Proc. NatL Acad. Sci. USA Vol. 80, pp. 5126-5128, August 1983 Neurobiology Carbachol can be released at a cholinergic ganglionic synapse as a false transmitter (acetylcholine/acetylcholinesterase/quantal release/intracellular injection) G. BAUX AND L. TAUC Laboratoire de Neurobiologie cellulaire, Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette, France Communicated by S. Hagiwara, May 3, 1983 ABSTRACT Carbachol was injected into a presynaptic cho- itory Cl--dependent postsynaptic responses in a nearby group linergic neuron in the buccal ganglion of Aplysia and the quantal of neurons (6). The pre- and postsynaptic cells were each pen- aspects of the ClP-dependent postsynaptic response to a pro- etrated by two separate low-resistance 3 M KCI-filled elec- longed stimulation were analyzed by a statistical fluctuation method. trodes (1-5 MW). The calculated amplitude of the miniature postsynaptic current Transmission was studied by using a method described by was increased with respect to control. Statistical fluctuation anal- Simonneau et aL (7). Both pre- and postsynaptic cells were si- ysis was also used to analyze the postsynaptic response obtained during ionophoretic application of acetylcholine and carbachol. multaneously voltage clamped to holding potentials of -50 mV The calculated unitary channel current was found to be greater and -80 mV, respectively. In the presence of TTX (0.1 mM), for carbachol than for acetylcholine. This increase could explain a 3-s step depolarization of the presynaptic neuron induced a the larger miniature postsynaptic current seen after intracellular postsynaptic response (LDIPSC, long depolarization-induced injection of carbachol into the presynaptic neuron if carbachol postsynaptic current), which showed fluctuations or "noise. " was released at the synapse as a false transmitter. This conclusion The amplitude of the miniature postsynaptic currents (MPSC) was supported by the observation that it was possible to restore was calculated by using Campbell's theorem, in which the size transmission at a synapse previously blocked by presynaptic in- of the unitary element or individual MPSC (i) is related to the tracellular injection of acetylcholinesterase with a presynaptic in- variance of the noise (E2) and the mean observed current change jection of carbachol. (I) by the equation: i = 2E2/I (8). Agonists were ionophoretically applied to the cell body of The specificity of the synaptic release mechanism is not ab- the postsynaptic neuron bearing AcCho receptors (9). The same solute and substances with molecular formulae relatively close equation was used to calculate the current crossing a single to the natural transmitter can be liberated. At cholinergic syn- channel opened by AcCho or carbachol, with the omission of apses, precursors like homocholine, monoethylcholine, and the factor 2, because of the pulse-like aspect of the elementary triethylcholine are taken up by cholinergic nerve terminals, current (8, 10). In all experiments, the null potential (-40 to acetylated, and released as false transmitters. This property has -55 mV) of the postsynaptic responses was continuously mon- been used as a tool to understand the mechanism of transmitter itored and the currents were expressed as conductance. release (1-4). A Fourier transform was used to calculate the power density We have investigated whether the well-known acetylcholine spectra from which the decay time constant of the MPSC and (AcCho) analogue, carbachol (carbamoylcholine chloride) can the channel open time were estimated (7). As previously shown, be released at a cholinergic synapse. Carbachol has the inter- the MPSC spectra could be approximated by single Lorentzian esting property of not being hydrolyzed by acetylcholinesterase curves (7), whereas spectra of Cl- channel noise could only be (AcChoE) and as such can be used as a pharmacological tool in fitted by double Lorentzians (11, 12). Thus, it may be hazard- the presence of large quantities of this enzyme. Carbachol is ous to give absolute values of the channel open time; the values not taken up by cholinergic terminals and having no acetyl group, reported here are indicative and concern only the slower com- it cannot be synthesized in the cytoplasm. We have overcome ponent. this difficulty by injecting carbachol into a presynaptic cholin- AcCho and carbachol were intracellularly injected by ion- ergic neuron of Aplysia buccal ganglion. We have found that it tophoresis with a constant current. Postsynaptic current re- can act as a false transmitter and can also replace AcCho at a sponses used for calculation and presented in the figures were synapse previously blocked by intracellular injection of Ac- obtained with 3-s depolarizations of the presynaptic neuron to ChoE (5). 0 mV. However, the difference in the actions of AcCho and car- bachol shown below was observed for other levels of presyn- MATERIALS AND METHODS aptic depolarization. AcChoE (Worthington) was injected as a 5% solution (wt/vol) Experiments were performed at room temperature (20-22°C) by an air pressure system on the buccal ganglion of Aplysia californica obtained from R. (5). Fay (Pacific Biomarine, Venice, CA). The ganglion was dis- sected free of connective tissue and perfused with artificial sea RESULTS water (460 mM NaCl/10 mM KCl/11 mM CaCJ2/25 mM MgCl2/ Fig. 1 illustrates the postsynaptic responses obtained when either 28 mM MgSO4/10 mM Tris-HC1, pH 7.8). Tetrodotoxin (TTX) AcCho or carbachol was injected into the presynaptic neuron. (Sigma) at 0.1 mM was added to block Na+ channels. Cells used After a delay, both drugs increase the amplitude of the mean were two cholinergic interneurons, which each induce inhib- postsynaptic current, and this increase is related to the amount The publication costs of this article were defrayed in part by page charge Abbreviations: AcCho, acetylcholine; AcChoE, acetylcholinesterase; TTX, payment. This article must therefore be hereby marked "advertise- tetrodotoxin; LDIPSC, long depolarization-induced postsynaptic cur- ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. rent; MPSC, miniature postsynaptic currents. 5126 Downloaded by guest on September 27, 2021 Neurobiology: Baux and Taue Proc. Natl. Acad. Sci. USA 80 (1983) 5127 A B to the enzymatic action of AcChoE (11). On the other hand, AcCho-activated channels may be differently affected by AcCho and carbachol. When AcCho and carbachol were ionophoresed at the same spot on the postsynaptic cell by using a double-bar- reled electrode, we found that the single channel conductances calculated for the (inhibitory) responses having the same mean current were different (Fig. 2). The mean (±SD) values ob- served in 15 experiments were 3.4 ± 0.8 pS for AcCho and 5.2 ± 1.1 pS for carbachol. In contrast, the calculated mean open time of the slower component of the Cl- channel was some- what shorter (by about 25%) with carbachol (10 ± 2 ms) than with AcCho (14 ± 2 ms). To ascertain that the difference in conductance of inhibitory channels activated by AcCho and carbachol was not an ex- -J perimental artefact, we used the same channel analysis in other Aplysia cells having excitatory receptors opening cation-selec- tive "sodium" channels (13). We found that these channels, whether opened by AcCho or carbachol, have essentially the same amplitudes but a shorter lifetime with carbachol, as al- ready shown by others (13). It should be mentioned that mean FIG. 1. Postsynaptic current response after sustained depolariza- single channel conductances and lifetimes computed from in- tions of the presynaptic neuron to 0 mV. Upper traces, DC recordings; lower traces, AC recordings at higher gain of the current fluctuations tegrated spectral densities for current fluctuations in frog end (calibrations, 4 nA for DC and 1 nA for AC recordings; time, 1 s). (A) plates were dependent on the agonists used (14). 1, test response; 2, response after a 30-min injection of carbachol with Another possible mechanism responsible for the increase in 50-nA constant current; 3, after another 30-min injection of carbachol. MPSC amplitude after carbachol injection could reside in a fa- The calculated amplitude of the MPSC was 1.09 nS in 1, 1.54 nS in 2, cilitatory action of a "leakage" or a nonquantal release of car- and 1.95 nS in 3. (B) In another preparation similar to the experiment bachol, shifting the sigmoid amplitude/concentration curve of inA, but AcCho was injected instead ofcarbachol. The amplitude ofthe MPSC remained unchanged (0.5 nS) throughout. The increase in am- released AcCho to the right (15). To test this possibility, we ana- plitude of the LDIPSC after AcCho injections is due exclusively to an lyzed the LDIPSC of noninjected preparations in the presence increase in the number of quanta released, whereas injected carbachol of carbachol (1 tkM to 0.1 mM) in the perfusing medium. In no increases both the quantal content and the size of MPSC. case was an increase in MPSC amplitude calculated. Thus, the larger unit conductance induced by carbachol on of the drug introduced into the presynaptic cell. Because in- the postsynaptic membrane explains the increase in amplitude tracellular ionophoresis increases the intracellular concentra- of the MPSC calculated after injection of carbachol into the tion of the drugs progressively, it is impossible to quantify the presynaptic neuron. After the injection of carbachol, each delay. Also, the final intracellular concentration reached after quantum released probably consists of a mixture of AcCho and injection cannot be precisely calculated, as the transfer number carbachol. The proportion of carbachol increases with the of the injection electrode is not known. It can be estimated as quantity of carbachol injected. being on the order of millimolar. The injected neuron showed The other method we used to show release of carbachol from a decrease in membrane potential, and when voltage clamped, the presynaptic neuron takes advantage of the fact that release the outward, presumably late K current was slightly decreased of AcCho is abolished by injection of AcChoE into the pre- during the depolarizing pulse.