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Ethanol suppresses collateral inhibition of the goldfish Mauthner cell
Brain Research, 104 (1976) 347-353
347
© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
Ethanol suppresses collateral inhibition of the goldfish Mauthner cell
DONALD S. FABER AND MANFRED R. KLEE* Research Institute on Alcoholism, New York State Department of Mental Hygiene, Buffalo, N.Y. 14203, and Department of Physiology, State University of New York at Buffalo, Buffalo, N.Y. 14214 (U.S.A.)
(Accepted November 25th, 1975)
It is well known that ethanol directly depresses the electrical excitability of a variety of vertebrate and invertebrate neurons, presumably through a relatively specific reduction in the early inward sodium current underlying spike initiation 1,s,zl (cf. reviews in ref. 24). In contrast, opposite actions of ethanol on junctional transmission have been described for the vertebrate and invertebrate nervous systems; although it depresses the acetylcholine sensitivity and excitatory postsynaptic potentials of molluscan neurons3,a, 6 and both inhibition and excitation of cat spinal motoneurons 9,17, it facilitates cholinergic transmission at the vertebrate neuromuscular junction15, 22. Furthermore, there is relatively little information concerning its effects on synaptic transmission in the vertebrate central nervous system, which, on the basis of psychophysical tests is appreciably more sensitive to ethanol than the peripheral nervous system (cf. ref. 24). A common criticism of electrophysiological experiments designed to elucidate the mechanisms of action of substances such as ethanol on specific neuronal systems is that the drug concentrations required to produce an effect, as well as the effects themselves, cannot readily be correlated with behavioral observations of the drug action. One preparation which appears suited for dealing with this problem is the goldfish medullary network involving the Mauthner cell (M-cell) and the startle reflex which it mediates, since both its neuronal organization and functional output are well-defined7,10-14,18,19, 26. In this paper we report that collateral inhibition of the M-cell is reduced or blocked completely at brain ethanol levels which have no significant effects on the cell's electrical excitability and which are appreciably lower than those which cause the fish to lose their righting reflexes. Rather, the depression of collateral inhibition occurs at brain levels associated with behavioral hyperexcitability. Furthermore, we present evidence suggesting that this effect of ethanol is due to an impaired release of transmitter, presumably acetylcholine, from M-cell axon collaterals. Common goldfish, 6-7 in. in length, were used for both the electrophysiological * Permanent address: Neurobiology Department, Max-Planck Institute for Brain Research, Frankfurt/M, G.F.R.
348 experiments and the behavioral observations. In the former series, tbe techniques utilized were similar to those described previously 12,18. The fish were curarized (1/~g/ g body wt.) and respired with a continuous flow of dechlorinated tap water through the mouth and over the gills. Bipolar electrodes on the spinal cord and the ipsi- or contralateral VlIIth nerves were used to activate the M-cell antidromically and orthodromically, respectively, with collateral inhibition of the cell also being evoked through the antidromic pathway (Fig. IA). Intra- and extracellular recordings were obtained from the M-cell with glass microelectrodes (2-6 m~2 resistance) filled with either 2.7 M KC1 or 0.6 M K2SO4. After obtaining the control responses, the solution perfusing the gills was switched to one containing 1-2 ~ ethanol (w/v), and the effects described here developed gradually during the next l-2 h. At the end of each experiment, the brain was removed and frozen rapidly in liquid nitrogen for subsequent gas chromatographic determination of the ethanol concentration. In parallel experiments, the effects of I ~o ethanol on freely swimming goldfish were initially determined by gross behavioral observations. Brain ethanol concentrations in these fish reached an equilibrium level of approximately 10 /~g/mg brain weight in approximately 2 h, whereas the ethanol concentration rose more slowly in the restrained fish used for the physiological experiments. During the first 30-60 min the fish undergo a phase of hyperexcitability characterized by hyperreflexia, including an enhanced startle reflex, poorly coordinated swimming and gulping of air at the water surface. These behavioral effects correlated with brain ethanol levels of 3-5 /zg/mg brain wt. As the ethanol concentration increased, the fish sank to the bottom of the aquarium, became grossly ataxic, overturned, and lost their righting reflexes. As shown in Fig. 1A, the startle reflex can be initiated by VIIIth nerve inputs to the M-cell lateral dendrite. Activation of the M-cell almost always results in contraction of the contralateral tail musculature, and the major inhibitory control of the reflex is mediated by medullary inhibitory interneurons which are excited by axon collaterals of both M-cells as well as by sensory afferents 11,~4,as. There are two components to the collateral inhibition: (1) a short latency electrical inhibition characterized by an extrinsic hyperpolarizing potential (EHP) which is best recorded as an extracellular positivity in the axon cap surrounding the initial segment of the M-cell axon (Fig. 1B1); and (2) a subsequent chemically mediated inhibitory postsynaptic potential (IPSP) 14. Since its equilibrium potential is close to the M-cell resting potential, the IPSP does not generally appear as a significant membrane potential change. It can, however, be quantified with double antidromic stimuli; the amplitude of the second antidromic action potential is reduced by the conductance increase associated with the IPSP 14 (Fig. 1C1). As described elsewherO ~,13,18, both the EHP and the IPSP are most likely mediated by the same inhibitory interneurons, the first being due to the flow of the extracellular currents generated by the action potentials in these neurons. It should be pointed out that the collateral inhibition illustrated in Fig. 1C1 is sufficiently strong to occasionally block in an all-or-none manner the major component of the second antidromic action potential, thereby unmasking a smaller spike component. Such a separation of the M-cell spike into components has not been pre-
349 A
C 1Control
C 2 Ethanol
i
i
2ms
D1Control
D2 Ethanol
M. o x o 0
~ Stim. 500~se¢
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,~f"
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B2 Ethanol
2ms
E2
200vsec
Fig. 1. Effect of ethanol on collateral inhibition of the M-cell and on M-cell electrogenesis. A: schematic illustration of the experimental arrangement and the neuronal model taken as the basis for analyzing the results. A microelectrode (ME) was inserted in the M-cell's soma or lateral dendrite (Lat. Dendr.) and intracellular potentials were monitored following stimulation (Stim.) of the M-cell's axon (M. axon) or the VIIIth nerve (VllIth N.). I represents a pool of inhibitory interneurons which are activated by M-cell axon collaterals and which in turn feed inhibition back onto the cell. Contralateral connections are not shown. B1 and B2 : effect of ethanol on the extracellular fields recorded in the axon cap. BI: control. Spinal cord stimulation at a rate of I/3 sec evoked a negative M-cell antidromic spike followed by a positive EHP (arrow). Bz: 35 min after starting perfusion with 1 ethanol, the EHP was strongly reduced. The experiment was terminated 20 min later, at which time the final ethanol level was 4.7/~g/mg brain wt. Three or more superimposed traces are shown in all records of this figure. C1 and C2: intracellular recordings from the M-cell soma in another experiment illustrate that ethanol blocks the collateral IPSP as well. C~: control. With paired antidromic stimuli the increased conductance associated with the IPSP evoked by the conditioning stimulus blocks completely or reduces by approximately 50 % the axon hillock component of the following test antidromic spike. Recording was with a K2SO4 microelectrode from the M-cell soma and there was no appreciable membrane potential change associated with the IPSP. Ca: after 60 min perfusion with 1 ~ ethanol, the IPSP conductance change was almost completely abolished. The ethanol brain concentration was 3.45 #g/rag. Dx and D2: a similar concentration of ethanol (4.51/~g/mg) had a minimal effect on the M-cell antidromic action potential as recorded in the cell's soma. Each record illustrates the simultaneously recorded M-cell spike (lower trace) and its electrically differentiated representation (upper trace). D~: control. Dz: after 86 min exposure to ethanol, the spike was slightly smaller and increased in duration; these changes were correlated with slower rates of rise and repolarization. Such changes in the M-cell spike often occur during 1-2 h continuous intracellular recording and may not be attributed to an action of ethanol. E1 and E2: at a higher brain ethanol level (8.2/,g/mg) distinct effects of ethanol on M-cell electrogenesis were occasionally observed. Both sets of records were obtained 1 h after starting perfusion with 2 ~ ethanol. E~: the spike initiated at the M-cell's axon hillock failed and unmasked a small axon spike. Ez: the M-cell was depolarized with a steady transmembrane current applied through the recording microelectrode; under these conditions the axon spike succeeded in bringing the cell's axon hillock to threshold and the latter initiated a spike as well.
350 viously reported. However, this phenomenon does not reflect M-cell injury since it can also be observed during extracellular recordings. Rather, we tentatively suggest that it represents a failure of active spike propagation from the M-cell axon to the axon hillock. In this context, the initial component would represent the antidromic spike generated by the medullated portion of the axon and the subsequent one the invasion of the axon hillock 8. Ethanol in a concentration of 3-5 #g/mg of brain tissue reduces both components of collateral inhibition (Fig. IB and C). In contrast, M-cell resting membrane potential (generally between --70 to --80 mV) remains unchanged even at levels as high as 20 ~g/mg and only minor changes in its antidromic action potential (Fig. 1D1 vs. Fig. 1 D2) occur at the lower ethanol levels. When the brain ethanol concentration was in the range of 8-15 #g/mg, direct effects on M-cell excitability were occasionally observed. In the example illustrated in Fig. l E1 the safety factor for transmission of the antidromic impulse to the axon hillock was reduced and invasion failed; under these conditions a full action potential could be restored by pairing a depolarizing current with the antidromic axon spike (Fig. 1E2). At higher ethanol levels, the M-cell axon spike may fail as well. Finally, we did not observe any significant effects of ethanol on either the excitatory or inhibitory inputs to the M-cell from the ipsi- and contralateral VIIIth nerves, respectively. This observation is significant in that the inhibitory components of these inputs are mediated by the same interneurons which are responsible for the collateral inhibition. Consequently, ethanol's effect cannot be attributed to a non-specific action on the inhibitory interneurons or to a blockage of inhibitory transmission onto the M-cell. The fact that both components of collateral inhibition are equally blocked by ethanol supports the above conclusions and indicates it acts at the level of the synapse between M-cell axon collaterals and the inhibitory interneurons. Three possible modes of action have been considered: (1) a presynaptic effect on spike propagation in the axon collaterals, (2) a depression of transmitter release, and (3) a reduction in the sensitivity of the subsynaptic receptors of the inhibitory interneurons to the M-cell transmitter. The first possibility seems unlikely since effects on the excitability of the M-cell axon only occurred at higher ethanol levels. Nevertheless, a reduced excitability of thinner axon collaterals cannot be ruled out. Although it is difficult to further localize the site of ethanol's action, the results of experiments on the time course of development of the effect described below strongly suggest that it acts presynaptically to impair transmitter release. Since the magnitude of collateral inhibition is very labile at stimulus frequencies above 1/sec in the curarized preparation 14, we routinely used a repetition rate of 1/3-1/7 sec in our experiments. Under these conditions, a stable maximal IPSP can be observed (Fig. 2A; note that the IPSP is reversed, i.e. depolarizing, in this figure since a KCI electrode was used for the intracellular recordings). At the lower repetition rate, no effect of 1 ethanol was seen after 25 min of perfusion (Fig. 2B) but there was typically a 50-70 reduction 10-15 min later (Fig. 2C). After ! h exposure, the IPSP appeared to be abolished (Fig. 2D). However, we observed at that time that a small 1PSP could be restored by switching to a lower stimulus frequency and that a full-sized one could be
351
B Ethanol ,25'
A Control
C 34, I
D 58'
F 69'; 1' rest
E 59'; 1'rest
,
~
G 74,; 5, rest
H 81'; 1' rest
~
i
--
-
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I" 86'; 5' rest
,
lms Fig. 2. Time course of the development of the ethanol effect on the collateral IPSP. The records are from the same experiment as in Fig. 1DI and Da and were obtained with a KCl-containing microelectrode. Three or more superimposed traces of the M-cell responses to spinal cord stimulation at a rate of 1/7 sec are shown in A-I. A: control. The antidromic action potential elicited by a single shock to the M-axon is followed by a depolarizing IPSP which causes the cell to initiate a second spike. B-I: records obtained at the indicated times after starting perfusion with 1% ethanol. The traces illustrated in E, F and H were preceded by 1 min rest periods during which no stimulation was employed, and those illustrated in G and I were preceded by 5 min rest periods. B-D: gradual reduction and, finally, blockage of the IPSP over a 58 min period. E: at 59 min ethanol, a 1 min rest restored the IPSP for the first stimulus in a train with a repetition rate of 1/7 sec. F: diminished effectiveness of the 1 min rest 10 min later. G: at 74 min, a 5 min rest restored the IPSP for one stimulus to a magnitude greater than that in control. H: 81 min after starting perfusion with ethanol, the 1 min rest was completely ineffective and no IPSP was evoked, h after an additional 5 rain rest, an IPSP less than 10 % the amplitude of that illustrated in control could be evoked by the first stimulus in the 1/7 sec train.
evoked for one stimulus after a 1 m i n period of rest w i t h o u t spinal cord s t i m u l a t i o n (Fig. 2E); a second stimulus 7 see later was completely ineffective (Fig. 2E). As the action o f e t h a n o l progressed further, the effectiveness of such 1 m i n rest periods d i m i n i s h e d appreciably (Fig. 2F) a n d longer rest periods in the range o f 5 m i n were necessary (Fig. 2G). Again, however, only the first in a t r a i n o f stimuli at a 1/7 see repetition rate evoked a n IPSP. Finally, after 80-90 m i n total perfusion time with ethanol, such rest periods were completely ineffective a n d the IPSP was completely blocked (Fig. 2 H a n d I). A t t h a t time, the e t h a n o l level in the experiment illustrated in Fig. 2 was 4.51/~g/mg b r a i n weight, a n d we estimate t h a t it was n o more t h a n 2-3 big/ m g b r a i n weight d u r i n g the first h o u r o f exposure. The evidence that such p r o l o n g e d rest periods are capable o f transiently restoring synaptic t r a n s m i s s i o n suggests that e t h a n o l acts o n the process of t r a n s m i t t e r release from the M-cell axon collaterals, a n d does n o t a p p e a r readily c o m p a t i b l e with effects o n collateral excitability or the sensitivity of the postsynaptic n e u r o n s to the M-cell t r a n s m i t t e r . It m a y be .*hat the M-cell p r e p a r a t i o n is especially sensitive to such effects, since t r a n s m i s s i o n readily fails at m o d e r a t e stimulus frequencies. A c o m p a r a ble situation exists at the cholinergic M-cell to giant fiber synapse in the hatchet fish z3.
352 A u e r b a c h and Bennett 2 have shown that the latter synapse also fatigues at moderate stimulus frequencies and have concluded that a single M-cell impulse releases most o f the readily available transmitter store. This is probably true o f the goldfish M-cell as well, which also appears to be cholinergic; l0 # M curare applied topically reversibly blocks both the E H P and the collateral IPSP (Faber and Klee, unpublished results). Consequently, it seems most probable that ethanol blocks transmitter synthesis or mobilization. These results are in agreement with the biochemical studies o f Kalant et al. 16, which indicated that incubation with ethanol inhibits acetylcholine release from unstimulated rat cerebral cortex slides. Finally, it is interesting to note that a somewhat similar presynaptic effect o f barbiturates on cat spinal synaptic transmission has been postulated20, 25. Our preliminary behavioral observations indicate that the initial phase of hyperexcitability following exposure to ethanol occurs at the same brain ethanol levels which rather selectively depress excitatory synaptic transmission from the M-cell collaterals onto the interneurons exerting a major inhibitory control o f the startle reflex. In addition, higher anesthetic levels m a y be associated with direct effects on excitability as well. Clearly, more quantitative behavioral experiments on the effects o f ethanol and additional experiments on the other components o f the neural network involved in the startle reflex are needed. Nevertheless, it is clear that this preparation offers a promising approach to the general problem o f correlating physiological and behavioral aspects o f drug actions. This work was supported in part by N I N C D S G r a n t No. NS12132-01 and by a travel grant to Manfred R. Kiee f r o m the Max-Planck Institute for Brain Research, Frankfurt, G.F.R. We gratefully acknowledge the technical assistance o f Alan Dash and the services o f the Analytical L a b o r a t o r y o f the Research Institute on Alcoholism. 1 ARMSTRONG,C. M., AND BINSTOCK, C., The effects of several alcohols on the properties of the squid giant axon, J. gen. Physiol., 48 (1964) 265-277. 2 AUERBACH, A. A., AND BENNETT, M. V. L., Chemically mediated transmission at a giant fiber synapse in the central nervous system of a vertebrate, J. gen. PhysioL, 53 (1969) 183-210. 3 BARKER,J. L., AND GAINER,H., Pentobarbital: selective depression of excitatory postsynaptic potentials, Science, 182 (1973) 720-722. 4 BERGMANN,M. C., FABER,D. S., AND KLEE, M. R., Reduction of the early inward sodium and calcium currents of Aplysia neurons by ethanol, Pfliigers Arch. ges. PhysioL, 332, Suppl. (1972) R66. 5 BERGMANN,M. C., KLEE,i . R., ANDFABER,n. S., Different sensitivities to ethanol of three early transient voltage clamp currents of Aplysia neurons, Pfliigers Arch. ges. PhysioL, 348 (1974) 139-153. 6 CHASE, R., The suppression of excitatory synaptic responses by ethyl alcohol in the nudibranch mollusc, Tritonia Diomedia, Comp. Biochem. PhysioL, 506 (1975) 37~10. 7 DIAMOND, J., The Mauthner cell. In W. S. HOAR AND n. J. RANDALL(Eds.), Fish Physiology, I/oL V, Academic Press, New York, 1971, pp. 265-346. 8 ECCLES,J. C., The central action of antidromic impulses in motor nerve fibres, Pfliigers Arch. ges. PhysioL, 260 (1955) 385415. 9 EIDELBERG,E., AND WOOLEY, D. F., Effects of ethyl alcohol upon spinal cord neurones, Arch. int. Pharmacodyn., 185 (1970) 388-396. 10 FABER, D. S., AND KORN, H., A neuronal inhibition mediated electrically, Science, 179 (1973) 577-578.
353 11 FABER, D. S., AND KORN, H., Inputs from the posterior lateral line nerves upon the goldfish Mauthner cell. lI. Evidence that the inhibitory components are mediated by interneurons of the recurrent collateral network, Brain Research, 96 (1975) 349-356. 12 FURSHPAN, E. J., AND FURUKAWA, T., Intracellular and extracellular responses of the several regions of the Mauthner cell of the goldfish, J. Neurophysiol., 25 (1962) 732-771. 13 FUROKAWA,T., Synaptic interaction at the Mauthner cell of goldfish. In T. TOKIZANEAND J. P. SCI-IADg (Eds.), Correlative Neurosciences. Part A: Fundamental Mechanisms, Vol. 21, Progr. Brain Res., Elsevier, Amsterdam, 1966, pp. 44-70. 14 FURUKAWA,T., AND FURSHPAN, E. J., TWO inhibitory mechanisms in the Mauthner neurons of goldfish, J. Neurophysiol., 26 (1963) 14{)-176. 15 GA~E, P. W., The effect of methyl, ethyl and n-propyl alcohol on neuromuscular transmission in the rat, J. Pharmacol. exp. Ther., 150 (1965) 236-243. 16 KALArqT, H., ISRAEL, Y., AND MAHON, M. A., The effect of ethanol on acetylcholine synthesis, release and degradation in brain, Canad. J. physiol. Pharmacol., 45 (1967) 172-176. 17 KL~E, M. R., LEE, K. C., AND PARK, M. R., Changes in cat motoneuron membrane properties with ethanol, Pfliigers Arch. ges. Physiol., 355 (1975) R85. 18 KORN, H., AND FABER, D. S., An electrically mediated inhibition in the goldfish medulla, J. Neurophysiol., 38 (1975) 452471. 19 KORN, H., AND FABER, D. S., Inputs from the posterior lateral line nerves upon the goldfish Mauthner cell. I. Prop:rties and synaptic localization of the excitatory component, Brain Research, 96 (1975) 342-348. 20 LOYNING, Y., OSHIMA, T., AND YOKOTA, T., Site of action of thiamytal sodium on the monosynaptic spinal reflex pathway in cats, J. Neurophysiol., 27 (1964) 408428. 21 MOORE,J. W., Effects of ethanol on ionic conductances in the squid axon membrane, Psychosom. Med., 28 (1966) 450-457. 22 OKADA,K., Effects of alcohols and acetone on the neuromuscular junction of frog, Jap. J. Physiol., 17 (1967) 245-261. 23 SPmA, M., MODEL, P. G., AND BENNETT, M. V. L., Cholinergic transmission at a vertebrate central synapse, J. Cell Biol., 47 (1970) 199a. 24 WALLGREN,H., AND BARRY,H., Cellular basis of ethanol action on the nervous system. In H. WALLGREN AND H. BARRY (Ed~.), Actions of Ethanol, Vol. 1, Biochemical, Physiological and Psychological Aspects, Ch. 5, Elsevier, New York, 1970, pp. 209-273. 25 WEAKLY,J. N., Effect of barbiturates on 'quantal' synoptic transmission in spinal motoneurones, J. Physiol. (Lond.), 204 (1969) 63-77. 26 WILSON, D. M., Function of giant Mauthner's neurons in the lungfish, Science, 129 (1959) 841-842.
347
© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
Ethanol suppresses collateral inhibition of the goldfish Mauthner cell
DONALD S. FABER AND MANFRED R. KLEE* Research Institute on Alcoholism, New York State Department of Mental Hygiene, Buffalo, N.Y. 14203, and Department of Physiology, State University of New York at Buffalo, Buffalo, N.Y. 14214 (U.S.A.)
(Accepted November 25th, 1975)
It is well known that ethanol directly depresses the electrical excitability of a variety of vertebrate and invertebrate neurons, presumably through a relatively specific reduction in the early inward sodium current underlying spike initiation 1,s,zl (cf. reviews in ref. 24). In contrast, opposite actions of ethanol on junctional transmission have been described for the vertebrate and invertebrate nervous systems; although it depresses the acetylcholine sensitivity and excitatory postsynaptic potentials of molluscan neurons3,a, 6 and both inhibition and excitation of cat spinal motoneurons 9,17, it facilitates cholinergic transmission at the vertebrate neuromuscular junction15, 22. Furthermore, there is relatively little information concerning its effects on synaptic transmission in the vertebrate central nervous system, which, on the basis of psychophysical tests is appreciably more sensitive to ethanol than the peripheral nervous system (cf. ref. 24). A common criticism of electrophysiological experiments designed to elucidate the mechanisms of action of substances such as ethanol on specific neuronal systems is that the drug concentrations required to produce an effect, as well as the effects themselves, cannot readily be correlated with behavioral observations of the drug action. One preparation which appears suited for dealing with this problem is the goldfish medullary network involving the Mauthner cell (M-cell) and the startle reflex which it mediates, since both its neuronal organization and functional output are well-defined7,10-14,18,19, 26. In this paper we report that collateral inhibition of the M-cell is reduced or blocked completely at brain ethanol levels which have no significant effects on the cell's electrical excitability and which are appreciably lower than those which cause the fish to lose their righting reflexes. Rather, the depression of collateral inhibition occurs at brain levels associated with behavioral hyperexcitability. Furthermore, we present evidence suggesting that this effect of ethanol is due to an impaired release of transmitter, presumably acetylcholine, from M-cell axon collaterals. Common goldfish, 6-7 in. in length, were used for both the electrophysiological * Permanent address: Neurobiology Department, Max-Planck Institute for Brain Research, Frankfurt/M, G.F.R.
348 experiments and the behavioral observations. In the former series, tbe techniques utilized were similar to those described previously 12,18. The fish were curarized (1/~g/ g body wt.) and respired with a continuous flow of dechlorinated tap water through the mouth and over the gills. Bipolar electrodes on the spinal cord and the ipsi- or contralateral VlIIth nerves were used to activate the M-cell antidromically and orthodromically, respectively, with collateral inhibition of the cell also being evoked through the antidromic pathway (Fig. IA). Intra- and extracellular recordings were obtained from the M-cell with glass microelectrodes (2-6 m~2 resistance) filled with either 2.7 M KC1 or 0.6 M K2SO4. After obtaining the control responses, the solution perfusing the gills was switched to one containing 1-2 ~ ethanol (w/v), and the effects described here developed gradually during the next l-2 h. At the end of each experiment, the brain was removed and frozen rapidly in liquid nitrogen for subsequent gas chromatographic determination of the ethanol concentration. In parallel experiments, the effects of I ~o ethanol on freely swimming goldfish were initially determined by gross behavioral observations. Brain ethanol concentrations in these fish reached an equilibrium level of approximately 10 /~g/mg brain weight in approximately 2 h, whereas the ethanol concentration rose more slowly in the restrained fish used for the physiological experiments. During the first 30-60 min the fish undergo a phase of hyperexcitability characterized by hyperreflexia, including an enhanced startle reflex, poorly coordinated swimming and gulping of air at the water surface. These behavioral effects correlated with brain ethanol levels of 3-5 /zg/mg brain wt. As the ethanol concentration increased, the fish sank to the bottom of the aquarium, became grossly ataxic, overturned, and lost their righting reflexes. As shown in Fig. 1A, the startle reflex can be initiated by VIIIth nerve inputs to the M-cell lateral dendrite. Activation of the M-cell almost always results in contraction of the contralateral tail musculature, and the major inhibitory control of the reflex is mediated by medullary inhibitory interneurons which are excited by axon collaterals of both M-cells as well as by sensory afferents 11,~4,as. There are two components to the collateral inhibition: (1) a short latency electrical inhibition characterized by an extrinsic hyperpolarizing potential (EHP) which is best recorded as an extracellular positivity in the axon cap surrounding the initial segment of the M-cell axon (Fig. 1B1); and (2) a subsequent chemically mediated inhibitory postsynaptic potential (IPSP) 14. Since its equilibrium potential is close to the M-cell resting potential, the IPSP does not generally appear as a significant membrane potential change. It can, however, be quantified with double antidromic stimuli; the amplitude of the second antidromic action potential is reduced by the conductance increase associated with the IPSP 14 (Fig. 1C1). As described elsewherO ~,13,18, both the EHP and the IPSP are most likely mediated by the same inhibitory interneurons, the first being due to the flow of the extracellular currents generated by the action potentials in these neurons. It should be pointed out that the collateral inhibition illustrated in Fig. 1C1 is sufficiently strong to occasionally block in an all-or-none manner the major component of the second antidromic action potential, thereby unmasking a smaller spike component. Such a separation of the M-cell spike into components has not been pre-
349 A
C 1Control
C 2 Ethanol
i
i
2ms
D1Control
D2 Ethanol
M. o x o 0
~ Stim. 500~se¢
B1
,~f"
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B2 Ethanol
2ms
E2
200vsec
Fig. 1. Effect of ethanol on collateral inhibition of the M-cell and on M-cell electrogenesis. A: schematic illustration of the experimental arrangement and the neuronal model taken as the basis for analyzing the results. A microelectrode (ME) was inserted in the M-cell's soma or lateral dendrite (Lat. Dendr.) and intracellular potentials were monitored following stimulation (Stim.) of the M-cell's axon (M. axon) or the VIIIth nerve (VllIth N.). I represents a pool of inhibitory interneurons which are activated by M-cell axon collaterals and which in turn feed inhibition back onto the cell. Contralateral connections are not shown. B1 and B2 : effect of ethanol on the extracellular fields recorded in the axon cap. BI: control. Spinal cord stimulation at a rate of I/3 sec evoked a negative M-cell antidromic spike followed by a positive EHP (arrow). Bz: 35 min after starting perfusion with 1 ethanol, the EHP was strongly reduced. The experiment was terminated 20 min later, at which time the final ethanol level was 4.7/~g/mg brain wt. Three or more superimposed traces are shown in all records of this figure. C1 and C2: intracellular recordings from the M-cell soma in another experiment illustrate that ethanol blocks the collateral IPSP as well. C~: control. With paired antidromic stimuli the increased conductance associated with the IPSP evoked by the conditioning stimulus blocks completely or reduces by approximately 50 % the axon hillock component of the following test antidromic spike. Recording was with a K2SO4 microelectrode from the M-cell soma and there was no appreciable membrane potential change associated with the IPSP. Ca: after 60 min perfusion with 1 ~ ethanol, the IPSP conductance change was almost completely abolished. The ethanol brain concentration was 3.45 #g/rag. Dx and D2: a similar concentration of ethanol (4.51/~g/mg) had a minimal effect on the M-cell antidromic action potential as recorded in the cell's soma. Each record illustrates the simultaneously recorded M-cell spike (lower trace) and its electrically differentiated representation (upper trace). D~: control. Dz: after 86 min exposure to ethanol, the spike was slightly smaller and increased in duration; these changes were correlated with slower rates of rise and repolarization. Such changes in the M-cell spike often occur during 1-2 h continuous intracellular recording and may not be attributed to an action of ethanol. E1 and E2: at a higher brain ethanol level (8.2/,g/mg) distinct effects of ethanol on M-cell electrogenesis were occasionally observed. Both sets of records were obtained 1 h after starting perfusion with 2 ~ ethanol. E~: the spike initiated at the M-cell's axon hillock failed and unmasked a small axon spike. Ez: the M-cell was depolarized with a steady transmembrane current applied through the recording microelectrode; under these conditions the axon spike succeeded in bringing the cell's axon hillock to threshold and the latter initiated a spike as well.
350 viously reported. However, this phenomenon does not reflect M-cell injury since it can also be observed during extracellular recordings. Rather, we tentatively suggest that it represents a failure of active spike propagation from the M-cell axon to the axon hillock. In this context, the initial component would represent the antidromic spike generated by the medullated portion of the axon and the subsequent one the invasion of the axon hillock 8. Ethanol in a concentration of 3-5 #g/mg of brain tissue reduces both components of collateral inhibition (Fig. IB and C). In contrast, M-cell resting membrane potential (generally between --70 to --80 mV) remains unchanged even at levels as high as 20 ~g/mg and only minor changes in its antidromic action potential (Fig. 1D1 vs. Fig. 1 D2) occur at the lower ethanol levels. When the brain ethanol concentration was in the range of 8-15 #g/mg, direct effects on M-cell excitability were occasionally observed. In the example illustrated in Fig. l E1 the safety factor for transmission of the antidromic impulse to the axon hillock was reduced and invasion failed; under these conditions a full action potential could be restored by pairing a depolarizing current with the antidromic axon spike (Fig. 1E2). At higher ethanol levels, the M-cell axon spike may fail as well. Finally, we did not observe any significant effects of ethanol on either the excitatory or inhibitory inputs to the M-cell from the ipsi- and contralateral VIIIth nerves, respectively. This observation is significant in that the inhibitory components of these inputs are mediated by the same interneurons which are responsible for the collateral inhibition. Consequently, ethanol's effect cannot be attributed to a non-specific action on the inhibitory interneurons or to a blockage of inhibitory transmission onto the M-cell. The fact that both components of collateral inhibition are equally blocked by ethanol supports the above conclusions and indicates it acts at the level of the synapse between M-cell axon collaterals and the inhibitory interneurons. Three possible modes of action have been considered: (1) a presynaptic effect on spike propagation in the axon collaterals, (2) a depression of transmitter release, and (3) a reduction in the sensitivity of the subsynaptic receptors of the inhibitory interneurons to the M-cell transmitter. The first possibility seems unlikely since effects on the excitability of the M-cell axon only occurred at higher ethanol levels. Nevertheless, a reduced excitability of thinner axon collaterals cannot be ruled out. Although it is difficult to further localize the site of ethanol's action, the results of experiments on the time course of development of the effect described below strongly suggest that it acts presynaptically to impair transmitter release. Since the magnitude of collateral inhibition is very labile at stimulus frequencies above 1/sec in the curarized preparation 14, we routinely used a repetition rate of 1/3-1/7 sec in our experiments. Under these conditions, a stable maximal IPSP can be observed (Fig. 2A; note that the IPSP is reversed, i.e. depolarizing, in this figure since a KCI electrode was used for the intracellular recordings). At the lower repetition rate, no effect of 1 ethanol was seen after 25 min of perfusion (Fig. 2B) but there was typically a 50-70 reduction 10-15 min later (Fig. 2C). After ! h exposure, the IPSP appeared to be abolished (Fig. 2D). However, we observed at that time that a small 1PSP could be restored by switching to a lower stimulus frequency and that a full-sized one could be
351
B Ethanol ,25'
A Control
C 34, I
D 58'
F 69'; 1' rest
E 59'; 1'rest
,
~
G 74,; 5, rest
H 81'; 1' rest
~
i
--
-
j2_
I" 86'; 5' rest
,
lms Fig. 2. Time course of the development of the ethanol effect on the collateral IPSP. The records are from the same experiment as in Fig. 1DI and Da and were obtained with a KCl-containing microelectrode. Three or more superimposed traces of the M-cell responses to spinal cord stimulation at a rate of 1/7 sec are shown in A-I. A: control. The antidromic action potential elicited by a single shock to the M-axon is followed by a depolarizing IPSP which causes the cell to initiate a second spike. B-I: records obtained at the indicated times after starting perfusion with 1% ethanol. The traces illustrated in E, F and H were preceded by 1 min rest periods during which no stimulation was employed, and those illustrated in G and I were preceded by 5 min rest periods. B-D: gradual reduction and, finally, blockage of the IPSP over a 58 min period. E: at 59 min ethanol, a 1 min rest restored the IPSP for the first stimulus in a train with a repetition rate of 1/7 sec. F: diminished effectiveness of the 1 min rest 10 min later. G: at 74 min, a 5 min rest restored the IPSP for one stimulus to a magnitude greater than that in control. H: 81 min after starting perfusion with ethanol, the 1 min rest was completely ineffective and no IPSP was evoked, h after an additional 5 rain rest, an IPSP less than 10 % the amplitude of that illustrated in control could be evoked by the first stimulus in the 1/7 sec train.
evoked for one stimulus after a 1 m i n period of rest w i t h o u t spinal cord s t i m u l a t i o n (Fig. 2E); a second stimulus 7 see later was completely ineffective (Fig. 2E). As the action o f e t h a n o l progressed further, the effectiveness of such 1 m i n rest periods d i m i n i s h e d appreciably (Fig. 2F) a n d longer rest periods in the range o f 5 m i n were necessary (Fig. 2G). Again, however, only the first in a t r a i n o f stimuli at a 1/7 see repetition rate evoked a n IPSP. Finally, after 80-90 m i n total perfusion time with ethanol, such rest periods were completely ineffective a n d the IPSP was completely blocked (Fig. 2 H a n d I). A t t h a t time, the e t h a n o l level in the experiment illustrated in Fig. 2 was 4.51/~g/mg b r a i n weight, a n d we estimate t h a t it was n o more t h a n 2-3 big/ m g b r a i n weight d u r i n g the first h o u r o f exposure. The evidence that such p r o l o n g e d rest periods are capable o f transiently restoring synaptic t r a n s m i s s i o n suggests that e t h a n o l acts o n the process of t r a n s m i t t e r release from the M-cell axon collaterals, a n d does n o t a p p e a r readily c o m p a t i b l e with effects o n collateral excitability or the sensitivity of the postsynaptic n e u r o n s to the M-cell t r a n s m i t t e r . It m a y be .*hat the M-cell p r e p a r a t i o n is especially sensitive to such effects, since t r a n s m i s s i o n readily fails at m o d e r a t e stimulus frequencies. A c o m p a r a ble situation exists at the cholinergic M-cell to giant fiber synapse in the hatchet fish z3.
352 A u e r b a c h and Bennett 2 have shown that the latter synapse also fatigues at moderate stimulus frequencies and have concluded that a single M-cell impulse releases most o f the readily available transmitter store. This is probably true o f the goldfish M-cell as well, which also appears to be cholinergic; l0 # M curare applied topically reversibly blocks both the E H P and the collateral IPSP (Faber and Klee, unpublished results). Consequently, it seems most probable that ethanol blocks transmitter synthesis or mobilization. These results are in agreement with the biochemical studies o f Kalant et al. 16, which indicated that incubation with ethanol inhibits acetylcholine release from unstimulated rat cerebral cortex slides. Finally, it is interesting to note that a somewhat similar presynaptic effect o f barbiturates on cat spinal synaptic transmission has been postulated20, 25. Our preliminary behavioral observations indicate that the initial phase of hyperexcitability following exposure to ethanol occurs at the same brain ethanol levels which rather selectively depress excitatory synaptic transmission from the M-cell collaterals onto the interneurons exerting a major inhibitory control o f the startle reflex. In addition, higher anesthetic levels m a y be associated with direct effects on excitability as well. Clearly, more quantitative behavioral experiments on the effects o f ethanol and additional experiments on the other components o f the neural network involved in the startle reflex are needed. Nevertheless, it is clear that this preparation offers a promising approach to the general problem o f correlating physiological and behavioral aspects o f drug actions. This work was supported in part by N I N C D S G r a n t No. NS12132-01 and by a travel grant to Manfred R. Kiee f r o m the Max-Planck Institute for Brain Research, Frankfurt, G.F.R. We gratefully acknowledge the technical assistance o f Alan Dash and the services o f the Analytical L a b o r a t o r y o f the Research Institute on Alcoholism. 1 ARMSTRONG,C. M., AND BINSTOCK, C., The effects of several alcohols on the properties of the squid giant axon, J. gen. Physiol., 48 (1964) 265-277. 2 AUERBACH, A. A., AND BENNETT, M. V. L., Chemically mediated transmission at a giant fiber synapse in the central nervous system of a vertebrate, J. gen. PhysioL, 53 (1969) 183-210. 3 BARKER,J. L., AND GAINER,H., Pentobarbital: selective depression of excitatory postsynaptic potentials, Science, 182 (1973) 720-722. 4 BERGMANN,M. C., FABER,D. S., AND KLEE, M. R., Reduction of the early inward sodium and calcium currents of Aplysia neurons by ethanol, Pfliigers Arch. ges. PhysioL, 332, Suppl. (1972) R66. 5 BERGMANN,M. C., KLEE,i . R., ANDFABER,n. S., Different sensitivities to ethanol of three early transient voltage clamp currents of Aplysia neurons, Pfliigers Arch. ges. PhysioL, 348 (1974) 139-153. 6 CHASE, R., The suppression of excitatory synaptic responses by ethyl alcohol in the nudibranch mollusc, Tritonia Diomedia, Comp. Biochem. PhysioL, 506 (1975) 37~10. 7 DIAMOND, J., The Mauthner cell. In W. S. HOAR AND n. J. RANDALL(Eds.), Fish Physiology, I/oL V, Academic Press, New York, 1971, pp. 265-346. 8 ECCLES,J. C., The central action of antidromic impulses in motor nerve fibres, Pfliigers Arch. ges. PhysioL, 260 (1955) 385415. 9 EIDELBERG,E., AND WOOLEY, D. F., Effects of ethyl alcohol upon spinal cord neurones, Arch. int. Pharmacodyn., 185 (1970) 388-396. 10 FABER, D. S., AND KORN, H., A neuronal inhibition mediated electrically, Science, 179 (1973) 577-578.
353 11 FABER, D. S., AND KORN, H., Inputs from the posterior lateral line nerves upon the goldfish Mauthner cell. lI. Evidence that the inhibitory components are mediated by interneurons of the recurrent collateral network, Brain Research, 96 (1975) 349-356. 12 FURSHPAN, E. J., AND FURUKAWA, T., Intracellular and extracellular responses of the several regions of the Mauthner cell of the goldfish, J. Neurophysiol., 25 (1962) 732-771. 13 FUROKAWA,T., Synaptic interaction at the Mauthner cell of goldfish. In T. TOKIZANEAND J. P. SCI-IADg (Eds.), Correlative Neurosciences. Part A: Fundamental Mechanisms, Vol. 21, Progr. Brain Res., Elsevier, Amsterdam, 1966, pp. 44-70. 14 FURUKAWA,T., AND FURSHPAN, E. J., TWO inhibitory mechanisms in the Mauthner neurons of goldfish, J. Neurophysiol., 26 (1963) 14{)-176. 15 GA~E, P. W., The effect of methyl, ethyl and n-propyl alcohol on neuromuscular transmission in the rat, J. Pharmacol. exp. Ther., 150 (1965) 236-243. 16 KALArqT, H., ISRAEL, Y., AND MAHON, M. A., The effect of ethanol on acetylcholine synthesis, release and degradation in brain, Canad. J. physiol. Pharmacol., 45 (1967) 172-176. 17 KL~E, M. R., LEE, K. C., AND PARK, M. R., Changes in cat motoneuron membrane properties with ethanol, Pfliigers Arch. ges. Physiol., 355 (1975) R85. 18 KORN, H., AND FABER, D. S., An electrically mediated inhibition in the goldfish medulla, J. Neurophysiol., 38 (1975) 452471. 19 KORN, H., AND FABER, D. S., Inputs from the posterior lateral line nerves upon the goldfish Mauthner cell. I. Prop:rties and synaptic localization of the excitatory component, Brain Research, 96 (1975) 342-348. 20 LOYNING, Y., OSHIMA, T., AND YOKOTA, T., Site of action of thiamytal sodium on the monosynaptic spinal reflex pathway in cats, J. Neurophysiol., 27 (1964) 408428. 21 MOORE,J. W., Effects of ethanol on ionic conductances in the squid axon membrane, Psychosom. Med., 28 (1966) 450-457. 22 OKADA,K., Effects of alcohols and acetone on the neuromuscular junction of frog, Jap. J. Physiol., 17 (1967) 245-261. 23 SPmA, M., MODEL, P. G., AND BENNETT, M. V. L., Cholinergic transmission at a vertebrate central synapse, J. Cell Biol., 47 (1970) 199a. 24 WALLGREN,H., AND BARRY,H., Cellular basis of ethanol action on the nervous system. In H. WALLGREN AND H. BARRY (Ed~.), Actions of Ethanol, Vol. 1, Biochemical, Physiological and Psychological Aspects, Ch. 5, Elsevier, New York, 1970, pp. 209-273. 25 WEAKLY,J. N., Effect of barbiturates on 'quantal' synoptic transmission in spinal motoneurones, J. Physiol. (Lond.), 204 (1969) 63-77. 26 WILSON, D. M., Function of giant Mauthner's neurons in the lungfish, Science, 129 (1959) 841-842.