- Email: [email protected]
Biochemical evidence for activation of specific monoanine pathways by ethanol
Alcohol, Vol. 5, pp. 295-299. ©Pergamon Press plc, 1988. Printed in the U.S.A.
0741-8329/88 $3.00 + .00
Biochemical Evidence for Activation of Specific Monoamine Pathways by Ethanol S. A. KHATIB, J. M. M U R P H Y A N D W. J. M c B R I D E 1 Departments of Psychiatry and Biochemistry, Institute of Psychiatric Research Indiana University School of Medicine, Indianapolis, IN 46223 R e c e i v e d 7 J a n u a r y 1988; A c c e p t e d 22 M a r c h 1988 KHATIB S. A., J. M. MURPHY AND W. J. McBRIDE. Biochemical evidencefor activation of specific rnonoamine pathways by ethanol. ALCOHOL 5(4) 295-299, 1988.--The effects of an acute intraperitoneal (IP) low (0.5 g/kg) or high (2.5 g/kg) dose of ethanol on the contents of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in 7 selected CNS regions of the rat were examined after 15, 30 and 60 minutes. The IP administration of 0.5 g/kg ethanol produced blood alcohol concentrations (BACs) of 41_+4, 40___4and 15___1 mg% (N=8 each) after 15, 30 and 60 minutes, respectively. This low dose of ethanol did not alter the levels of DA, DOPAC, HVA, 5-HT and 5-HIAA in any of the 7 CNS regions at any of the time points examined, The dose of 2.5 g/kg ethanol produced BACs of 254_+26, 268_+20 and 282_+10 mg%(N=8 each) after 15, 30 and 60 minutes, respectively. This high dose of ethanol did not alter the contents of DA and 5-HT in any of the regions examined at any of the times, except for a 30% increase in the content of DA in the posterior striatum after 60 minutes. The administration of 2.5 g ethanol/kg elevated the levels of DOPAC and/or HVA 25 to 70% over saline control values in the (a) nucleus accumbens (ACC) and hypothalamus (HYPO) after 15, 30 and 60 minutes, and (b) posterior striatum (PSTR), lateral septal nucleus (LSN) and frontal cortex (FCTX) after 60 minutes. The contents of DOPAC and/or HVA were not altered by the high dose of ethanol in either the thalamus or olfactory bulbs. The administration of 2.5 g ethanoi/kg increased the contents of 5-HIAA 20 to 25% above control values in the ACC and PSTR only after 60 minutes. The high dose of ethanol did not alter the level of 5-HIAA in any of the other 5 regions examined. The higher CNS levels of DOPAC and/or HVA at BACs of approximately 250 mg% suggest enhanced activity of DA projections (a) from the ventral tegmental area to the ACC, LSN and FCTX; (b) from the substantia nigra to the PSTR; and (c) within the incertohypothalamic system. The elevated contents of 5-HIAA in the ACC and PSTR suggest increased activity of the 5-HT dorsal ascending pathway from the dorsal raphe nucleus. Acute ethanol administration Serotonin metabolism Dopamine metabolism 5-Hydroxyindoleacetic acid levels 3,4-Dihydroxyphenylacetic acid levels Homovanillic acid levels
ally reported by these investigators to accompany the changes in the acid metabolites. Fewer studies have been reported regarding the effects of acute administration of ethanol on 5-HT metabolism, but the results are similar to those reported for DA. Elevated levels of the 5-HT metabolite, 5-hydroxyindoleacetic acid (5-HIAA), in whole brain have been reported following the IP administration of 1.5 to 4 g ethanol/kg to rats [11,24]. A relationship between the activity of certain DA and 5-HT neuronal pathways and the levels of their respective acid metabolites has been demonstrated. For example, Roth et al. [26] showed that electrical stimulation of the nigrostriatal or mesolimbic DA neurons enhanced the formation of DOPAC in the striatum and olfactory tubercles of rats. Conversely, when impulse flow decreased, the level of DOPAC in both areas was reduced [26]. In the case of the 5-HT neuronal pathways, Aghajanian et al. [1] showed that elec-
T H E objective of the present study was to determine if either a low or high dose of ethanol could activate specific dopamine (DA) and/or serotonin (5-HT) pathways in the CNS of the rat. Ethanol generally has a biphasic action on behavior in both humans and experimental animals, with low doses producing stimulation and high doses causing sedation [23]. Although there have been a number of biochemical studies in which the effects of acute ethanol on DA and 5-HT metabolism were examined, none was designed to take into account distinct monoamine pathways projecting from specific CNS nuclei. With regard to DA metabolism, studies have demonstrated that acute administration of high doses of ethanol elevated the levels of the DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and/or homovanillic acid (HVA), in rat whole brain [2,16], rat striatum [5, 10, 14, 20, 25], mouse whole brain [2], and mouse striatum and hypothalamus [8]. Alterations in the levels o f DA were not gener-
~Requests for reprints should be addressed to Dr. W. J. McBride, Institute of Psychiatric Research, 791 Union Drive, Indianapolis, IN 46223.
295
KHATIB, MURPHY AND McBRIDE
296 TABLE 1
CONTENTS OF DOPAMINE. DOPAC AND HVA IN SEVERAL CNS REGIONS OF THE RAT 15, 30 AND 60 MINUTES AFTER AN IP INJECTION OF 2.5 g ETHANOL/kg nmol/g Wet wt. Region
N. Accumbens SAL EtOH-15 EtOH-30 EtOH-60 Post. Striatum SAL EtOH-15 EtOH-30 EtOH-60 Lat. Septal N. SAL EtOH-15 EtOH-30 EtOH-60 Front. Cortex SAL EtOH-15 EtOH-30 EtOH-60 Hypothalamus SAL EtOH-15 EtOH-30 EtOH-60 Thalamus SAL EtOH-15 EtOH-30 EtOH-60 O. Bulbs SAL EtOH-15 EtOH-30 EtOH-60
DA
43 39 35 43
± ± ± ±
39 36 32 52
± 2 ± 3 _+ 4 + 5*
17 11 13 19
DOPAC
I 2 2 3
4.6 5.5 5.7 6.8
_+ 0.1 ± 0.3* ± 0.4* + 0.4*
HVA
2.5 2.9 3.1 4.4
± ± + ±
0.2 0.2 0.2 0.2*
3.3 ± 0.1 3.2 -+ 0.2 (7) 3.5 ± 0.5 5.7 ± 0.2*
2.6 ± 0.2 2.5 ± 0.2 (7) 2.6 -+ 1/.4 4.5 +- 0.4*
_+ 2 ± 3 ± 2 ± 3
1.8 1.8 2.1 2.8
-+ 0.2 ± 0.3 -+ 0.3 ± 0.3*
0.9 0.8 1.0 1.8
± + ± ±
± 0.2 ± 0.10 (7) _+ 0.2 ± 0.05
0.29 0.22 0.32 0.34
± ± ± ±
0.49 0.42 0.55 0.67
-+ 0.03 ± 0.02 (7) ± 0.05 ± 0.09*
3.1 +_ 0.1 3.1 ± 0.2 3.2 ± 0.2 3.6 ÷ 0.2
0.41 0.53 0.53 0.66
± 0.02 _+ 0.03* ± 0.05* ± 0.05*
0.23 0.25 0.29 0.35
+ ± ± ±
1.9 1.4 1.4 2.0
_+ 0.2 ± 0.2 ± 0.2 ± 0.6
0.28 0.28 0.51 0.32
_+ 0.02 _+ 0.03 ± 0.18 ± 0.04 (7)
0.22 0.22 0.19 0.28
_+ 0.02 _+ 0.03 +_ 0.01 (6) ± 0.02 (7)
0.69 0.66 0.63 0.63
_+ 0.03 _+ 0.03 ± 0.04 ± 0.05
0.34 0.38 0.32 0.30
_+ 0.04 _+ 0.08 (7) _+ 0.07 (6) ± 0.11 (4)
0.53 0.48 0.47 0.48
± 0.02 + 0.03 (7) ± 0.02 +_ 0.03
1.0 0.46 1.0 0.48
(7)
0.03 0.02 (7) 0.04 (7) 0.08
0.1 0.1 0. I 0.2*
0.01 0.02 0.04 0.04*
Data are the means + SEM of N=19-24 for saline group and N=8 (unless otherwise noted) for the ethanol group. *p<0.05 by ANOVA and Duncan's tests.
trical stimulation o f the m i d b r a i n r a p h e nuclei i n c r e a s e d the level o f 5 - H I A A in the forebrain region o f the rat. Findings that i n c r e a s e d activity o f the 5-HT n e u r o n s p r o d u c e d elev a t e d levels o f 5 - H I A A in C N S regions receiving their proj e c t i o n s have b e e n c o n f i r m e d by o t h e r s [7,27]. The D A and 5-HT p a t h w a y s in the rat C N S have b e e n well studied and t h e r e is a m p l e e v i d e n c e that m o n o a m i n e n e u r o n s in specific nuclei p r o j e c t to s e l e c t e d C N S regions [17,28]. A s cited a b o v e , t h e r e have b e e n a n u m b e r o f studies indicating that acute a d m i n i s t r a t i o n o f ethanol e l e v a t e s the levels o f 5 - H I A A and D O P A C in the C N S , p r e s u m a b l y indicating i n c r e a s e d n e u r o n a l activity, but t h e s e studies h a v e b e e n d o n e primarily with s e d a t i v e - h y p n o t i c d o s e s o f ethanol and h a v e u s e d m o s t l y w h o l e brain or only striatal s a m p l e s . T h e r e f o r e , the p r e s e n t study w a s u n d e r t a k e n to e x a m i n e the effects o f a low (0.5 g/kg) and a s e d a t i v e - h y p n o t i c (2.5 g/kg)
d o s e o f e t h a n o l on the levels o f D O P A C , H V A and 5-H1AA in several C N S regions w h i c h receive p r o j e c t i o n s f r o m distinct nuclei. METHOD A d u l t male Wistar rats (Harlan, Indianapolis; weighing a p p r o x i m a t e l y 300 g) w e r e u s e d in t h e s e studies. T h e y w e r e individually h o u s e d with free a c c e s s to H20 and s t a n d a r d rat c h o w (Purina No. 5001) on a 12-hour light-dark c y c l e (0600 hr lights on). R a t s w e r e h a n d l e d daily and a d a p t e d to a liquid N2 dipping a p p a r a t u s for at least 5 d a y s before killing. C o n t r o l rats w e r e injected IP with sterile saline (0.9% NaC1) in a v o l u m e e q u i v a l e n t to that n e e d e d for the highest d o s e o f ethanol. A solution o f e t h a n o l (12 g/100 ml saline) w a s p r e p a r e d f r e s h daily f r o m s t o c k 95% ethanol. Rats w e r e
E T H A N O L E F F E C T S ON M O N O A M I N E P A T H W A Y S injected with saline, 0.5 or 2.5 g ethanol/kg body wt. and were killed by the near-freezing technique [31] 15, 30 or 60 minutes later. Following decapitation, trunk blood was collected into heparinized capillary tubes and the brain was dissected in a cold box at - 2 0 °C into the following regions: nucleus accumbens, posterior striatum, lateral septal nucleus, frontal cortex, hypothalamus, thalamus and olfactory bulbs. The regional brain samples were stored at - 7 0 ° C until assayed by an established H P L C - E C procedure for the contents of DA, DOPAC, HVA, 5-HT and 5-HIAA [19]. Plasma obtained by centrifugation of the trunk blood samples was stored at - 2 0 ° C until assayed for ethanol content by published GC methods [32]. All data are expressed as means_+S.E.M. Statistical comparisons among the groups were done with A N O V A and Duncan's tests. RESULTS The contents of DA were highest in the accumbens, posterior striatum and lateral septal nucleus and lowest in the olfactory bulbs (Table 1). Intermediate values for DA were found in the frontal cortex, thalamus and hypothalamus. The three regions with the highest content of DA also had the highest levels of DOPAC and H V A (Table 1). The highest content of 5-HT was observed in the accumbens and hypothalamus and was nearly 3-fold greater than the lowest value found in the olfactory bulbs (Table 2). Values in the remaining 4 regions were in the intermediate range of 3.5 to 4.8 nmol/g (Table 2). The distribution of the contents of 5-HIAA did not necessarily parallel the distribution of the levels of 5-HT (Table 2). For example, the content of 5-HT in the accumbens is nearly the highest but the level of 5-HIAA in this region ranks in the middle and is below regions with lower amounts of 5-HT (Table 2). The IP injection of 0.5 g ethanol/kg produced blood alcohol concentrations (BACs) of 41---4, 40_+4 and 15_+1 mg% ( N = 8 each) after 15, 30 and 60 minutes, respectively. This low dose of ethanol produced no significant changes in the levels of DA, DOPAC, HVA, 5-HT and 5-HIAA in any of the 7 CNS regions examined at any of the time points (data not shown). The IP administration of 2.5 g ethanol/kg produced BACs of 254_+26, 268_+20 and 282_+ 10 mg% ( N = 8 each) after 15, 30 and 60 minutes, respectively. Behavioral sedation was evident within 5 minutes after injection and continued for all rats throughout the entire 15, 30 or 60 minute period. This dose of 2.5 g ethanol/kg did not significantly alter the levels of DA in any of the 7 regions examined at any of the time points, with the one exception of a 3(1% increase of DA content in the posterior striatum 60 minutes after ethanol administration (Table 1). However, this high dose of ethanol did cause a 40 to 70% increase in the contents of DOPAC and/or H V A in 5 of the 7 CNS regions 60 minutes after injection (Table I). The levels of the DA metabolites were also significantly elevated in the accumbens (20 to 25% above saline values) and hypothalamus (approximately 30% above saline values) at the 15 and 30 minute time points, as well as after 60 minutes (Table 1). Increased levels of DA metabolites were observed in the posterior striatum, lateral septal nucleus and frontal cortex only at the 60 minute time point after ethanol injection (Table I). The 2.5 g/kg dose of ethanol did not alter the contents of DOPAC or H V A in the thalamus or olfactory bulbs (Table 1). The contents of 5-HT were not altered in any of the 7
297
TABLE 2 CONTENTS OF SEROTONINAND 5-HIAAIN SEVERALCNS REGIONS OF THE RAT 15.30 AND 60 MINUTESAFTER AN IP INJECTION OF 2.5 g ETHANOL/kg nmol/g Wet wt. 5-HT N. Accumbens SAL EtOH-15 EtOH-30 EtOH-60 Post. Striatum SAL EtOH-15 EtOH-30 EtOH-60 Lat. Septal N. SAL EtOH-15 EtOH-30 EtOH-60 Front. Cortex SAL EtOH-15 EtOH-30 EtOH-60 Hypothalamus SAL EtOH-15 EtOH-30 EtOH-60 Thalamus SAL EtOH-15 EtOH-30 EtOH-60 O. Bulbs SAL EtOH-15 EtOH-30 EtOH-60
5-HIAA
6.6 6.0 6.0 6.1
± ± ± ±
0.3 0.3 (7) 0.2 0.3 (7)
3.5 3.9 3.6 4.4
± ± ± ±
0.2 0.2 0.1 0.4*
4.2 4.0 4.1 4.5
± ± ± ±
0.2 0.2 0.4 0.2
3.9 4.0 4.2 4.7
± 0.1 _+ 0.2 ± 0.4 ± 0.2*
3.5 2.8 4.0 3.3
_+ 0.2 ± 0.3 ± 0.4 - 0.3
2.9 2.5 3.2 3.2
± 0.2 ± 0.2 ± 0.4 -+ 0.2
4.5 4.3 4.2 4.2
± ± ± ±
0.1 0.1 0.2 0.1
2.6 2.7 2.7 2.9
± ± ± ±
6.9 6.6 7.2 6.8
± ± ± ±
0.1 0.3 0.2 0.1
6.2 6.1 6.3 6.4
_+ 0.1 ± 0.3 ± 0.2 ± 0.1
4.8 4.7 5.1 4.8
± 0.2 ± 0.2 _+ 0.3 ± 0.2
5.2 5.1 5.1 5.7
± 0.2 ± 0.3 ± 0.4 _+ 0.3
2.4 2.1 2.3 2.1
± 0.1 ___0.1 ± 0.1 -+ 0.1
1.5 1.5 1.5 1.6
± 0.1 ± 0.2 ± 0.1 ___0.2
0.1 0.1 0.1 0.1
Data are the means ± SEM of N=23, 24 for saline group and N=8 (unless otherwise noted) for the ethanol group. *p <0.05 by ANOVA and Duncan's tests.
CNS regions examined at any of time points following administration of the 2.5 g/kg dose of ethanol (Table 2). However, in two of the 7 CNS regions studied, the level of 5-HIAA was significantly elevated in the ethanol-treated group with respect to the saline group (Table 2). In the nucleus accumbens and posterior striatum, the content of 5-HIAA was 20-25% higher in the ethanol group 60 minutes after injection. Significant differences in the contents of 5-HIAA were not observed at the earlier time points between ethanol- and saline-injected animals. DISCUSSION The levels of DOPAC and/or H V A were elevated in 5 of the 7 CNS regions examined 60 minutes after injection of 2.5
298
KHATIB, MURPHY AND McBRIDE
g ethanol/kg (Table 1), presumably indicating that this high dose of ethanol increased DA metabolism by enhancing release of DA from selectively activated pathways. The higher levels of DA metabolites in these regions suggest that BACs of approximately 250 mg%, produced by IP administration of 2.5 g ethanol/kg, can enhance the activity of DA projections from (a) the ventral tegmental area to the nucleus accumbens, lateral septal nucleus and frontal cortex and (b) the substantia nigra to the posterior striatum. In addition, the DA incertohypothalamic system also appears to be activated by the high dose of alcohol, as indicated by the higher levels of DOPAC and/or HVA in the hypothalamus of the ethanoltreated rats (Table 1). On the other hand, since the contents of the DA metabolites in the thalamus and olfactory bulbs were the same in the saline and ethanol-injected groups (Table 1), this would indicate that the DA periventricular system that sends projections to the thalamus and periglomerular DA neurons within the olfactory bulbs are not affected by intoxicating levels of alcohol. The present findings of elevated levels of DOPAC and/or HVA after high-dose ethanol are generally in agreement with published reports on rats [2, 5, 16, 20, 25] and mice [2,8] which all found that acute high doses of ethanol can increase the levels of DA metabolites in the CNS. Results from electrophysiological and in vivo release experiments support the concept that the elevated levels of DOPAC and/or HVA observed following sedative-hypnotic doses of alcohol are due to increased activity of DA neurons. The IV administration of 0.5-2.0 g ethanol/kg increased neuronal activity in the ventral tegmental area and substantia nigra of rats [12,18] while other reports indicated that IP doses of 2.5 g ethanol/kg and lower increased the in vivo release of DA in the accumbens and striatum of the rat [9,15]. Moreover, the above electrophysiological and in vivo release data indicated that the accumbens was more sensitive to ethanol than was the striatum. The present findings of elevated levels of DOPAC and/or HVA in the accumbens but not the posterior striatum at 15 and 30 minutes (Table 1) after the 2.5 g ethanoi/kg injection provides additional evidence that the accumbens is more sensitive to alcohol than is the striatum. The present data did not indicate that the 0.5 g/kg dose of ethanol enhanced DA metabolism or neuronal activity in the accumbens whereas findings from in vivo release [15] and electrophysiological [12] studies indicate otherwise. This apparent disagreement is likely due to in vivo release and electrophysiological measurements being a more sensitive indicator of changes in neuronal activity than can be obtained by measuring tissue contents of the monoamine metabolites. The content of DA was not altered by the 2.5 g/kg dose of ethanol in any of the regions at any of the time points, except
for an increase in the posterior striatum after 60 minutes (Table 1). One possible explanation is that autoreceptor activation by extracellular DA has reduced release while synthesis of DA continues at an accelerated level. The effects of the 2.5 g/kg dose of ethanol on the 5-HT systems were very limited (Table 2). The elevated contents of 5-HIAA in the nucleus accumbens and posterior striatum suggest specific increased activity of the 5-HT dorsal ascending pathway from the dorsal raphe nucleus to these two CNS regions. Similar findings of an elevated level of 5-HIAA in rat whole brain after a high dose of ethanol have been reported [11, 24, 29]. Tabakoff et al. [30] observed that ethanol reduced the transport of [14C] 5-HIAA from whole brain of mice given an IP injection of 3 g ethanol/kg. The results of the present study for the 5-HT acid metabolite are not likely due exclusively to inhibition of transport of 5-H1AA from the brain, If inhibition of transport was the major cause, then a general elevation of 5-HIAA in all the CNS regions examined (with the possible exception of the hypothalamus) should have been observed. However, this was not the case for 5-HIAA (Table 2). On the other hand, it is possible that there is some inhibition of 5-HIAA transport from the brain produced by the high dose of alcohol. This effect on transport would be experimentally observed as elevated levels of 5-HIAA only when sufficiently enhanced 5-HT neuronal activity also occurs. There is some evidence that the higher level of 5-H1AA in the accumbens and striatum of the ethanol-treated rats observed in the present study (Table 2) may be due to increased 5-HT neuronal activity. Holman and Snape[ 13] reported that the IP administration of ethanol, which raised BACs to approximately 250 mg%, increased the release of 5-HT as reflected by the higher concentration of 5-HIAA in the rat striatal dialysis perfusate. Chu and Keenan [6], using the brain stem slice preparation, reported that 100-200 mgCA ethanol produced more excitation than inhibition of neurons in the midbrain dorsal raphe. The data for the CNS distribution of DA, 5-HT and their metabolites found in the present study (Tables 1 and 2) are generally in agreement with results of others [3, 4, 1%211. In conclusion, this study provides neurochemical evidence supporting the findings of others that intoxicating doses of alcohol can selectively activate specific populations of monoaminergic neurons in the CNS of the rat.
ACKNOWLEDGEMENTS The technical advice of Mr. Steven Cunningham is deeply appreciated. Supported in part by AA 03243 and AA 07462.
REFERENCES 1. Aghajanian, G. K., J. A. Rosencrans and M. H. Sheard. Serotonin: release in the forebrain by stimulation of midbrain raphe. Science 156: 402-403, 1967. 2. Alari, L., T. Lewander and 13. Sjoquist. The effect of ethanol on the brain catecholamine systems in female mice, rats, and guinea pigs. Alcohol: Clin Exp Res 11: 144-149, 1987. 3. Bacopoulos, N. G., R. K. Bhatnager and L. S. Van Order, 111. The effects of subhypnotic doses of ethanol on regional catecholamine turnover. J Pharmacol Exp Ther 204: 1-10, 1978.
4. Brownstein, M. J. and M. Palkovits. Catecholamines, serotonin, acetylcholine, and y-aminobutyric acid in the rat brain: biochemical studies. In: Handbook of Chemical Neuroanatomy. Vol 2: Classical Transmitters in the CNS. Part 1, edited by A. Bjorklund and T. Hokfelt. Amsterdam: Elsevier, 1984, pp. 23-54. 5. Bustos, G. and R. H. Roth. Effect of acute ethanol treatment on transmitter synthesis and metabolism in central dopaminergic neurons. J Pharm Pharmacol 28: 580-582, 1976.
ETHANOL EFFECTS ON MONOAMINE PATHWAYS
6. Chu, N.-S. and L. Keenan. Responses ofmidbrain dorsal raphe neurons to ethanol studied in brainstem slices. Alcohol 4: 373374, 1987. 7. Curzon, G., J. C. R. Fernando and C. A. Marsden. 5-Hydroxytryptamine: the effects of impaired synthesis on its metabolism and release in rat. Br J Pharmacol 63: 627-634, 1978. 8. Dar, M. S. and W. R. Wooles. The effect of acute ethanol on dopamine metabolism and other neurotransmitters in the hypothalamus and the corpus striatum of mice. J Neural Transm 60: 283-294, 1984. 9. DiChiara, G. and A. lmperato. Ethanol preferentially stimulates dopamine release in the nucleus accumbens of freely moving rats. Eur J Pharmacol 115: 131-132, 1985. 10. Fadda, F., A. Argiolas, M. R. Melis, G. Serra and G. L. Gessa. Differential effects of acute and chronic ethanol on dopamine metabolism in frontal cortex, caudate nucleus and substantia nigra. LiJ~ Sci 27: 979-986, 1980. 11. Fukumori, R., A. Minegisti, T. Satoh, H. Kitagawa and S. Yanaura. Changes in the serotonin and 5-hydroxyindoleacetic acid contents in rat brain after ethanol and disulfiram treatments. Eur J Pharmacol 61: 199-202, 1980. 12. Gessa, G. L., F. Muntoni, M. Collu, L. Vargiu and G. Mereu. Low doses of ethanol activate dopaminergic neurons in the ventral tegmental area. Brain Res 348: 201-203, 1985. 13. Holman, R. B. and B. M, Snape. Effects of ethanol on 5-hydroxytryptamine release from rat corpus striatum in vivo. Alcohol 2: 249-253, 1985. 14. Hunt, W.A. and E. Majchrowicz. Studies of neurotransmitter interactions after acute and chronic ethanol administration. Pharmacol Biochem Behav 18: 371-374, 1983. 15. lmperato, A. and G. DiChiara. Preferential stimulation of dopamine release in the nucleus accumbens of freely moving rats by ethanol. J Pharmacol Exp Ther 239: 21%228, 1986. 16. Karoum, F., R. J. Wyatt and E. Majchrowicz. Brain concentrations of biogenic amine metabolites in acutely treated and ethanol-dependent rats. Br J Pharmaco! 56:403-411, 1976. 17. Lindvall, O. and A. Bjorklund. Dopamine- and norepinephrinecontaining neuron systems: Their anatomy in the rat brain. In: Chemical Neuroanatomy, edited by P, C. Emson. New York: Raven Press, 1983, pp. 22%255. 18. Mereu, G., F. Fadda and G. L. Gessa. Ethanol stimulates the firing rate of nigral dopaminergic neurons in unanesthetized rats. Brain Res 292: 63-69, 1984. 19. Murphy, J. M., W. J. McBride, L. Lumeng and T.-K. Li. Regional brain levels of monoamines in alcohol-preferring and -nonpreferring lines of rats. Pharmacol Biochem Behav 16: 145-149, 1982.
299
20. Murphy, J. M., W. J. McBride, L. Lumeng and T.-K. Li. Monoamine and metabolite levels in CNS regions of the P line of alcohol-preferring rats after acute and chronic ethanol treatment. Pharmacol Biochem Behav 19: 849-856, 1983. 2 I. Murphy, J. M., W. J. McBride, L. Lumeng and T.-K. Li. Alcohol preference and regional brain monoamine contents of N/Nih heterogeneous stock rats. Alcohol Drug Res 7: 33-39, 1986. 22. Murphy, J. M., W. J. McBride, L. Lumeng and T.-K. Li. Contents of monoamines in forebrain regions of alcohol-preferring (P) and -nonpreferring (NP) lines of rats. Pharmacol Biochem Behav 26: 38%392, 1987. 23. Pohorecky, L. A. Biphasic action of ethanol. Biobehav Rev !: 231-240, 1977. 24. Pohorecky, L. A., B. Newman, J. Sun and W. H. Bailey. Acute and chronic ethanol ingestion and serotonin metabolism in rat brain. J Pharmacol Exp Ther 204: 424-432, 1978. 25. Reggiani, A., M. L. Barbaccia, P. F. Spano and M. Trabucchi. Dopamine metabolism and receptor function after acute and chronic ethanol. J Neurochem 35: 34-37, 1980. 26. Roth, R. H., L. C. Murrin and J. R. Waiters. Central dopaminergic neurons: effects of alterations in impulse flow on the accumulation of dihydroxyphenylacetic acid. Fur J Pharmacol 36: 163-171, 1976. 27. Shannon, N. J., J. W. Gunnet and K. E. Moore. A comparison of biochemical indices of 5-hydroxytryptaminergic neuronal activity following electrical stimulation of the dorsal raphe nucleus. J Neurochem 47: 958-965, 1986. 28. Steinbusch, H. W. M. and R. Nieuwenhuys. The Raphe nuclei of the rat brainstem: A cytoarchitectonic and immunohistochemical study. In: Chemical Neuroanatomy, edited by P. C. Emson. New York: Raven Press, 1983, pp, 131-207. 29. Tabakoff, B. and W. O. Boggan. Effects of ethanol on serotonin metabolism in brain. J Neurochem 22: 75%764, 1974. 30. Tabakoff, B., R. F. Ritzman and W. O. Boggan. Inhibition of the transport of 5-hydroxyindoleacetic acid from brain by ethanol. J Neurochem 24: 1043-1051, 1975. 31. Takahashi, R. and M. H. Aprison. Acetylcholine content of discrete areas of the brain obtained by a near-freezing method. J Neurochem 11: 887-898, 1964. 32. Waller, M. B., W. J. McBride, L. Lumeng and T.-K, Li. Initial sensitivity and acute tolerance to ethanol in the P and NP lines of rats. Pharmacol Biochem Behav 19: 683-686, 1983.
0741-8329/88 $3.00 + .00
Biochemical Evidence for Activation of Specific Monoamine Pathways by Ethanol S. A. KHATIB, J. M. M U R P H Y A N D W. J. M c B R I D E 1 Departments of Psychiatry and Biochemistry, Institute of Psychiatric Research Indiana University School of Medicine, Indianapolis, IN 46223 R e c e i v e d 7 J a n u a r y 1988; A c c e p t e d 22 M a r c h 1988 KHATIB S. A., J. M. MURPHY AND W. J. McBRIDE. Biochemical evidencefor activation of specific rnonoamine pathways by ethanol. ALCOHOL 5(4) 295-299, 1988.--The effects of an acute intraperitoneal (IP) low (0.5 g/kg) or high (2.5 g/kg) dose of ethanol on the contents of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in 7 selected CNS regions of the rat were examined after 15, 30 and 60 minutes. The IP administration of 0.5 g/kg ethanol produced blood alcohol concentrations (BACs) of 41_+4, 40___4and 15___1 mg% (N=8 each) after 15, 30 and 60 minutes, respectively. This low dose of ethanol did not alter the levels of DA, DOPAC, HVA, 5-HT and 5-HIAA in any of the 7 CNS regions at any of the time points examined, The dose of 2.5 g/kg ethanol produced BACs of 254_+26, 268_+20 and 282_+10 mg%(N=8 each) after 15, 30 and 60 minutes, respectively. This high dose of ethanol did not alter the contents of DA and 5-HT in any of the regions examined at any of the times, except for a 30% increase in the content of DA in the posterior striatum after 60 minutes. The administration of 2.5 g ethanol/kg elevated the levels of DOPAC and/or HVA 25 to 70% over saline control values in the (a) nucleus accumbens (ACC) and hypothalamus (HYPO) after 15, 30 and 60 minutes, and (b) posterior striatum (PSTR), lateral septal nucleus (LSN) and frontal cortex (FCTX) after 60 minutes. The contents of DOPAC and/or HVA were not altered by the high dose of ethanol in either the thalamus or olfactory bulbs. The administration of 2.5 g ethanoi/kg increased the contents of 5-HIAA 20 to 25% above control values in the ACC and PSTR only after 60 minutes. The high dose of ethanol did not alter the level of 5-HIAA in any of the other 5 regions examined. The higher CNS levels of DOPAC and/or HVA at BACs of approximately 250 mg% suggest enhanced activity of DA projections (a) from the ventral tegmental area to the ACC, LSN and FCTX; (b) from the substantia nigra to the PSTR; and (c) within the incertohypothalamic system. The elevated contents of 5-HIAA in the ACC and PSTR suggest increased activity of the 5-HT dorsal ascending pathway from the dorsal raphe nucleus. Acute ethanol administration Serotonin metabolism Dopamine metabolism 5-Hydroxyindoleacetic acid levels 3,4-Dihydroxyphenylacetic acid levels Homovanillic acid levels
ally reported by these investigators to accompany the changes in the acid metabolites. Fewer studies have been reported regarding the effects of acute administration of ethanol on 5-HT metabolism, but the results are similar to those reported for DA. Elevated levels of the 5-HT metabolite, 5-hydroxyindoleacetic acid (5-HIAA), in whole brain have been reported following the IP administration of 1.5 to 4 g ethanol/kg to rats [11,24]. A relationship between the activity of certain DA and 5-HT neuronal pathways and the levels of their respective acid metabolites has been demonstrated. For example, Roth et al. [26] showed that electrical stimulation of the nigrostriatal or mesolimbic DA neurons enhanced the formation of DOPAC in the striatum and olfactory tubercles of rats. Conversely, when impulse flow decreased, the level of DOPAC in both areas was reduced [26]. In the case of the 5-HT neuronal pathways, Aghajanian et al. [1] showed that elec-
T H E objective of the present study was to determine if either a low or high dose of ethanol could activate specific dopamine (DA) and/or serotonin (5-HT) pathways in the CNS of the rat. Ethanol generally has a biphasic action on behavior in both humans and experimental animals, with low doses producing stimulation and high doses causing sedation [23]. Although there have been a number of biochemical studies in which the effects of acute ethanol on DA and 5-HT metabolism were examined, none was designed to take into account distinct monoamine pathways projecting from specific CNS nuclei. With regard to DA metabolism, studies have demonstrated that acute administration of high doses of ethanol elevated the levels of the DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and/or homovanillic acid (HVA), in rat whole brain [2,16], rat striatum [5, 10, 14, 20, 25], mouse whole brain [2], and mouse striatum and hypothalamus [8]. Alterations in the levels o f DA were not gener-
~Requests for reprints should be addressed to Dr. W. J. McBride, Institute of Psychiatric Research, 791 Union Drive, Indianapolis, IN 46223.
295
KHATIB, MURPHY AND McBRIDE
296 TABLE 1
CONTENTS OF DOPAMINE. DOPAC AND HVA IN SEVERAL CNS REGIONS OF THE RAT 15, 30 AND 60 MINUTES AFTER AN IP INJECTION OF 2.5 g ETHANOL/kg nmol/g Wet wt. Region
N. Accumbens SAL EtOH-15 EtOH-30 EtOH-60 Post. Striatum SAL EtOH-15 EtOH-30 EtOH-60 Lat. Septal N. SAL EtOH-15 EtOH-30 EtOH-60 Front. Cortex SAL EtOH-15 EtOH-30 EtOH-60 Hypothalamus SAL EtOH-15 EtOH-30 EtOH-60 Thalamus SAL EtOH-15 EtOH-30 EtOH-60 O. Bulbs SAL EtOH-15 EtOH-30 EtOH-60
DA
43 39 35 43
± ± ± ±
39 36 32 52
± 2 ± 3 _+ 4 + 5*
17 11 13 19
DOPAC
I 2 2 3
4.6 5.5 5.7 6.8
_+ 0.1 ± 0.3* ± 0.4* + 0.4*
HVA
2.5 2.9 3.1 4.4
± ± + ±
0.2 0.2 0.2 0.2*
3.3 ± 0.1 3.2 -+ 0.2 (7) 3.5 ± 0.5 5.7 ± 0.2*
2.6 ± 0.2 2.5 ± 0.2 (7) 2.6 -+ 1/.4 4.5 +- 0.4*
_+ 2 ± 3 ± 2 ± 3
1.8 1.8 2.1 2.8
-+ 0.2 ± 0.3 -+ 0.3 ± 0.3*
0.9 0.8 1.0 1.8
± + ± ±
± 0.2 ± 0.10 (7) _+ 0.2 ± 0.05
0.29 0.22 0.32 0.34
± ± ± ±
0.49 0.42 0.55 0.67
-+ 0.03 ± 0.02 (7) ± 0.05 ± 0.09*
3.1 +_ 0.1 3.1 ± 0.2 3.2 ± 0.2 3.6 ÷ 0.2
0.41 0.53 0.53 0.66
± 0.02 _+ 0.03* ± 0.05* ± 0.05*
0.23 0.25 0.29 0.35
+ ± ± ±
1.9 1.4 1.4 2.0
_+ 0.2 ± 0.2 ± 0.2 ± 0.6
0.28 0.28 0.51 0.32
_+ 0.02 _+ 0.03 ± 0.18 ± 0.04 (7)
0.22 0.22 0.19 0.28
_+ 0.02 _+ 0.03 +_ 0.01 (6) ± 0.02 (7)
0.69 0.66 0.63 0.63
_+ 0.03 _+ 0.03 ± 0.04 ± 0.05
0.34 0.38 0.32 0.30
_+ 0.04 _+ 0.08 (7) _+ 0.07 (6) ± 0.11 (4)
0.53 0.48 0.47 0.48
± 0.02 + 0.03 (7) ± 0.02 +_ 0.03
1.0 0.46 1.0 0.48
(7)
0.03 0.02 (7) 0.04 (7) 0.08
0.1 0.1 0. I 0.2*
0.01 0.02 0.04 0.04*
Data are the means + SEM of N=19-24 for saline group and N=8 (unless otherwise noted) for the ethanol group. *p<0.05 by ANOVA and Duncan's tests.
trical stimulation o f the m i d b r a i n r a p h e nuclei i n c r e a s e d the level o f 5 - H I A A in the forebrain region o f the rat. Findings that i n c r e a s e d activity o f the 5-HT n e u r o n s p r o d u c e d elev a t e d levels o f 5 - H I A A in C N S regions receiving their proj e c t i o n s have b e e n c o n f i r m e d by o t h e r s [7,27]. The D A and 5-HT p a t h w a y s in the rat C N S have b e e n well studied and t h e r e is a m p l e e v i d e n c e that m o n o a m i n e n e u r o n s in specific nuclei p r o j e c t to s e l e c t e d C N S regions [17,28]. A s cited a b o v e , t h e r e have b e e n a n u m b e r o f studies indicating that acute a d m i n i s t r a t i o n o f ethanol e l e v a t e s the levels o f 5 - H I A A and D O P A C in the C N S , p r e s u m a b l y indicating i n c r e a s e d n e u r o n a l activity, but t h e s e studies h a v e b e e n d o n e primarily with s e d a t i v e - h y p n o t i c d o s e s o f ethanol and h a v e u s e d m o s t l y w h o l e brain or only striatal s a m p l e s . T h e r e f o r e , the p r e s e n t study w a s u n d e r t a k e n to e x a m i n e the effects o f a low (0.5 g/kg) and a s e d a t i v e - h y p n o t i c (2.5 g/kg)
d o s e o f e t h a n o l on the levels o f D O P A C , H V A and 5-H1AA in several C N S regions w h i c h receive p r o j e c t i o n s f r o m distinct nuclei. METHOD A d u l t male Wistar rats (Harlan, Indianapolis; weighing a p p r o x i m a t e l y 300 g) w e r e u s e d in t h e s e studies. T h e y w e r e individually h o u s e d with free a c c e s s to H20 and s t a n d a r d rat c h o w (Purina No. 5001) on a 12-hour light-dark c y c l e (0600 hr lights on). R a t s w e r e h a n d l e d daily and a d a p t e d to a liquid N2 dipping a p p a r a t u s for at least 5 d a y s before killing. C o n t r o l rats w e r e injected IP with sterile saline (0.9% NaC1) in a v o l u m e e q u i v a l e n t to that n e e d e d for the highest d o s e o f ethanol. A solution o f e t h a n o l (12 g/100 ml saline) w a s p r e p a r e d f r e s h daily f r o m s t o c k 95% ethanol. Rats w e r e
E T H A N O L E F F E C T S ON M O N O A M I N E P A T H W A Y S injected with saline, 0.5 or 2.5 g ethanol/kg body wt. and were killed by the near-freezing technique [31] 15, 30 or 60 minutes later. Following decapitation, trunk blood was collected into heparinized capillary tubes and the brain was dissected in a cold box at - 2 0 °C into the following regions: nucleus accumbens, posterior striatum, lateral septal nucleus, frontal cortex, hypothalamus, thalamus and olfactory bulbs. The regional brain samples were stored at - 7 0 ° C until assayed by an established H P L C - E C procedure for the contents of DA, DOPAC, HVA, 5-HT and 5-HIAA [19]. Plasma obtained by centrifugation of the trunk blood samples was stored at - 2 0 ° C until assayed for ethanol content by published GC methods [32]. All data are expressed as means_+S.E.M. Statistical comparisons among the groups were done with A N O V A and Duncan's tests. RESULTS The contents of DA were highest in the accumbens, posterior striatum and lateral septal nucleus and lowest in the olfactory bulbs (Table 1). Intermediate values for DA were found in the frontal cortex, thalamus and hypothalamus. The three regions with the highest content of DA also had the highest levels of DOPAC and H V A (Table 1). The highest content of 5-HT was observed in the accumbens and hypothalamus and was nearly 3-fold greater than the lowest value found in the olfactory bulbs (Table 2). Values in the remaining 4 regions were in the intermediate range of 3.5 to 4.8 nmol/g (Table 2). The distribution of the contents of 5-HIAA did not necessarily parallel the distribution of the levels of 5-HT (Table 2). For example, the content of 5-HT in the accumbens is nearly the highest but the level of 5-HIAA in this region ranks in the middle and is below regions with lower amounts of 5-HT (Table 2). The IP injection of 0.5 g ethanol/kg produced blood alcohol concentrations (BACs) of 41---4, 40_+4 and 15_+1 mg% ( N = 8 each) after 15, 30 and 60 minutes, respectively. This low dose of ethanol produced no significant changes in the levels of DA, DOPAC, HVA, 5-HT and 5-HIAA in any of the 7 CNS regions examined at any of the time points (data not shown). The IP administration of 2.5 g ethanol/kg produced BACs of 254_+26, 268_+20 and 282_+ 10 mg% ( N = 8 each) after 15, 30 and 60 minutes, respectively. Behavioral sedation was evident within 5 minutes after injection and continued for all rats throughout the entire 15, 30 or 60 minute period. This dose of 2.5 g ethanol/kg did not significantly alter the levels of DA in any of the 7 regions examined at any of the time points, with the one exception of a 3(1% increase of DA content in the posterior striatum 60 minutes after ethanol administration (Table 1). However, this high dose of ethanol did cause a 40 to 70% increase in the contents of DOPAC and/or H V A in 5 of the 7 CNS regions 60 minutes after injection (Table I). The levels of the DA metabolites were also significantly elevated in the accumbens (20 to 25% above saline values) and hypothalamus (approximately 30% above saline values) at the 15 and 30 minute time points, as well as after 60 minutes (Table 1). Increased levels of DA metabolites were observed in the posterior striatum, lateral septal nucleus and frontal cortex only at the 60 minute time point after ethanol injection (Table I). The 2.5 g/kg dose of ethanol did not alter the contents of DOPAC or H V A in the thalamus or olfactory bulbs (Table 1). The contents of 5-HT were not altered in any of the 7
297
TABLE 2 CONTENTS OF SEROTONINAND 5-HIAAIN SEVERALCNS REGIONS OF THE RAT 15.30 AND 60 MINUTESAFTER AN IP INJECTION OF 2.5 g ETHANOL/kg nmol/g Wet wt. 5-HT N. Accumbens SAL EtOH-15 EtOH-30 EtOH-60 Post. Striatum SAL EtOH-15 EtOH-30 EtOH-60 Lat. Septal N. SAL EtOH-15 EtOH-30 EtOH-60 Front. Cortex SAL EtOH-15 EtOH-30 EtOH-60 Hypothalamus SAL EtOH-15 EtOH-30 EtOH-60 Thalamus SAL EtOH-15 EtOH-30 EtOH-60 O. Bulbs SAL EtOH-15 EtOH-30 EtOH-60
5-HIAA
6.6 6.0 6.0 6.1
± ± ± ±
0.3 0.3 (7) 0.2 0.3 (7)
3.5 3.9 3.6 4.4
± ± ± ±
0.2 0.2 0.1 0.4*
4.2 4.0 4.1 4.5
± ± ± ±
0.2 0.2 0.4 0.2
3.9 4.0 4.2 4.7
± 0.1 _+ 0.2 ± 0.4 ± 0.2*
3.5 2.8 4.0 3.3
_+ 0.2 ± 0.3 ± 0.4 - 0.3
2.9 2.5 3.2 3.2
± 0.2 ± 0.2 ± 0.4 -+ 0.2
4.5 4.3 4.2 4.2
± ± ± ±
0.1 0.1 0.2 0.1
2.6 2.7 2.7 2.9
± ± ± ±
6.9 6.6 7.2 6.8
± ± ± ±
0.1 0.3 0.2 0.1
6.2 6.1 6.3 6.4
_+ 0.1 ± 0.3 ± 0.2 ± 0.1
4.8 4.7 5.1 4.8
± 0.2 ± 0.2 _+ 0.3 ± 0.2
5.2 5.1 5.1 5.7
± 0.2 ± 0.3 ± 0.4 _+ 0.3
2.4 2.1 2.3 2.1
± 0.1 ___0.1 ± 0.1 -+ 0.1
1.5 1.5 1.5 1.6
± 0.1 ± 0.2 ± 0.1 ___0.2
0.1 0.1 0.1 0.1
Data are the means ± SEM of N=23, 24 for saline group and N=8 (unless otherwise noted) for the ethanol group. *p <0.05 by ANOVA and Duncan's tests.
CNS regions examined at any of time points following administration of the 2.5 g/kg dose of ethanol (Table 2). However, in two of the 7 CNS regions studied, the level of 5-HIAA was significantly elevated in the ethanol-treated group with respect to the saline group (Table 2). In the nucleus accumbens and posterior striatum, the content of 5-HIAA was 20-25% higher in the ethanol group 60 minutes after injection. Significant differences in the contents of 5-HIAA were not observed at the earlier time points between ethanol- and saline-injected animals. DISCUSSION The levels of DOPAC and/or H V A were elevated in 5 of the 7 CNS regions examined 60 minutes after injection of 2.5
298
KHATIB, MURPHY AND McBRIDE
g ethanol/kg (Table 1), presumably indicating that this high dose of ethanol increased DA metabolism by enhancing release of DA from selectively activated pathways. The higher levels of DA metabolites in these regions suggest that BACs of approximately 250 mg%, produced by IP administration of 2.5 g ethanol/kg, can enhance the activity of DA projections from (a) the ventral tegmental area to the nucleus accumbens, lateral septal nucleus and frontal cortex and (b) the substantia nigra to the posterior striatum. In addition, the DA incertohypothalamic system also appears to be activated by the high dose of alcohol, as indicated by the higher levels of DOPAC and/or HVA in the hypothalamus of the ethanoltreated rats (Table 1). On the other hand, since the contents of the DA metabolites in the thalamus and olfactory bulbs were the same in the saline and ethanol-injected groups (Table 1), this would indicate that the DA periventricular system that sends projections to the thalamus and periglomerular DA neurons within the olfactory bulbs are not affected by intoxicating levels of alcohol. The present findings of elevated levels of DOPAC and/or HVA after high-dose ethanol are generally in agreement with published reports on rats [2, 5, 16, 20, 25] and mice [2,8] which all found that acute high doses of ethanol can increase the levels of DA metabolites in the CNS. Results from electrophysiological and in vivo release experiments support the concept that the elevated levels of DOPAC and/or HVA observed following sedative-hypnotic doses of alcohol are due to increased activity of DA neurons. The IV administration of 0.5-2.0 g ethanol/kg increased neuronal activity in the ventral tegmental area and substantia nigra of rats [12,18] while other reports indicated that IP doses of 2.5 g ethanol/kg and lower increased the in vivo release of DA in the accumbens and striatum of the rat [9,15]. Moreover, the above electrophysiological and in vivo release data indicated that the accumbens was more sensitive to ethanol than was the striatum. The present findings of elevated levels of DOPAC and/or HVA in the accumbens but not the posterior striatum at 15 and 30 minutes (Table 1) after the 2.5 g ethanoi/kg injection provides additional evidence that the accumbens is more sensitive to alcohol than is the striatum. The present data did not indicate that the 0.5 g/kg dose of ethanol enhanced DA metabolism or neuronal activity in the accumbens whereas findings from in vivo release [15] and electrophysiological [12] studies indicate otherwise. This apparent disagreement is likely due to in vivo release and electrophysiological measurements being a more sensitive indicator of changes in neuronal activity than can be obtained by measuring tissue contents of the monoamine metabolites. The content of DA was not altered by the 2.5 g/kg dose of ethanol in any of the regions at any of the time points, except
for an increase in the posterior striatum after 60 minutes (Table 1). One possible explanation is that autoreceptor activation by extracellular DA has reduced release while synthesis of DA continues at an accelerated level. The effects of the 2.5 g/kg dose of ethanol on the 5-HT systems were very limited (Table 2). The elevated contents of 5-HIAA in the nucleus accumbens and posterior striatum suggest specific increased activity of the 5-HT dorsal ascending pathway from the dorsal raphe nucleus to these two CNS regions. Similar findings of an elevated level of 5-HIAA in rat whole brain after a high dose of ethanol have been reported [11, 24, 29]. Tabakoff et al. [30] observed that ethanol reduced the transport of [14C] 5-HIAA from whole brain of mice given an IP injection of 3 g ethanol/kg. The results of the present study for the 5-HT acid metabolite are not likely due exclusively to inhibition of transport of 5-H1AA from the brain, If inhibition of transport was the major cause, then a general elevation of 5-HIAA in all the CNS regions examined (with the possible exception of the hypothalamus) should have been observed. However, this was not the case for 5-HIAA (Table 2). On the other hand, it is possible that there is some inhibition of 5-HIAA transport from the brain produced by the high dose of alcohol. This effect on transport would be experimentally observed as elevated levels of 5-HIAA only when sufficiently enhanced 5-HT neuronal activity also occurs. There is some evidence that the higher level of 5-H1AA in the accumbens and striatum of the ethanol-treated rats observed in the present study (Table 2) may be due to increased 5-HT neuronal activity. Holman and Snape[ 13] reported that the IP administration of ethanol, which raised BACs to approximately 250 mg%, increased the release of 5-HT as reflected by the higher concentration of 5-HIAA in the rat striatal dialysis perfusate. Chu and Keenan [6], using the brain stem slice preparation, reported that 100-200 mgCA ethanol produced more excitation than inhibition of neurons in the midbrain dorsal raphe. The data for the CNS distribution of DA, 5-HT and their metabolites found in the present study (Tables 1 and 2) are generally in agreement with results of others [3, 4, 1%211. In conclusion, this study provides neurochemical evidence supporting the findings of others that intoxicating doses of alcohol can selectively activate specific populations of monoaminergic neurons in the CNS of the rat.
ACKNOWLEDGEMENTS The technical advice of Mr. Steven Cunningham is deeply appreciated. Supported in part by AA 03243 and AA 07462.
REFERENCES 1. Aghajanian, G. K., J. A. Rosencrans and M. H. Sheard. Serotonin: release in the forebrain by stimulation of midbrain raphe. Science 156: 402-403, 1967. 2. Alari, L., T. Lewander and 13. Sjoquist. The effect of ethanol on the brain catecholamine systems in female mice, rats, and guinea pigs. Alcohol: Clin Exp Res 11: 144-149, 1987. 3. Bacopoulos, N. G., R. K. Bhatnager and L. S. Van Order, 111. The effects of subhypnotic doses of ethanol on regional catecholamine turnover. J Pharmacol Exp Ther 204: 1-10, 1978.
4. Brownstein, M. J. and M. Palkovits. Catecholamines, serotonin, acetylcholine, and y-aminobutyric acid in the rat brain: biochemical studies. In: Handbook of Chemical Neuroanatomy. Vol 2: Classical Transmitters in the CNS. Part 1, edited by A. Bjorklund and T. Hokfelt. Amsterdam: Elsevier, 1984, pp. 23-54. 5. Bustos, G. and R. H. Roth. Effect of acute ethanol treatment on transmitter synthesis and metabolism in central dopaminergic neurons. J Pharm Pharmacol 28: 580-582, 1976.
ETHANOL EFFECTS ON MONOAMINE PATHWAYS
6. Chu, N.-S. and L. Keenan. Responses ofmidbrain dorsal raphe neurons to ethanol studied in brainstem slices. Alcohol 4: 373374, 1987. 7. Curzon, G., J. C. R. Fernando and C. A. Marsden. 5-Hydroxytryptamine: the effects of impaired synthesis on its metabolism and release in rat. Br J Pharmacol 63: 627-634, 1978. 8. Dar, M. S. and W. R. Wooles. The effect of acute ethanol on dopamine metabolism and other neurotransmitters in the hypothalamus and the corpus striatum of mice. J Neural Transm 60: 283-294, 1984. 9. DiChiara, G. and A. lmperato. Ethanol preferentially stimulates dopamine release in the nucleus accumbens of freely moving rats. Eur J Pharmacol 115: 131-132, 1985. 10. Fadda, F., A. Argiolas, M. R. Melis, G. Serra and G. L. Gessa. Differential effects of acute and chronic ethanol on dopamine metabolism in frontal cortex, caudate nucleus and substantia nigra. LiJ~ Sci 27: 979-986, 1980. 11. Fukumori, R., A. Minegisti, T. Satoh, H. Kitagawa and S. Yanaura. Changes in the serotonin and 5-hydroxyindoleacetic acid contents in rat brain after ethanol and disulfiram treatments. Eur J Pharmacol 61: 199-202, 1980. 12. Gessa, G. L., F. Muntoni, M. Collu, L. Vargiu and G. Mereu. Low doses of ethanol activate dopaminergic neurons in the ventral tegmental area. Brain Res 348: 201-203, 1985. 13. Holman, R. B. and B. M, Snape. Effects of ethanol on 5-hydroxytryptamine release from rat corpus striatum in vivo. Alcohol 2: 249-253, 1985. 14. Hunt, W.A. and E. Majchrowicz. Studies of neurotransmitter interactions after acute and chronic ethanol administration. Pharmacol Biochem Behav 18: 371-374, 1983. 15. lmperato, A. and G. DiChiara. Preferential stimulation of dopamine release in the nucleus accumbens of freely moving rats by ethanol. J Pharmacol Exp Ther 239: 21%228, 1986. 16. Karoum, F., R. J. Wyatt and E. Majchrowicz. Brain concentrations of biogenic amine metabolites in acutely treated and ethanol-dependent rats. Br J Pharmaco! 56:403-411, 1976. 17. Lindvall, O. and A. Bjorklund. Dopamine- and norepinephrinecontaining neuron systems: Their anatomy in the rat brain. In: Chemical Neuroanatomy, edited by P, C. Emson. New York: Raven Press, 1983, pp. 22%255. 18. Mereu, G., F. Fadda and G. L. Gessa. Ethanol stimulates the firing rate of nigral dopaminergic neurons in unanesthetized rats. Brain Res 292: 63-69, 1984. 19. Murphy, J. M., W. J. McBride, L. Lumeng and T.-K. Li. Regional brain levels of monoamines in alcohol-preferring and -nonpreferring lines of rats. Pharmacol Biochem Behav 16: 145-149, 1982.
299
20. Murphy, J. M., W. J. McBride, L. Lumeng and T.-K. Li. Monoamine and metabolite levels in CNS regions of the P line of alcohol-preferring rats after acute and chronic ethanol treatment. Pharmacol Biochem Behav 19: 849-856, 1983. 2 I. Murphy, J. M., W. J. McBride, L. Lumeng and T.-K. Li. Alcohol preference and regional brain monoamine contents of N/Nih heterogeneous stock rats. Alcohol Drug Res 7: 33-39, 1986. 22. Murphy, J. M., W. J. McBride, L. Lumeng and T.-K. Li. Contents of monoamines in forebrain regions of alcohol-preferring (P) and -nonpreferring (NP) lines of rats. Pharmacol Biochem Behav 26: 38%392, 1987. 23. Pohorecky, L. A. Biphasic action of ethanol. Biobehav Rev !: 231-240, 1977. 24. Pohorecky, L. A., B. Newman, J. Sun and W. H. Bailey. Acute and chronic ethanol ingestion and serotonin metabolism in rat brain. J Pharmacol Exp Ther 204: 424-432, 1978. 25. Reggiani, A., M. L. Barbaccia, P. F. Spano and M. Trabucchi. Dopamine metabolism and receptor function after acute and chronic ethanol. J Neurochem 35: 34-37, 1980. 26. Roth, R. H., L. C. Murrin and J. R. Waiters. Central dopaminergic neurons: effects of alterations in impulse flow on the accumulation of dihydroxyphenylacetic acid. Fur J Pharmacol 36: 163-171, 1976. 27. Shannon, N. J., J. W. Gunnet and K. E. Moore. A comparison of biochemical indices of 5-hydroxytryptaminergic neuronal activity following electrical stimulation of the dorsal raphe nucleus. J Neurochem 47: 958-965, 1986. 28. Steinbusch, H. W. M. and R. Nieuwenhuys. The Raphe nuclei of the rat brainstem: A cytoarchitectonic and immunohistochemical study. In: Chemical Neuroanatomy, edited by P. C. Emson. New York: Raven Press, 1983, pp, 131-207. 29. Tabakoff, B. and W. O. Boggan. Effects of ethanol on serotonin metabolism in brain. J Neurochem 22: 75%764, 1974. 30. Tabakoff, B., R. F. Ritzman and W. O. Boggan. Inhibition of the transport of 5-hydroxyindoleacetic acid from brain by ethanol. J Neurochem 24: 1043-1051, 1975. 31. Takahashi, R. and M. H. Aprison. Acetylcholine content of discrete areas of the brain obtained by a near-freezing method. J Neurochem 11: 887-898, 1964. 32. Waller, M. B., W. J. McBride, L. Lumeng and T.-K, Li. Initial sensitivity and acute tolerance to ethanol in the P and NP lines of rats. Pharmacol Biochem Behav 19: 683-686, 1983.