Ethanol and other CNS depressants decrease GABA synthesis in mouse cerebral cortex and cerebellum in vivo

Life Sciences, Vol. 27, pp. 1035-1040 Printed in the U.S.A.

Pergamon Press

ETHANOL AND OTHER CNS DEPRESSANTS DECREASE GABA SYNTHESIS IN MOUSE CEREBRAL CORTEX AND CEREBELLUM IN VIVO Porntlp Supavilai and Manfred Karobath Department of Biochemical Psychiatry Psychiatrische Universit~tsklinik Vienna, Austria A-I090 (Received in final form July ii, 1980) Summer 7 GABA synthesis in mouse brain in vivo was estimated by measuring the rate of GABA accumulation one hour after inhibition of GABA degradation using the selective and irreversible antagonism of GABA-transaminase by gabaculine. Using this method we found that acute and repeated ethanol administration lead to a potent depression of gabaculine induced enhancement of GABA levels in mouse brain cerebellum and cerebral cortex. Alcohol, in the absence of gabaculine had no effect on steady state GABA levels. These results demonstrate potent effects of ethanol on the dynamics of GABA metabolism which are compatible with a GABA like effect of ethanol. Although ethanol has important behavioural effects in the central nervous system (CNS), little is known of its biochemical mode of action. Ethanol is a CNS depressant, and during withdrawal hyperexcitability and seizures can be observed (1). Thus, ethanol may act by altering excitatory or inhibitory synaptic activity in the CNS. Recently, Redos et al. (2) have demonstrated that ethanol, even in a dose of I g/kg can lead to a decrease of cerebellar guanosine 3'5'-cyclic monophosphate (cGMP) levels; these results have been recently reproduced (3). Several other lines of evidence suggest that in cerebellum cGMP levels correlate inversely with the activity of the GABA system. Thus, stimulation or GABAreceptot activity by muscimol or by benzodiazepines leads to a large decrease of cerebellar cGMP levels while convulsants have the opposite effect (4). It is therefore reasonable to assume that alcohol may exert its depressant action on cGMP levels by affecting the functional state of the GABA system. We therefore determined the effects of alcohol on GABA synthesis in vivo using a newly developed method (5) which permits to study the rate of GABA accumulation after selective, quantitative and irreversible inhibition of GABA transemlnase by gabaculine. Methods GABA synthesis was estimated by measuring the rate of GABA accumulation one hour after inhibition of GABA degradation by gabacullne (5,6). swiss Albino mice (20-30 g, Porschungsinstltut ffir Versuchstierzucht, Himberg, Austria) were treated with 0.9 % saline or ethanol (20 % w/v solution in 0.9 % saline, i.p.) at

s t a t e d t i m e p o i n t s b e f o r e k i l l i n g , and g a b a c u l i n e ( 0 . I g / k g , i . p . ) was i n j e c t e d 60 min b e f o r e s a c r i f i c e . The a n i m a l s were then k i l l e d by f o c u s s e d mlcrowave i r r a d i a t i o n using a modified Husqvarna electronic oven (I kW for 3 sec). After cooling, the brains were dissected and GABA levels were determined by gaschromatography (7). For repeated ethanol administration mice were fed for 14 days a liquid diet to which (10 % w/v) ethanol was added whereas the corresponding 0024-3205/80/381035-06502.00/0 Copyright (c) 1980 Pergamon Press Ltd.

Ethanol on GABA Synthesis In V4uo

1036

Vol. 27, No. 12, 1980

control mice received the same diet without the addition of ethanol to which isocaloric sucrose was added (8). Blood ethanol levels were determined using the NAD-NADH alcoholdehydrogenase method (9). Glutamic acid decarboxylase was d e t e r m i n e d as d e s c r i b e d (10). Results As shown in Figure l, ethanol administered 75 urn before sacrifice, even in moderate doses decreased the gabaculine induced accumulation of GABAin mouse cerebral cortex and cerebellum. This decrease of the rate of GABA synthesis was dose dependent and related to blood ethanol levels. In the cerebellum a dose of I g/kg ethanol reduced the gabaculine induced accumulation of GABA by more than 50 %, while a dose of 4 g/kg virtually abolished GABA synthesis (Figure l).

CORTEX

CEREBELLUM

BLOOD- ETOH

2~

_

E w

&,

IE

I °

a~

~2

0

ETHANOL DOSAGE glkg OABACULINE DOSAGE gJkg

0 0

0 0.1

,1

2 3 4, 0,1

0 0

0 0,1

,1 2 3 4 0,1

,1 2 3 ~,, 0

FIGURE ! Gabaculine induced accumulation of GABA levels in mouse brain cerebral cortex or cerebellum and its inhibition by ethanol: Saline or ethanol (20 % w/v solution in 0.9 % saline) were injected i.p. 75 m£n before killing and gabaculine was administered i.p. 60 min before sacrifice. Data shown are mean values of GABA levels ± s.e.m. (n~ 5). In both regions GABA levels in all groups treated with ethanol and gabaculine differed significantly (p
Vol. 27, No. 12, 1980

Ethanol on GABA Synthesisln V~vo

1037

The s l o p e o f t h e dose r e s p o n s e c u r v e f o r c e r e b e l l u m l o o k s d i f f e r e n t t h a n c o r t e x , w i t h a l l doses more e f f e c t i v e , b u t h i g h e r doses n o t so much than t h e l o w e s t . The time c o u r s e o f t h e e f f e c t o f 3 E/kg e t h a n o l i n c e r e b e l l u m ( F i g u r e 2) and c e r e b r a l c o r t e x ( d a t a n o t shown) d e m o n s t r a t e d t h a t a l c o h o l induced i t s s h o r t l a s t i n g b u t p r o f o u n d d e c r e a s e i n GABA s y n t h e s i s r a t e s o n l y a t t h o s e time p o i n t s where e t h a n o l l e v e l s c o u l d be d e t e c t e d . A s i m i l a r e t h a n o l induced d e c r e a s e o f i n v i v o r a t e s of GABA s y n t h e s i s was a l s o o b s e r v e d i n hippocampus ( d a t a n o t shown).

2

a .J

o z

~

2,

1 .2 m

I HOURS FIGURE 2 Time c o u r s e o f the e f f e c t of a s i n g l e dose o f e t h a n o l on t h e g ab ac u l i n e induced accummlation of GABA i n mouse b r a i n c e r e b e l l u m : mice were i n j e c t e d w i t h 3 g / k g e t h a n o l i . p . a t z e r o time and k i l l e d a t t h e i n d i c a t e d time p o i n t s . One hour b e f o r e s a c r i f i c e they were t r e a t e d w i t h 0.1 g / k g g a b a c u l i n e . Shown a r e g a b a c u l i n e i n d u c e d e n hancements of GABA l e v e l s (~) and b l o o d e t h a n o l l e v e l s ( o ) . Each v a l u e r e p r e s e u t s t h e mean ± s . e . m . ( n ~ 5 ) . I n o r d e r t o d e t e r m i n e w h e t h e r r e p e a t e d a d m i n i s t r a t i o n of a l c o h o l which has been shown t o produce t o l e r a n c e t o t h e h y p o t h e r m l c e f f e c t of a l c o h o l ( | 1 ) , a l s o d e c r e a s e d GABA s y n t h e s i s i n v i v o , mice were f ed f o r 14 days a l i q u i d d i e t t o which e t h a n o l was added (8~. I n t h e s e mice which were p r o b a b l y n o t t o l e r a n t t o e t h a n o l , a l s o a s i g n i f i c a n t r e d u c t i o n of 8 a b a c u l i n e i n d u ced enhancement o f GABA l e v e l s was found when compared t o c o n t r o l groups (Table I ) . I n c o n f i r m a t i o n o f p r e v i o u s r e p o r t s ( 5 ,6 ) we found t h a t a f t e r t r e a t m e n t w i t h d i a z e p a m o r ~ r l t h p e n t o b a r b i t a l GABA s y n t h e s i s i n v l v o was markedly r e d u c e ~ I n a d d i t i o n , muscimol, a GAB&-mlmetic, and e t h y l e n e g l y c o l - which i s f r e q u e n t l y used as s o l v e n t f o r d r u g s - had a l s o p o t e n t d e p r e s s a n t e f f e c t s on g a b a c u l i n e indu ced a c c u m u l a t i o n o f CABA l e v e l s i n v i t r o (Table I ) .

1038

Ethanol on GABA Synthesis In V~uo

Vol. 27, No. 12, 1980

TABLE I Effect of Pretreatment with Various Drugs on Steady State GABA Levels and on Gabaculine Induced Accumulation of GABA in Mouse Cerebellum

Pretreatment

steady state GABA levels ~moles/g tissue

gabaculine induced accumulation of GABA ~moles/g tissue (% of control)

Liquid diet Liquid diet containing lO % ethanol

1.O5 ± 0.33 0.99 ± 0.08

3.36 ± O.18 1.62 ~ 0.25

(|00) (48)

Saline Diazepam 2 mg/kg Pentobarbital 50 mg/kg Muscimol 2 mg/kg Ethylene glycol 2 ml/kg

1.27 1.19 !.29 1.25 1.24

4.00 1.43 1.12 1.O2 2.70

(lO0) (36) (28) ( 261 (68)

± ± ± ~ ±

0.08 0.07 0.05 O.11 0.06

~ ~ ± i ±

O.19 0.19 0.12 0.24 0.22

Saline or drugs in saline were injected i.p. 75 min before sacrifice and gabaculine (O.| g/kg i.p.) when administered, was given 60 mln before mice were killed. Data are mean values ~ s.e.m. (n ffi 4) of steady state GABA levels or of gabaculine induced enhancement of GABA levels. Chronic ethanol treated mice were fed for 14 days a liquid diet to which (|O % w/v) ethanol was added. Blood ethanol levels in these mice at sacrifice were 2.23 ~ 0.22 mg/ml (mean ± s.e.m.; n = 5).

In some experiments we studied whether ethanol alone or in combination with gabacullne could interfere with GABA synthesis in vivo by decreasing glutamic acid decarboxylase activity. Pretreatment of mice in vivo with ethanol had no effect on the activity of glutamic acid decarboxylase determined subsequently in vitro (Table II). Furthermore, coincubation with ethanol in the absence or presence of gabaculine had also no effect on glutamic acid decarboxylase activity (Table II).

TABLE II Effect of Ethanol on Glutamic Acid Decarboxylase Activity in vitro Condition

GAD activity (% of control) (mean ~ s.e.m.; n = 31

Experiment 1 Pretreatment in vivo x) with Ethanol (3 g/kg) Ethanol (3 g/kg) + gabaculine (O.1 g/kg) Experiment 2 Coincubation with Ethanol (0.2 M) Ethanol (0.2 M) + gabaculine (O.! m M )

101.3~2.4 98.3~4.4

98.1~1.4 99.6±1.8

X)ethanol and gabaculine were injected 75 and 60 min before sacrifice.

Vol. 27, No. 12, 1980

Ethanol on GABA Syntheslsln V~vo

1039

Discussion Our results clearly show that the single or repeated administration of ethanol has potent depressant effects on the rate of GABA biosynthesis in mouse brain in vivo. It appears unlikely that ethanol exerts this action by a direct inhibi~-o~-~of glutamlc acid decarboxylase, since we were unable to observe effects on the activity of this enzyme in vitro, which is in agreement with previous results (IOt. In the method of Bernasconi and Martin (5) which we used for the estimation of GABA synthesis in vivo, the rate of GABA accumulation after specific and irreversible inhibit-~on of GABA degradation is estimated. When gabaculine is administered, GABA levels increase linearly for more than 2 hours, whereas the levels of all other amino acids remain unaffected (5,12,13). While this method has the advantage to permit the estimation of the dynamics of GABA metabolism in vivo, it has also limitations since (i) steady state conditions are not main'~ai--~d and since (ii) "metabolic" and "transmitter" compartments of GABA cannot be distinguished. Thus, the rates of GABA accumulation estimated with this method do not necessarily reflect the amount of GABA released at synapses. Ethanol and a number of other CNS depressants such as benzodiazepines, barbiturates and muscimol potently depress GABA synthesis in vivo, although these drugs have no effect on steady state GABA levels. A specificity of the effect of benzodiazepines is suggested by the observations that their potency exhibits stereospecificity and reflects their affinity for benzodiazepine receptors (6). Since muscimol, benzodiazepines (6), barbiturates (5) and other antiepileptic drugs (5) which have been shown to depress GABA synthesis in vivo have pharmacological effects which are compatible with a GABA-mimetic efficacy, it appears that GABA synthesis rates may be downregulated in response to drugs which enhance GABA-ergic activity. In addition, these results suggest that steady state GABA levels can be maintained even under conditions where the flux of GABA, as revealed by the rate of accumulation after blockade of GABA degradation, is greatly diminished or even abolished. There are several similarities between functional and biochemical CNS effects of ethanol and those of other CNS depressants such as benzodiazepines and barbiturates. Thus, these drugs potentiate each other in their CNS effects and they produce ataxia, probably cerebellar in origin (14). They all have anticonvulsive effects when given acutely and enhanced susceptibility to seizures is observed during withdrawal (I,15,16). Recently, it has been shown that polyethylene glycol has also anticonvulsant effects in monkeys (17). Furthermore, alcohol (2,3), benzodiazepines (4) or muscimol (4) have been found to depress cerebellar cGMP levels and these drugs decrease GABA synthesis in vivo. These similarities are compatible with the hypothesis that alcohol may exert at least some of its CNS effects by a direct or indirect activation of the GABA system. This notion is consistent with the observation that GABA antagonists produce symptoms similar to those seen during ethanol withdrawal while GABA-mimetics can attenuate ethanol symptoms (18-20). Furthermore, acute and chronic treatment with ethanol has recently been shown to alter GABA receptor sensitivity (21). In addition, neurophysiological findings also suggest that ethanol enhances GABA-ergic activity since alcohol increases transmission in synapses where the transmitter appears to be GABA (22-24). However, there are alternative mechanisms by which ethanol could depress GABA synthesis rates in vivo. For example, by general metabolic effects (25) ethanol could lead to-~d--~-~-nished availability of glutamate in a compartment essential for GABA synthesis. In addition, local anaesthetic effects of ethanol including effects on membrane fluidity (267 could reduce the firing rate of neurons and thus, secondarily lead to a decrease of GABAsynthesis. However, if a local anaesthetic effect of ethanol is responsible for the depression of GABA synthesis, then one would expect that the biosynthesis of other transmitter substances, unless GABA neurons are preferentially inhibited, would be similarly

1040

Ethanol on GABA Synthesis In V~uo

Vol. 27, No. 12, 1980

affected, which has been shown not to be the case (25). Furthermore, local anaesthetic effects are unlikely to occur at the low blood levels we observed. In conclusion, the present results support and extend previous observations (5,6) that CNS depressant drugs which appear to enhance the functional activity of GABA neurons can decrease GABA biosynthesis under conditions where steady state levels remain unchanged. Similarly ethanol, even in low doses, can also depress GABA biosynthesis, suggesting that alcohol may exert at least some of its CNS effects by directly or indirectly altering the functional state of the GABA system. Acknowledgements We thank R. Bernasconi for the help during the introduction of his method, G. Siggins for his help during the preparation of the manuscript, and J. Reisenhofer for secretarial help. Supported by "Ponds zur FSrderung der wissenschaftlichen Forschung in ~sterreich". References 1. M. VICTOR, P o s t g r a d . Med. 47 68-72 (1970). 2. J . D . REDOS, G.N. CATRAVA8 and W.A. HUNT, S c i e n c e 193 58-59 (1976). 3. R.B. MAILMAN, G.D. FYRE, R.A. HUELLER and G.R. BREESE, Nature 272 832-833 (1978). 4. G. BIGGIO, B. BRODIE, E. COSTA and A. GUIDOTTI, P r o c . n a t n . Acad. S c i . USA 74 3592-3596 (1977). 5. R. BERNASCONI and P. MARTIN, Naunyn Schmledebergs Arch. Pharmacol. 302 R58 (1978). 6. R. BERNASCONI and P. MARTIN, Naunyn Schmiedebergs Arch. Pharmacol. 307 R63 (1979). 7. R. SCHHID and M. KAROBATH, J . Chromat. 139 i 0 1 - I 0 9 (1977). 8. H. OGATA, F. OGATO, J.H. MENDELSON and N.K. MELLO, J. Pharmacol. Exp. Therap. 180216-230 (1972). 9. F. LUNDQVIST, Methods Biochem. Anal. ~ 240-25l (1977). lO. I. SUTTON and M.A. SIMMONDS, Biochem. Pharmac. 2__221685-1692 (1973). 11. J.C. CRABBE, H. RIGTER, J. UIJLEN and C. STRIJBOS, J. Pharmacol. Exp. T h er ap . 208 128-133 (1979). 12. Y. MATSUI and T. DEGUCHI, L i f e S c i . 2 0 1291-1296 (1977). 13. P . J . SCHECHTER, Y. TRANIER and J . GROVE, L i f e S c i . 2 4 1173-1182 (1979). 14. E. EIDELBERG, M.L. BOND and A. KELTER, Arch. I n t . Pharmacodyn. Therap. 192 2 | 3 - 2 1 9 (1971). 15. B. SANDERS and S.K. SHARPLESS, L i f e S c i . 2 3 2493-2500 (1978). 16. B.R. COOPER, K. VIIK, R.M. FERRIS and H.L. WHITE, J . Pharmacol. Exp. Therap. 209 396-403 (1979). 17. J . S . LOCKARD and R.H. LERY, L i f e S c i . 2 3 2 4 9 9 - 2 5 0 2 (1978). 18. D.B. GOLDSTEIN, J . Pharmacol. Exp. Therap. 186 I - 9 (1973). 19. B. BISWAS and A. CARLSSON, P s y c h o p h a r m a c o l o g y 5 9 91-94 (1978). 20. J. COTT, A. CARLSSON, J. ENGEL and M. LINDQVIST, Naunyn Schmiedebergs Arch. Pharmacol. 295203-209 (1976). 21. M.A. TICKU and T. BURCH, J. Neurochem. 34417-423 (1980). 22. N.R. BANNA, Experientia 2 5 6 1 9 - 6 2 0 (1969). 23. R.A. DAVIDOFF, Arch. Neurol. 2 8 6 0 - 6 3 (1973). 24. J.T. MIYAHARA, D.W. ESPLIN and B. ZABLOCKA, J. Pharmacol. Exp. Therap. 154 I19-127 (1966). 25. A.K. RAWAT, Int. Rev. Neuroblol. 19 124-162 (1976). 26. P. SEEMAN, Pharmacol. Rev. 24 583-655 (1972).