Psychopharmacology
Psychopharmacology61,251 - 254 (1979)
9 by Springer-Verlag 1979
Cholinergic Involvement in Ethanol Intoxication and Withdrawal-Induced Seizure Susceptibility A. Ebel* **, R. Vigran, G. Mack, T. Durkin, and P, Mandel Centre de Neurochimiedu CNRS, and Institut de Chimie Biologique,Facult6 de M6decine,11 rue Humann, 67085 Strasbourg Cedex, France
Abstract. The enzymes of the cholinergic system have been investigated in discrete brain areas in alcoholdependent rats, which were still intoxicated or were undergoing withdrawal. The ethanol intoxication resulted in a slight, but significant increase in choline acetyltransferase (CAT) activity in the caudate nucleus both 1 and 7 h after the last dose of ethanol. We also found a significant decrease in CAT activity in the temporal limbic cortex while rats were highly intoxicated. All other brain regions investigated, e.g., cerebellum, pons-medulla, frontoparietal cortex, hypothalamus and septum showed unchanged CAT activity. Rats were also analysed immediately following the onset of a withdrawal-induced audiogenic convulsive seizure where, in addition to the striatum, depressed CAT activity was observed in the hippocampus. In all the analysed situations acetylcholinesterase activity remained unchanged. These results show that ethanol intoxication leads to a perturbation in the synthetic capacity of acetylcholine in certain defined brain structures and that this may have some correlation to the observed behavioural impairments.
Key words: Rat - Alcohol - Withdrawal Audiogenic seizure - Cholinergic enzymes - Nucleus caudatus - Hippocampus
Evidence for a cholinergic involvement in ethanol intoxication has been provided by studies that have demonstrated that ethanol decreases acetylcholine (ACh) output (Kalant et al., 1967; Kalant and Grose, * Charg6e de Rechercheau CNRS ** To whom offprint requests should be sent
1967); contradictory results exist for its effect on ACh levels in brain (Erikson and Graham, 1973; Rawat, 1974; Hunt and Dalton, 1976). Moreover, in a study of the overall effect of ethanol on putative CNS neurotransmitters, Carmichael and Israel (1975) found that of all transmitters studied, ACh was the most sensitive to ethanol in pharmacologically relevant concentrations. However, strong controversy remains as to the mechanism of the interactions between ethanol and the cholinergic transmission system in brain. Many factors could account for the decrease in brain ACh output induced by ethanol. It has been suggested that the depressant effect of ethanol on ACh output is exerted primarily on the release process (Kalant et al., 1966) and that this may be a consequence of altered ion permeabilities, particularly the changes in Na § conductance induced by ethanol (Carmichael and Israel, 1975). Ethanol may also inhibit ACh synthesis (Rawat, 1974) or increase its rate of degradation. It has also been suggested that the variation of free ACh would be a reflection of decreased overall neuronal activity (Graham and Erickson, 1974). Little is known about the response of the enzymes of the cholinergic system in brain following alcohol administration. Thus, in the present report we have investigated the response of the cholinergic system in several brain regions by studying choline acetyltransferase (CAT, EC. 2.3.1.6) and acetylcholinesterase (ACHE, EC. 3.1.1.7) activities following a prolonged alcohol administration in which rats were constantly maintained under alcohol intoxication for a period of 4 days. We also investigated these cholinergic enzymes in susceptibility to audiogenic seizure, a common feature of the ethanol withdrawal syndrome in animals, which is thought to reflect changes in neural excitability. Further, this investigation was designed to test the hypothesis that the withdrawal seizures are a manifestation of increased susceptibility of the brain to seizure
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252
activity following a period of depression by the sedative action of alcohol and an attempt is made to describe the potential cholinergic involvement in this withdrawalinduced seizure susceptibility.
Psychopharmacology 61 (1979) Proteins were measured in every sample by the method of Lowry et al., with bovine serum albumin as standard (Lowry et al., 1951). Blood ethanol levels were determined using the Boehringer Alcohol Test Combination.
Results Materials and Methods Male Wistar rats ( 2 0 0 - 300 g) were housed four per cage and had unlimited access to rodent pellets and water throughout the entire experimental period. Ethanol dependence was induced according to the procedure of Majchrowicz (1975). Ethanol aqueous solution (20 % v/v of absolute ethyl alcohol) was administered for 4 days by intragastric intubation. In order to maintain a continuous elevation of blood, ethanol concentration (300-400mg/dl) doses of 15 g/kg were administrated in three fractions daily for a period of 4 days. At the doses given, the animals were unconscious with 1 5 30 rain after each intubation. The intubations were performed every 6 - 8 h according to the behaviour of the animal concerned. No animal ever became fully conscious as they were intubated at a stage presenting moderate to severe behavioural depression. Therefore the animals were maintained in a state of either stupor of complete unconsciousness. The time course of the behaviour was as follows: at 1 h after the intubation the animals were completely unconscious with respiration visibly slowed. at 4 h the animals were unconscious or slightly conscious, but unable to move in their cages. at 7 h the animals were conscious, however they were still in a depressed state showing low vigilance and obvious ataxia. at 12 h the animals were behaving normally, beginning to manifest the first signs of the abstinence syndrome, i.e. tremor, rigidity and hyperexcitability. at 18 h the animals showed a full-blown withdrawal syndrome with gross tremor, rigidity of the body and tail and hyperexcitability manifested by increased restlessness and susceptibility to induced audiogenic seizures. The animals were killed after 1, 7 or 18 h following the last dose of ethanol. Eighteen hours after the last ethanol administration the withdrawal syndrome is apparent. For the study of the withdrawal-induced audiogenic seizure, the rats were exposed to the ringing of a bell in a sound-attenuated chamber for 60 s. The sound intensity was approximately 90 dB and the animals were sacrificed immediately following the onset of convulsive seizure. For enzyme activity determinations the animals were killed by decapitation, the brains exised and the following areas dissected out: cerebellum, pons-medulla, hypothalamus, frontoparietal and temporal limbic cortex, septum and caudate nucleus. Immediately after dissection the samples were frozen on dry ice and stored at 20 ~ C. i~or biochemical determinations the samples were homogenized at 0 ~ ~ in 0.5 % Triton X-100 (100 pg tissue/1 ~tl) using Potter Elvejhem glass-teflon homogenizers. Samples were diluted with 0.05 % bovine serum albumin. Choline acetyltransferase was measured according to the microtechnique of McCaman and Hunt (1965) as modified by Goldberg et al. (1969) by following the incorporation of (1-14C) acetate from acetyl-CoA into acetylcholine. The (1-1~C - acetylcholine formed was precipitated with potassium periodide and the amount of radioactivity present was counted in a Packard scintillation counter. The enzyme activity was calculated from the known specific activity of the (1-14C) acetyl CoA (NEN Chemical, 59.2 mCi/mmol). Acetylcholinesterase was determined by the method of Ellman et al. (1961) by following the increase of yellow colour produced from thiocholine when it reacts with dithio-bisnitrobenzoate ion (DTNB) at pH 7.6 and 30 ~ C.
Alcohol Intoxication (4 days) - Analysis 1 h After the Last Alcohol Administration
The ethanol intoxication resulted in a slight (15 %), but significant increase (P < 0.01, using Student's t-test) in CAT activity in the caudate nucleus as compared to controls (Table 1). We also found a significant decrease (P < 0.01) in CAT activity in the temporal limbic cortex. All other brain regions investigated: cerebellum, ponsmedulla, frontoparietal cortex, septum, hippocampus and hypothalamus showed unchanged CAT activity. Table 1. Choline acetyltransferase activity in discrete CNS areas 1 and 7 h after 4 days prolonged alcohol intoxication A Control Cerebellum 1.84_+0.16 Pons-medulla 26.90 _+ 2.49 Frontoparietal cortex 11.63 • 0.38 Temporal limbic cortex 16.16_+0.40 Caudatus nucleus 46.07 _+ 1.53 Septum 17.32 _+ 0.30 Hippocampus 16.44 • 1.81 Hypothalamus 13.81 • 1.41
B C Alc. intoxicated Alc. intoxicated after 7 h after 1 h 1.91 _+0.14 25.62 • 2.36
1.84 + 0.15 27.31 + 0.72
11.60 • 1.00
10.57 • 1.23
14.43 +_0.85"
16.22_+1.37
52.18 17.31 15.79 15.06
51.16 18.35 15.63 12.72
+ 4.358 _+ 1.60 • 2.15 _+ 1.58
-+ 3.74 c _+ 0.77 • 2.21 + 0.92
Mean values of six experiments • SD " A/B P
Psychopharmacology61,251 - 254 (1979)
9 by Springer-Verlag 1979
Cholinergic Involvement in Ethanol Intoxication and Withdrawal-Induced Seizure Susceptibility A. Ebel* **, R. Vigran, G. Mack, T. Durkin, and P, Mandel Centre de Neurochimiedu CNRS, and Institut de Chimie Biologique,Facult6 de M6decine,11 rue Humann, 67085 Strasbourg Cedex, France
Abstract. The enzymes of the cholinergic system have been investigated in discrete brain areas in alcoholdependent rats, which were still intoxicated or were undergoing withdrawal. The ethanol intoxication resulted in a slight, but significant increase in choline acetyltransferase (CAT) activity in the caudate nucleus both 1 and 7 h after the last dose of ethanol. We also found a significant decrease in CAT activity in the temporal limbic cortex while rats were highly intoxicated. All other brain regions investigated, e.g., cerebellum, pons-medulla, frontoparietal cortex, hypothalamus and septum showed unchanged CAT activity. Rats were also analysed immediately following the onset of a withdrawal-induced audiogenic convulsive seizure where, in addition to the striatum, depressed CAT activity was observed in the hippocampus. In all the analysed situations acetylcholinesterase activity remained unchanged. These results show that ethanol intoxication leads to a perturbation in the synthetic capacity of acetylcholine in certain defined brain structures and that this may have some correlation to the observed behavioural impairments.
Key words: Rat - Alcohol - Withdrawal Audiogenic seizure - Cholinergic enzymes - Nucleus caudatus - Hippocampus
Evidence for a cholinergic involvement in ethanol intoxication has been provided by studies that have demonstrated that ethanol decreases acetylcholine (ACh) output (Kalant et al., 1967; Kalant and Grose, * Charg6e de Rechercheau CNRS ** To whom offprint requests should be sent
1967); contradictory results exist for its effect on ACh levels in brain (Erikson and Graham, 1973; Rawat, 1974; Hunt and Dalton, 1976). Moreover, in a study of the overall effect of ethanol on putative CNS neurotransmitters, Carmichael and Israel (1975) found that of all transmitters studied, ACh was the most sensitive to ethanol in pharmacologically relevant concentrations. However, strong controversy remains as to the mechanism of the interactions between ethanol and the cholinergic transmission system in brain. Many factors could account for the decrease in brain ACh output induced by ethanol. It has been suggested that the depressant effect of ethanol on ACh output is exerted primarily on the release process (Kalant et al., 1966) and that this may be a consequence of altered ion permeabilities, particularly the changes in Na § conductance induced by ethanol (Carmichael and Israel, 1975). Ethanol may also inhibit ACh synthesis (Rawat, 1974) or increase its rate of degradation. It has also been suggested that the variation of free ACh would be a reflection of decreased overall neuronal activity (Graham and Erickson, 1974). Little is known about the response of the enzymes of the cholinergic system in brain following alcohol administration. Thus, in the present report we have investigated the response of the cholinergic system in several brain regions by studying choline acetyltransferase (CAT, EC. 2.3.1.6) and acetylcholinesterase (ACHE, EC. 3.1.1.7) activities following a prolonged alcohol administration in which rats were constantly maintained under alcohol intoxication for a period of 4 days. We also investigated these cholinergic enzymes in susceptibility to audiogenic seizure, a common feature of the ethanol withdrawal syndrome in animals, which is thought to reflect changes in neural excitability. Further, this investigation was designed to test the hypothesis that the withdrawal seizures are a manifestation of increased susceptibility of the brain to seizure
0033-3158/79/0061/0251/$ 01.00
252
activity following a period of depression by the sedative action of alcohol and an attempt is made to describe the potential cholinergic involvement in this withdrawalinduced seizure susceptibility.
Psychopharmacology 61 (1979) Proteins were measured in every sample by the method of Lowry et al., with bovine serum albumin as standard (Lowry et al., 1951). Blood ethanol levels were determined using the Boehringer Alcohol Test Combination.
Results Materials and Methods Male Wistar rats ( 2 0 0 - 300 g) were housed four per cage and had unlimited access to rodent pellets and water throughout the entire experimental period. Ethanol dependence was induced according to the procedure of Majchrowicz (1975). Ethanol aqueous solution (20 % v/v of absolute ethyl alcohol) was administered for 4 days by intragastric intubation. In order to maintain a continuous elevation of blood, ethanol concentration (300-400mg/dl) doses of 15 g/kg were administrated in three fractions daily for a period of 4 days. At the doses given, the animals were unconscious with 1 5 30 rain after each intubation. The intubations were performed every 6 - 8 h according to the behaviour of the animal concerned. No animal ever became fully conscious as they were intubated at a stage presenting moderate to severe behavioural depression. Therefore the animals were maintained in a state of either stupor of complete unconsciousness. The time course of the behaviour was as follows: at 1 h after the intubation the animals were completely unconscious with respiration visibly slowed. at 4 h the animals were unconscious or slightly conscious, but unable to move in their cages. at 7 h the animals were conscious, however they were still in a depressed state showing low vigilance and obvious ataxia. at 12 h the animals were behaving normally, beginning to manifest the first signs of the abstinence syndrome, i.e. tremor, rigidity and hyperexcitability. at 18 h the animals showed a full-blown withdrawal syndrome with gross tremor, rigidity of the body and tail and hyperexcitability manifested by increased restlessness and susceptibility to induced audiogenic seizures. The animals were killed after 1, 7 or 18 h following the last dose of ethanol. Eighteen hours after the last ethanol administration the withdrawal syndrome is apparent. For the study of the withdrawal-induced audiogenic seizure, the rats were exposed to the ringing of a bell in a sound-attenuated chamber for 60 s. The sound intensity was approximately 90 dB and the animals were sacrificed immediately following the onset of convulsive seizure. For enzyme activity determinations the animals were killed by decapitation, the brains exised and the following areas dissected out: cerebellum, pons-medulla, hypothalamus, frontoparietal and temporal limbic cortex, septum and caudate nucleus. Immediately after dissection the samples were frozen on dry ice and stored at 20 ~ C. i~or biochemical determinations the samples were homogenized at 0 ~ ~ in 0.5 % Triton X-100 (100 pg tissue/1 ~tl) using Potter Elvejhem glass-teflon homogenizers. Samples were diluted with 0.05 % bovine serum albumin. Choline acetyltransferase was measured according to the microtechnique of McCaman and Hunt (1965) as modified by Goldberg et al. (1969) by following the incorporation of (1-14C) acetate from acetyl-CoA into acetylcholine. The (1-1~C - acetylcholine formed was precipitated with potassium periodide and the amount of radioactivity present was counted in a Packard scintillation counter. The enzyme activity was calculated from the known specific activity of the (1-14C) acetyl CoA (NEN Chemical, 59.2 mCi/mmol). Acetylcholinesterase was determined by the method of Ellman et al. (1961) by following the increase of yellow colour produced from thiocholine when it reacts with dithio-bisnitrobenzoate ion (DTNB) at pH 7.6 and 30 ~ C.
Alcohol Intoxication (4 days) - Analysis 1 h After the Last Alcohol Administration
The ethanol intoxication resulted in a slight (15 %), but significant increase (P < 0.01, using Student's t-test) in CAT activity in the caudate nucleus as compared to controls (Table 1). We also found a significant decrease (P < 0.01) in CAT activity in the temporal limbic cortex. All other brain regions investigated: cerebellum, ponsmedulla, frontoparietal cortex, septum, hippocampus and hypothalamus showed unchanged CAT activity. Table 1. Choline acetyltransferase activity in discrete CNS areas 1 and 7 h after 4 days prolonged alcohol intoxication A Control Cerebellum 1.84_+0.16 Pons-medulla 26.90 _+ 2.49 Frontoparietal cortex 11.63 • 0.38 Temporal limbic cortex 16.16_+0.40 Caudatus nucleus 46.07 _+ 1.53 Septum 17.32 _+ 0.30 Hippocampus 16.44 • 1.81 Hypothalamus 13.81 • 1.41
B C Alc. intoxicated Alc. intoxicated after 7 h after 1 h 1.91 _+0.14 25.62 • 2.36
1.84 + 0.15 27.31 + 0.72
11.60 • 1.00
10.57 • 1.23
14.43 +_0.85"
16.22_+1.37
52.18 17.31 15.79 15.06
51.16 18.35 15.63 12.72
+ 4.358 _+ 1.60 • 2.15 _+ 1.58
-+ 3.74 c _+ 0.77 • 2.21 + 0.92
Mean values of six experiments • SD " A/B P
