138

Brain Research, 504 (1991) 138-142 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939124911Z

BRES 24911

Ethanol enhances G,&.m4~A ~ ~ cerebral cortical

CUn 'Tt8 in chick rons

J.N. Reynolds and A. Prasad Faculty of Medicine, Memorial University of Newfoundland, St. John's, Nfld. (Canada) (Accepted 30 July 1991)

Key words: Ethanol; y-Aminobutyricacid; Cerebral cortical neuron; Neuromodulation; Voltage clamp; Chloride current

Primary cultures of cerebral cortical neurons were prepared from 7- to 8-day-oldchick embryos. The effect of ethanol on GABA-activated membrane current was examined using whole-celt voltage-clamp recording in cells maintained for 3-25 days in vitro. In approximately 60% of neurons examined ethanol caused a potentiation of the membrane current elicited by GABA. The threshold concentration:of ethanol was i mM, and the potentiating effect of ethanol on GABA-activated currents was maximal at 10 mM. In many ceils higher concentrations (40-50 mM) of ethanol inhibited GABA-activated currents. These effects of ethanol were all reversible. The anxiolytic, sedative and hypnotic effects of ethanol have frequently been attributed to a selective enhancement of inhibitory synaptic transmission mediated by y-aminobutyric acid (GABA), through an interaction at the G A B A A receptor-chloride channel complex. Ethanol shares several pharmacologic properties (sedation, motor incoordination, anticonvulsant) with both barbiturates and benzodiazepines, suggesting that at least some of the actions of these compounds may be mediated by common mechanisms. Benzodiazepines and barbiturates have been shown to selectively enhance the activity of G A B A via an interaction with specific recognition sites on the G A B A A receptor-chloride channel complex 21'29. Electrophysiological studies have consistently demonstrated that both benzodiazepines and barbiturates potentiate membrane currents activated by G A B A 5'24'29. However, there is conflicting evidence from electrophysiological studies concerning the interaction of ethanol with the G A B A A receptor-chloride channel complex (reviewed in ref. 20). While some authors report an enhancement by ethanol of GABAA receptor-activated chloride currents (IGABA)1'8'23, others have failed to find any interaction between ethanol and G A B A 7'24'27'33. The reason for this discrepancy is unclear, since different results have been reported even when similar methodologies were employed 1'4'8~33. It has been reported that 10-s, but not 30-s, applications of ethanol could potentiate IOABA in cultured chick spinal cord neurons 8, whereas IOABAin cultured rat spinal cord neurons was not potentiated by ethanol 4. Primary cul-

tures of rat dorsal root ganglion neurons exhibit a transient and a sustained IGABA, and ethanol could selectively potentiate the transient component 23. In contrast. ethanol did not alter/GABA in acutely dissociated dorsal root ganglion neurons from the adult rat 33. Aguayo ~ reported that ethanol could potentiate /GABA in primary cultures of mouse cortical and hippocampal neurons, However. others have reported that ethanol does not potentiate irGABAin cultured rat hippocampal neurons L6' 18. The present study was undertaken to further study the potential interaction between ethanol and IGABA in neurons of the central nervous system. Primary cultures of cerebral cortical neurons were prepared from chick embryos using modifications of the technique reported by Huettner and Baughman 17 Briefly, fertile eggs from domestic chickens were obtained from a local supplier and maintained in a humidified, 37 °C incubator. After 7-8 days of incubation (embryonic stage 31-34 as determined by the criteria of Hamburger and Hamilton'3), embryos were removed from the egg, decapitated, and the cerebral hemispheres dissected into ice-cold CaZ+/Mg2+-free Tyrode's buffer. Cortical tissue was minced and then incubated for 30-40 min in Earle's Balanced Salt Solution (EBSS, Sigma Chemical Co.) containing 30 U/ml papain (Boehringer Mannheim) prewarmed to 37 °C. The tissue was rinsed with fresh EBSS and then triturated with fire polished Pasteur pipettes. Cells were resuspended in the growth medium which consisted of Minimum Essential Medium (Gibco) supplemented with 1 mM u-glutamine, 4.5 g/1

Correspondence: J.N. Reynolds, Faculty of Medicine, Memorial University of Newfoundland. St. John's. Nfld.. Canada AIB 3V6

139 D-glucose, 50 U/ml penicillin, 50 #g/ml streptomycin, 10% calf serum (Seru-Max 3, Sigma Chemical Co.) and 2% Nu-serum (Collaborative Research Inc.). Trypan blue exclusion was used to assess cell viability and cell number. Cells were plated at a density of 0.5 × 106 cells/dish into 35 mm-culture dishes (Nunc) coated with poly-Dlysine (Sigma Chemical Co.) and grown in a humidified atmosphere containing 5% CO2 at 37 °C. Tissue from a single embryo yielded sufficient cells to plate more than 20 culture dishes, so tissue from different embryos was not pooled. After 3-4 days in vitro fresh growth media was added, and thereafter the medium was changed every week. Whole-cell voltage-clamp recordings were obtained from neurons grown for 3-25 days in vitro. Individual neurons were visualized on the stage of an inverted microscope (Zeiss IM35). For electrophysiological recording the cells were continuously perfused (1-2 mi/min) with a physiological saline containing (in raM) 140 NaCl, 5 KC1, 2 CaCl2, 1 MgC12, 10 HEPES, 10 glucose, and 0.5 /~M tetrodotoxin, pH 7.3. Ethanol was diluted in the extracellular solution to a final concentration of 0.5-50 mM and applied by bath perfusion. G A B A (10-50 /~M) was applied by brief (20-100 ms) pressure pulses (Picospritzer II) directly to the soma of the neuron under study. The concentration of G A B A in the pressure pipette did not affect the results. Whole-cell voltage-clamp recordings were obtained using glass micropipettes (1.5 ram; TW150F-4; World Precision Instruments) pulled in two stages on a Narashige PB-7 electrode puller. Recording electrodes had tip resistances of 3-5 Mff2 when filled with (in mM) 140 KC1, 2 MgC12, 10 HEPES, 4 Na-ATP, pH 7.3. All recordings were obtained using an Axoclamp-2A amplifier in discontinuous single-electrode voltage-clamp mode (switching frequency 10 kHz). Data acquisition and analysis were performed using pClamp software (Axon Instruments). Unless otherwise described, all recordings were performed at a holding potential o f - 6 0 mV. Brief pressure pulses of G A B A elicited a membrane current which reversed at 0 mV and was reversibly blocked by 50/~M bicuculline (Fig. 1). Thus, membrane currents activated by G A B A in these neurons reversed at the Cl- equilibrium potential imposed by the recording conditions (symmetrical C1 concentrations across the cell membrane) and were blocked by a competitive antagonist at the G A B A A receptor. The presence or absence of calcium chelators in the intracellular recording solution did not affect the amplitude or stability of 1GABA"Stable recordings could be maintained for 40-60 rain under these conditions. Calcium chelators were routinely omitted from the intracellular recording solution for the following reasons: (i) ethanol is reported to release calcium from intracellular stores in several neu-

ronal preparations 9'1°'25, and increased intracellular calcium levels may underlie at least some of the actions of ethano16'32; (ii) high concentrations (5 mM) of the calcium chelator BAPTA attenuate the ability of the volatile anesthetic halothane to potentiate GABA-mediated spontaneous inhibitory synaptic currents in adult rat hippocampal CA1 and dentate granule neurons 22, suggesting that IGABAis sensitive to small changes in the intracellular Ca2+ concentration. Recordings were obtained from a total of 80 cells derived from 12 different chick embryos, with 4-10 cells sampled from each set of cultures. In approximately 60% of cells examined bath perfusion of ethanol (1-20 mM) produced a reversible potentiation of IGABA (Fig. 2A). This effect on G A B A responses was maximal at 5-10 rnM ethanol. Increasing the ethanol concentration did not further potentiate IGABA" In fact, higher ethanol concentrations ( > 10 raM) tended to produce less enhancement of IGABA as compared to lower concentrations ( < 10 mM) of ethanol (Fig. 2A). In many cells a

O~__omO0 pA

50 luM Bi~'tdline

Fig. 1. GABA receptor-activated currents in cultured chick cerebral cortical neurons have the characteristics of GABA A receptoractivated chloride currents. Upper: a series of current traces generated by GABA applied to a neuron voltage-clamped at different membrane potentials (-80 mV to +40 mV). At negative holding potentials the GABA-activated membrane currents are inward (downward directed current traces), but reverse to outward currents at positive holding potentials. Lower: in a different neuron voltage-clamped at -60 mV, the GABA-activated membrane current was reversibly blocked by 50/~M bicucullinc.

140

A

B

mM F.,a1~

Fig. 2. Low concentrations of ethanol potentiate membrane currents elicited by GABA. A: brief (50 ms) pressure pulse application of 50/~M GABA to a chick cerebral cortical neuron generated a large inward current (Control). Bath perfusion of 5 mM ethanol resulted in a substantial potentiation of IGABA"Perfusion with 20 mM ethanol also resulted in potentiation of /GABA,but to a lesser degree compared to 5 mM ethanol. This effect of ethanol on /GABA WaS revelT,ed after switching back to the control solution (Wash). B: in a different cell, a 100 ms pulse of GABA (50/~M) generated an inward current (Control) which was enhanced by bath perfusion with 10 mM ethanol. However. in the same cell, increasing the ethanol concentration to 40 mM resulted in inhibition of IGABA, which reversed after wash. In this same cell. a subsequent exposure to 10 mM ethanol resulted in a second potentiation of IGAaA'

biphasic effect of ethanol was observed. A n initial enh a n c e m e n t of I~ABA at low ethanol concentrations (5-20 m M ) was often followed by inhibition of/GABA at higher e t h a n o l concentrations (40-50 raM) (Fig. 2B). These effects of ethanol were all reversible. In s o m e cells the m e m b r a n e current elicited by G A B A was not potentiated by ethanol (Fig. 3). H o w e v e r , in these cells bath perfusion o f the hypnotic b e n z o d i a z e p i n e flurazepam (1 /~M) p r o d u c e d a strong and reversible p o t e n t i a t i o n of /GABA(Fig. 3). In addition, in cells which failed to show

an e n h a n c e m e n t of the G A B A response at low (1-10 m M ) ethanol concentrations, higher concentrations o f ethanol (20-50 raM) could still produce an a p p a r e n t inhibition of the G A B A response (not shown). Cells obtained from different e m b r y o s varied widely in their sensitivity to ethanol. In 4 sets of cultures, all o f the cells s a m p l e d exhibited a potentiation of I~AaA by low (1-10 raM) concentrations of ethanol. However, in 3 sets of cultures none of the cells tested s h o w e d this effect of ethanol. In the r e m a i n d e r of the cultures, an ethanol-in-

141 duced potentiation of IGABAwas seen in 50-80% of cells examined. The results of this study support previous reports 1,8,23 that low concentrations (< 20 mM) of ethanol can potentiate/GABA in neurons of the central nervous system. The reversal potential (0 mV) and sensitivity to bicuculline suggest that the membrane current enhanced by ethanol is a CI- current elicited by G A B A acting at the G A B A A receptor. However, potentiation of /GABA by ethanol did not occur in all neurons tested. The fraction of cells (60%) which showed an ethanol enhancement of 1GABA in the present study is strikingly similar to that reported by Celentano et al. s in cultured chick spinal cord neurons. Similarly, a large fraction (80%) of cultured mouse cerebral cortical neurons showed an enhancement by ethanol of IGABA1. Recent studies have demonstrated the remarkable molecular heterogeneity of G A B A A receptor subunits, and a corresponding variation in modulation of receptor function by drugs such as benzodiazepines and neurosteroids 26'28'3°. Thus, it seems reasonable to suggest that one or more specific configurations of the GABAA receptor-chloride channel complex exist which convey sensitivity of the complex to ethanol. This idea is supported by neurochemical studies on the effects of ethanol on G A B A A receptor-activated 36C1- flUX into microsac and synaptoneurosome preparations of cerebral cortex and cerebellum. Mouse and rat

II~ Co.~rol

~l ~ l a ~! mr Fig. 3. GABA-activated currents in a chick cerebral cortical neuron which did not respond to ethanol. In this cell a 100-ms application of GABA (25/~M) generated an inward current which was unaffected by ethanol. The control response to GABA and the response after perfusion with 10 mM ethanol are shown superimposed. In the same cell, subsequent perfusion with 1 /tM flurazepam resulted in a reversible potentiation of IC~ABA"

lines have been selectively bred for a high (HS) or low (LS) sensitivity to an acute hypnotic dose of ethanol. Ethanol potentiates G A B A A receptor-activated 36Clflux in membrane preparations obtained from HS, but not LS, animals 3"14"19. Similarly, messenger RNA isolated from the brains of HS mice and expressed in Xenopus oocytes produces functional GABAA receptors which are potentiated by ethanol, whereas LS messenger R N A produces G A B A a receptors which are not potentiated by ethanol 31. Furthermore, ethanol enhancement of G A B A A receptor-activated 36Cl- flux appears to cosegregate with an increased sensitivity of this receptor mechanism to both benzodiazepines and barbiturates TM, suggesting that the genetically determined differences seen in response to ethanol are a result of differences in the G A B A A receptor-chloride channel complex. Since the number and affinity of benzodiazepine binding sites is not different between these two lines, the genetic variability appears to lie in the functional coupling between G A B A and benzodiazepine binding sites and the chloride channel. Ethanol produced a biphasic effect on IGABA" Maximal potentiation of the G A B A response occurred at low (5-10 raM) concentrations of ethanol, whereas higher ethanol concentrations (40-50 mM) usually inhibited IGABA (Fig. 2B). A bell-shaped concentration-response relationship has been reported for ethanol using both electrophysiologicall and neurochemical 2 measures of/GABA, with the peak effect of ethanol occurring around 10 raM. Ethanol alters membrane fluidity ~5 and therefore, at higher concentrations, may perturb the membrane environment surrounding the G A B A receptor-chloride channel complex, leading to a decrease in ion flux. Conflicting evidence concerning the interaction of ethanol with the GABAA receptor-channel complex (see ref. 20) may therefore arise for several reasons. First, the genetic variability, and consequent pharmacologic variability, in G A B A A receptor subunits will affect the frequency of observing an ethanol enhancement of IGABA" There are significant variations in the expression of G A B A A receptor subunits in different regions of the CNS and at different developmental stages ~ which may contribute to the pharmacologic variability of this receptor-channel complex. Certainly, there are distinct regional and interanimal differences in the modulation of G A B A A receptor activity by ethanol 3'~'~4"19. However, an interaction between ethanol and I~;ABA does not appear to be restricted to any particular developmental stage of the CNS. Ethanol enhances IC;ABAin some, but not all, neurons cultured from embryonic tissue obtained from chick, mouse and rat 1'4"8"16"18'23. However, messenger RNA isolated from the brains of adult mice and expressed in Xenopus oocytes produces functional G A B A A

142 r e c e p t o r s w h i c h a r e p o t e n t i a t e d by e t h a n o l 3~. Similarly,

b e l l - s h a p e d n a t u r e of the e t h a n o l - G A B A c o n c e n t r a t i o n -

ethanol

r e s p o n s e r e l a t i o n s h i p suggests that h i g h e r ( 2 0 - 1 0 0 m M )

enhancement

of

GABA A receptor-activated

36C1- flux has b e e n o b s e r v e d in m e m b r a n e p r e p a r a t i o n s

c o n c e n t r a t i o n s o f e t h a n o l , which h a v e b e e n m o s t fre-

o b t a i n e d f r o m adult a n i m a l s 3't4'19. W e are c u r r e n t l y car-

q u e n t l y utilized, m a y give a false n e g a t i v e result unless

rying o u t studies to c o m p a r e the effects o f e t h a n o l o n

l o w e r c o n c e n t r a t i o n s of e t h a n o l are also tested.

IGABA in n e u r o n s d e r i v e d f r o m (1) d i f f e r e n t r e g i o n s o f the c e n t r a l n e r v o u s system in the s a m e species, and (2) the s a m e b r a i n r e g i o n o f d i f f e r e n t species. S e c o n d , the

This work was supported by the Medical Research Council of Canada. Flurazepam HC1 was supplied by Hoffmann-La Roche Ltd.

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