Acta pharmacol. et toxicol. 1975, 36, 289-298.
From the Department of Pharmacology, University of Helsinki, Siltavuorenpenger 10, SF-00170 Helsinki 17, Finland
Effect of Ethanol Dependence and Withdrawal on the Catecholamines in Rat Brain and Heart BY Liisa Abtee and Margareta Svartstrom-Fraser (Received October 9, 1974; Accepted November 19, 1974)
Abstract: To study whether ethanol dependence is accompanied by changes in the brain monoamines, male Sprague-Dawley rats were intubated intragastrically with 10% (w/v) ethanol solution 3 times daily (daily dose 8-11 g/kg) for 7-10 days. Control rats were intubated with similar volumes of tap water. The rats were killed 4-6 hrs (intoxicated group) or 16-18 hrs (withdrawal group) after the last administration of ethanol. At this time the withdrawn rats showed a stiff tail, spastic rigidity, piloerection, tremor, hyperexcitability, stereotyped head movements as well as convulsions. The dopamine and homovanillic acid concentrations as well as the disappearance rate of dopamine after synthesis blockade (a-methyl-ptyrosine 250 mg/kg intraperitoneally 3 hrs) were similar in the brain of control, ethanol intoxicated and withdrawn rats. Chronic ethanol administration did not alter the concentration of noradrenaline in the brain or the heart of the rats. However, the disappearance rate of noradrenaline after synthesis blockade was accelerated by about 30-50% in the brain and heart of rats withdrawn from ethanol. Moreover, the concentration of noradrenaline was decreased by 40-60 % in the brain and heart of rats which died in withdrawal convulsions. The experiments suggest that the noradrenergic mechanisms but not the dopaminergic mechanisms may be associated with the occurrence of withdrawal symptoms. Key-words: Ethanol withdrawal - dopamine
- noradrenaline
- homovanillic acid.
Administration of intoxicating amounts of alcohol twice daily or more for several days produces physical dependence in rats, shown by withdrawal symptoms at the termination of the treatment (MAJCHROWICZ 1973; WALLGREN et al. 1972 & 1973). Ethanol withdrawal after chronic administration induces symptoms which suggest that the nervous transmission in the central and possibly also in the peripheral nervous system is altered. These symptoms consist of stiffness of the tail and rigidity of the body as well as stereotyped head
290
L. AHTEE AND M. SVARTSTRUM-FRASER
movements which are suggestive of changes in the extrapyramidal motor system. Another group of symptoms such as hyperexcitability and convulsions also suggests that during chronic ethanol administration changes are induced in the central nervous system. However, certain symptoms such as piloerection could be induced by changes in the peripheral sympathetic system. In an earlier experiment (WALLGREN el al. 1972 & 1973) we did not find any changes in the concentration of noradrenaline in the brain of ethanol or tertiary butanol dependent rats during intoxication or withdrawal. In the present experiments the possible association of ethanol dependence with brain catecholamines has been studied more extensively. In a few experiments the heart noradrenaline concentration was also determined.
Materials and Methods Administration of ethanol. Male Sprague-Dawley rats (250-300 g) were housed one to a cage on a standard laboratory diet and water ad libitum under a lighting schedule with lights on from 7 a.m. to 7 p.m. The rats were intubated intragastrically with 10% (w/v) ethanol solution 3 times daily, at 8 a.m., 3 p. m. and 10 p. m. for 7-10 days. The rats were weighed daily and the ethanol dose was calculated according to the body weight. The daily dose increased from 8 to 11 g/kg. Control rats were intubated with similar volumes of tap water. The ethanol treated rats lost 50-100 g of their weight during the treatment. Moreover the control rats which were repeatedly intubated intragastrically (30-45 ml tap water daily) tended to lose weight (10-20 g) during the treatment. During the chronic ethanol treatment 20-30% of the rats died. The deaths were most probably due to a too large dose of ethanol, because the rats which died were often still intoxicated during the last ethanol intubation. Estiniation of dopamine, hornovanillic acid and noradrenaline. The rats were killed by decapitation between 12 noon and 2 p.m. either 4-6 hrs (intoxicated group) or 16-18 hrs (withdrawal group) after the last administration of ethanol (either at 8 p.m. or at 8 a.m., 4-5 g/kg). The blood ethanol concentrations measured (ERIKSSON 1973) at this time were: 91.5+4.9 mmol/l (mean kS.E.M., n = 6; intoxicated group) and 10.2k4.8 mmol/l (n = 6; withdrawal group). The brain was rapidly removed and dissected according to Craigie’s Neuroanatomy of the Rat (ZEMAN& INNES1965). In most experiments the cerebellum was discarded and thereafter the brain was divided into three parts: 1) pons+medulla oblongata (mean weight+_S.D.: 176+21 mg, n = 51), 2) mesencephalon+diencephalon+ telencephalon except cortex (536k45 mg, n = 51), and 3) cortex (635+50 mg, n = 51). For the estimation of homovanillic acid the striata from two brains (99f 4 mg, 20 samples) were pooled. The heart was rapidly removed and rinsed with ice-cold 0.9% NaCl solution. Dopamine and noradrenaline were estimated in the same tissue sample by the method of SHELLENBERGER & GORDON (1971). After precipitation of the proteins with 0.4 N perchloric acid the amines were adsorbed to activated alumina and thereafter eluted with 0.05 N perchloric acid. The amines were then oxidized with iodine and the fluorescence was read in an Aminco-Bowman spectrophotofluorimeter. The results were corrected for the recovery of standard carried through the method. The recovery for dopamine was 88 k 2 % (mean f S.E.M., n = 15) and for noradrenaline 82.5 k 1 % (n = 15). Homovanillic acid (HVA) was estimated as described by PORTIGet al. (1968). The tissue
CATECHOLAMINES IN ETHANOL WITHDRAWAL
291
samples were homogenized in 0.1 N-HCI, proteins were precipitated with perchloric acid, and HVA was extracted into n-butylacetate. From n-butylacetate HVA was extracted into Tris solution and estimated in an Aminco-Bowman spectrofluorimeter. Recovery for added HVA was 71 k 2 % (mean k S.E.M. from 4 estimations). Results have been corrected for losses. Rectal temperalure was measured with an electric thermocouple (Ellab Instruments, Copenhagen). The rats were tested for their response to electric footshock by using a chamber with a 25 x 27.5 cm grid floor (EVANS1961). Rats were given a series of 0.3 sec. shocks in increasing intensity (0.10-0.12-0.15-0.18-0.22-0.27-0.33-0.39-0.47-0.56-0.680.82-1.0-1.2-1.5-1.8-2.2-2.7 mA). The shocks were given at 30 sec. intervals. The rats’ paws were wiped with a glycerine-saline solution to allow good conductance. At the end of each shock the squealing and jumping responses of the rats were observed. DL-a-methyl-p-tyrosine methylester HCI (H 44/68, Labkemi Ab, Gothenburg) was dissolved in saline and injected intraperitoneally in a volume of 2 ml/kg. The dose refers to the base. The statistical significance of differences between means was calculated by Student’s t-test.
Results Behaviour of rats during ethanol withdrawal. After three -four days of repeated ethanol administration, the rats showed withdrawal symptoms after the longest interval before the next ethanol administration (10 hrs, between 10 p.m. and 8 a.m.). These signs were most often stiff tail, piloerection and tremor as well as stereotyped head movements. When the rats were withdrawn from ethanol more severe symptoms developed. Table 1 shows the occurrence of these
Table 1. Occurrence of withdrawal symptoms in 35 rats which had been treated with 8-11 g/kg of ethanol daily for 7-10 days, 15-16 hrs after administration of the last dose of ethanol (4-5 g/kg). Symptom
Occurrence (%)
Stiff tail Stereotypic head movements Spastic rigidity Piloerection Tremor Not able to walk on laboratory bench Stays on vertical grid over 30 sec. Squealing o n touching Tonic clonic convulsions
77 80 54 51 63 23 25 9 11
L. AHTEE AND M. SVARTSTROM-FXASER
292
Table 2. Thresholds for squealing and jumping responses to electric foot shock in control rats and in rats treated with 8-1 1 g/kg of ethanol daily for 7-10 days. The last dose of ethanol (4-5 g/kg) was administered either 3-4 hrs (intoxicated group) or 15-16 hrs (withdrawal group) before testing. Number of rats tested
Treatment Control Ethanol intoxication* Ethanol withdrawal
*
34 15 25
Threshold (mA) 5 S.E.M. for squealing jumping 0.26 f 0.02 1.06 k 0.16 0.36 f 0.06
0.54 k 0.03 1.35 f 0.14 0.51 5 0.07
Only rats with positive righting reflex were tested.
symptoms in 35 rats at 15-16 hrs after ethanol withdrawal when the symptoms were at their peak. Rats with a fully developed withdrawal syndrome remained in a characteristic posture with paws spread out and shaking continuously. Even a slight disturbance such as lifting the lid of their cage made them jump and run around the cage. However, they were not able to walk on a smooth laboratory bench and once put on a vertical grid they stayed there for an abnormally long time. Tonic clonic convulsions were also observed and the rats occasionally died during these convulsions. In the present experiments ethanol withdrawal did not alter the threshold for squealing and jumping responses to electric foot shock (table 2). However, in a few rats the jumping response developed into a convulsive state and
Table 3. Effect of a-methyl-p-tyrosine (aMT, 250 mg/kg, intraperitoneally) on the rectal ternperature of control rats and of rats treated with 8-11 g/kg of ethanol daily for 7-10 days. The last dose of ethanol (4-5 g/kg) was administered either 1-3 hrs (intoxicated group) or 13-15 hrs (withdrawal group) before aMT (or saline) administration. Rectal temperature Chronic treatment
Control Intoxication Withdrawal
Of
S.E.M. (n)
Saline
aMT
0 hrs
3 hrs
0 hrs
3 hrs
36.9k0.1 (10) 35.8 f0.2 (6) 37.0k0.6 (6)
37.0k0.1 (10) 36.3 f 0.8 (6) 36.9f0.5 (6)
36.8k0.1 (10) 35.9 f 0.5 (9) 37.1 f0.3 (9)
35.3k0.2 (10) 34.4 f 0.6 (8) 35.1 k0.3 (9)
CATECHOLAMINES IN ETHANOL WITHDRAWAL 2.0 -
T
293
MESENCEPHALON + DIENCEPHALON + TELENCEPHALON- CORTEX
1.81.61.1, -
1.2m
1
m 3 1.0-
w
I I: 0.8a a 0
0.60.L -
INTOXICATION
0.20-
WITHDRAWAL ALINE
dMT
Fig. 1. Effect of a-methyl-p-tyrosine (aMT, 250 mg/kg, intraperitoneally, 3 hrs) on the concentration of dopamine in the mesencephalon diencephalon telencephalon except cortex of rats treated with 8-11 g/kg of ethanol daily for 7-10 days. The rats were killed either 4-6 hrs (intoxicated group) or 16-1 8 hrs (withdrawal group) following administration of the last dose of ethanol (4-5 g/kg). Mean values from 6 to 10 rats; vertical bars indicate the S.E.M. The control group was treated chronically with water.
+
+
Table 4. Homovanillic acid (HVA) concentration in the striatum of rats treated with 8-1 1 g/kg of ethanol daily for 7-10 days. The rats were killed either 4-6 hrs (intoxicated group) or 16-18 hrs (withdrawal group) after administration of the last ethanol dose (4-5 g/kg). Treatment Control Ethanol intoxication Ethanol withdrawal
HVA pg/g+S.E.M. (n) 0.77 k 0.05 (5) 0.78+0.11 (6) 0.73 k 0.03 (6)
294
L. AHTEE A N D M. SVARTSTRdM-FRASER
one rat died in such a convulsion. It should be noted that we tested the responses very quickly by giving only one series of electric shocks of increasing intensity to each rat and noting the intensity in which the rat first squealed or jumped. Table 3 shows that ethanol intoxication decreased the rectal temperature of the rats by about lo, but withdrawal from ethanol did not alter the rectal temperature of the rats. Inhibition of the synthesis of catecholamines by 250 mg/kg of a-methyl-p-tyrosine (aMT) decreased the rectal temperature similarly in the control and ethanol treated rats.
0.4 0.2
0 MESENCEPHALON+ DIENCEPHALON+
MEDULLA OBLONGATA
INTOXICATION
SALINE
d MT
Fig. 2. Effect of a-methyl-p-tyrosine (aMT, 250 mg/kg, intraperitoneally, 3 hrs) on the concentration of noradrenaline in 1) cortex, 2) mesencephalon + diencephalon + telencephalon except cortex, and 3) pons+medulla oblongata of rats treated with 8-11 g/kg of ethanol daily for 7-10 days. The rats were killed either 4-6 hrs (intoxicated group) or 16-18 hrs (withdrawal group) after administration of the last dose of ethanol (4-5 g/kg). Mean values from 6-10 rats, vertical bars indicate the S.E.M. The asterisks indicate significant change (P < 0.001) from the corresponding control group treated chronically with water.
CATECHOLAMINES IN ETHANOL WITHDRAWAL
295
HEART
0
CONTROL
a
ETHANOL INTOXICATION
SALINE
ETHANOL WITHDRAWAL
dMT
Fig. 3. Effect of a-methyl-p-tyrosine (aMT, 250 mg/kg, intraperitoneally, 3 hrs) on the concentration of noradrenaline in the hearts of rats treated with 8-11 g/kg of ethanol daily for 7-10 days. The rats were killed either 4-6 hrs (intoxicated group) or 16-18 hrs (withdrawal group) after administration of the last dose of ethanol (4-5 g/kg). Mean values from 4 to 10 rats; vertical bars indicate the S.E.M. The asterisk indicates significant change (P < 0.01) from the corresponding control group treated chronically with water.
E f j i t of chronic ethanol administration on brain dopamine. Chronic ethanol administration did not change the concentration of dopamine in the brain of rats (fig. 1). Moreover, the rate of aMT-induced disappearance of dopamine was similar in the brain of control rats, of ethanol intoxicated rats and of rats withdrawn from ethanol. Table 4 shows that chronic ethanol administration did not alter the concentration of homovanillic acid in the striata of rats. E e c t of chronic ethanol administration on brain and heart noradrenaline. Fig. 2 shows that the noradrenaline concentrations were similar in all parts of the brain studied, 1) cortex, 2) mesencephalon diencephalon telencephalon except cortex, 3) ponsfmedulla oblongata, in control rats and in ethanol intoxicated rats as well as in rats withdrawn from ethanol. No were any differences found in the noradrenaline concentrations of the hearts of these rats (fig. 3). However, the three rats, which died in withdrawal convulsions (and the braiu and heart of which were dissected immediately after death) had significantly reduced brain and heart noradrenaline contents (brain concentration = 0.30 f 0.01 pg/g, normal mean f S.E.M.=0.53 k 0.01 pg/g; heart concentration= 0.32 f0.03 pg/g, normal = 0.92 k 0.03 pg/g). The dopamine, 5-hydroxytryptamine and 5-hydroxyindoleacetic acid concentrations in the brain of the same rats did not differ from those of the control rats. When the synthesis of noradrenaline was blocked by treating the rats with 250 mg/kg of a M T intraperitoneally 3 hrs before killing, the rate of disap-
+
+
L. AHTEE AND M. SVARTSTROM-FRASER
296
pearance of noradrenaline was increased by 40 to 50 YOin all parts of the brain of rats withdrawn from ethanol. The noradrenaline concentration in the cortex of uMT-treated control rats was decreased in three hours by 43 %, in the cortex of ethanol intoxicated rats by 42Y0, and in the cortex of rats withdrawn from ethanol by 65 YO (P
From the Department of Pharmacology, University of Helsinki, Siltavuorenpenger 10, SF-00170 Helsinki 17, Finland
Effect of Ethanol Dependence and Withdrawal on the Catecholamines in Rat Brain and Heart BY Liisa Abtee and Margareta Svartstrom-Fraser (Received October 9, 1974; Accepted November 19, 1974)
Abstract: To study whether ethanol dependence is accompanied by changes in the brain monoamines, male Sprague-Dawley rats were intubated intragastrically with 10% (w/v) ethanol solution 3 times daily (daily dose 8-11 g/kg) for 7-10 days. Control rats were intubated with similar volumes of tap water. The rats were killed 4-6 hrs (intoxicated group) or 16-18 hrs (withdrawal group) after the last administration of ethanol. At this time the withdrawn rats showed a stiff tail, spastic rigidity, piloerection, tremor, hyperexcitability, stereotyped head movements as well as convulsions. The dopamine and homovanillic acid concentrations as well as the disappearance rate of dopamine after synthesis blockade (a-methyl-ptyrosine 250 mg/kg intraperitoneally 3 hrs) were similar in the brain of control, ethanol intoxicated and withdrawn rats. Chronic ethanol administration did not alter the concentration of noradrenaline in the brain or the heart of the rats. However, the disappearance rate of noradrenaline after synthesis blockade was accelerated by about 30-50% in the brain and heart of rats withdrawn from ethanol. Moreover, the concentration of noradrenaline was decreased by 40-60 % in the brain and heart of rats which died in withdrawal convulsions. The experiments suggest that the noradrenergic mechanisms but not the dopaminergic mechanisms may be associated with the occurrence of withdrawal symptoms. Key-words: Ethanol withdrawal - dopamine
- noradrenaline
- homovanillic acid.
Administration of intoxicating amounts of alcohol twice daily or more for several days produces physical dependence in rats, shown by withdrawal symptoms at the termination of the treatment (MAJCHROWICZ 1973; WALLGREN et al. 1972 & 1973). Ethanol withdrawal after chronic administration induces symptoms which suggest that the nervous transmission in the central and possibly also in the peripheral nervous system is altered. These symptoms consist of stiffness of the tail and rigidity of the body as well as stereotyped head
290
L. AHTEE AND M. SVARTSTRUM-FRASER
movements which are suggestive of changes in the extrapyramidal motor system. Another group of symptoms such as hyperexcitability and convulsions also suggests that during chronic ethanol administration changes are induced in the central nervous system. However, certain symptoms such as piloerection could be induced by changes in the peripheral sympathetic system. In an earlier experiment (WALLGREN el al. 1972 & 1973) we did not find any changes in the concentration of noradrenaline in the brain of ethanol or tertiary butanol dependent rats during intoxication or withdrawal. In the present experiments the possible association of ethanol dependence with brain catecholamines has been studied more extensively. In a few experiments the heart noradrenaline concentration was also determined.
Materials and Methods Administration of ethanol. Male Sprague-Dawley rats (250-300 g) were housed one to a cage on a standard laboratory diet and water ad libitum under a lighting schedule with lights on from 7 a.m. to 7 p.m. The rats were intubated intragastrically with 10% (w/v) ethanol solution 3 times daily, at 8 a.m., 3 p. m. and 10 p. m. for 7-10 days. The rats were weighed daily and the ethanol dose was calculated according to the body weight. The daily dose increased from 8 to 11 g/kg. Control rats were intubated with similar volumes of tap water. The ethanol treated rats lost 50-100 g of their weight during the treatment. Moreover the control rats which were repeatedly intubated intragastrically (30-45 ml tap water daily) tended to lose weight (10-20 g) during the treatment. During the chronic ethanol treatment 20-30% of the rats died. The deaths were most probably due to a too large dose of ethanol, because the rats which died were often still intoxicated during the last ethanol intubation. Estiniation of dopamine, hornovanillic acid and noradrenaline. The rats were killed by decapitation between 12 noon and 2 p.m. either 4-6 hrs (intoxicated group) or 16-18 hrs (withdrawal group) after the last administration of ethanol (either at 8 p.m. or at 8 a.m., 4-5 g/kg). The blood ethanol concentrations measured (ERIKSSON 1973) at this time were: 91.5+4.9 mmol/l (mean kS.E.M., n = 6; intoxicated group) and 10.2k4.8 mmol/l (n = 6; withdrawal group). The brain was rapidly removed and dissected according to Craigie’s Neuroanatomy of the Rat (ZEMAN& INNES1965). In most experiments the cerebellum was discarded and thereafter the brain was divided into three parts: 1) pons+medulla oblongata (mean weight+_S.D.: 176+21 mg, n = 51), 2) mesencephalon+diencephalon+ telencephalon except cortex (536k45 mg, n = 51), and 3) cortex (635+50 mg, n = 51). For the estimation of homovanillic acid the striata from two brains (99f 4 mg, 20 samples) were pooled. The heart was rapidly removed and rinsed with ice-cold 0.9% NaCl solution. Dopamine and noradrenaline were estimated in the same tissue sample by the method of SHELLENBERGER & GORDON (1971). After precipitation of the proteins with 0.4 N perchloric acid the amines were adsorbed to activated alumina and thereafter eluted with 0.05 N perchloric acid. The amines were then oxidized with iodine and the fluorescence was read in an Aminco-Bowman spectrophotofluorimeter. The results were corrected for the recovery of standard carried through the method. The recovery for dopamine was 88 k 2 % (mean f S.E.M., n = 15) and for noradrenaline 82.5 k 1 % (n = 15). Homovanillic acid (HVA) was estimated as described by PORTIGet al. (1968). The tissue
CATECHOLAMINES IN ETHANOL WITHDRAWAL
291
samples were homogenized in 0.1 N-HCI, proteins were precipitated with perchloric acid, and HVA was extracted into n-butylacetate. From n-butylacetate HVA was extracted into Tris solution and estimated in an Aminco-Bowman spectrofluorimeter. Recovery for added HVA was 71 k 2 % (mean k S.E.M. from 4 estimations). Results have been corrected for losses. Rectal temperalure was measured with an electric thermocouple (Ellab Instruments, Copenhagen). The rats were tested for their response to electric footshock by using a chamber with a 25 x 27.5 cm grid floor (EVANS1961). Rats were given a series of 0.3 sec. shocks in increasing intensity (0.10-0.12-0.15-0.18-0.22-0.27-0.33-0.39-0.47-0.56-0.680.82-1.0-1.2-1.5-1.8-2.2-2.7 mA). The shocks were given at 30 sec. intervals. The rats’ paws were wiped with a glycerine-saline solution to allow good conductance. At the end of each shock the squealing and jumping responses of the rats were observed. DL-a-methyl-p-tyrosine methylester HCI (H 44/68, Labkemi Ab, Gothenburg) was dissolved in saline and injected intraperitoneally in a volume of 2 ml/kg. The dose refers to the base. The statistical significance of differences between means was calculated by Student’s t-test.
Results Behaviour of rats during ethanol withdrawal. After three -four days of repeated ethanol administration, the rats showed withdrawal symptoms after the longest interval before the next ethanol administration (10 hrs, between 10 p.m. and 8 a.m.). These signs were most often stiff tail, piloerection and tremor as well as stereotyped head movements. When the rats were withdrawn from ethanol more severe symptoms developed. Table 1 shows the occurrence of these
Table 1. Occurrence of withdrawal symptoms in 35 rats which had been treated with 8-11 g/kg of ethanol daily for 7-10 days, 15-16 hrs after administration of the last dose of ethanol (4-5 g/kg). Symptom
Occurrence (%)
Stiff tail Stereotypic head movements Spastic rigidity Piloerection Tremor Not able to walk on laboratory bench Stays on vertical grid over 30 sec. Squealing o n touching Tonic clonic convulsions
77 80 54 51 63 23 25 9 11
L. AHTEE AND M. SVARTSTROM-FXASER
292
Table 2. Thresholds for squealing and jumping responses to electric foot shock in control rats and in rats treated with 8-1 1 g/kg of ethanol daily for 7-10 days. The last dose of ethanol (4-5 g/kg) was administered either 3-4 hrs (intoxicated group) or 15-16 hrs (withdrawal group) before testing. Number of rats tested
Treatment Control Ethanol intoxication* Ethanol withdrawal
*
34 15 25
Threshold (mA) 5 S.E.M. for squealing jumping 0.26 f 0.02 1.06 k 0.16 0.36 f 0.06
0.54 k 0.03 1.35 f 0.14 0.51 5 0.07
Only rats with positive righting reflex were tested.
symptoms in 35 rats at 15-16 hrs after ethanol withdrawal when the symptoms were at their peak. Rats with a fully developed withdrawal syndrome remained in a characteristic posture with paws spread out and shaking continuously. Even a slight disturbance such as lifting the lid of their cage made them jump and run around the cage. However, they were not able to walk on a smooth laboratory bench and once put on a vertical grid they stayed there for an abnormally long time. Tonic clonic convulsions were also observed and the rats occasionally died during these convulsions. In the present experiments ethanol withdrawal did not alter the threshold for squealing and jumping responses to electric foot shock (table 2). However, in a few rats the jumping response developed into a convulsive state and
Table 3. Effect of a-methyl-p-tyrosine (aMT, 250 mg/kg, intraperitoneally) on the rectal ternperature of control rats and of rats treated with 8-11 g/kg of ethanol daily for 7-10 days. The last dose of ethanol (4-5 g/kg) was administered either 1-3 hrs (intoxicated group) or 13-15 hrs (withdrawal group) before aMT (or saline) administration. Rectal temperature Chronic treatment
Control Intoxication Withdrawal
Of
S.E.M. (n)
Saline
aMT
0 hrs
3 hrs
0 hrs
3 hrs
36.9k0.1 (10) 35.8 f0.2 (6) 37.0k0.6 (6)
37.0k0.1 (10) 36.3 f 0.8 (6) 36.9f0.5 (6)
36.8k0.1 (10) 35.9 f 0.5 (9) 37.1 f0.3 (9)
35.3k0.2 (10) 34.4 f 0.6 (8) 35.1 k0.3 (9)
CATECHOLAMINES IN ETHANOL WITHDRAWAL 2.0 -
T
293
MESENCEPHALON + DIENCEPHALON + TELENCEPHALON- CORTEX
1.81.61.1, -
1.2m
1
m 3 1.0-
w
I I: 0.8a a 0
0.60.L -
INTOXICATION
0.20-
WITHDRAWAL ALINE
dMT
Fig. 1. Effect of a-methyl-p-tyrosine (aMT, 250 mg/kg, intraperitoneally, 3 hrs) on the concentration of dopamine in the mesencephalon diencephalon telencephalon except cortex of rats treated with 8-11 g/kg of ethanol daily for 7-10 days. The rats were killed either 4-6 hrs (intoxicated group) or 16-1 8 hrs (withdrawal group) following administration of the last dose of ethanol (4-5 g/kg). Mean values from 6 to 10 rats; vertical bars indicate the S.E.M. The control group was treated chronically with water.
+
+
Table 4. Homovanillic acid (HVA) concentration in the striatum of rats treated with 8-1 1 g/kg of ethanol daily for 7-10 days. The rats were killed either 4-6 hrs (intoxicated group) or 16-18 hrs (withdrawal group) after administration of the last ethanol dose (4-5 g/kg). Treatment Control Ethanol intoxication Ethanol withdrawal
HVA pg/g+S.E.M. (n) 0.77 k 0.05 (5) 0.78+0.11 (6) 0.73 k 0.03 (6)
294
L. AHTEE A N D M. SVARTSTRdM-FRASER
one rat died in such a convulsion. It should be noted that we tested the responses very quickly by giving only one series of electric shocks of increasing intensity to each rat and noting the intensity in which the rat first squealed or jumped. Table 3 shows that ethanol intoxication decreased the rectal temperature of the rats by about lo, but withdrawal from ethanol did not alter the rectal temperature of the rats. Inhibition of the synthesis of catecholamines by 250 mg/kg of a-methyl-p-tyrosine (aMT) decreased the rectal temperature similarly in the control and ethanol treated rats.
0.4 0.2
0 MESENCEPHALON+ DIENCEPHALON+
MEDULLA OBLONGATA
INTOXICATION
SALINE
d MT
Fig. 2. Effect of a-methyl-p-tyrosine (aMT, 250 mg/kg, intraperitoneally, 3 hrs) on the concentration of noradrenaline in 1) cortex, 2) mesencephalon + diencephalon + telencephalon except cortex, and 3) pons+medulla oblongata of rats treated with 8-11 g/kg of ethanol daily for 7-10 days. The rats were killed either 4-6 hrs (intoxicated group) or 16-18 hrs (withdrawal group) after administration of the last dose of ethanol (4-5 g/kg). Mean values from 6-10 rats, vertical bars indicate the S.E.M. The asterisks indicate significant change (P < 0.001) from the corresponding control group treated chronically with water.
CATECHOLAMINES IN ETHANOL WITHDRAWAL
295
HEART
0
CONTROL
a
ETHANOL INTOXICATION
SALINE
ETHANOL WITHDRAWAL
dMT
Fig. 3. Effect of a-methyl-p-tyrosine (aMT, 250 mg/kg, intraperitoneally, 3 hrs) on the concentration of noradrenaline in the hearts of rats treated with 8-11 g/kg of ethanol daily for 7-10 days. The rats were killed either 4-6 hrs (intoxicated group) or 16-18 hrs (withdrawal group) after administration of the last dose of ethanol (4-5 g/kg). Mean values from 4 to 10 rats; vertical bars indicate the S.E.M. The asterisk indicates significant change (P < 0.01) from the corresponding control group treated chronically with water.
E f j i t of chronic ethanol administration on brain dopamine. Chronic ethanol administration did not change the concentration of dopamine in the brain of rats (fig. 1). Moreover, the rate of aMT-induced disappearance of dopamine was similar in the brain of control rats, of ethanol intoxicated rats and of rats withdrawn from ethanol. Table 4 shows that chronic ethanol administration did not alter the concentration of homovanillic acid in the striata of rats. E e c t of chronic ethanol administration on brain and heart noradrenaline. Fig. 2 shows that the noradrenaline concentrations were similar in all parts of the brain studied, 1) cortex, 2) mesencephalon diencephalon telencephalon except cortex, 3) ponsfmedulla oblongata, in control rats and in ethanol intoxicated rats as well as in rats withdrawn from ethanol. No were any differences found in the noradrenaline concentrations of the hearts of these rats (fig. 3). However, the three rats, which died in withdrawal convulsions (and the braiu and heart of which were dissected immediately after death) had significantly reduced brain and heart noradrenaline contents (brain concentration = 0.30 f 0.01 pg/g, normal mean f S.E.M.=0.53 k 0.01 pg/g; heart concentration= 0.32 f0.03 pg/g, normal = 0.92 k 0.03 pg/g). The dopamine, 5-hydroxytryptamine and 5-hydroxyindoleacetic acid concentrations in the brain of the same rats did not differ from those of the control rats. When the synthesis of noradrenaline was blocked by treating the rats with 250 mg/kg of a M T intraperitoneally 3 hrs before killing, the rate of disap-
+
+
L. AHTEE AND M. SVARTSTROM-FRASER
296
pearance of noradrenaline was increased by 40 to 50 YOin all parts of the brain of rats withdrawn from ethanol. The noradrenaline concentration in the cortex of uMT-treated control rats was decreased in three hours by 43 %, in the cortex of ethanol intoxicated rats by 42Y0, and in the cortex of rats withdrawn from ethanol by 65 YO (P
