Proc. Nat. Acad. Sci. USA Vol. 72, No. 8, pp. 3097-3101, August 1975 Biophysics 1-Pyrene-butyrylcholine: A fluorescent probe for the system (long-lifetime fluorescent probe/neuromuscular junction/ /visualization of synapse) F. J. BARRANTES, B. SAKMANN, R. BONNER, H. EIBL, AND T. M. JOVIN Departments of Molecular Biology, Neurobiology, and Biochemical Kinetics, Max-Planck-Institut fur Biophysikalische Chemie, D-3400 Gottingen-Nikolausberg, West Germany Communicated by Manfred Eigen, May 19,1975

ABSTRACT The action of I-pyrene-butyrylcholine, a probes having intrinsically long excited-state lifetimes along new cholinergic fluorescent probe, as been studied at the with a high sensitivity to environmental factors. With this in cellular level using electrophysiological and fluorescence mind, 1-pyrene-butyrylcholine (PBC) was synthesized, and techniques. The spectroscopic properties of the probe were its action studied at the cellular level under physiological found to be similar to those of pyrene-butyric acid, the ex- cited-state lifetime in air-saturated aqueous solutions being conditions. 92 nsec. At micromolar concentrations the probe was found The fluorescent probe behaves as a nondepolarizing, re- to exert a nondepolarizing, reversible blocking action at the versible blocking agent in a nicotinic synapse, it specifically neuromuscular junction of the frog. The same cholinolytic ef- labels the neuromuscular junction, and it possesses a long ex- fect was observed in hypersensitive denervated muscles. cited-state lifetime. The -like cholinergic activity of The synaptic localization of the probe could be observed make it therefore ap- with fluorescence microscopy using sub- and micromolar PBC and its spectroscopic properties concentrations. Treatment of the nerve-muscle preparations propriate for studies of the rotational mobility and intermo- with proteolytic enzymes, resulting in the separation of the lecular interactions of the solubilized and membrane-bound nerve ending from the muscle end-plate, enabled a distinc- AChR and the -ester hydrolases, as well as the kinet- tion to be made between the fluorescence arising from these ics of binding to all these macromolecular entities. two parts of the synapse. Intense presynaptic fluorescence was observed, and was not altered by micromolar concentra- tions of a-, d-tubocurarine, hemicholinium, or MATERIALS AND METHODS inhibitors. Faint reversible staining of the end-plate region was observed in enzymically treated mus- Synthesis of 1-Pyrene-butyrylcholine (PBC). (i) 1-Py- cles and was inhibited by prior treatment with a-bungarotox- rene-butyrylchloride (): A solution of 576 mg (2 mmol) of in. Fluorescent a-toxin revealed similar patterns of fluores- 1-pyrene-butyric acid (Eastman) in 15 ml of dry chloroform cence in the end-plate of enzyme-treated muscles. The post- (refluxed for 1 hr over P205 and distilled) and 1.5 g (12 synaptic localization of the fluorescent probe is therefore tentatively identified as the one producing the cholinolytic mmol) of oxalylchloride were refluxed for 4 hr. Benzene, 45 effect upon binding to sites. ml, was added to the reaction mixture. The solvents were evaporated at 300, together with the excess oxalylchloride. Fluorescence techniques have been widely applied to the The residue, a mixture of I and oxalic acid, was treated with study of biomembranes and model systems (1). In the partic- 10 ml of dry chloroform to dissolve the formed 1-pyrene- ular case of excitable membranes, considerable attention has butyrylchloride. been paid to changes of fluorescence intensity occurring (ii) 1-Pyrene-butyric acid-bromoethyl ester (II): The solu- during nerve conduction (2, 3) or electrical excitation of the tion of I (2 mmol) in chloroform was added dropwise at 200 electroplax (4). In all cases, however, nonspecific fluorescent to a thoroughly stirred mixture of 1 g (8 mmol) of 2-bro- probes have been used, with consequent limitations on the moethanol (freshly distilled), 15 ml of dry chloroform, and 1 biological interpretability of the findings. g (10 mmol) of triethylamine. Stirring of the reaction mix- The introduction of a specific cholinergic fluorescent ture was continued for 1 hr at 200. The solvents were evapo- probe (1-dimethylaminonaphthalene-5-sulfonamidoethyltri- rated at 300, and the residue was dissolved in 30 ml of di-iso- methylammonium perchlorate, DNETMA, ref. 5) has great- propyl ether and treated with 30 ml of 0.5 M HCl to transfer ly contributed to the study of acetylcholine receptor (AChR) traces of triethylamine into the aqueous phase. The ether (6, 7). This dansyl derivative, however, has a short fluores- phase was dried over sodium phosphate and filtered. The fil- cence lifetime (Barrantes, unpublished results) and a rela- trate was evaporated to dryness at 300 and the residue (II) tively complex pharmacological activity (7). Some of the was dissolved in 20 ml of acetone. physico-chemical properties of large macromolecular en- (iii) 1-Pyrene-butyrylcholine (III): The solution of II in tities, such as AChR or acetylcholine hydrolase (EC 3.1.1.7) acetone was mixed with 10 ml of trimethylamine (a 33% so- (AChE), can be more conveniently studied with fluorescent lution in ethanol) and allowed to react at 500 for 12 hr (8). The mixture was evaporated to dryness and the residue was Abbreviations: PBC, 1-pyrene-butyrylcholine (1-pyrene-butyric recrystallized from acetone and dried under reduced pres- acid-choline ester); ACh, acetylcholine; AChR, acetylcholine recep- product, PBC, was dissolved in chlo- tor; AChE, acetylcholine hydrolase (EC 3.1.1.7); BuChE, acylcho- sure. The recrystallized line acyl-hydrolase (EC 3.1.1.8); a-BT, a-bungarotoxin; FITC-a-BT roform/methanol/water (200:15:1 by vol) and chromato- fluorescein isothiocyanate a-bungarotoxin; DNETMA, 1-dimethyl- graphed through a silicic acid column (Silicar CCR 7, Mal- aminonaphthalene-5-sulfonamidoethyltrimethylammonium per- linckrodt) using solvents of increasing polarity. PBC eluted chlorate; m.e.p.ps, miniature endplate potentials. with a 65:15:1 (v/v) mixture, with an overall yield of 65%. 3097 Downloaded by guest on October 4, 2021 3098 Biophysics: Barrantes et al. Proc. Nat. Acad. Sci. USA 72 (1975)

c Q25- 0 I 276.5 S/CH3 !1 CH 2(?CH2CH 2CCH2CH 2N-CH3 Br" In- I) 1- 3 z w (I W&A0MOAif-, -Ml %1%44**14- r 342.5 378 -1.0 -z G (t) cIc- ITT "m Ir-ll-"---W"- Alul A w l 0 -ccX) wz cn z 0.15- Il I, I' I1 327 Z, 265 1 III1 pi w IL-) 0. I A I I I 0 0 - 311 F(t) co -0.5 -L w loel 1, I Jo I I1 50 NSEC I 1 311 1 6- CD Q05- w I I

-- CD_1- t It106 212 318 424 530 636 74282 8 CHRNNEL 240 260 280 300 320 340 360 380 400 420 440 460 FIG. 1. Structure of 1-pyrene-butyrylcholine bromide (MW 472.4), together with the absorption and fluorescence spectra of a 5.3 MM solution of the probe in 100 mM phosphate buffer (pH 7.0) 0 10 21 3~~18 42 50 3 72 4 (20°). The technical fluorescence emission spectrum ( ) of the same solution is also shown (excitation wavelength, 342.5 nm). The dotted line corresponds to the absorption spectrum. STD]. 0EV I T I ON FIG. 2. Nanosecond fluorescence decay of 5 MM PBC mn air- All steps of the synthesis and the purity of the final deriva- equilibrated frog's Ringer solution. Excitation wavelength: 337 nm. tive were assessed by thin-layer chromatography on silica A Schott KV 360 filter was used on the emission. The upper graph gel using various chloroform/methanol/water mixtures. shows the time histogram of the detected fluorescence photons F(t) and the scattered excitation photons G(t) relative to the tim- C25H3003NBr (PBC monohydrate) ing-pulse of the excitation flash. The smooth curve fitting the ex- Calcd.: C 63.56; H 6.40; N 2.96; Br 16.92 perimental response decay F(t) is the reconstituted convolution of Found: 63.53; 6.54; 2.84; 17.02. the flash and a single exponential fluorescence decay of 92 nsec. The lower graph (in expected sigma) shows the deviation D(t), Interaction of PBC with Choline-esterases. A purified normalized to the expected counting error. The upper insert (A-C) preparation of the specific AChE from Torpedo californica corresponds to the autocorrelation function of the error A(t) = f (kindly provided by Dr. P. Taylor, Univ. of California at San D(T) D(r + t)dr. This function is a sensitive measure of any sys- Diego) and horse serum acylcholine acyl-hydrolase (EC tematic errors~in the fit. In the present case, the fit to the single 3.1.1.8) (BuChE) (Sigma Type IV) were assayed according exponential seems to be appropriate to a high degree of accuracy. to Ellman et al. (9) in the presence of PBC. Spectroscopic Determinations and Data Analysis. Ab- nonoverlap of the thick and thin filaments, as assessed by sorption spectra were recorded with a Cary 16 spectrometer. measurement of the optical diffraction patterns of the mus- Fluorescence spectra were made with a FICA 55 spectroflu- cle fibers using a He-Ne laser. Fluorescence was observed orimeter, which gives quantum-corrected excitation (220- with a Zeiss Universal microscope using incident illumina- 550 nm) and emission (200-800 rm) spectra. Fluorescence tion of the surface muscle fibers with a XBO 150 W/i lifetime determinations were made by the single-photon lamp. A UG5 (Arabs33 nm) and a BG3 (X,na,360 nm) time correlation method with an Ortec 9200 nsec decay ap- Schott filter combination was used for exciting PBC fluores- paratus and a Fabritek 1074 multichannel analyzer, con- cence, and a 418 rim long-pass filter was placed on the emis- nected on-line to a PDP 11-20 computer. Single-exponential sion side. Photomicrographs were taken using Kodak Tri-X decay analyses were performed using a phase-plane method and Ektachrome films. (10). Multi-exponential analyses were done using a method Enzymic Treatment of the preparations was done using 1 of modulating functions (ref. 11 and G. Striker, in prepara- mg/mI of collagenase (Sigma Type I) for 45 mmn followed tion). by 0.1 mg/ml of protease (Sigma Type VIII) for 15-40 mmn Electrophysiological Measurements. Nerve-muscle (16). The histochemical staining for AChE was performed preparations, and in some experiments 6-week denervated according to ref. 17. muscles (cutaneous pectoris from the frog Rana esculenta) RESULTS were used. Preparations were bathed in normal frog's Ring- er-solution [114 mM NaCl, 2.5 mM KC1, 1.8 mM CaCI2, 5 Structure and spectroscopic properties of PBC mM sodium phosphate buffer (pH 7.1), 5 mM glucose] at The structure of PBC and its absorption and fluorescence room temperature. In some experiments neostigmine methyl spectra are shown in Fig. 1. The characteristic absorption sulfate (3 gM) was added to inhibit AChE activity. End- bands of the pyrene moiety are conserved in the intermedi- plates from the medial edge of the muscle were localized by ate 1-pyrene-butyric acid (18) as well as in the final tri- the rise-time of intracellularly recorded miniature endplate methylammonium derivative. Both compounds also have potentials (m.e.p.ps). Membrane potentials were recorded similar fluorescence emission bands, at 378 and 398 nm. with 3 M KCl-filled glass microelectrodes of 10 MO resis- PBG fluorescence decays monotonically in aqueous media. tance. Acetylcholine (ACh)-sensitivity of endplate regions In frog's Ringer solution equilibrated with air, mono- and was measured by iontophoresis of ACh (12). multi-exponential decay analyses yielded an excited-state Fluorescence Labeling of the Synaptic Region. a-Bun- lifetime of about 92 nsec (Fig. 2). garotoxin was purified from lyophilized venom of Bungarus Pharmacological action of PBC on neuromuscular multinctus (13). Fluorescein isothiocyanate a-bungarotox- transmission in (FITC-a-BT) was prepared according to ref. 14. The Upon incubation of the nerve-muscle preparation with 8 AM nerve-muscle preparation was stretched to the region of PBG, m.e.p.ps, due to the spontaneous release of A~h (19), Downloaded by guest on October 4, 2021 Biophysics: Barrantes et al. Proc. Nat. Acad. Sci. USA 72 (1975) 3099 a

FIG. 3. Recording of membrane potential at a neuromuscular FIG. 5. Visualization of the neuromuscular junction of the frog junction before, during, and after bath application of PBC (2 uM with 0.8 MM PBC applied for 15 min and followed by 2-min wash- for 15 min). Neostigmine methyl sulfate (3 MM) was present ing with Ringer solution (a). The histochemical reaction for AChE throughout. Before application of PBC (top 3 traces) m.e.p.ps with (17) was subsequently carried out. The copper ferrocyanide pre- an amplitude of 0.48 4 0.21 mV (mean i SE, n = 55) and a mean cipitate, the final reaction product of the enzymic hydrolysis of frequency of 2.5/sec were recorded. Application of PBC rendered acetylthiocholine, outlines the edges of the same synapse (b). Cali- m.e.p.ps undetectable within 15 min of application (middle 3 trac- bration bars indicate 20Mgm. es). After washing out the probe, m.e.p.ps could again be detected and had a mean amplitude of 0.51 I 0.21 mV (n = 53) and a mean frequency of 1.9/sec (bottom 3 traces). Time calibration: 400 msec; Interaction of PBC with voltage calibration: 1 mV. In view of the lower PBC concentrations needed to block decreased in amplitude progressively with time, without al- m.e.p.ps in the presence of neostigmine, the susceptibility of tering their frequency, and within 10-15 min became unde- the probe to enzymatic hydrolysis was investigated. When 1 tectable. In the presence of 3 MM neostigmine, 2 MM PBC mM PBC was incubated with 50 units/ml of AChE from produced the same reversible effect (Fig. 3). Concomitantly, Torpedo californica, an amount of enzyme sufficient to a decrease of the sensitivity of the postsynaptic membrane to completely hydrolyze acetylthiocholine at comparable con- iontophoretically-applied ACh was observed. The sensitivity centrations within 3 sec, no perceptible hydrolysis of PBC was also restored upon washing the preparation. Resting po- was detected. Although not a substrate, PBC was found to be tentials and input resistances were not affected by 1-8 ;&M an inhibitor of AChE; 15 IiM PBC reduced the initial rate of PBC, and normal action potentials could be elicited by di- hydrolysis of 37 uM acetylthiocholine by 50%. Complete hy- rect electrical stimulation of the muscle fibers (Fig. 4). drolysis of 20 1AM PBC to pyrene-butyric acid and choline The ACh-sensitivity of hypersensitive denervated. fibers was observed upon incubation of the probe with BuChE (1 was measured for different concentrations of PBC at the for- mg/ml) for 1 min at 37'. mer end-plate region and at hypersensitive extra-junctional Visualization of the synaptic region with fluorescence areas. Junctional as well as extra-junctional sensitivity of all microscopy muscle fibers was reduced to less than 10% of the control values after treatment with 8 MM PBC for 15 min. The ACh- In parallel with the electrophysiological experiments, the lo- sensitivity was fully restored after washing the preparations. calization of the fluorescent probe was studied in the nerve- Application of PBC for longer periods (30 min- hr) re- muscle preparation using fluorescence microscopy. After sulted in a 2- to 10-fold increase in the frequency of the identification of the final unmyelinated nerve branches with m.e.p.ps, which persisted for at least 1 hr after washout. interference contrast (15), 0.1-1.0 uM PBC was applied; Concentrations of PBC higher than 25 AM produced a de- within 2-15 min a specific pattern of fluorescence devel- crease of resting membrane potentials and input resistances oped (Fig. S a) which coincided with the end-plate region as of the muscle fibers. Local contractions were also observed revealed by the histochemical staining for AChE (17) (Fig. 5 at high concentrations. b). Similar patterns of fluorescence were observed when muscles were treated with 2 ,M FITC-a-BT for 45 min or with 2-5 AM DNETMA for 15 min, except that glial cells were often intensely stained with PBC or DNETMA but not with the fluorescent a-toxin (Fig. 6). Resolution of the synaptic fluorescence into its pre- and postsynaptic components Enzyme treatment of the nerve-muscle preparations re- sulted in the dissociation of the nerve endings from their at- tachment on the end-plate (16). Under such conditions, 0.7 M&M PBC depicted intense pre- and faint postsynaptic fluo- FIG. 4. Resting and action potentials in a muscle fiber exposed rescence. Since enzyme treatment eliminates AChE activity to PBC (8 AM for 15 min). The muscle fiber was stimulated with a from the synaptic region (16, 20, 21), the end-plate fluores- second intracellular electrode near the recording site. Square puls- cence should correspond to AChR sites and should account es of current of increasing amplitude were applied and elicited subthreshold electrotonic potentials. A suprathreshold stimulus for the found blocking action. In order to test this hypothe- evoked a normal muscle action potential. Time calibration: 2 msec; sis, enzymically digested preparations were incubated with voltage calibration: 50 mV. 1-2 MM native a-bungarotoxin (a-BT) before PBC (1-5 MM) Downloaded by guest on October 4, 2021 3100 Biophysics: Barrantes et al. Proc. Nat. Acad. Sci. USA 72 (1975) cal activity of' PBC and its localization. Presynaptic fluores- cence was not inhibited by a-toxin, hemicholinium, or cho- linesterase inhibitors, and persisted for at least 24 hr after washing PBC. This long-term fluorescence may be related to the increase in the frequency of the m.e.p.ps observed after long applications and/or high concentrations of PBC. Since the frequency of the m.e.p.ps is a function of the presynap- tic membrane polarization (23, 24), the frequency increase probably reflects the irreversible binding of PBC to this membrane. Whether the probe is also incorporated into the nerve terminal is not known, although preliminary results (M. J. Dowdall, personal communication) indicate a marked inhibitory effect of PBC on the postulated choline uptake mech nsm (25) in isolated nerve-endings from Torpedo. Preliminary experiments from our laboratory also showed FIG. 6. Longitudinal branches of a neuromuscular junction high-affinity binding for AChR- and AChE-rich membrane after application of 8 I'M PBC for 15 min in the presence of 1 #M neostigmine (a). FITC-a-BT (2 MM) rendered similar patterns of fragments from the same source. The localization of the fluorescence. (b) Another view of a neuromuscular junction where PBC fluorescence in the intact tissue may thus be the result the longitudinal and transversal branches are observed, together of multiple binding to various synaptic structures. That the with the Schwann cells (arrows). (8 MM PBC, 20 min). DNETMA latter include putative AChR sites on the presynaptic mem- (2-5 MM, 15 min), the dansyl cholinergic derivative (5), produced brane and the Schwann cells cannot be ruled out by our ex- the same pattern of fluorescence, also staining the glial cells. Cali- periments; however, no inhibitory effect of a-BT nor stain- bration bar, 20 Mm. ing with FITC-a-BT was observed in these structures. The application. No fluorescence was observed in the end-plate fluorescence of the Schwann cells may be accounted for by of these preparations. Further indication that the end-plate their high content of BuChE (26). fluorescence corresponds to AChR sites stemmed from the The postsynaptic effect of PBC presents a clearer picture; use of FITC-a-BT (1-2 AM), which produced similar though the blocking activity observed in the electrophysiological ex- more intense patterns of fluorescence in enzyme-treated periments and the inhibition of the end-plate localization of muscles. The presynaptic fluorescence, on the other hand, the probe upon incubation with a-BT are fully consistent was not inhibited by prior treatment with 2 MM a-BT, 6AM with binding to AChR sites. That these sites are conserved in neostigmine, 2 AM hemicholinium, 100 AM tetramonopro- the postsynapse of enzyme-treated muscles can be inferred pyl-pyrophosphortetramide (a BuChE inhibitor), or by a from the similar localization of FITC-a-BT. combination of all these compounds, applied for 1 hr before In conclusion, the properties of the pyrene derivative in- addition of 2 AM PBC. The presynaptic fluorescence re- troduced in the present work suggest its applicability to mained unchanged for at least 24 hr after washing the prep- structural or kinetic studies exploiting the sensitivity and aration. versatility of fluorescence techniques. Although initially de- Concentrations of PBC above 25 MM and/or incubations signed to gain information about the solubilized and mem- for more than 1 hr produced extra-synaptic fluorescence, brane-bound AChR, PBC is likely to be a useful fluorescent mainly in the myelinated nerve trunks. In view of the hy- probe in the study of cholinesterase-ligand interactions. drophobic nature of the pyrene ring and the preferential ac- cumulation of PBC in membranous structures, 1-pyrene- We are extremely indebted to G. Striker for the analysis of nano- butyric acid was applied to some preparations. Intense non- second fluorescence decay data. synaptic fluorescence was observed, but the synaptic region did not become apparent even at 0.1 mM I-pyrene-butyric 1. Radda, G. K. & Vanderkooi, J. M. (1972) Biochim. Biophys. acid (1 hr). Interestingly, typical synaptic fluorescence was Acta 285,,509-549. 2. Tasaki, L (1968) Nerve Excitation. A Macromolecular Ap- observed when the same preparations were subsequently in- proach (C. C Thomas, Springfield III.), 201 p. cubated with 5 AM PBC, in spite of the strong background 3. Cohen, L. B., Salzberg, B. M., Davila, H. V., Ross, W. N., Lan- fluorescence. downe, D., Waggoner, A. S. & Wang, C. H. (1974) J. Membr. Biol. 19,1-36. DISCUSSION 4. Patrick, J., Valeur, B., Monnerie, L. & Changeux, J. P. (1971) J. Membr. Biol. 5,10-12o.0 1-Pyrene-butyrylcholine possesses several properties which 5. Weber, G., Borris, D., De Robertis, E., Barrantes, F. J., La make it particularly attractive for studies of the cholinergic Torre, J. L. & Carlin, M. C. L. (1971) Mol. Pharmacol. 7, system. The reported effect of PBC on the m.e.p.ps and the 530-537. ACh-induced depolarization indicates that the probe has a 6. Cohen, J. B. & Changeux, J. P; (1973) Biochemistry 12, reversible blocking action on a nicotinic postsynaptic mem- 485-4864. brane, i.e., it acts as a curare-like antagonist. Since butyric 7. Cohen, J. B., Weber, M. & Changeux, J. P. (1974) Mol. Phar- acid-choline esters generally act as (22), the an- macol. 10, 904-932. tagonistic effect is probably due to the large pyrene ring. 8. Eibl, H., Arnold, D., Weltzien, H. V. & Westphal, 0. (1967) In addition, even submicromolar concentrations of PBC ren- Justus Liebigs Ann. Chem. 709,226-230. & der a specific pattern of fluorescence in the neuromuscular 9. Ellman, G. L., Courtney, K. D., Andres, V. Featherstone, R. M. (1961) Biochem. Pharmacol. 7,88-95. As on junction. expected, the specificity depends the tri- 10. Demas, J. N. & Adamson, A. W. (1971) J. Phys. Chem. 75, methylammonium end of the molecule, since pyrene-butyr- 2463-2466. ic acid fails to depict the synaptic region. 11. Valeur, B. & Moirez, J. (1973) J. Chim. Phys. 70,500-506. The enzymatic treatment of the synapse enabled a corre- 12. Del Castillo, J. & Katz, B. (1955) J. Physiol. (London) 128, lation to be established between the observed pharmacologi- 157-181. Downloaded by guest on October 4, 2021 Biophysics: Barrantes et al. Proc. Nat. Acad. Sci. USA 72 (1975) 3101

13. Clark, D. G., Macmurchie, D. D., Elliott, E., Wolcott, R. G., 20. Albuquerque, E. X., Sokall, M. D., Sonesson, B. & Thesleff, S. Landel, A. M. & Raftery, M. A. (1972) Biochemistry 11, (1968) Eur. J. Pharmacol. 4,40-46. 1663-1668. 21. Hall, Z. W. & Kelly, R. B. (1971) Nature New Blol. 232, 62- 14. Anderson, M. J. & Cohen, M. W. (1974) J. Physiol. (London) 63. 237,385-40. 22. Chang, H. C. & Gaddum, J. H. (1933) J. Physiol. (London) 15. McMahan, U. J., Spitzer, N. C. & Peper, K. (1972) Proc. Roy. 79,255-285. Soc. London Ser. B. 181, 421-430. 16. Betz, W. & Sakmann, B. (1973) J. Physiol. (London) 230, 23. Liley, A. W. (1956) J. Physlol. (London) 134,42744. 673-688. 24. Del Castillo, J. & Katz, B. (1954) J. Physiol. (London) 124, 17. Karnovsky, M. J. (1964) J. Cell Biol. 23,217-222. 586-604. 18. Knopp, J. A. & Weber, G. (1969) j. Biol. Chem. 244, 6309- 25. Schueler, F. W. (1960) Int. Rev. Neuroblol. 2,77-97. 6315. 26. Michelson, M. J. & Zeimal, E. V. (1973) Acetylcholine. An 19. Katz, B. (1971) The Release of Neural Transmitter Sub- Approach to the Molecular Mechanism of Action (Pergamon stances (Liverpool University Press, Liverpool, U.K.). Press, Oxford, U.K.), p. 18. Downloaded by guest on October 4, 2021