Alcohol, Vo|. 7, pp. 159-163. ©Pergamon Press plc, 1990. Printed in the U.S.A.
0741-8329/90 $3.00 + .00
Effect of Ethanol on Brain Catecholamines in Rat Lines Developed for Differential Ethanol-Induced Motor Impairment KAISA HELLEVUO,
KALERVO KIIANMAA 1
Research Laboratories, Alko Ltd., POB 350, SF-O0101 Helsinki, Finland AND CHUL KIM
Addiction Research Foundation o f Ontario, Toronto, Ontario M5S 2S1, Canada R e c e i v e d 2 4 A u g u s t 1989; A c c e p t e d 30 N o v e m b e r 1989
HELLEVUO, K., K. KIIANMAA AND C. KIM. Effect of ethanol on brain catecholamines in rat lines developed for differential ethanol-induced motor impairment. ALCOHOL 7(2) 159-163, 1990.--The importance of the central catecholamines, with the emphasis on the noradrenergic neurons in the differential sensitivity to ethanol between the AT (alcohol-tolerant) rats selected for low and the ANT (alcohol-nontolerant) rats selected for high sensitivity to ethanol-induced (2 g/kg) motor impairment, was clarified by studying the effects of ethanol (2 and 4 g/kg, IP) on the utilization of norepinephrine (NA) and dopamine (DA), and on the metabolism of NA. The utilization of the catecholamines was estimated from the disappearance of the amines after inhibition of the brain tyrosine hydroxylase by et-methyl-p-tyrosine (200 mg/kg, IP), given 15 rain after the administration of ethanol. The formation of 3-methoxy-4-hydroxy-phenylglycol (MHPG) was used as an estimate of NA metabolism, and was measured 30 min after the administration of ethanol. The basal utilization rate of NA and DA was similar between the two rat lines, but the increased formation of MHPG suggested that the naive AT rats had a higher noradrenergic activity in the limbic forebrain, hypothalamus, and cerebellum than did ANT rats. In the brain of both lines, ethanol accelerated the utilization and metabolism of NA in the same manner. Ethanol also increased the utilization of DA in the limbic forebrain of the AT and ANT rats. The higher sensitivity of the ANT rats' DA neurons to ethanol in the limbic forebrain and striatum was revealed by the significant rat line x ethanol interaction. The present findings suggest that the AT and ANT rats differ in the dopaminergic, but not in the noradrenergic responses to ethanol. Ethanol
Motor impairment
Catecholamines
MHPG
SEVERAL rodent lines have been developed that differ in their ethanol-related behavior as a result of their differential central nervous system sensitivity to ethanol; they are used as tools for studying the mechanisms of the actions of ethanol [cf. (9)]. For instance, the AT (alcohol-tolerant) rats have been selected for low and the ANT (alcohol-nontolerant) rats for high sensitivity to the motor-impairing effects of 2 g/kg of ethanol [cf. (7)]. The selection seems to have been relatively specific for the motor impairment task, since the rat lines do not differ in ethanolinduced hypothermia (6,21), although the loss of righting reflex has recently been shown to last for a shorter time in the ethanol-insensitive AT than in the ethanol-sensitive ANT rats (21). It has been suggested that the central catecholamine neurons have a role in mediating the effects of ethanol, since it is known that the catecholamines are affected by ethanol [cf. (24,26)].
Selected rat lines
Genetics
Furthermore, there is evidence for both dopaminergic and noradrenergic involvement in ethanol-induced motor impairment (15-17, 22). In line with this view, the AT and ANT rats are known to have several differences in their central catecholaminergic systems. The concentration of dopamine (DA) in the striatum and in the limbic forebraln, the rate of synthesis of catecholamines in the stfiatum, and the activity of brain aromatic amino acid decarboxylase is higher in the ANT than in the AT rats (1, 19, 25). The contribution of these differences between naive AT and ANT rats to the differential sensitivity of the lines to ethanol has remained unclear. To evaluate the sensitivity of the central catecholaminergic neurons in the AT and ANT rats to ethanol, the effect of ethanol on the synthesis of catecholamines and metabolism of DA (10,19) has been studied in these animals. These previous studies have not
tRequests for reprints should be addressed to Kalervo Kiiannma.
159
160
HELLEVUO, KIIANMAA AND KIM
been able to assess the effect of ethanol on the noradrenergic functions• The functioning of the noradrenergic systems in the two lines, however, needs to be determined, since the findings that levels of brain noradrenaline (NA) lowered by the neurotoxin 6-hydroxydopamine potentiates the ethanol-induced motor impairment in rats (17), and that alpha2-adrenoreceptor antagonists atipamezole and idazoxan reverse the ataxic effects of ethanol in mice (22) suggest that central noradrenergic neurons play a role in ethanol intoxication. The aim of the present study was to clarify the role of the noradrenergic neurons in the differential sensitivity to ethanol between the AT and ANT rats by measuring the effect of ethanol on the utilization and metabolism of NA in different brain parts. The utilization of NA was estimated from the changes in the disappearance rate of NA after inhibition of brain tyrosine hydroxylase with c~-methyl-p-tyrosine (etMPT) [cf. (2)]. This method also allowed us to monitor the utilization of DA. The effect of ethanol on NA metabolism was estimated from the formation of 3-methoxy-4-hydroxy-phenylglycol (MHPG) [cf. (23)]•
NA
[-7 SAL 100
E 8 "5
40.
,
/x /x
?
x~
x /x
20.
~ ~ ~ ~
x x
0 AT
ANT
AT
100" CEREBRAL CORTEX
6oi 401 2oi
The concentrations of NA and DA were analyzed with high performance liquid chromatography, using electrochemical detection as described earlier (10). The chromatograph system consisted of a Waters Model 510 pump, a Waters Intelligent Sample Processor with a thermostated sample unit, and an amperometric detector LC-4B (Bioanalytical Systems Inc., West Lafayette, IN) with a glassy carbon working electrode. The sample components were separated with a column Nucleosil 5 C18, 200 x 4 mm i.d. (Macherey-Nagel, Duren, F.R.G.) in an isocratic reversed phase
HYPOTHALAMUS
_T_
Male AT and ANT rats of generations F26 and F29 were used in the experiments. The animals were 14 weeks old. The rats were housed in stainless steel cages in groups of 5--6 animals; they had free access to tap water and standard R3 rat food (Ewos Ab, S6dertalje, Sweden). The ambient temperature was 22 ± 2°C, the relative humidity 50-55%, and the light/dark cycle 12 hr/12 hr (lights on at 6:00 a.m.).
Biochemical Assays
EION4 g/kg
60.
80"
The effect of ethanol on the utilization of NA and DA was estimated from the disappearance of the catecholamines after inhibition of brain tyrosine hydroxylase with DL-tx-methyl-ptyrosine-methyl-ester-HC1 (otMPT) [cf. (2)]. Ethanol [2 g/kg (12% v/v in saline) or 4 g/kg (15% v/v)] was administered intraperitoneally 15 min prior to ctMPT (200 mg/kg, IP, in a volume of 3 ml/kg in saline). The controls received etMPT and saline, or only saline. The rats were decapitated 120 min after the administration of ~xMPT. The effect of ethanol on the metabolism of NA was estimated from the formation of MHPG [cf. (23)]. The rats were injected intraperitoneally with saline or ethanol [2 g/kg (12% v/v in saline) or 4 g/kg (15% v/v)] and decapitated 30 rain later. After decapitation the brain was immediately dissected, on ice, into the dorsal part of the cerebral cortex, the frontal cortex, the striatum, the limbic forebrain (containing the tuberculum olfactorium, the nucleus accumbens and the septum), the hypothalamus, the lower brain stem (containing the pons and the medulla), and the cerebellum. The rest was discarded. The tissue samples were frozen on dry CO2 and stored at - 8 0 ° C until the catecholamine concentrations were assayed with high performance liquid chromatography.
LIMBIC FOREBRAIN
80
METHOD
Drug Treatment and Dissection of the Brain
r ~ EtOH2 g/kg [ ]
AN'q
FRONTAL CORTEX
O" AT 100
ANT
CEREBELLUM
AT
ANT
LOWER BRAIN STEM
80 7-
60
FA~ F/b~
40
20
V/k/~
0 AT
ANT
AT
ANT
FIG. 1. Effect of ethanol (EtOH 2 g/kg and 4 g/kg, IP) or saline (SAL) on the etMPT-induced (200 mg/kg, IP) disappearance of NA in different parts of the brain of AT and ANT rats. The results are expressed as the percent of the saline-saline control (mean ± SEM, N = 6-12). **p
0741-8329/90 $3.00 + .00
Effect of Ethanol on Brain Catecholamines in Rat Lines Developed for Differential Ethanol-Induced Motor Impairment KAISA HELLEVUO,
KALERVO KIIANMAA 1
Research Laboratories, Alko Ltd., POB 350, SF-O0101 Helsinki, Finland AND CHUL KIM
Addiction Research Foundation o f Ontario, Toronto, Ontario M5S 2S1, Canada R e c e i v e d 2 4 A u g u s t 1989; A c c e p t e d 30 N o v e m b e r 1989
HELLEVUO, K., K. KIIANMAA AND C. KIM. Effect of ethanol on brain catecholamines in rat lines developed for differential ethanol-induced motor impairment. ALCOHOL 7(2) 159-163, 1990.--The importance of the central catecholamines, with the emphasis on the noradrenergic neurons in the differential sensitivity to ethanol between the AT (alcohol-tolerant) rats selected for low and the ANT (alcohol-nontolerant) rats selected for high sensitivity to ethanol-induced (2 g/kg) motor impairment, was clarified by studying the effects of ethanol (2 and 4 g/kg, IP) on the utilization of norepinephrine (NA) and dopamine (DA), and on the metabolism of NA. The utilization of the catecholamines was estimated from the disappearance of the amines after inhibition of the brain tyrosine hydroxylase by et-methyl-p-tyrosine (200 mg/kg, IP), given 15 rain after the administration of ethanol. The formation of 3-methoxy-4-hydroxy-phenylglycol (MHPG) was used as an estimate of NA metabolism, and was measured 30 min after the administration of ethanol. The basal utilization rate of NA and DA was similar between the two rat lines, but the increased formation of MHPG suggested that the naive AT rats had a higher noradrenergic activity in the limbic forebrain, hypothalamus, and cerebellum than did ANT rats. In the brain of both lines, ethanol accelerated the utilization and metabolism of NA in the same manner. Ethanol also increased the utilization of DA in the limbic forebrain of the AT and ANT rats. The higher sensitivity of the ANT rats' DA neurons to ethanol in the limbic forebrain and striatum was revealed by the significant rat line x ethanol interaction. The present findings suggest that the AT and ANT rats differ in the dopaminergic, but not in the noradrenergic responses to ethanol. Ethanol
Motor impairment
Catecholamines
MHPG
SEVERAL rodent lines have been developed that differ in their ethanol-related behavior as a result of their differential central nervous system sensitivity to ethanol; they are used as tools for studying the mechanisms of the actions of ethanol [cf. (9)]. For instance, the AT (alcohol-tolerant) rats have been selected for low and the ANT (alcohol-nontolerant) rats for high sensitivity to the motor-impairing effects of 2 g/kg of ethanol [cf. (7)]. The selection seems to have been relatively specific for the motor impairment task, since the rat lines do not differ in ethanolinduced hypothermia (6,21), although the loss of righting reflex has recently been shown to last for a shorter time in the ethanol-insensitive AT than in the ethanol-sensitive ANT rats (21). It has been suggested that the central catecholamine neurons have a role in mediating the effects of ethanol, since it is known that the catecholamines are affected by ethanol [cf. (24,26)].
Selected rat lines
Genetics
Furthermore, there is evidence for both dopaminergic and noradrenergic involvement in ethanol-induced motor impairment (15-17, 22). In line with this view, the AT and ANT rats are known to have several differences in their central catecholaminergic systems. The concentration of dopamine (DA) in the striatum and in the limbic forebraln, the rate of synthesis of catecholamines in the stfiatum, and the activity of brain aromatic amino acid decarboxylase is higher in the ANT than in the AT rats (1, 19, 25). The contribution of these differences between naive AT and ANT rats to the differential sensitivity of the lines to ethanol has remained unclear. To evaluate the sensitivity of the central catecholaminergic neurons in the AT and ANT rats to ethanol, the effect of ethanol on the synthesis of catecholamines and metabolism of DA (10,19) has been studied in these animals. These previous studies have not
tRequests for reprints should be addressed to Kalervo Kiiannma.
159
160
HELLEVUO, KIIANMAA AND KIM
been able to assess the effect of ethanol on the noradrenergic functions• The functioning of the noradrenergic systems in the two lines, however, needs to be determined, since the findings that levels of brain noradrenaline (NA) lowered by the neurotoxin 6-hydroxydopamine potentiates the ethanol-induced motor impairment in rats (17), and that alpha2-adrenoreceptor antagonists atipamezole and idazoxan reverse the ataxic effects of ethanol in mice (22) suggest that central noradrenergic neurons play a role in ethanol intoxication. The aim of the present study was to clarify the role of the noradrenergic neurons in the differential sensitivity to ethanol between the AT and ANT rats by measuring the effect of ethanol on the utilization and metabolism of NA in different brain parts. The utilization of NA was estimated from the changes in the disappearance rate of NA after inhibition of brain tyrosine hydroxylase with c~-methyl-p-tyrosine (etMPT) [cf. (2)]. This method also allowed us to monitor the utilization of DA. The effect of ethanol on NA metabolism was estimated from the formation of 3-methoxy-4-hydroxy-phenylglycol (MHPG) [cf. (23)]•
NA
[-7 SAL 100
E 8 "5
40.
,
/x /x
?
x~
x /x
20.
~ ~ ~ ~
x x
0 AT
ANT
AT
100" CEREBRAL CORTEX
6oi 401 2oi
The concentrations of NA and DA were analyzed with high performance liquid chromatography, using electrochemical detection as described earlier (10). The chromatograph system consisted of a Waters Model 510 pump, a Waters Intelligent Sample Processor with a thermostated sample unit, and an amperometric detector LC-4B (Bioanalytical Systems Inc., West Lafayette, IN) with a glassy carbon working electrode. The sample components were separated with a column Nucleosil 5 C18, 200 x 4 mm i.d. (Macherey-Nagel, Duren, F.R.G.) in an isocratic reversed phase
HYPOTHALAMUS
_T_
Male AT and ANT rats of generations F26 and F29 were used in the experiments. The animals were 14 weeks old. The rats were housed in stainless steel cages in groups of 5--6 animals; they had free access to tap water and standard R3 rat food (Ewos Ab, S6dertalje, Sweden). The ambient temperature was 22 ± 2°C, the relative humidity 50-55%, and the light/dark cycle 12 hr/12 hr (lights on at 6:00 a.m.).
Biochemical Assays
EION4 g/kg
60.
80"
The effect of ethanol on the utilization of NA and DA was estimated from the disappearance of the catecholamines after inhibition of brain tyrosine hydroxylase with DL-tx-methyl-ptyrosine-methyl-ester-HC1 (otMPT) [cf. (2)]. Ethanol [2 g/kg (12% v/v in saline) or 4 g/kg (15% v/v)] was administered intraperitoneally 15 min prior to ctMPT (200 mg/kg, IP, in a volume of 3 ml/kg in saline). The controls received etMPT and saline, or only saline. The rats were decapitated 120 min after the administration of ~xMPT. The effect of ethanol on the metabolism of NA was estimated from the formation of MHPG [cf. (23)]. The rats were injected intraperitoneally with saline or ethanol [2 g/kg (12% v/v in saline) or 4 g/kg (15% v/v)] and decapitated 30 rain later. After decapitation the brain was immediately dissected, on ice, into the dorsal part of the cerebral cortex, the frontal cortex, the striatum, the limbic forebrain (containing the tuberculum olfactorium, the nucleus accumbens and the septum), the hypothalamus, the lower brain stem (containing the pons and the medulla), and the cerebellum. The rest was discarded. The tissue samples were frozen on dry CO2 and stored at - 8 0 ° C until the catecholamine concentrations were assayed with high performance liquid chromatography.
LIMBIC FOREBRAIN
80
METHOD
Drug Treatment and Dissection of the Brain
r ~ EtOH2 g/kg [ ]
AN'q
FRONTAL CORTEX
O" AT 100
ANT
CEREBELLUM
AT
ANT
LOWER BRAIN STEM
80 7-
60
FA~ F/b~
40
20
V/k/~
0 AT
ANT
AT
ANT
FIG. 1. Effect of ethanol (EtOH 2 g/kg and 4 g/kg, IP) or saline (SAL) on the etMPT-induced (200 mg/kg, IP) disappearance of NA in different parts of the brain of AT and ANT rats. The results are expressed as the percent of the saline-saline control (mean ± SEM, N = 6-12). **p
