H2 did not affect transmitter release. Although the situation is unclear in mammalian nerves, these collective findings indicate that blockade of transmitter release in Aplysia neurons requires the intracellular presence of light chain and H1 (by inference), whilst H2 contributes to the internalization step.

Acetylcholine↗

PubMed1989

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.

Acetylcholine↗

PubMed1989

Demonstration of functional acetylcholinesterase on the soma of individual neurones of Aplysia by in vivo microspectrophotometry.

The presence of functional acetylcholinesterase is demonstrated in vivo on somatic membranes of single ganglionic neurones of Aplysia using concurrently microspectrophotometry and electrophysiology. The similarity of the effects of an irreversible blocker of acetylcholinesterase and of phospholipase C from Bacillus cereus suggests that acetylcholinesterase is anchored in the membrane via phosphatidylinositol.

Acetylcholinesterase↗

PubMed1988

Involvement of the constituent chains of botulinum neurotoxins A and B in the blockade of neurotransmitter release.

1. The abilities of botulinum neurotoxins, types A and B (single and two-chain forms) to inactivate an intraneuronal component required for transmitter release were quantified in a phrenic-nerve-diaphragm preparation, cerebrocortical synaptosomes or the buccal ganglion of Aplysia californica and compared with the mouse toxicity assay. 2. Homogeneous preparations of the individually renatured polypeptide chains of both toxin types showed low residual toxicity in the whole animal and had no effect on neurotransmission in all three systems, when tested singly. 3. Mixtures of individually renatured heavy chain, from type A or B, and either light chain proved very effective in blocking the evoked release of acetylcholine when bath-applied to the buccal ganglion of Aplysia whilst they were relatively inactive on mammalian nerve terminals, indicating a less efficient uptake of the polypeptides in the latter. 4. When renatured together, the homologous, but not the heterologous, chains of each toxin type yielded toxic, disulphide-linked two-chain species. 5. A role for the heavy chain alone in acceptor recognition and membrane translocation was implicated by the blockade of acetylcholine release produced when light chain was applied to a ganglion of Aplysia previously bathed in heavy chain and washed extensively. No blockade was observed when the order of application of the two chains was reversed. 6. These findings are discussed in the context of the intracellular requirement for both the constituent toxin chains for toxicity, and in the apparent need for these chains to be linked via a disulphide bond for uptake in rodents but not in Aplysia.

Acetylcholine↗

PubMed1988

Both presynaptic nicotinic-like and muscarinic-like autoreceptors regulate acetylcholine release at an identified neuro-neuronal synapse of Aplysia.

The possible involvement of cholinergic presynaptic receptors regulating evoked quantal acetylcholine (ACh) release was investigated at an identified cholinergic neuro-neuronal synapse in the buccal ganglion of Aplysia, using cholinergic agonists (carbachol, pilocarpine, oxotremorine) and/or antagonists (curare, atropine, hexamethonium). Bath applied carbachol or pilocarpine (10(-8) M to 10(-4) M) induced a decrease in the evoked quantal release of ACh. As the effects of carbachol were prevented by atropine (5.10(-6) M) and not by curare (10(-5) M), it was concluded that carbachol activated presynaptic muscarinic-like receptors implicated in a negative feed-back on ACh release. On the contrary, oxotremorine (up to 10(-4) M) induced a potentiation of ACh release which was suppressed by curare (4.10(-6) M) or hexamethonium (10(-5) M) but not by atropine (5.10(-6) M) pointing to the activation of presynaptic nicotinic-like receptors implicated in a positive feed-back on ACh release. Moreover, in the presence of curare, oxotremorine decreased ACh release: this suggested that oxotremorine also activated the presynaptic muscarinic-like receptors. These results revealed the conjoint presence, on the same terminal, of both muscarinic-like and nicotinic-like autoreceptors.

Acetylcholine↗

PubMed1988

Neurotransmitter release is blocked intracellularly by botulinum neurotoxin, and this requires uptake of both toxin polypeptides by a process mediated by the larger chain.

Botulinum neurotoxins (types A and B), which are microbial proteins consisting of two disulfide-linked chains, inhibit specifically and with high potency the release of acetylcholine from peripheral nerve terminals. As a prerequisite for a long-term development of effective treatments for botulism, the internalization and inhibitory action of the toxin and its constituent chains were examined by electrophysiological methods at identified synapses in Aplysia preparations that allow both intracellular and bath application of the neurotoxins. Intracellular recordings from cholinergic cells of the buccal ganglion demonstrated that extra- or intracellular application of low doses of botulinum neurotoxin results in a specific blockade of evoked transmitter release, without changing the quantal size; an intraneuronal site of action has thus been established. In contrast, release from noncholinergic neurons of cerebral ganglion was prevented by the neurotoxin only after injection into the cell. Purified preparations of the individual renatured chains, shown to be nontoxic in a mouse bioassay, failed to affect acetylcholine release when applied extra- or intracellularly. However, inhibition of release was observed after intracellular administration of both chains or when the light chain was injected and the heavy chain was bath-applied. These findings show that both chains are required on the cytosolic side of the neuronal plasma membrane for expression of toxicity and that the cholinergic specificity of the neurotoxin is attributable to its heavy chain, which mediates targeting and subsequent neuronal uptake.

Acetylcholine↗

PubMed1988

[Ganglionic synapses of Aplysia as a model for the study of the mechanism of action of botulinum neurotoxins].

The action of type A and type B botulinum neurotoxin on neurotransmitter release was studied on identified ganglionic synapses of Aplysia. Using this model, we have shown that botulinum neurotoxins at concentrations used in vertebrate preparations had the same specificity of action and that both heavy and light chains of these toxins are intracellularly required to inhibit neurotransmitter release.

Action Potentials↗

PubMed1987

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.

Acetylcholine↗

PubMed1987

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.

Aplysia↗

PubMed1987

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.

Animals↗

PubMed1987

Properties of miniature postsynaptic currents during depolarization-induced release at a cholinergic neuroneuronal synapse.

1. Miniature postsynaptic currents were analyzed at an inhibitory cholinergic neuroneuronal synapse in the buccal ganglion of Aplysia. Under double voltage-clamp, it was possible to induce postsynaptic currents by long-duration depolarizations of the presynaptic neuron and to analyze these as the linear summation of individual miniature postsynaptic currents (MPSCs). The amplitude of these miniature currents (imin) was calculated from the ratio of the variance of the noise (E2) to the mean of the postsynaptic current (Im), according to Campbell's theorem, with imin = 2E2/Im. Their decay time (tau min) was obtained from the cutoff frequencies of the power spectra obtained from the noise. 2. Neither the conductance nor the decay time of MPSCs was voltage dependent. However, imin appeared to decrease when the quantal content of the response increased. Meanwhile, tau min increased slightly with Imin. 3. Carbamylcholine was injected into the neuropile and this led to a decrease in imin and a slight increase in tau min. 4. Power spectra obtained after the application of inhibitors of acetylcholinesterase (AChE), with or without curare, suggested that acetylcholine (ACh) does not accumulate during large depolarizations. 5. The possible origin of the nonlinear relationship between the variance and the mean of the postsynaptic currents is discussed.

Acetylcholine↗

PubMed1987

Modulation of an acetylcholine receptor responsiveness by filipin and chlorpromazine studied in neurons of Aplysia californica.

The responsiveness of Aplysia acetylcholine receptors (AChR) was studied using a polyene antibiotic, filipin, which specifically complexes cholesterol, and another compound, chlorpromazine (CPZ), which inserts at the proteolipidic interface. Both substances enhanced the evoked postsynaptic responses or responses to iontophoretic application of carbachol only on the H-type receptor (opening a Cl-permeability), whereas at the same concentrations filipin was without effect on the D-type receptor (opening a cationic permeability) while CPZ depressed the D-type response. The facilitation observed specifically for the H-type receptor was similar to that previously described after acetylcholinesterase (AChE) inhibition or when low concentrations of detergents were applied to this preparation. No additive effect was obtained after the addition of chlorpromazine following a maximal potentiation obtained with an anticholinesterase agent. Since at Aplysia central neurons, AChE is a membranal protein, we propose that the facilitation of H-type responses is attributable to the removal of a modulatory action of AChE on AChR. Filipin or chlorpromazine might disrupt the interaction between AChR and AChE.

Animals↗

PubMed1987

Presynaptic actions of curare and atropine on quantal acetylcholine release at a central synapse of Aplysia.

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.

Acetylcholine↗

PubMed1986

Acetylcholine-activated currents in mouse neuroblastoma cells.

The nicotine and muscarinic responses of differentiated mouse neuroblastoma cells from the clonal line N1E 115 to applied cholinergic agents were recorded using single channel and whole cell patch clamp techniques. An inward macroscopic current induced by acetylcholine (ACh) at the resting potential was blocked by curare; cell-attached recordings revealed a single channel conductance of 18 pS and a lifetime of 36 ms at 30 degrees C, with 200 nM ACh. The zero current potential was close to 0 mV. The kinetics of these nicotinic currents were described by multiexponential functions for both the open and closed time distributions. An outward single channel current, present at resting and slightly depolarized potentials, was also observed and has been tentatively described as being dependent on muscarinic receptor activation, as it was usually blocked by atropine. Under our conditions of whole cell clamp, no macroscopic outward current sensitive to ACh was observed.

Acetylcholine↗

PubMed1986

Fasciculin II, a protein inhibitor of acetylcholinesterase, tested on central synapses of Aplysia.

Fasciculin II, a potential inhibitor of acetylcholinesterase (AChE), was tested on two types of Aplysia cholinergic receptors: H type, opening Cl- channels; and D type, opening cationic channels. Evoked postsynaptic inhibitory responses and responses to ionophoretic application of acetylcholine (ACh) or carbachol onto H-type receptors were potentiated in the presence of fasciculin II at 10(-9) M, whereas the same concentration of this drug was without effect on the evoked postsynaptic excitatory responses or on the application of ACh or carbachol on D-type receptors. The observed effects of fasciculin II were identical to those obtained with other inhibitors of AChE on the same preparation. The facilitatory effect on the carbachol response in H-type cells indicates that fasciculin II, as other AChE inhibitors, does not act on H-type synapses solely by blocking the hydrolysis of ACh. We concluded that fasciculin II was a good inhibitor of acetylcholinesterase on neuronal preparations in vivo. The results are further discussed as a new element in favor of a previously proposed hypothesis of a molecular interaction between AChE and ACh H-type receptors.

Acetylcholine↗

PubMed1986

Presynaptic transmitter content controls the number of quanta released at a neuro-neuronal cholinergic synapse.

In the buccal ganglion of Aplysia the overloading of the cholinergic presynaptic neuron by exogenous acetylcholine (AcCho) led to an enhancement of the postsynaptic response. The deprivation of choline in the presynaptic neuron by extra- and/or intracellularly applied choline oxidase to prevent AcCho synthesis resulted in a decrease of the postsynaptic response. In both cases, the size of the calculated miniature postsynaptic current (i.e., the size of the quantum) remained unchanged. It was concluded that, for a given stimulation, the number of quanta released (i.e., the quantal content) is directly related to the quantity of AcCho available for release in the presynaptic neuron.

Acetylcholine↗

PubMed1986

Quantal analysis of action of hemicholinium-3 studied at a central cholinergic synapse of Aplysia.

The effects of hemicholinium-3 (HC-3) on cholinergic transmission were studied on central identified inhibitory (H-type post-synaptic cell, Cl- channels) and on excitatory (D-type post-synaptic cell, cationic channels) synapses of Aplysia californica. In the H-type post-synaptic cell, the amplitude and the decay time of miniature post-synaptic currents (m.p.s.c.s.) were calculated by statistical analysis of long duration induced post-synaptic current (l.d.i.p.s.c.) due to 3 s depolarizations of the presynaptic neurone in the presence of tetrodotoxin. On H-type receptors, with respect to acetylcholine (ACh), HC-3 acted as an agonist and a blocker whereas on D-type receptors, it acted only as a blocker. At low concentration of bath-applied HC-3, in the H-type synapse, the decay time of the evoked inhibitory post-synaptic current (i.p.s.c.) as well as that of the m.p.s.c. was lengthened. These changes were rapidly reversible by wash. The decay time of excitatory post-synaptic current (e.p.s.c.) at the D-type synapse was not affected. On the inhibitory synapse, HC-3 applied in the bath at the concentration of 10(-5) M, reduced considerably the size of the m.p.s.c.s whereas the evoked i.p.s.c.s and the l.d.i.p.s.c.s were only slightly affected pointing to an increase of the quantal content of both responses. After wash, both i.p.s.c.s and l.d.i.p.s.c.s showed a clear facilitation which persisted for several tens of minutes. The presence of presynaptic receptors was considered. Similar facilitation of e.p.s.c.s by HC-3 was observed at the D-type synapse. The comparison of the degree of depression by HC-3 of the m.p.s.c.s and of the responses to ionophoretically applied ACh, indicated that the size of the quantum was not changed. Intracellular injection of HC-3 into the presynaptic neurone of the H-type synapse led to a decrease of transmitter release which affected solely the quantal content of the responses. As the synaptic transmission could not be restored by injection of exogenous ACh into the presynaptic neurone, it was concluded that the depression of transmission was not due to a decrease of ACh synthesis.

Acetylcholine↗