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Ethanol suppresses hippocampal cell firing through a calcium and cyclic AMP-sensitive mechanism
European Journal of Pharmacology, 164 (1989) 591-594
591
Elsevier EJP 20382 Short communication
Ethanol suppresses hippocampal cell firing through a calcium and cyclic AMP-sensitive mechanism D e b o r a h M. B e n s o n 2,4, R o b e r t D. Blitzer 2 a n d E m m a n u e l M. L a n d a u 1,2,3,* i Department of Psychiatry, Bronx VA Medical Center, Bronx, N Y 10468, and Departments of 2 Psychiatry and 3 Pharmacology, Mr. Sinai School of Medicine, New York, N Y 10029, and 4 Department of Psychology, Queens College of The City University of New York, Flushing, N Y 11367, U.S.A.
Received 28 March 1989, accepted 4 April 1989
The effects of ethanol were studied intracellularly in hippocampal pyramidal cells in vitro. Ethanol, 50-100 mM, produced a marked suppression of neuronal firing. This effect was blocked by treating the cell with cyclic 3% 5'-adenosine monophosphate (cAMP) or cadmium ions. Ethanol had no effect on the after-hyperpolarizing current. It is concluded that the ethanol-induced reduction of firing rate is due to a calcium-dependent process, and modulated by cAMP. Ethanol; Hippocampus; cAMP;
Ca2+; After-hyperpolarization; (Neuronal firing)
1. Introduction Ethanol is a major psychoactive drug, with widespread use leading to substantial medical and social consequences. A commonly reported central nervous system effect of ethanol is a suppression of neuronal firing (Harris and Sinclair, 1984; Shefner and Tabakoff, 1985), although mixed, biphasic or excitatory effects have also been reported (Siggins et al., 1987). The aim of the present study was to investigate the mechanism of this suppressant effect of ethanol in hippocampal slices in vitro. Our major finding is that ethanol's suppression of hippocampal neuronal firing is mediated through a calcium- and cyclic-AMP (cAMP)-sensitive mechanism. This mechanism does not involve enhancement of the calcium-dependent potassium (after-hyperpolarizing) current, which was not affected by ethanol.
* To whom all correspondence should be addressed: Dept. Psychiatry, Bronx VA Hospital, 130 W. Kingsbridge Road, Bronx, NY 10468, U.S.A.
2. Materials and methods Standard intracellular recording methods (Nicoll and Alger, 1981) were used to record from guinea pig hippocampal slices maintained in vitro. Experiments were performed at 23-25°C and at 30-32°C. Most slices were taken from chloral hydrate-anesthetized guinea pigs, although several guinea pigs were instantly decapitated without anesthesia, to rule out the possibility that chloral hydrate contributed to the effects seen. Following decapitation, the brains from all animals were immediately transferred into ice-cold Ringer solution. CA 1 pyramidal cells were impaled with glass microelectrodes filled with either 2 M potassium methylsulfate (KMeSO4) , 3 M potassium chloride (KC1), or, in a few experiments, 100-200 mM cAMP. To measure firing rate, the cell membrane was depolarized through a bridge circuit to a predetermined membrane potential with 30 s depolarizing current pulses of 0.1 to 0.4 nA (to achieve a regular rate of firing throughout the train). To test the membrane input impedance, brief (100 ms) hyperpolarizing pulses (0.2-0.4 nA) were passed through the bridge circuit. For volt-
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
592
age-clamp experiments, a single-electrode voltage clamp amplifier was used (Axoclamp-2), with a switching frequency of 2-3 kHz and a 30% duty cycle. For our studies, we accepted cells with resting potentials of < - 5 5 mV, action potential amplitudes of > 70 mV and membrane resistances of > 20 M~2. Typical values were resting potential: - 6 4 . 9 + 7 mV (n = 38), spike amplitude: 92.4 + 10 mV (n = 37), input resistance: 67.9 + 15 M$2 (n = 34). For statistical analyses, comparisons were made within cells using two-tailed t-tests. Error values referred to in the text are S.E.M.
3. R e s u l t s
Ethanol (50-100 mM; 0.25-0.5 g / d l ) consistently produced a suppression of neuronal firing, causing fewer spikes to be elicited upon stimulation by a 30 s long depolarizing current pulse, as illustrated in fig. 1A-C. This ethanol-induced suppression of firing was found in all cells tested. No differences were found
A.
28
B.
between cells impaled with either KCI- or KMeSOa-containing electrodes, between cells recorded at room or elevated temperatures, or between slices obtained from anesthetized or unanesthetized animals. In view of this, the data from all of these conditions were pooled. The average number of spikes during a 30 s depolarizing pulse decreased to 65 _+ 2% (n = 36) of the control value in ethanol. The effect was statistically significant, t(35)= 7.65, P <0.0005. The ethanol effect was easily reversible, and spike frequencies recovered to 109 _+ 5% (n = 36) of control (not significantly different) after washing out the drug. There was no relationship between the number of spikes generated during the pulse and the percentage reduction by ethanol. The suppressant effect of ethanol was probably not due to changes in passive m e m b r a n e properties, since it produced only a small (1-2 mV) hyperpolarization of the cell m e m b r a n e and a 5% decrease in the membrane input impedance. The depolarizing currents employed to elicit neuronal firing were adjusted to compensate for these
C,
1
i
i
I
I
F----[
___
i
D.
I0 mV T
F,
E.
0.5 n A ~ 4 0 0 ms
44
58
46
i I
I
Fig. 1. Cadmium blocks the ethanol suppressant effect. Chart records of the membrane response to 30 s depolarizing current pulses. Numbers over spike trains denote the number of spikes in the train. Broken vertical line through each trace denotes break in the record. In each condition, the pulse brought the membrane potential to the same level ( - - 5 5 mV). Left panels: control; middle panels: 25 min into 100 mM ethanol; right panels: 10 min after washout of ethanol. (A-C) Ethanol effects in the control medium. (D-F) Ethanol effects in the presence of'300 p.M cadmium chloride. Note the failure of ethanol to suppress spike frequency in cadmium. Spikes truncated due to frequency characteristics of chart recorder. Vertical calibration 10 mV. Horizontal calibration 400 ms. Recording electrode: KC1.
593 changes so as to d e p o l a r i z e the cells to the same level as in the c o n t r o l situation. W e next e x a m i n e d the effect of e t h a n o l on the a f t e r - h y p e r p o l a r i z i n g ( A H P ) current, which has p r e v i o u s l y b e e n r e p o r t e d to b e e n h a n c e d b y e t h a n o l (Carlen et al., 1982; however, see Siggins et al., 1987), a n d is a m a j o r d e t e r m i n a n t of neuronal firing r a t e ( L a n c a s t e r a n d A d a m s , 1986; M a d i son a n d Nicoll, 1982). F o u r cells were voltagec l a m p e d in the presence of t e t r o d o t o x i n (TTX) at - 6 0 m V a n d s t e p p e d for 2 s to - 3 5 mV. T h e resulting ' t a i l ' c u r r e n t ( A H P current) was m e a sured 200 m s after the e n d of the d e p o l a r i z i n g pulse. T h e value of the A H P current in the c o n t r o l c o n d i t i o n was 106 + 23 p A a n d in 100 m M ethanol, the A H P c u r r e n t was 99 + 26 p A . T h e r e was no significant difference b e t w e e n these values. I n o r d e r further to investigate the m e c h a n i s m of the s u p p r e s s a n t effect of e t h a n o l on n e u r o n a l firing, we e x a m i n e d the effects of c a d m i u m (which b l o c k s c a l c i u m channels) a n d c A M P on the e t h a n o l effect. Both c a l c i u m a n d c A M P have b e e n imp l i c a t e d in the cellular effects of e t h a n o l (Messing et al., 1986; G o r d o n et al., 1986). Both drugs b l o c k e d a n d in s o m e cases, reversed, the e t h a n o l i n d u c e d s u p p r e s s i o n of firing. F i g u r e 1 A - C shows a cell in which e t h a n o l p r o d u c e d a typical reduction in firing. However, after a d d i t i o n of 3 0 0 / ~ M c a d m i u m ( D - F ) there was a small increase in the n u m b e r of spikes elicited in ethanol. This effect of c a d m i u m (0.2-0.3 m M ) was seen in five cells, the n u m b e r of spikes in e t h a n o l averaging 127 + 17% of c o n t r o l (no significant difference). In these e x p e r i m e n t s , the n u m b e r of spikes elicited d u r i n g the d e p o l a r i z i n g pulse in the c o n t r o l m e d i u m was 39 + 5 (n = 5). This is n o t significantly different f r o m the c o r r e s p o n d i n g value o b t a i n e d with no c a d m i u m ions p r e s e n t (49 ___5, n = 36). Therefore, the b l o c k i n g b y c a d m i u m of the ethanol suppress a n t effect c a n n o t be e x p l a i n e d b y changes in n e u r o n a l firing rates in the presence of c a d m i u m ions. Similar results were f o u n d with c A M P a p p l i e d in the m i c r o e l e c t r o d e (n = 4) or in the b a t h as the 8 - b r o m o a n a l o g u e (0.5 m M , n = 4). A s shown in fig. 2 D - F , c A M P a p p l i e d in the r e c o r d i n g elect r o d e p r e v e n t e d the e t h a n o l - i n d u c e d decrease in firing rate. F o r c o m p a r i s o n , the typical effect of
A.
D.
~
B,
47
E.
56
31
IOmV~ los
82
C,
46
46
,,~,~ _
Fig. 2. The suppressant effect of ethanol is blocked by cAMP. Shown are chart records of the membrane response to 30 s depolarizing current pulses in a cell impaled with a 3 M KCl-containing electrode (A-C) and in a different cell impaled with a 200 mM cAMP/3 M KCl-containing electrode (D-F). Numbers over spike trains denote the number of spikes in the train (spikes truncated due to frequency characteristics of chart recorder). (A,D) Control; (B,E) 100 mM ethanol; (C,F) washout. Calibration: vertical, 10 mV; horizontal, 10 s (except following the pulse in A-C:2 s).
e t h a n o l is shown in fig. 2 A - C (different cell). O n the average, in the presence of c A M P , the n u m b e r of spikes in e t h a n o l increased to 107 + 8% (n = 8) of control. This effect was statistically significant, (t(7) = 2.13, P < 0.05). T h e e t h a n o l - i n d u c e d increase in firing rate seen in the p r e s e n c e of c A M P m a y reflect an u n d e r l y i n g e x c i t a t o r y effect of e t h a n o l which can be revealed w h e n the s u p p r e s sant effect is b l o c k e d . T h e n u m b e r of spikes elicited u p o n d e p o l a r i z a t i o n in the p r e s e n c e of c A M P was significantly greater t h a n in it's absence (93 + 25, n = 8 vs. 49 + 5, n = 36; t(42) = 2.89, P < 0.005). However, the i n c r e a s e d n e u r o n a l r e s p o n s i v i t y in c A M P is n o t likely to e x p l a i n the o b s e r v e d i n t e r a c t i o n b e t w e e n c A M P a n d ethanol, since this r e s p o n s i v i t y was n o t c o r r e l a t e d with the m a g n i t u d e of the e t h a n o l effect (see above).
4. Discussion W e have f o u n d that c o n c e n t r a t i o n s of e t h a n o l in the range of 50-100 m M (0.25-0.5 g / d l ) consistently suppress the firing rate of h i p p o c a m p a l neurons. These c o n c e n t r a t i o n s cause s e d a t i o n a n d a t a x i a in rats (Majchrowicz, 1975), a n d are c o n s i d -
594
ered to be in the high-intoxicating range. This finding differs somewhat from that reported by Siggins et al. (1987), who found more variable effects of ethanol on excitability in C A 1 and C A 3 neurons. They found more consistent ethanoldepressant effects on spontaneous firing of CA 1 neurons than on depolarizing current-evoked spikes. It can be noted, however, that our protocol of administering 30 s long depolarizing pulses may, in fact, better approximate a spontaneously firing neuron than one responding to a brief depolarizing current pulse. Ethanol had no effect on the A H P current in this study, in contrast to the ethanol-induced enhancement of the A H P current reported by Carlen et al. (1982) in current-clamped cells. The present experiments were done in voltage-clamped neurons, however, allowing a more reliable measurement of the A H P current than in current-clamped cells, in which the number of spikes generated and the amplitude of the depolarizing pulse are difficult to control. Since the A H P current was not influenced by ethanol in these experiments, the ethanol effect on neuronal firing cannot be mediated through an enhancement of this current. An alternative hypothesis is that the ethanol suppressant effect may be due to increasing the rate of spontaneous inhibitory post-synaptic potentials. However, the ethanol effect was the same in both KC1- and KMeSOa-containing electrodes. Thus, this possibility can be ruled out. The suppressant effect of ethanol was blocked by cadmium and cAMP. While these agents both block the A H P current, this blockade cannot be the common mechanism by which they exert their effects, since this current was not influenced by ethanol in the present study. Therefore, the reduction in firing produced by ethanol must be due to another process which is both modulated by c A M P and dependent on calcium entrance into the cell. Further work is needed to elucidate the precise nature of this mechanism which underlies the
ethanol suppressant effect on hippocampal pyramidal neurons.
Acknowledgements This work was supported in part by N I A A A Grant No. 5-R23-AA06659 and N I H - N I A Grant No. 5-P50-AG05138.
References Carlen, P.L., N. Gurevich and D. Durand, 1982, Ethanol in low doses augments calcium-mediated mechanisms measured intracellularly in hippocampal neurons, Science 215, 306. Gordon, A.S., K. Collier and I. Diamond, 1986, Ethanol regulation of adenosine receptor-stimulated c A M P levels in a clonal neural cell line: an in vitro model of cellular tolerance to ethanol, Proc. Nat. Acad. Sci. U.S.A., 83, 2105. Harris, D.P. and J.G. Sinclair, 1984, Ethanol depresses inferior olive neurons and reduces purkinje cell complex spike activity evoked by cerebral cortical stimulation, Gen. Pharmacol. 15, 455. Lancaster, B. and P.R. Adams, 1986, Calcium-dependent current generating the after hyperpolarization of hippocampal neurons, J. Neurophysiol. 55, 1268. Madison, D.V. and R.A. Nicoll, 1982, Noradrenaline blocks accommodation of pyramidal cell discharge in the hippocampus, Nature 299, 636. Majchrowicz, E., 1975, Induction of physical dependence upon ethanol and the associated behavioral changes in rats, Psychopharmacologia 43, 245. Messing, R.O., C.L. Carpenter, I. D i a m o n d and D.A. Greenberg, 1986, Ethanol regulates calcium channels in clonal neural ceils, Proc. Nat. Acad. Sci. U.S.A., 83, 6213. Nicoll, R.A. and B.E. Alger, 1981, A simple chamber for recording from submerged brain slices, J. Neurosci. Meth. 4, 153. Shefner, S.A. and B. Tabakoff, 1985, Basal firing rate of rat locus coeruleus neurons affects sensitivity to ethanol, Alcohol 2, 239. Siggins, G.R., Q.J. Pittman and E.D. French, 1987, Effects of ethanol on CA 1 and CA 3 pyramidal cells in the hippocampai slice preparation: an intracellular study, Brain Res. 414, 22.
591
Elsevier EJP 20382 Short communication
Ethanol suppresses hippocampal cell firing through a calcium and cyclic AMP-sensitive mechanism D e b o r a h M. B e n s o n 2,4, R o b e r t D. Blitzer 2 a n d E m m a n u e l M. L a n d a u 1,2,3,* i Department of Psychiatry, Bronx VA Medical Center, Bronx, N Y 10468, and Departments of 2 Psychiatry and 3 Pharmacology, Mr. Sinai School of Medicine, New York, N Y 10029, and 4 Department of Psychology, Queens College of The City University of New York, Flushing, N Y 11367, U.S.A.
Received 28 March 1989, accepted 4 April 1989
The effects of ethanol were studied intracellularly in hippocampal pyramidal cells in vitro. Ethanol, 50-100 mM, produced a marked suppression of neuronal firing. This effect was blocked by treating the cell with cyclic 3% 5'-adenosine monophosphate (cAMP) or cadmium ions. Ethanol had no effect on the after-hyperpolarizing current. It is concluded that the ethanol-induced reduction of firing rate is due to a calcium-dependent process, and modulated by cAMP. Ethanol; Hippocampus; cAMP;
Ca2+; After-hyperpolarization; (Neuronal firing)
1. Introduction Ethanol is a major psychoactive drug, with widespread use leading to substantial medical and social consequences. A commonly reported central nervous system effect of ethanol is a suppression of neuronal firing (Harris and Sinclair, 1984; Shefner and Tabakoff, 1985), although mixed, biphasic or excitatory effects have also been reported (Siggins et al., 1987). The aim of the present study was to investigate the mechanism of this suppressant effect of ethanol in hippocampal slices in vitro. Our major finding is that ethanol's suppression of hippocampal neuronal firing is mediated through a calcium- and cyclic-AMP (cAMP)-sensitive mechanism. This mechanism does not involve enhancement of the calcium-dependent potassium (after-hyperpolarizing) current, which was not affected by ethanol.
* To whom all correspondence should be addressed: Dept. Psychiatry, Bronx VA Hospital, 130 W. Kingsbridge Road, Bronx, NY 10468, U.S.A.
2. Materials and methods Standard intracellular recording methods (Nicoll and Alger, 1981) were used to record from guinea pig hippocampal slices maintained in vitro. Experiments were performed at 23-25°C and at 30-32°C. Most slices were taken from chloral hydrate-anesthetized guinea pigs, although several guinea pigs were instantly decapitated without anesthesia, to rule out the possibility that chloral hydrate contributed to the effects seen. Following decapitation, the brains from all animals were immediately transferred into ice-cold Ringer solution. CA 1 pyramidal cells were impaled with glass microelectrodes filled with either 2 M potassium methylsulfate (KMeSO4) , 3 M potassium chloride (KC1), or, in a few experiments, 100-200 mM cAMP. To measure firing rate, the cell membrane was depolarized through a bridge circuit to a predetermined membrane potential with 30 s depolarizing current pulses of 0.1 to 0.4 nA (to achieve a regular rate of firing throughout the train). To test the membrane input impedance, brief (100 ms) hyperpolarizing pulses (0.2-0.4 nA) were passed through the bridge circuit. For volt-
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
592
age-clamp experiments, a single-electrode voltage clamp amplifier was used (Axoclamp-2), with a switching frequency of 2-3 kHz and a 30% duty cycle. For our studies, we accepted cells with resting potentials of < - 5 5 mV, action potential amplitudes of > 70 mV and membrane resistances of > 20 M~2. Typical values were resting potential: - 6 4 . 9 + 7 mV (n = 38), spike amplitude: 92.4 + 10 mV (n = 37), input resistance: 67.9 + 15 M$2 (n = 34). For statistical analyses, comparisons were made within cells using two-tailed t-tests. Error values referred to in the text are S.E.M.
3. R e s u l t s
Ethanol (50-100 mM; 0.25-0.5 g / d l ) consistently produced a suppression of neuronal firing, causing fewer spikes to be elicited upon stimulation by a 30 s long depolarizing current pulse, as illustrated in fig. 1A-C. This ethanol-induced suppression of firing was found in all cells tested. No differences were found
A.
28
B.
between cells impaled with either KCI- or KMeSOa-containing electrodes, between cells recorded at room or elevated temperatures, or between slices obtained from anesthetized or unanesthetized animals. In view of this, the data from all of these conditions were pooled. The average number of spikes during a 30 s depolarizing pulse decreased to 65 _+ 2% (n = 36) of the control value in ethanol. The effect was statistically significant, t(35)= 7.65, P <0.0005. The ethanol effect was easily reversible, and spike frequencies recovered to 109 _+ 5% (n = 36) of control (not significantly different) after washing out the drug. There was no relationship between the number of spikes generated during the pulse and the percentage reduction by ethanol. The suppressant effect of ethanol was probably not due to changes in passive m e m b r a n e properties, since it produced only a small (1-2 mV) hyperpolarization of the cell m e m b r a n e and a 5% decrease in the membrane input impedance. The depolarizing currents employed to elicit neuronal firing were adjusted to compensate for these
C,
1
i
i
I
I
F----[
___
i
D.
I0 mV T
F,
E.
0.5 n A ~ 4 0 0 ms
44
58
46
i I
I
Fig. 1. Cadmium blocks the ethanol suppressant effect. Chart records of the membrane response to 30 s depolarizing current pulses. Numbers over spike trains denote the number of spikes in the train. Broken vertical line through each trace denotes break in the record. In each condition, the pulse brought the membrane potential to the same level ( - - 5 5 mV). Left panels: control; middle panels: 25 min into 100 mM ethanol; right panels: 10 min after washout of ethanol. (A-C) Ethanol effects in the control medium. (D-F) Ethanol effects in the presence of'300 p.M cadmium chloride. Note the failure of ethanol to suppress spike frequency in cadmium. Spikes truncated due to frequency characteristics of chart recorder. Vertical calibration 10 mV. Horizontal calibration 400 ms. Recording electrode: KC1.
593 changes so as to d e p o l a r i z e the cells to the same level as in the c o n t r o l situation. W e next e x a m i n e d the effect of e t h a n o l on the a f t e r - h y p e r p o l a r i z i n g ( A H P ) current, which has p r e v i o u s l y b e e n r e p o r t e d to b e e n h a n c e d b y e t h a n o l (Carlen et al., 1982; however, see Siggins et al., 1987), a n d is a m a j o r d e t e r m i n a n t of neuronal firing r a t e ( L a n c a s t e r a n d A d a m s , 1986; M a d i son a n d Nicoll, 1982). F o u r cells were voltagec l a m p e d in the presence of t e t r o d o t o x i n (TTX) at - 6 0 m V a n d s t e p p e d for 2 s to - 3 5 mV. T h e resulting ' t a i l ' c u r r e n t ( A H P current) was m e a sured 200 m s after the e n d of the d e p o l a r i z i n g pulse. T h e value of the A H P current in the c o n t r o l c o n d i t i o n was 106 + 23 p A a n d in 100 m M ethanol, the A H P c u r r e n t was 99 + 26 p A . T h e r e was no significant difference b e t w e e n these values. I n o r d e r further to investigate the m e c h a n i s m of the s u p p r e s s a n t effect of e t h a n o l on n e u r o n a l firing, we e x a m i n e d the effects of c a d m i u m (which b l o c k s c a l c i u m channels) a n d c A M P on the e t h a n o l effect. Both c a l c i u m a n d c A M P have b e e n imp l i c a t e d in the cellular effects of e t h a n o l (Messing et al., 1986; G o r d o n et al., 1986). Both drugs b l o c k e d a n d in s o m e cases, reversed, the e t h a n o l i n d u c e d s u p p r e s s i o n of firing. F i g u r e 1 A - C shows a cell in which e t h a n o l p r o d u c e d a typical reduction in firing. However, after a d d i t i o n of 3 0 0 / ~ M c a d m i u m ( D - F ) there was a small increase in the n u m b e r of spikes elicited in ethanol. This effect of c a d m i u m (0.2-0.3 m M ) was seen in five cells, the n u m b e r of spikes in e t h a n o l averaging 127 + 17% of c o n t r o l (no significant difference). In these e x p e r i m e n t s , the n u m b e r of spikes elicited d u r i n g the d e p o l a r i z i n g pulse in the c o n t r o l m e d i u m was 39 + 5 (n = 5). This is n o t significantly different f r o m the c o r r e s p o n d i n g value o b t a i n e d with no c a d m i u m ions p r e s e n t (49 ___5, n = 36). Therefore, the b l o c k i n g b y c a d m i u m of the ethanol suppress a n t effect c a n n o t be e x p l a i n e d b y changes in n e u r o n a l firing rates in the presence of c a d m i u m ions. Similar results were f o u n d with c A M P a p p l i e d in the m i c r o e l e c t r o d e (n = 4) or in the b a t h as the 8 - b r o m o a n a l o g u e (0.5 m M , n = 4). A s shown in fig. 2 D - F , c A M P a p p l i e d in the r e c o r d i n g elect r o d e p r e v e n t e d the e t h a n o l - i n d u c e d decrease in firing rate. F o r c o m p a r i s o n , the typical effect of
A.
D.
~
B,
47
E.
56
31
IOmV~ los
82
C,
46
46
,,~,~ _
Fig. 2. The suppressant effect of ethanol is blocked by cAMP. Shown are chart records of the membrane response to 30 s depolarizing current pulses in a cell impaled with a 3 M KCl-containing electrode (A-C) and in a different cell impaled with a 200 mM cAMP/3 M KCl-containing electrode (D-F). Numbers over spike trains denote the number of spikes in the train (spikes truncated due to frequency characteristics of chart recorder). (A,D) Control; (B,E) 100 mM ethanol; (C,F) washout. Calibration: vertical, 10 mV; horizontal, 10 s (except following the pulse in A-C:2 s).
e t h a n o l is shown in fig. 2 A - C (different cell). O n the average, in the presence of c A M P , the n u m b e r of spikes in e t h a n o l increased to 107 + 8% (n = 8) of control. This effect was statistically significant, (t(7) = 2.13, P < 0.05). T h e e t h a n o l - i n d u c e d increase in firing rate seen in the p r e s e n c e of c A M P m a y reflect an u n d e r l y i n g e x c i t a t o r y effect of e t h a n o l which can be revealed w h e n the s u p p r e s sant effect is b l o c k e d . T h e n u m b e r of spikes elicited u p o n d e p o l a r i z a t i o n in the p r e s e n c e of c A M P was significantly greater t h a n in it's absence (93 + 25, n = 8 vs. 49 + 5, n = 36; t(42) = 2.89, P < 0.005). However, the i n c r e a s e d n e u r o n a l r e s p o n s i v i t y in c A M P is n o t likely to e x p l a i n the o b s e r v e d i n t e r a c t i o n b e t w e e n c A M P a n d ethanol, since this r e s p o n s i v i t y was n o t c o r r e l a t e d with the m a g n i t u d e of the e t h a n o l effect (see above).
4. Discussion W e have f o u n d that c o n c e n t r a t i o n s of e t h a n o l in the range of 50-100 m M (0.25-0.5 g / d l ) consistently suppress the firing rate of h i p p o c a m p a l neurons. These c o n c e n t r a t i o n s cause s e d a t i o n a n d a t a x i a in rats (Majchrowicz, 1975), a n d are c o n s i d -
594
ered to be in the high-intoxicating range. This finding differs somewhat from that reported by Siggins et al. (1987), who found more variable effects of ethanol on excitability in C A 1 and C A 3 neurons. They found more consistent ethanoldepressant effects on spontaneous firing of CA 1 neurons than on depolarizing current-evoked spikes. It can be noted, however, that our protocol of administering 30 s long depolarizing pulses may, in fact, better approximate a spontaneously firing neuron than one responding to a brief depolarizing current pulse. Ethanol had no effect on the A H P current in this study, in contrast to the ethanol-induced enhancement of the A H P current reported by Carlen et al. (1982) in current-clamped cells. The present experiments were done in voltage-clamped neurons, however, allowing a more reliable measurement of the A H P current than in current-clamped cells, in which the number of spikes generated and the amplitude of the depolarizing pulse are difficult to control. Since the A H P current was not influenced by ethanol in these experiments, the ethanol effect on neuronal firing cannot be mediated through an enhancement of this current. An alternative hypothesis is that the ethanol suppressant effect may be due to increasing the rate of spontaneous inhibitory post-synaptic potentials. However, the ethanol effect was the same in both KC1- and KMeSOa-containing electrodes. Thus, this possibility can be ruled out. The suppressant effect of ethanol was blocked by cadmium and cAMP. While these agents both block the A H P current, this blockade cannot be the common mechanism by which they exert their effects, since this current was not influenced by ethanol in the present study. Therefore, the reduction in firing produced by ethanol must be due to another process which is both modulated by c A M P and dependent on calcium entrance into the cell. Further work is needed to elucidate the precise nature of this mechanism which underlies the
ethanol suppressant effect on hippocampal pyramidal neurons.
Acknowledgements This work was supported in part by N I A A A Grant No. 5-R23-AA06659 and N I H - N I A Grant No. 5-P50-AG05138.
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