Effects of chronic alcohol administration on synaptic membrane Na+Ca2+ exchange activity

Brain Research, 414 (1987) 239-244

239

Elsevier BRE 12655

Effects of chronic alcohol administration on synaptic membrane Na+-Ca 2+ exchange activity M.L. Michaelis 1, E.K. Michaelis 1'2, E.W. Nunley I and N. Galton 2 1Centerfor Biochemical Research and 2Departmentof Human Development, Universityof Kansas, Lawrence, KS 66046 (U.S.A.) (Accepted 11 November 1986)

Key words: C a 2+ transport; Ethanol; Synaptic membrane; Na+-Ca 2+ antiporter; Chronic alcohol; Ion transport

We have recently reported that ethanol and other n-alkanols added to in vitro assays inhibit the activity of the Na+-Ca 2+ exchange system in brain synaptic plasma membrane vesicles. The present studies were undertaken to determine whether in vivo chronic ethanol administration leads to alterations in this Na+-Ca 2+ antiporter that might be indicative of an adaptive response to alcohol. The Na+-dependent Ca 2÷ transport activity in the plasma-membrane fractions obtained was measured at various Ca2+ concentrations. Resuits of these experiments revealed that a 3-week ethanol regimen brought about a significant increase in the Na+-dependent Ca2+ transport activity only in the membrane fraction enriched in synaptic junctional complexes. These membranes showed a near doubling in the maximal transport activity of the antiporter in alcohol-treated compared with the control animals. Changes in the kinetic parameters were reversible as the Na+-Ca 2÷ exchange activity in these membranes from animals maintained on alcohol for 3 weeks and then withdrawn for 1 week was indistinguishable from that of membranes from control animals. Thus it appears that ethanol-treated animals make a reversible adaptation in their neuronal cell membranes to compensate for the acute effects of ethanol on the Na +Ca2÷ antiporter.

INTRODUCTION Drugs that act on the central nervous system to p r o d u c e neuronal depression have b e e n shown by several investigators to affect Ca 2÷ fluxes across nerve cell m e m b r a n e s . F o r e x a m p l e , depolarizationinduced Ca 2+ channel activation is inhibited by barbiturates and ethanol 7,11,13. B a s e d on the involvement of Ca z+ in the initiation of exocytotic release of m a n y neurotransmitters, decreases in depolarization-induced Ca 2÷ conductance would be expected to cause diminished n e u r o t r a n s m i t t e r release and, secondarily, a failure in synaptic transmission. D e c r e a s e s in n e u r o t r a n s m i t t e r release have been o b s e r v e d following acute administration of some d e p r e s s a n t drugs in vivo or following the in vitro application of these agents to neuronal p r e p a r a t i o n s 5'8'21'25. H o w e v e r , in several o t h e r p r e p a r a t i o n s such as isolated striatal nerve endings, the n e u r o m u s c u l a r junction, or hip-

p o c a m p a l slices, ethanol was r e p o r t e d to increase the release of e n d o g e n o u s transmitter substances 2'4'9'24'26. Such a p p a r e n t discrepancies in the actions of ethanol are likely to be due to the existence of multiple targets for its actions, and these sites m a y include intracellular entities as well as p l a s m a m e m brane systems which maintain free intraneuronal calcium ion concentrations at a very low level (10 -7 M). Interference with the activity of any of the Ca2+-regulating systems in neurons could lead to progressive intracellular accumulation of this cation, causing alterations in n e u r o t r a n s m i t t e r release, in the conductance properties of Ca2+-sensitive K ÷ channels, and in several Ca2+-regulated metabolic activities such as protein p h o s p h o r y l a t i o n or c a l m o d u l i n - d e p e n d e n t activation of enzymes and t r a n s p o r t systems 2'1°'2°'22. W e have previously r e p o r t e d that a brain synaptic m e m b r a n e exchange carrier which has a high transport capacity for Ca 2+ is very sensitive to inhibition

Correspondence: M.L. Michaelis, University of Kansas, Center for Biomedical Research, 2099 Constant Avenue, Lawrence, KS 66046, U.S.A. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B .V. (Biomedical Division)

240 by concentrations of ethanol that are physiologically relevant to intoxication in humans 17'19. This synaptic plasma membrane N a ÷ - C a 2÷ exchange carrier is thought to be the most active, relatively high-affinity Ca2÷-transporting system in neuronal membranes I. On the other hand, we have found that the high affinity, ATP-dependent Ca 2+ transport activity in synaptic membranes was very resistant to the effects of ethanol TM. Relatively high concentrations of ethanol (>600 mM) were required to bring about any detectable inhibition of the ATP-dependent synaptic membrane Ca 2+ transport system TM. The marked sensitivity of the N a ÷ - C a 2÷ exchange carrier to the effects of acute in vitro exposure of synaptic membranes to ethanol led us to examine the effects of chronic in vivo ethanol administration on this Ca 2÷ transport activity. In particular, we were interested in determining whether chronic alcohol treatment leads to an enhanced activity of synaptic membrane N a + - C a 2÷ exchange in animals that have developed tolerance to the effects of ethanol. Such a change might develop as an adaptive response of the organism to the inadequate functioning of the carriers in the presence of high CNS ethanol concentrations during continuous exposure to alcohol. Furthermore, we examined the possibility that changes in N a ÷ - C a 2+ exchange activity brought about by chronic intake of ethanol might be most prominent in membrane subffactions that are highly enriched in synaptic junctions. Our observations were indicative of a marked increase in the maximal transport activity of the exchange carrier in synaptic membranes obtained from chronically ethanol-treated rats compared with membranes from their respective controls. Furthermore, these changes in carrier activity were confined to the plasma membrane fraction that is enriched in synaptic junctions. MATERIALS AND METHODS

Animals and diets Adult male Sprague-Dawley rats (CrI:CD) obtained from Charles River Breeding Labs., Wilminton, MA, were used for all experiments. The rats were maintained on Purina laboratory chow until they reached a body wt. of 250-300 g. They were pair-fed a diet consisting of 71% Slender, 8% (w/v) ethanol for the experimental animals or an isocaloric

amount of sucrose for the control animal of each pair. The dietary regimen and the amounts of ethanol consumed have been described in detail in a previous publication 15 with the only difference here being that the animals were maintained on the ethanol for a period of 3 rather than 2 weeks.

Preparation of brain subcellular fractions The brains from a pair of control and ethanoltreated or withdrawn animals were always processed in parallel, from homogenization to activiy measurements. The scheme used for the preparation of various brain subcellular fractions was identical to that described in detail previously TM. The synaptosomeenriched fraction obtained by Ficoll-sucrose density gradient centrifugation 3 was osmotically lysed and subjected to differential centrifugation and flotationsedimentation 23 to obtain the highly purified synaptic plasma membrane subfractions. The bands at the interface of 10% and 28.5% (w/v) sucrose and at the interface of 28.5% and 34% sucrose were pelleted, resuspended in 0.25 M sucrose-50/~M MgCI z (6-9 mg protein/ml), and frozen in small aliquots in liquid N 2. Protein concentrations of the various subffactions were determined by the method of Lowry 12.

Electron microscopy of brain subfractions The particulate fractions obtained by sucrose density flotation-sedimentation were pelleted at top speed for 15 min in a Beckman microfuge. The fixative, consisting of 0.1 M cacodylate, pH 7.4, 3 mM CaC12, 4% paraformaldehyde, 2.5% glutaraldehyde, and 0.5% tannic acid, was layered over the pellets for overnite fixation at 4 °C. The pellets were washed 3 times with cacodylate-calcium buffer, postfixed with 1% OsO4 for 1 h and rinsed 3 times with distilled water. Following staining with 1% uranyl acetate for 1 h, the pellets were dehydrated in an ethanol series and embedded in Spurr resin. Thin sections were stained with 2% aqueous uranyl acetate and lead citrate, and viewed and photographed using a Philips 300 transmission electron microscope.

Measurement of Na +-dependent Ca2+fluxes The Na+-dependent Ca 2+ transport activity was measured essentially as described previously 16a7 with some modifications. Membrane vesicles (10-20 /~g protein) were loaded internally with 150 mM

241 NaC1 and diluted 20-fold into an incubation medium containing either 150 mM KCI ( + N a ÷ gradient in out) or 150 mM NaC1 ( - N a ÷ gradient) plus the indicated concentrations of isotopically diluted 45CAC12 (0.07/xCi). Incubations were carried out at 26 °C for 8 s (linear phase) and were terminated by the addition of 2 ml of ice-cold 160 mM KC1-25 mM T r i s - H C l - l m M E G T A , pH 7.4, and rapid filtration through Whatman GF/B filters under moderate vacuum. Filters were then washed with 3 ml of the icecold KCI solution, dried, and assayed for radioactivity. Background adsorption of 45Ca to the filters and the membranes was determined in samples to which 2 ml of the 'stop' solution were added prior to addition of the membranes. These samples were immediately filtered and washed identically to the others, and the values obtained in this way were subtracted from the values for the incubated samples in the calculation of Ca 2+ influx. The specific activity of Ca 2÷ transport (mol/mg protein) was calculated for both the + N a ÷ gradient and the - N a ÷ gradient conditions. The no gradient-dependent transport was approximately 10% of the gradient-dependent transport for Ca 2+ concentrations up to 25/tM and increased to - 2 5 % of the gradient-dependent transport at 50 and 75 p M Ca 2+. This - N a ÷ gradient activity was subtracted from the activity in the presence of a gradient for each Ca 2+ concentration in order to estimate the actual Na÷-dependent transport. Membranes from each pair of control and ethanol-treated animals were processed and tested togehter.

pendent Ca 2+ transport 16'17. Further subfractionation of these synaptosomal membranes by means of sucrose density flotation-sedimentation procedures led to the isolation of a light synaptic plasma membrane subfraction (interface of 10% and 28.5% sucrose), which under electron microscopic examiniation did not contain recognizable synaptic junctions, and a synaptic membrane fraction (interface of 28.5% and 34% sucrose) that was highly enriched in synaptic junctions (manuscript submitted). The protein concentration of the various subfractions obtained throughout the isolation of the membranes did not reveal any differences in the recoveries between the control and ethanol-treated animals. For example, the yields on the membrane fractions used in these studies were as follows: synaptic junctional membranes: 2.0 + 0.71 for the controls and 2.3 + 0.52 for the chronically treated animals, n = 6; light synaptic membranes: 0.99 + 0.44 for the controls and 0.77 + 0.14 for the chronically ethanol treated animals, n = 6. The Ca 2+ concentration-dependent activity of the N a + - C a 2÷ exchange transport was essentially identical in the two membrane subfractions (Figs 1 and 2). The estimated Kac t for Ca 2+ was 28.2pM for the light synaptic membrane fraction and 30.3/xM for the synaptic junction-enriched membranes obtained from the untreated animals. In addition, the Vmax values

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Analysis of data Transport data were analyzed by a curve fitting procedure involving computer-assisted optimization of a weighted least squares fit to the Michaelis-Menten equation. The weighting function used was the reciprocal of the variance of the Ca 2÷ transport activity. Statistical analysis of the significance of differences between control and drug-treated animals was performed using Student's t-test for unpaired sampies. RESULTS Isolated synaptic plasma membrane vesicles obtained following osmotic rupturing of brain synaptosomes exhibit high activity of the Na+-gradient de-

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Fig. 1. Ca 2+ concentration dependence of Na÷-Ca2÷ exchange activity in the light synaptic plasma membranes obtained from control and alcohol-treated animals. Animals were maintained on the control or ethanol-containing diets for 21 days as described in Materials and Methods. Each point is the mean of 10-12 determinations from 6 experiments. A total of 6 animals per condition were processed as pairs. The lines drawn were obtained by computer fitting of the data to the Michaelis-Menten equation and were identical for the two groups.

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transport activity and the chronic ethanol administration was d e m o n s t r a t e d by allowing one series of experimental animals to go through a 7-day withdrawal period prior to the d e t e r m i n a t i o n of the exchange carrier activity. A s is shown in Fig. 3, after 21 days of

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Fig. 2. Ca2+ concentration dependence of the Na+-Ca 2÷ exchange activity in isolated synaptic junctional membranes from controls and ethanol-treated animals. The animals were maintained on the control or ethanol-containing diet for 21 days as described in Materials and Methods. Each point is the mean of 28-30 determinations from 11 experiments. A total of 6 animals per condition were processed as pairs. Significant differences *P < 0.05 and **P < 0.01 are indicated. The lines drawn were obtained by computer fitting to the Michaelis-Menten equation.The estimated Kact values for Ca2+ were 30.3 #M for the controls and 29.2 #M for the alcohol-treated animals.

calculated for the transport activity in these two m e m b r a n e fractions from the control animals were 2.96 nmol/mg protein/8 s and 2.65 nmol/mg protein/ 8s. A f t e r the animals had been t r e a t e d with ethanol for 21 days as described in Materials and M e t h o d s , there was a significant increase in the Ca 2+ concent r a t i o n - d e p e n d e n t activity of the N a + - C a 2+ exchange carrier in the synaptic-junction enriched subfraction (Fig. 2), whereas the antiporter activity in the light plasma m e m b r a n e fraction was unchanged (Fig. 1). The N a + - d e p e n d e n t Ca 2+ transport in the ethanol-treated animals was significantly greater at most concentrations in the range from 1 to 75 # M Ca 2÷ in the junctional m e m b r a n e fraction (Fig. 2). Computer-assisted analysis of the data for the N a + Ca 2+ exchange activity in the m e m b r a n e s from control and alcohol-treated animals indicated that there was a 50% increase in the Vmax of this transport carrier in m e m b r a n e s from chronically e t h a n o l - t r e a t e d animals, 3.9 nmol/mg protein for the alcohol-treated group vs 2.65 nmol/mg protein for the controls. Chronic ethanol t r e a t m e n t did not p r o d u c e any significant changes in the activation constant for Ca 2÷ as indicated in the legend of Fig 2. The relationship between the increase in Ca 2÷-

ethanol t r e a t m e n t followed by 7 days of withdrawal, the junction-enriched m e m b r a n e s from control and experimental animals exhibited no significant differences in the c o n c e n t r a t i o n - d e p e n d e n t activity of this carrier. A s indicated in the legend of Fig. 3, the estim a t e d gac t and Vmax for the N a + - C a 2÷ exchange activity in m e m b r a n e s obtained from chronicallytreated animals were very similar to those estimated for the carrier activity in the same m e m b r a n e s obtained from the respective control animals suggesting a return of the N a + - C a 2+ exchange activity to baseline levels following withdrawal from ethanol. In this series of studies the protein recoveries achieved during the isolation of the m e m b r a n e s were lower than those we had previously o b t a i n e d for the controls and chronically e t h a n o l - t r e a t e d animals, possibly reflecting a somewhat higher degree of purification of synaptosomes and of their m e m b r a n e s (synaptic junctional m e m b r a n e s : 1.1 + 0.42 and 1.2 + 0.38 for controls and ethanol-withdrawn animals, n = 5; light synaptic membranes: 0.41 + 0.12 and 0.59 + 0.23 for controls and ethanol-withdrawn animals, n = 5). The

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Fig. 3. Na+-Ca 2+ exchange activity in synaptic junctional membrane fraction obtained from control and ethanol-treated and then withdrawn animals. Following 21 days of maintenance on the control or ethanol-containing liquid diet, the animals were placed on the lab chow for 7 days. The analysis of the data is identical to that described for Fig. 2. The estimated gac t for Ca2+ was 21.6#M for the controls and 19.5#M for the ethanolwithdrawn animals. The respective V~,axvalues were 3.4 and 3.3 nmol/mg protein. Each point is the mean of 11-13 determinations from 5 experiments. A total of 5 animals per condition were used.

243 higher activity detected for the synaptic junctional membranes from control animals for this series compared with the activity determined in the membranes from controls in the initial series may be due to our having performed the last series of studies with more highly purified membranes. DISCUSSION The studies described in this paper were undertaken on the basis of a prediction that, if acute in vivo ethanol produces inhibition of the N a ÷ - C a 2+ exchange carriers in brain neurons, then a component of physiological tolerance to the effects of chronically administered ethanol might involve the appearance of enhanced activity of these carriers in neuronal membranes. In our studies we did observe such an apparent adaptive response to the chronic administration of ethanol, which took the form of increased maximal activity of the N a + - C a 2+ exchange carriers without any concomitant change in the activation constant of these carriers by Ca 2÷. These changes in the maximal transport activity of synaptic membrane carriers were detectable only while the experimental animals were under the continuous influence of ethanol. Sodium-dependent Ca 2÷ transport in membranes from withdrawn animals was indistinguishable from that of their appropriate cohorts. Based on these observations, it seems reasonable to suggest that the increased maximal activity of the synaptic membrane N a ÷ - C a 2+ exchange transport may represent an adaptive response to chronic exposure of brain neurons to ethanol. The fact that ethanol-induced changes in the carrier activity were observed only in the junction-enriched membrane subfraction might indicate that acute exposure to ethanol was more disruptive to the function of nerve endings than to the function of other parts of the nerve cell. Some supportive evidence for this conclusion has been obtained by Carlen et al. 2 who observed a net accumulation of intracellular Ca 2÷ in hippoccampal neurons, an increased release of both excitatory and inhibitory neurotransmitters, and a prolongation of after-hyperpolarization due to a CaZ+-mediated increase in K ÷ efflux. The link between inhibition of the N a + - C a 2÷ exchange carrier activity by acute ethanol intake and increased transmitter release or prolongation of the after-hyperpolarization is tentative at this time. There are no spe-

cific inhibitors for this transport system, and thus it is not yet possible to demonstrate that inhibition of this transport system alone actually produces such changes in neuronal or nerve ending activity. The nerve ending region may be quite susceptible to the disruptive effects of an inhibitor of Ca 2÷ extrusion processes since this region contains a very high density of voltage-sensitive Ca 2+ channels 6. The intracellular environment of the nerve ending region is repeatedly subjected to large increases in Ca 2÷ influx leading to substantial elevations of intraterminal free (Ca2+).Even a moderate inhibition of one of the Ca2+-extruding systems such as the N a ÷ - C a 2+ antiporter might have profound effects on nerve terminal membrane potential, ion channel function, transmitter release, and long-term regulation of enzyme activities. Therefore it is not surprising that an adaptive cellular response to chronic ethanol exposure could involve the expression of an enhanced transport capacity in this cellular region. The yield of highly purified synaptic plasma membranes that could be obtained from the brain of each animal was very low, usually in the order of 1% of the protein in whole brain homogenate TM. Such a low yield of total protein made it impossible to pursue a regional localization of neurons or brain areas that might be unusually susceptible to the actions of ethanol. Thus, although we succeeded in identifying which subcellular fractions may be undergoing changes in the activity of the N a + - C a 2+ exchange carriers, we could not provide any clues to possible differential responses of neurons in specific brain regions to chronic ethanol exposure. A more selective characterization of the molecular changes that underly the observed alterations in N a + - C a 2+ exchange activity of synaptic membranes should be possible once the macromolecular complex of these membrane carriers has been indentified and purified. ACKNOWLEDGEMENTS We thank Linda Kunkle for excellent assistance in the preparation of the manuscript, and we acknowledge the support provided by the Center for Biomedical Research, University of Kanses. This work was supported by National Institute of Alcoholism and Alcohol Abuse Grant A A 04732, National Institute on Aging Grant A G 04762, and Biomedical Research Support Grant R R 5606.

244 REFERENCES 1 Blaustein, M.P., Ratzlaff, R.W. and Kendrick, W.K., The regulation of intraeellular calcium in presynaptic nerve terminals, Ann. N. Y. Acad. Sci., 28 (1978) 195-212. 2 Carlen, P.L., Gurevich, N. and Durand, D., Ethanol in low doses augments calcium-mediated mechanisms measured intracellularly in hippocampal neurons, Science, 215 (1982) 306-309. 3 Cotman, C.W. and Matthews, D.A., Synaptic plasma membranes from rat brain synaptosomes: isolation and partial characterization, Biochim. Biophys. Acta, 249 (1971) 380-394. 4 Curran, M. and Seeman, P., Alcohol tolerance in a cholinergic nerve terminal: relation to the membrane expansionfluidization theory of ethanol action, Science, 197 (1977) 910-911. 5 Erickson, C.K. and Graham, D.T., Alteration of cortical and reticular acetylchofine release by ethanol in vivo, J. Pharmacol. Exp. Ther., 185 (1973) 583-593. 6 Hagiwara, S. and Byerly, L., Calcium channel. Annu. Rev. Neurosci., 4 (1981) 69-125. 7 Harris, R.A. and Bruno, P., Membrane disordering by anesthetic drugs: relationship to synaptosomal sodium and calcium fluxes, J. Neurochem, 44 (1985) 1274-1281. 8 Haycock, J.W., Levy, W.B. and Cotman, C.W., Pentobarbital depression of stimulus-secretion coupling in brain-selective inhibition of depolarization-induced calcium dependent release, Biochem Pharmacol., 26 (1977)159-161. 9 Inoue, F. and Frank, G.B., Effects of ethyl alcohol on excitability and on neuromuscular transmission in frog skeletal muscle, Br. J. Pharmacol. Chemother., 30 (1967) 186-193. 10 Kennedy, M.B., Expermental approaches to understanding the role of protein phosphorylation in the regulation of neuronal functions, Annu. Rev. Neurosci., 6 (1983) 493-525. 11 Leslie, S.W., Barr, E., Chandler, J. and Farrar, R.P., Inhibition of fast- and slow-phase depolarization-dependent synaptosomal calcium uptake by ethanol, J. Pharmacol. Exp. Ther., 225 (1983) 571-575. 12 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951)265-275. 13 Messing, R.O., Carpenter, C.L. and Greenberg, D.A., Soc. Neurosci. Abstr., 11 (1985) 794.

14 Michaelis, E.K., Michaelis, M.L., Chang, H.H. and Kitos, T.E., High affinity Ca2+-stimulated Mg2+-dependent ATPase in rat brain synaptosomes, synaptic membranes and microsomes, J. Biol. Chem., 258 (1983) 6101-6108. 15 Michaelis, E.K., Mulvaney, M.J. and Freed, W.J., Effects of acute and chronic ethanol intake on synaptosomal glutamate binding activity, Biochem. Pharmacol., 27 (1978) 1685-1691. 16 Michaelis, M.L. and Michaelis, E.K., Ca 2+ fluxes in resealed synaptic plasma membrane vesicles, Life Sci., 28 (1981) 37-45. 17 Michaelis, M.L. and Michaelis, E.K., Alcohol and local anesthetic effects on Na+-dependent Ca 2÷ fluxes in brain synaptic membrane vesicles, Biochem. Pharmacol., 32 (1983) 963-969. 18 Michaelis, M.L., Kitos, T.E. and Tehan, T., Differential effects of ethanol on two synaptic membrane Ca 2+ transport systems, Alcohol 2 (1985) 129-132. 19 Michaelis, M.L., Michaelis, E.K. and Tehan, T., Alcohol effects on synaptic membrane calcium ion fluxes, Pharmacol. Biochem. Behav., 18, Suppl. 1, (1983) 19-23. 20 Nairn, A.C., Hemmings Jr.,H.C. and Greengard, P., Protein kinases in the brain, Annu. Rev. Biochem., 54 (1985) 931-976. 21 Phillis, J.W. and Jhamandas, K., The effects of chlorpromazine and ethanol on in vivo release of acetylcholine from the cerebral cortex, Comp. Gen. Pharmacol., 2 (1971) 306310. 22 Rahaminoff, R., Meiri, H., Erulkar, S.D. and Borenholz, Y., Changes in transmitter releases induced by ion-containing liposomes, Proc. Natl. Acad. Sci. U.S.A., 307 (1978) 5214-5216. 23 Salvaterra, P.M. and Matthews, D.A., Isolation of rat brain subcellular fraction enriched in putative neurotransmitter receptors and synaptic junctions Neurochem. Res., 5 (1980) 181-194. 24 Seeman, P. and Lee, T., The dopamine-releasing actions of neuroleptics and ethanol, J. Pharmacol. Exp. Ther., 190 (1974) 131-140. 25 Sunahara, G.I. and Kalant, H., Effect of ethanol on potassium-stimulated and electrically-stimulated acetylcholine release in vitro from rat cortical slices, Can. J. Physiol. Pharmacol., 58 (1980) 706-711. 26 Velussi, C., Daneli-Betto, D. and Boschiero, R., Effects of two synaptic activators, calcium and ethanol, on MEPP distribution in time, Am. J. Physiol., 237 (1979) c264-c268.