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Dopamine and salsolinol levels in rat hypothalami and striatum after schedule-induced self-injection (SISI) of ethanol and acetaldehyde
122
Brain Research, 358 (1985) 122-128 Elsevier
BRE 11182
Dopamine and Salsolinol Levels in Rat Hypothalami and Striatum After ScheduleInduced Self-Injection (SISI) of Ethanol and Acetaldehyde WENDY D. MYERS l, KIM T. NG 1, GEORGE SINGER1, GEORGE A. SMYTHE 2 and MARK W. DUNCAN: 1Department of Psychology and Brain-Behaviour Research Institute, La Trobe University, Bundoora, Vic. 3083 and 2Garvan Institute of Medical Research, St. Vincents Hospital, Sydney N. S. W. 2010 (Australia)
(Accepted February 25th, 1985) Key words: salsolinol - - dopamine - - hypothalamus - - striatum - - schedule-induced self-injection - - acetaldehyde - - ethanol
Endogenous levels of salsolinol and dopamine were measured by a gas chromatography/mass spectrometry (GC/MS) - - selected ion monitoring technique using deuterated internal standards in rats allowed to self-inject either acetaldehyde, ethanol or saline control solution over 20 days for 1 h/day. Sil~fificant increases in medial basal hypothalamic (MBH) and striatal salsolinol concentrations were found in animals exposed to acetaldehyde but not ethanol, whereas dopamine concentrations for these animals did not differ significantly from rats exposed to control conditions. The data provides further support for the in vivo formation of salsolinol following acetaldehyde exposure in experimental animals.
INTRODUCTION Evidence suggests that acetaldehyde may play a significant role in the psychopharmacological effects of ethanol3-5,7, 20. Acetaldehyde interacts with biogenic amines to form Pictet-Spengler condensation products that have been implicated in the mediation of ethanol reinforcementS,9,30. The proposal that the in vivo formation of mammalian alkaloids due to a Pictet-Spengler condensation between biogenic monoamines such as dopamine and an aldehyde may play a role in alcoholism has been an controversial issue for over a decade. It has been difficult to test this hypothesis because of a lack of assays sufficiently specific or sensitive to detect the formation of putative mammalian alkaloids following the ingestion of alcohol in either man or experimental animalst2,13. The dopamine-acetaldehyde-derived alkaloid, salsolinol, has attracted much attention as a candidate for a possible role in alcoholism. Salsolinol has been suggested to exert marked behavioural effects with respect to alcohol preference in rats 19. Furthermore rats with a prior history of schedule-induced acetaldehyde self-injection have been shown to consume significantly more ethanol than rats with a prior history of schedule-induced saline self-injection22, 23.
So far radioenzymatic 10, high-performance liquid chromatographic6,25, 27 and combined gas chromatography/mass spectrometric (GC/MS)12a3, 3~ techniques have been applied to the assay of salsotinol. Much of the controversy surrounding reports of the detection of saisolinol in mammalian tissue and fluids has been directed at the analytical methods usedt~, 13. A lack of assay specificity and possible artifactual formation have been suggested as being largely responsible for relatively high levels of salsolinol reported in some studies12, t3. A n o t h e r concern is the finding that alkaloids might arise in mammals directly from dietary sources 11-13. Duncan and colleagues 11-13 recently reported the presence of salsotinol in a variety of fermented, non-distilled beverages and food stuffs including bananas, soy sauce and chocolate. In an attempt to demonstrate the formation of salsolinol by an in vivo Pictet-Spengler condensation, Smythe and Nicholson (unpublished observations) used a variety of procedures aimed at increasing endogenous brain levels of dopamine and acetaldehyde in the rat and then examined brain tissue for salsotinol. The acute treatment of normal rats with pharmacological doses of L - D O P A , acetaldehyde or ethyl alcohol, either alone or in various combinations, failed
0006-8993/85/$03.30© 1985 Elsevier Science Publishers B.V. (Biomedical Division)
123 to induce increases in hypothalamic or other brain regional levels of salsolinol. However, the possibility that circumstances might exist whereby salsolinol is formed in vivo was supported recently by data in which we found marked increases in salsolinol concentrations in the brain tissue of rats that had been exposed to ethanol at high doses over extended periods 21. In the present report we use a simple, highly sensitive and specific method (GC/MS with appropriate deuterated internal standards) to examine the effects of schedule-induced self-injection of ethanol and acetaldehyde on dopamine and salsolinol levels in dopamine-rich areas of the rat brain (striatum and hypothalamus) that have been implicated in alcohol consumption21,3o,3a. MATERIALS AND METHODS The study was carried out using a Hewlett-Packard 5993A GC/MS data system (Hewlett-Packard Australia, North Ryde, NSW). Glass columns (2 m x 2 mm i.d.) were treated with dichlorodimethylsilane and packed with a 3% OV-17 on Supelcoport 100120 mesh (Supelco, Bellefonte, PA, U.S.A.). Ultrahigh purity helium at a flow rate of 30 ml'min -1 was used as carrier gas. An initial GC oven temperature of 146 °C was held for the first 1 rain of the analysis and then raised to 200 °C at 26 °C'min -1. The injection port temperature was 220 °C. A membrane separator was used at the GC/MS interface. Both dopamine and salsolinol were derivatized using trifluoroacetic anhydride and for maximum specificity both the molecular ion and the major fragment ion (base peak) were scanned. Rat brain tissue was collected and extracted according to our established method 21,33. Deuterated internal standards were incorporated into all samples before extraction. The mean wet weight of medial basal hypothalamic (MBH) and striatal samples for animals in the acetaldehyde group was, respectively, 22.58 mg and 56.23 mg; for animals in the ethanol group, 18.90 mg and 54.45 mg, respectively; for analysis in the saline group, 19.70 mg and 49.15 mg, respectively, and in the treatment control group 18.18 mg and 49.73 rag, respectively.
Experimentalprocedure The animals weighing 330-360 g at the start of the
study were housed individually in wire mesh cages (26 x 34 x 20 cm) and were adapted to a 12:12 h light/dark cycle (lights off 07.00 h) for two weeks prior to the commencement of experimentation. Intravenous catheters (SP 28 Dural Plastic, o.d. 0.88 mm, i.d. 0.40 mm) were surgically implanted into the jugular vein of the animals under anaesthesia (pentobarbitone sodium 60 mg/ml; 1 ml/kg, i.p.) according to our established procedures 22.23,26,28,29.35. After reduction to 90% of their free-feeding body weights, the rats were randomly assigned to one of four treatment groups: schedule induced acetaldehyde self-injection (AcH SISI), schedule-induced ethanol selfinjection (etoh SISI), schedule-induced saline self-injection control (saline SISI control) and treatment control. Rats in the treatment control group were not removed from the home-cage evironment nor exposed to drug or saline control treatment. Animals in the schedule-induced self-injection (SISI) groups were given the opportunity to individually self-infuse either drug or saline control solution for 1 h/day over 20 consecutive days between 09.00 and 12.00 h. On day 20, after 60 min in the operant test chambers each rat was removed and immediately decapitated. Details of our procedure for schedule-induced intravenous self-injection of acetaldehyde, ethanol and other drugs are well established and have been published elsewhere 22-24,26,28,29,32,35. In brief the schedule of acetaldehyde self-injection was as follows. When an animal pressed the operant bar, a syringe infusion pump (Sage Instruments, model 341) was activated for 5 s and an infusion of fluid (0.07 ml) was delivered into the jugular vein. During the 5-s infusion interval, additional bar presses did not reactivate the pump and were not recorded. All infusions during each 1-h test session were automatically monitored on cumulative recorders. In operation, the fixed-time 1 min (FT-1) schedule delivered Noyes food pellets (45 rag) non-contingently at the rate of one pellet per minute, into the food pellet dispensing units attached to one side wall of the modified operant test chambers. A 1% v/v acetaldehyde solution (2.32 mg/kg/infusion) was used. The procedure used for schedule-induced ethanol self-injection was identical to that used for the AcH SISI group and has been reported elsewhere24,32. A 20% ethanol solution (i.e. 44.33 mg/kg/infusion) was chosen, as this has been found to be the optimum dose for schedule-
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125 induced self-injection of ethanol in our laboratory24.3L Animals in the saline SISI control group were allowed to self-inject a 0.9% (v/v) NaC1 solution which was also the vehicle control for the acetaldehyde and ethanol solutions. Animals in the treatment control group were maintained in their homecages without exposure to the test situation or drug treatment for the entire duration of the experiment. As in previous reports22.23 the acetaldehyde (99.5%, B.D.H. Chemicals, Australia) was freshly prepared for intravenous administration prior to each test session by diluting it in 0.9% NaC1. The acetaldehyde sample was regularly distilled before use and then diluted and stored at 4 °C. The purity of the acetaldehyde sample was analyzed regularly by NMR spectroscopy (Perkin-Elmer, R32A). For ethanol self-injection 95% ethanol (Ajax-Chemicals, Sydney, Australia) was prepared for intravenous administration prior to each test session by mixing it in 0.9% NaC1 to make up the 20% v/v ethanol solution. RESULTS
Self-injection data For purposes of statistical analysis, the overall mean number of self-infusions for each animal over 20 days in the SISI treatment groups were compared in order to test the hypothesis that the population frequency distributions differ. Results of the KruskalWallis one-way analysis of variance by ranks indicated no significant differences between the SISI treatment groups (H = 4.731, df = 2, P > 0.05). The overall mean number of self-infusions for animals in the different treatment groups are as follows: Acetaldehyde ~ = 15.99, Ethanol ~ = 9.25 and Saline ~ = 14.18.
Dopamine and salsolinol levels Levels of dopamine and salsolinol were obtained from the medial basal hypothalamus and striatum of rats in each of the four treatment groups. The Kruskal-Wallis test was conducted on the obtained concentrations of dopamine (pmol/mg) and salsolinol (pmol/mg). The means for levels of MBH salsolinol and dopamine for animals in the four treatment groups are as follows in pmol/mg wet brain tissue: acetaldehyde (0.72 and 3.90), ethanol (0.01 and 2.87), saline (0.01 and 3.84) and treatment control
(0.02 and 3.34). The respective means for levels of striatal salsolinol and dopamine (pmol/mg wet brain tissue) are as follows: acetaldehyde (3.36 and 55.50), ethanol (0.02 and 52.37), saline (0.02 and 65.48), and treatment control (0.01 and 63.97). A Bonferroni adjustment was used 16to adjust the critical values of the individual tests so that the Type 1 error rate per analysis was set at a i = 0.0125 with an overall experimentwise error rate of E = 0.05 to allow for the fact that four separate analyses were performed in the same experiment. Results of the Kruskal-Wallis test comparing the salsolinol levels for medial basal hypothalamus (MBH) of rats showed a significant difference between treatment groups (H = 13.186, df = 3, P < 0.005). However, the Kruskal-Wallis performed on dopamine levels from medial basal hypothalamus of rats did not support the test hypothesis for difference among treatment groups (H = 7.742, df = 3, P > 0.05). Paired comparisons of frequency distributions for salsolinol from MBH were carried out by means of the Mann-Whitney U-test. Findings showed there was significantly more salsolinol for the acetaldehyde SISI group in comparison with the ethanol SISI (U = 0, P < 0.002), and saline SISI control group (U = 0, P < 0.004) but not Treatment control (U = 3.5, P > 0.015) group (Fig. 1). Results of the Kruskal-Wallis one-way analysis of variance comparing the four different treatment groups on striatal salsolinol levels and dopamine levels are shown in Fig. 1. There was a significant difference between treatment groups on salsolinol (H = 12.823, df = 3, P < 0.005) but not on dopamine (H = 8.836, df = 3, P > 0.05) levels. Mann-Whitney U-test comparisons showed animals in the acetaldehyde SISI group to have significantly more striatal salsolinol than animals in all the other treatment groups (U = 0, P < 0.002). (See Fig. 1). DISCUSSION The data from this experiment demonstrate the presence of the dopamine-acetaldehyde condensation product, salsolinol, in the medial basal hypothalamus (MBH) and striatum of all experimental animals (Fig. 1). The finding of: (i)a significantly greater concentration of salsolinol in the MBH of animals in the acetaldehyde SISI group relative to the ethanol
126 and saline treatment groups: and (ii) a significantly greater concentration of salsolinol in the striatum of animals in the acetaldehyde SISI group relative to the ethanol SISI, saline SISI and Treatment control groups, provides evidence for the possibility of an acetaldehyde-mediated mechanism involving salsolinol in schedule-induced acetaldehyde self-injection. The finding of trace levels of salsolinol in the hypothalami and striatum of the ethanol SISI, saline SISI control and Treatment control rats agree with other reports on control animals31,34, although it is noteworthy that different strains of rats were used in each of the studies referred to, including the present experiment (Sprague-Dawley3t, Wistar34, Long-Evans, in the present report). The finding of low saisolinol levels in animals in the ethanol SISI and saline SISI control groups in comparison with the acetaldehyde SISI group is interesting in this context (Fig. 1) for a number of reasons. First, it was demonstrated that although there was no significant difference between behavioural measures (rate of self-infusion) for animals in the acetaldehyde SISI, ethanol SISI and saline SISI control groups these groups differed significantly in salsolinol levels. Second, the significant difference in salsolinol levels for rats in the acetaldehyde SISI group relative to the ethanol S1SI group adds evidence to other reports of differences between the effects of these two compounds 5.7.36. However, it is relevant to note that while the overall amount of ethanol consumed was nearly 10 times that of acetaldehide, acetaldehyde is reported to be at least 10-30 times more potent than ethanol due to its chemically reactive aldehyde group 1.2.17. Previous findings show that increased hypothalamic and striatal salsolinoi levels have been found in rats after 10 months chronic ethanol exposure 21 and in rat limbic forebrain after 150 days chronic exposure to ethanoP 0. Whereas acute treatment of rats with pharmacological doses of ethanol failed to increase hypothalamic or other brain regional levels of salsolinol (Smythe and Nicholson, unpublished observation). The possibility must therefore be entertained that the 20 days of ethanol self-injection may not have been long enough to lead to a disturbance in dopamine status and resultant rise in salsolino121. Other studies have shown that rats would only self-administer acetaldehyde intraventricularly but not ethanoF suggesting that acetaldehyde rather
than ethanol itself may mediate the reinforcing effects of ethanol in the brain 3,5,7. Further reports have also found that for some pharmacological actions, acetaldehyde and ethanol are antagonistic14,1L These factors may have some bearing on why no salsolinol was formed in rats given ethanol. It is of interest to note that there was considerable variability among the salsolinol concentrations in MBH and striatum for animals in the acetaldehyde SISI group, whereas the same cannot be said for animals in the ethanol SISI group (Fig. 1). Since neither acetaldehyde levels nor enzyme activity was measured, no firm conclusions can be drawn. However, large individual variation in sensitivity to acetaldehyde has been reported by other researchers 3~. It has been suggested that such individual differences may result from variability in the metabolism of acetaldehyde, which in turn could lead to higher circulating concentrations, associated perhaps with an increased release of catecholamines 36. This, in turn, could promote formation of condensation products t8. It is reported that elevated acetaldehyde levels can result from faster production, slower removal or a combination of both 36. It is not possible on the basis of the present findings to claim a specific association between the increased salsolinol levels observed in MBH and striatum of animals in the acetaldehyde SISI group and dopamine levels for these same animals. No significant difference was shown between the acetaldehyde SISI group and the saline SISI control, ethanol SISI and treatment control groups for MBH dopamine concentrations (Fig. 1). The overall MBH dopamine levels from all treatment groups in the present study (~ = 3.487) are in keeping with those reported previously for control rats 34. Striatal dopamine levels for the acetaldehyde SISI group were not significantly different from those of the other three treatment groups. However, the pattern of high dopamine concentrations in striatum of animals from all four treatment groups in the present study (~ = 59.330) relative to the lower MBH dopamine concentrations (~ = 3.487), agrees with previous findings 30 (see Fig. 1.). These data suggest a role for salsolinol in scheduleinduced acetaldehyde self-injection and provide support for speculation 22,23 regarding involvement of acetaldehyde mediated mechanisms in this behavioural phenomenon.
127 ACKNOWLEDGEMENT
tional Health and Medical Research Council (NH and MRC) of Australia.
This work was supported by a grant from the Na-
REFERENCES 1 Akabane, J., Pharmacological aspects of manifestation of the acute after-effects of alcoholic beverages: a role of acetaldehyde in alcoholism, Med. J. Shinsu Univ., 5 (1960) 113-122. 2 Akabane, J., Aldehydes and related compounds in alcohols and derivatives. In J. Tremolieres (Ed), International Encyclopedia of Pharmacology and Therapeutics Section 20, Vol. 2, Pergamon Press, Oxford, 1970, p. 523-560. 3 Amir, S., Brown, Z. and Amit, Z., The role of acetaldehyde in the pharmacological effects of ethanol. In H. Rigter and J.C. Crabbe Jr. (Eds.), Alcohol Tolerance and Dependence, Elsevier, Amsterdam, 1980, pp. 317-337. 4 Amit, Z., Brown, Z.W. and Rockman, G.E., Possible involvement of acetaldehyde, norepinephrine and their tetrahydroisoquinoline derivatives in the regulation of ethanol self-administration, Drug Alcohol Depend., 2 (1977) 495-500. 5 Amit, Z., Brown, Z.W., Rockman, G.E., Smith, B. and Amir, S., Acetaldehyde: a positive reinforcer mediating ethanol consumption. In H. Begleiter (Ed.), Biological Effects of Alcohol, Plenum Press, New York, 1980, pp. 413-423. 6 Brien, J.F., Andrews, P.J., Loomis, C.W, and Page, J.A., Gas-liquid chromatographic determination of salsolinol in the striatum of rat brain using the calcium carbimide-ethanol interaction, Can. J. Physiol. Pharmacol., 61 (1983) 632. 7 Brown, Z.W., Amit, Z. and Rockman, G.E., Intraventricular self-administration of acetaldehyde, but not ethanol in naive laboratory rats, Psychopharmacology, 64 (1979) 271-276. 8 Cohen, G. and Collins, M.A., Alkaloids from catecholamines in adrenal tissue: possible role in alcoholism, Science, 167 (1970) 1749-1751. 9 Davis, V.E. and Walsh, M.J., Alcohol, amines and alkaloids: a possible biochemical basis for alcohol addiction, Science, 167 (1970) 1005-1007. 10 Dean, R.A., Henry, D.P., Bowsher, R.R. and Furnay, R.B., A sensitive radioenzymatic assay for the simultaneous determination of salsolinol and dopamine, Life Sci., 27 (1980) 403. 11 Duncan, M.W. and Smythe, G.A., Salsolinol and dopamine in alcohol beverages, Lancet, i (1982) 904. 12 Duncan, M.W., Smythe, G.A. and Clezy, P.S., GC/MS methodology for simultaneous assay of salsolinol, dopamine, norepinephrine, dihydroxyphenylacetic acid and dihydroxyphenylethanol, Biorned. Mass Spec., in press. 13 Duncan, M.W., Smythe, G.A., Nicholson, M.V. and Clezy, P.S., Comparison.of HPLC/ED and GC/mass fragmentographic methods for the assay of salsolinol, dopamine and dopamine metabolites in food and beverage samples, J. Chromatogr., in press'. 14 Friedman, H.S., Matsuzaki, S,, Choe, S.-S., Fernando, H.A., Celis, A., Zaman, Q. and Lieber, C.S., Demonstration of dissimilar acute haemodynamic effects of ethanol and acetaldehyde, Cardiovasc. Res., 13 (1979) 477-487.
15 Hald, J., Jacobsen, E. and Larsen, V., The antagonism between alcohol and acetaldehyde in rabbits, Acta Pharmacol. Toxicol., 8 (1952) 164-170. 16 Harris, R.J., A Primer of Multivariate Statistics, Academic Press, New York, 1975, p. 98. 17 Holtzman, S.G. and Schneider, F.H., Comparison of acetaldehyde and ethanol: depression of motor activity in mice, Life Sci., 14 (1974) 1143-1250. 18 Lindros, K.O., Acetaldehyde - its metabolism and role in the actions of alcohol. In Y. Israel, F.B. Glaser, H. Kalant, R.E. Popham, W. Schmidt and R.G. Smart (Eds.), Research Advances in Alcohol and Drug Problems, Vol. 4, Plenum Press, New York, 1978, pp. 111-176. 19 Myers, R.D. and Melchior, C.L., Differential actions on voluntary alcohol intake of tetrahydroisoquinolines or a flcarboline infused chronically in the ventricle of the rat, Pharmacol. Biochem. Behav., 7 (1977) 381-392. 20 Myers, R.D. and Veale, W.L., Alterations in volitional alcohol intake produced in rats by chronic intraventricular infusions of acetaldehyde, paraldehyde or methanol, Archs. Int. Pharmacodyn., 180 (1969) 100-113. 21 Myers, W., McKenzie, L., Ng, K.T., Singer, G., Smythe, G.A. and Duncan, M.W., Salsolinol and dopamine in rat medial basal hypothalamus after chronic ethanol exposure, Life Sci., 36 (1985) 309-314. 22 Myers, W.D., Ng, K.T., Marzuki, S., Myers, R.D. and Singer, G., Alteration of alcohol drinking in the rat by peripherally self-administered acetaldehyde, Alcohol, 1 (1984) 229-236. 23 Myers, W.D., Ng, K.T. and Singer, G., Ethanol preference in rats with a prior history of acetaldehyde self-administration, Experientia, 40 (1984) 1008-1010. 24 Oei, T.P.S. and Singer, G., Effects of a fixed time schedule and body weight on ethanol self-administration, Pharmacol. Biochem. Behav., 10 (1979) 767-770. 25 O'Neill, P.J. and Rahwan, R.G. Modified electron-capture GLC assay for salsolinol in brain tissue. J. Pharmacol. Sci., 66 (1977) 893-895. 26 Papasava, M., Oei, T.P.S. and Singer, G., Low dose cocaine self-administration by naive rats: Effects of body weight and a fixed time one minute food delivery schedule, Pharmacol. Biochem. Behav., 15 (1981) 485-488. 27 Riggin, R.M. and Kissinger, P.T., Determination of tetrahydroisoquinoline alkaloids in biological materials with high performance liquid chromatography, Anal. Chem., 49 (1977) 530-533. 28 Singer, G., Oei, T.P.S. and Wallace, M., Schedule-induced self-injection of drugs, Neurosci. Biobehav. Rev., 6 (1982) 77'--83. 29 Singer, G., Simpson, F. and Lang, W.J., Schedule-induced self-injection of nicotine with recovered body weight, Pharmacol. Biochem, Behav., 9 (1978)387-389. 30 Sjoquist, B., Liljequist, S. and Engel, J., Increased salsolinol levels in rat striatum and limbic forebrain following chronic ethanol treatment, J. Neurochem., 39 (1982) 259-262. 31 Sjoquist, B. and Magnusson, E., Analysis of salsolinol and
128 salsoline in biological samples using deuterium-labelled internal standards and gas chromatography-mass spectrometry, J. Chromatogr., 183 (1980) 17-24. 32 Smith, D., Oei, T.P.S., Ng, K.T. and Armstrong, S., Rat self-administration of ethanol: enhancement by darkness and exogenous melatonin, Physiol. Behav., 25 (1980) 449-455. 33 Smythe, G.A., Bradshaw, J.E. and Vining, R.F., Hypothalamic monoamine control of stress-induced adrenocorticotropin release in the rat, Endocrinology, 113 (1983) 1062-1071.
34 Smythe, G.A., Duncan, M.W. and Bradshaw, .I.E., Salsolinol is a potent stimulus for prolactin secretion in the rat: evidence for hypothalamic dopamine blockade, IRCS Med. Sci. (Biochem.), 9 (1981) 472-473. 35 Takahashi, R.N. and Singer, G., Effects of body weight levels on cannabis self-injection, Pharmacol. Biochem. Behay., 13 (1980) 877-881. 36 von Wartburg, J.P., Acetaldehyde. In M. Sandier (Ed.), Psychopharmacology of Alcohol, Raven Press, New York, 1980, pp. 137-147.
Brain Research, 358 (1985) 122-128 Elsevier
BRE 11182
Dopamine and Salsolinol Levels in Rat Hypothalami and Striatum After ScheduleInduced Self-Injection (SISI) of Ethanol and Acetaldehyde WENDY D. MYERS l, KIM T. NG 1, GEORGE SINGER1, GEORGE A. SMYTHE 2 and MARK W. DUNCAN: 1Department of Psychology and Brain-Behaviour Research Institute, La Trobe University, Bundoora, Vic. 3083 and 2Garvan Institute of Medical Research, St. Vincents Hospital, Sydney N. S. W. 2010 (Australia)
(Accepted February 25th, 1985) Key words: salsolinol - - dopamine - - hypothalamus - - striatum - - schedule-induced self-injection - - acetaldehyde - - ethanol
Endogenous levels of salsolinol and dopamine were measured by a gas chromatography/mass spectrometry (GC/MS) - - selected ion monitoring technique using deuterated internal standards in rats allowed to self-inject either acetaldehyde, ethanol or saline control solution over 20 days for 1 h/day. Sil~fificant increases in medial basal hypothalamic (MBH) and striatal salsolinol concentrations were found in animals exposed to acetaldehyde but not ethanol, whereas dopamine concentrations for these animals did not differ significantly from rats exposed to control conditions. The data provides further support for the in vivo formation of salsolinol following acetaldehyde exposure in experimental animals.
INTRODUCTION Evidence suggests that acetaldehyde may play a significant role in the psychopharmacological effects of ethanol3-5,7, 20. Acetaldehyde interacts with biogenic amines to form Pictet-Spengler condensation products that have been implicated in the mediation of ethanol reinforcementS,9,30. The proposal that the in vivo formation of mammalian alkaloids due to a Pictet-Spengler condensation between biogenic monoamines such as dopamine and an aldehyde may play a role in alcoholism has been an controversial issue for over a decade. It has been difficult to test this hypothesis because of a lack of assays sufficiently specific or sensitive to detect the formation of putative mammalian alkaloids following the ingestion of alcohol in either man or experimental animalst2,13. The dopamine-acetaldehyde-derived alkaloid, salsolinol, has attracted much attention as a candidate for a possible role in alcoholism. Salsolinol has been suggested to exert marked behavioural effects with respect to alcohol preference in rats 19. Furthermore rats with a prior history of schedule-induced acetaldehyde self-injection have been shown to consume significantly more ethanol than rats with a prior history of schedule-induced saline self-injection22, 23.
So far radioenzymatic 10, high-performance liquid chromatographic6,25, 27 and combined gas chromatography/mass spectrometric (GC/MS)12a3, 3~ techniques have been applied to the assay of salsotinol. Much of the controversy surrounding reports of the detection of saisolinol in mammalian tissue and fluids has been directed at the analytical methods usedt~, 13. A lack of assay specificity and possible artifactual formation have been suggested as being largely responsible for relatively high levels of salsolinol reported in some studies12, t3. A n o t h e r concern is the finding that alkaloids might arise in mammals directly from dietary sources 11-13. Duncan and colleagues 11-13 recently reported the presence of salsotinol in a variety of fermented, non-distilled beverages and food stuffs including bananas, soy sauce and chocolate. In an attempt to demonstrate the formation of salsolinol by an in vivo Pictet-Spengler condensation, Smythe and Nicholson (unpublished observations) used a variety of procedures aimed at increasing endogenous brain levels of dopamine and acetaldehyde in the rat and then examined brain tissue for salsotinol. The acute treatment of normal rats with pharmacological doses of L - D O P A , acetaldehyde or ethyl alcohol, either alone or in various combinations, failed
0006-8993/85/$03.30© 1985 Elsevier Science Publishers B.V. (Biomedical Division)
123 to induce increases in hypothalamic or other brain regional levels of salsolinol. However, the possibility that circumstances might exist whereby salsolinol is formed in vivo was supported recently by data in which we found marked increases in salsolinol concentrations in the brain tissue of rats that had been exposed to ethanol at high doses over extended periods 21. In the present report we use a simple, highly sensitive and specific method (GC/MS with appropriate deuterated internal standards) to examine the effects of schedule-induced self-injection of ethanol and acetaldehyde on dopamine and salsolinol levels in dopamine-rich areas of the rat brain (striatum and hypothalamus) that have been implicated in alcohol consumption21,3o,3a. MATERIALS AND METHODS The study was carried out using a Hewlett-Packard 5993A GC/MS data system (Hewlett-Packard Australia, North Ryde, NSW). Glass columns (2 m x 2 mm i.d.) were treated with dichlorodimethylsilane and packed with a 3% OV-17 on Supelcoport 100120 mesh (Supelco, Bellefonte, PA, U.S.A.). Ultrahigh purity helium at a flow rate of 30 ml'min -1 was used as carrier gas. An initial GC oven temperature of 146 °C was held for the first 1 rain of the analysis and then raised to 200 °C at 26 °C'min -1. The injection port temperature was 220 °C. A membrane separator was used at the GC/MS interface. Both dopamine and salsolinol were derivatized using trifluoroacetic anhydride and for maximum specificity both the molecular ion and the major fragment ion (base peak) were scanned. Rat brain tissue was collected and extracted according to our established method 21,33. Deuterated internal standards were incorporated into all samples before extraction. The mean wet weight of medial basal hypothalamic (MBH) and striatal samples for animals in the acetaldehyde group was, respectively, 22.58 mg and 56.23 mg; for animals in the ethanol group, 18.90 mg and 54.45 mg, respectively; for analysis in the saline group, 19.70 mg and 49.15 mg, respectively, and in the treatment control group 18.18 mg and 49.73 rag, respectively.
Experimentalprocedure The animals weighing 330-360 g at the start of the
study were housed individually in wire mesh cages (26 x 34 x 20 cm) and were adapted to a 12:12 h light/dark cycle (lights off 07.00 h) for two weeks prior to the commencement of experimentation. Intravenous catheters (SP 28 Dural Plastic, o.d. 0.88 mm, i.d. 0.40 mm) were surgically implanted into the jugular vein of the animals under anaesthesia (pentobarbitone sodium 60 mg/ml; 1 ml/kg, i.p.) according to our established procedures 22.23,26,28,29.35. After reduction to 90% of their free-feeding body weights, the rats were randomly assigned to one of four treatment groups: schedule induced acetaldehyde self-injection (AcH SISI), schedule-induced ethanol selfinjection (etoh SISI), schedule-induced saline self-injection control (saline SISI control) and treatment control. Rats in the treatment control group were not removed from the home-cage evironment nor exposed to drug or saline control treatment. Animals in the schedule-induced self-injection (SISI) groups were given the opportunity to individually self-infuse either drug or saline control solution for 1 h/day over 20 consecutive days between 09.00 and 12.00 h. On day 20, after 60 min in the operant test chambers each rat was removed and immediately decapitated. Details of our procedure for schedule-induced intravenous self-injection of acetaldehyde, ethanol and other drugs are well established and have been published elsewhere 22-24,26,28,29,32,35. In brief the schedule of acetaldehyde self-injection was as follows. When an animal pressed the operant bar, a syringe infusion pump (Sage Instruments, model 341) was activated for 5 s and an infusion of fluid (0.07 ml) was delivered into the jugular vein. During the 5-s infusion interval, additional bar presses did not reactivate the pump and were not recorded. All infusions during each 1-h test session were automatically monitored on cumulative recorders. In operation, the fixed-time 1 min (FT-1) schedule delivered Noyes food pellets (45 rag) non-contingently at the rate of one pellet per minute, into the food pellet dispensing units attached to one side wall of the modified operant test chambers. A 1% v/v acetaldehyde solution (2.32 mg/kg/infusion) was used. The procedure used for schedule-induced ethanol self-injection was identical to that used for the AcH SISI group and has been reported elsewhere24,32. A 20% ethanol solution (i.e. 44.33 mg/kg/infusion) was chosen, as this has been found to be the optimum dose for schedule-
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125 induced self-injection of ethanol in our laboratory24.3L Animals in the saline SISI control group were allowed to self-inject a 0.9% (v/v) NaC1 solution which was also the vehicle control for the acetaldehyde and ethanol solutions. Animals in the treatment control group were maintained in their homecages without exposure to the test situation or drug treatment for the entire duration of the experiment. As in previous reports22.23 the acetaldehyde (99.5%, B.D.H. Chemicals, Australia) was freshly prepared for intravenous administration prior to each test session by diluting it in 0.9% NaC1. The acetaldehyde sample was regularly distilled before use and then diluted and stored at 4 °C. The purity of the acetaldehyde sample was analyzed regularly by NMR spectroscopy (Perkin-Elmer, R32A). For ethanol self-injection 95% ethanol (Ajax-Chemicals, Sydney, Australia) was prepared for intravenous administration prior to each test session by mixing it in 0.9% NaC1 to make up the 20% v/v ethanol solution. RESULTS
Self-injection data For purposes of statistical analysis, the overall mean number of self-infusions for each animal over 20 days in the SISI treatment groups were compared in order to test the hypothesis that the population frequency distributions differ. Results of the KruskalWallis one-way analysis of variance by ranks indicated no significant differences between the SISI treatment groups (H = 4.731, df = 2, P > 0.05). The overall mean number of self-infusions for animals in the different treatment groups are as follows: Acetaldehyde ~ = 15.99, Ethanol ~ = 9.25 and Saline ~ = 14.18.
Dopamine and salsolinol levels Levels of dopamine and salsolinol were obtained from the medial basal hypothalamus and striatum of rats in each of the four treatment groups. The Kruskal-Wallis test was conducted on the obtained concentrations of dopamine (pmol/mg) and salsolinol (pmol/mg). The means for levels of MBH salsolinol and dopamine for animals in the four treatment groups are as follows in pmol/mg wet brain tissue: acetaldehyde (0.72 and 3.90), ethanol (0.01 and 2.87), saline (0.01 and 3.84) and treatment control
(0.02 and 3.34). The respective means for levels of striatal salsolinol and dopamine (pmol/mg wet brain tissue) are as follows: acetaldehyde (3.36 and 55.50), ethanol (0.02 and 52.37), saline (0.02 and 65.48), and treatment control (0.01 and 63.97). A Bonferroni adjustment was used 16to adjust the critical values of the individual tests so that the Type 1 error rate per analysis was set at a i = 0.0125 with an overall experimentwise error rate of E = 0.05 to allow for the fact that four separate analyses were performed in the same experiment. Results of the Kruskal-Wallis test comparing the salsolinol levels for medial basal hypothalamus (MBH) of rats showed a significant difference between treatment groups (H = 13.186, df = 3, P < 0.005). However, the Kruskal-Wallis performed on dopamine levels from medial basal hypothalamus of rats did not support the test hypothesis for difference among treatment groups (H = 7.742, df = 3, P > 0.05). Paired comparisons of frequency distributions for salsolinol from MBH were carried out by means of the Mann-Whitney U-test. Findings showed there was significantly more salsolinol for the acetaldehyde SISI group in comparison with the ethanol SISI (U = 0, P < 0.002), and saline SISI control group (U = 0, P < 0.004) but not Treatment control (U = 3.5, P > 0.015) group (Fig. 1). Results of the Kruskal-Wallis one-way analysis of variance comparing the four different treatment groups on striatal salsolinol levels and dopamine levels are shown in Fig. 1. There was a significant difference between treatment groups on salsolinol (H = 12.823, df = 3, P < 0.005) but not on dopamine (H = 8.836, df = 3, P > 0.05) levels. Mann-Whitney U-test comparisons showed animals in the acetaldehyde SISI group to have significantly more striatal salsolinol than animals in all the other treatment groups (U = 0, P < 0.002). (See Fig. 1). DISCUSSION The data from this experiment demonstrate the presence of the dopamine-acetaldehyde condensation product, salsolinol, in the medial basal hypothalamus (MBH) and striatum of all experimental animals (Fig. 1). The finding of: (i)a significantly greater concentration of salsolinol in the MBH of animals in the acetaldehyde SISI group relative to the ethanol
126 and saline treatment groups: and (ii) a significantly greater concentration of salsolinol in the striatum of animals in the acetaldehyde SISI group relative to the ethanol SISI, saline SISI and Treatment control groups, provides evidence for the possibility of an acetaldehyde-mediated mechanism involving salsolinol in schedule-induced acetaldehyde self-injection. The finding of trace levels of salsolinol in the hypothalami and striatum of the ethanol SISI, saline SISI control and Treatment control rats agree with other reports on control animals31,34, although it is noteworthy that different strains of rats were used in each of the studies referred to, including the present experiment (Sprague-Dawley3t, Wistar34, Long-Evans, in the present report). The finding of low saisolinol levels in animals in the ethanol SISI and saline SISI control groups in comparison with the acetaldehyde SISI group is interesting in this context (Fig. 1) for a number of reasons. First, it was demonstrated that although there was no significant difference between behavioural measures (rate of self-infusion) for animals in the acetaldehyde SISI, ethanol SISI and saline SISI control groups these groups differed significantly in salsolinol levels. Second, the significant difference in salsolinol levels for rats in the acetaldehyde SISI group relative to the ethanol S1SI group adds evidence to other reports of differences between the effects of these two compounds 5.7.36. However, it is relevant to note that while the overall amount of ethanol consumed was nearly 10 times that of acetaldehide, acetaldehyde is reported to be at least 10-30 times more potent than ethanol due to its chemically reactive aldehyde group 1.2.17. Previous findings show that increased hypothalamic and striatal salsolinoi levels have been found in rats after 10 months chronic ethanol exposure 21 and in rat limbic forebrain after 150 days chronic exposure to ethanoP 0. Whereas acute treatment of rats with pharmacological doses of ethanol failed to increase hypothalamic or other brain regional levels of salsolinol (Smythe and Nicholson, unpublished observation). The possibility must therefore be entertained that the 20 days of ethanol self-injection may not have been long enough to lead to a disturbance in dopamine status and resultant rise in salsolino121. Other studies have shown that rats would only self-administer acetaldehyde intraventricularly but not ethanoF suggesting that acetaldehyde rather
than ethanol itself may mediate the reinforcing effects of ethanol in the brain 3,5,7. Further reports have also found that for some pharmacological actions, acetaldehyde and ethanol are antagonistic14,1L These factors may have some bearing on why no salsolinol was formed in rats given ethanol. It is of interest to note that there was considerable variability among the salsolinol concentrations in MBH and striatum for animals in the acetaldehyde SISI group, whereas the same cannot be said for animals in the ethanol SISI group (Fig. 1). Since neither acetaldehyde levels nor enzyme activity was measured, no firm conclusions can be drawn. However, large individual variation in sensitivity to acetaldehyde has been reported by other researchers 3~. It has been suggested that such individual differences may result from variability in the metabolism of acetaldehyde, which in turn could lead to higher circulating concentrations, associated perhaps with an increased release of catecholamines 36. This, in turn, could promote formation of condensation products t8. It is reported that elevated acetaldehyde levels can result from faster production, slower removal or a combination of both 36. It is not possible on the basis of the present findings to claim a specific association between the increased salsolinol levels observed in MBH and striatum of animals in the acetaldehyde SISI group and dopamine levels for these same animals. No significant difference was shown between the acetaldehyde SISI group and the saline SISI control, ethanol SISI and treatment control groups for MBH dopamine concentrations (Fig. 1). The overall MBH dopamine levels from all treatment groups in the present study (~ = 3.487) are in keeping with those reported previously for control rats 34. Striatal dopamine levels for the acetaldehyde SISI group were not significantly different from those of the other three treatment groups. However, the pattern of high dopamine concentrations in striatum of animals from all four treatment groups in the present study (~ = 59.330) relative to the lower MBH dopamine concentrations (~ = 3.487), agrees with previous findings 30 (see Fig. 1.). These data suggest a role for salsolinol in scheduleinduced acetaldehyde self-injection and provide support for speculation 22,23 regarding involvement of acetaldehyde mediated mechanisms in this behavioural phenomenon.
127 ACKNOWLEDGEMENT
tional Health and Medical Research Council (NH and MRC) of Australia.
This work was supported by a grant from the Na-
REFERENCES 1 Akabane, J., Pharmacological aspects of manifestation of the acute after-effects of alcoholic beverages: a role of acetaldehyde in alcoholism, Med. J. Shinsu Univ., 5 (1960) 113-122. 2 Akabane, J., Aldehydes and related compounds in alcohols and derivatives. In J. Tremolieres (Ed), International Encyclopedia of Pharmacology and Therapeutics Section 20, Vol. 2, Pergamon Press, Oxford, 1970, p. 523-560. 3 Amir, S., Brown, Z. and Amit, Z., The role of acetaldehyde in the pharmacological effects of ethanol. In H. Rigter and J.C. Crabbe Jr. (Eds.), Alcohol Tolerance and Dependence, Elsevier, Amsterdam, 1980, pp. 317-337. 4 Amit, Z., Brown, Z.W. and Rockman, G.E., Possible involvement of acetaldehyde, norepinephrine and their tetrahydroisoquinoline derivatives in the regulation of ethanol self-administration, Drug Alcohol Depend., 2 (1977) 495-500. 5 Amit, Z., Brown, Z.W., Rockman, G.E., Smith, B. and Amir, S., Acetaldehyde: a positive reinforcer mediating ethanol consumption. In H. Begleiter (Ed.), Biological Effects of Alcohol, Plenum Press, New York, 1980, pp. 413-423. 6 Brien, J.F., Andrews, P.J., Loomis, C.W, and Page, J.A., Gas-liquid chromatographic determination of salsolinol in the striatum of rat brain using the calcium carbimide-ethanol interaction, Can. J. Physiol. Pharmacol., 61 (1983) 632. 7 Brown, Z.W., Amit, Z. and Rockman, G.E., Intraventricular self-administration of acetaldehyde, but not ethanol in naive laboratory rats, Psychopharmacology, 64 (1979) 271-276. 8 Cohen, G. and Collins, M.A., Alkaloids from catecholamines in adrenal tissue: possible role in alcoholism, Science, 167 (1970) 1749-1751. 9 Davis, V.E. and Walsh, M.J., Alcohol, amines and alkaloids: a possible biochemical basis for alcohol addiction, Science, 167 (1970) 1005-1007. 10 Dean, R.A., Henry, D.P., Bowsher, R.R. and Furnay, R.B., A sensitive radioenzymatic assay for the simultaneous determination of salsolinol and dopamine, Life Sci., 27 (1980) 403. 11 Duncan, M.W. and Smythe, G.A., Salsolinol and dopamine in alcohol beverages, Lancet, i (1982) 904. 12 Duncan, M.W., Smythe, G.A. and Clezy, P.S., GC/MS methodology for simultaneous assay of salsolinol, dopamine, norepinephrine, dihydroxyphenylacetic acid and dihydroxyphenylethanol, Biorned. Mass Spec., in press. 13 Duncan, M.W., Smythe, G.A., Nicholson, M.V. and Clezy, P.S., Comparison.of HPLC/ED and GC/mass fragmentographic methods for the assay of salsolinol, dopamine and dopamine metabolites in food and beverage samples, J. Chromatogr., in press'. 14 Friedman, H.S., Matsuzaki, S,, Choe, S.-S., Fernando, H.A., Celis, A., Zaman, Q. and Lieber, C.S., Demonstration of dissimilar acute haemodynamic effects of ethanol and acetaldehyde, Cardiovasc. Res., 13 (1979) 477-487.
15 Hald, J., Jacobsen, E. and Larsen, V., The antagonism between alcohol and acetaldehyde in rabbits, Acta Pharmacol. Toxicol., 8 (1952) 164-170. 16 Harris, R.J., A Primer of Multivariate Statistics, Academic Press, New York, 1975, p. 98. 17 Holtzman, S.G. and Schneider, F.H., Comparison of acetaldehyde and ethanol: depression of motor activity in mice, Life Sci., 14 (1974) 1143-1250. 18 Lindros, K.O., Acetaldehyde - its metabolism and role in the actions of alcohol. In Y. Israel, F.B. Glaser, H. Kalant, R.E. Popham, W. Schmidt and R.G. Smart (Eds.), Research Advances in Alcohol and Drug Problems, Vol. 4, Plenum Press, New York, 1978, pp. 111-176. 19 Myers, R.D. and Melchior, C.L., Differential actions on voluntary alcohol intake of tetrahydroisoquinolines or a flcarboline infused chronically in the ventricle of the rat, Pharmacol. Biochem. Behav., 7 (1977) 381-392. 20 Myers, R.D. and Veale, W.L., Alterations in volitional alcohol intake produced in rats by chronic intraventricular infusions of acetaldehyde, paraldehyde or methanol, Archs. Int. Pharmacodyn., 180 (1969) 100-113. 21 Myers, W., McKenzie, L., Ng, K.T., Singer, G., Smythe, G.A. and Duncan, M.W., Salsolinol and dopamine in rat medial basal hypothalamus after chronic ethanol exposure, Life Sci., 36 (1985) 309-314. 22 Myers, W.D., Ng, K.T., Marzuki, S., Myers, R.D. and Singer, G., Alteration of alcohol drinking in the rat by peripherally self-administered acetaldehyde, Alcohol, 1 (1984) 229-236. 23 Myers, W.D., Ng, K.T. and Singer, G., Ethanol preference in rats with a prior history of acetaldehyde self-administration, Experientia, 40 (1984) 1008-1010. 24 Oei, T.P.S. and Singer, G., Effects of a fixed time schedule and body weight on ethanol self-administration, Pharmacol. Biochem. Behav., 10 (1979) 767-770. 25 O'Neill, P.J. and Rahwan, R.G. Modified electron-capture GLC assay for salsolinol in brain tissue. J. Pharmacol. Sci., 66 (1977) 893-895. 26 Papasava, M., Oei, T.P.S. and Singer, G., Low dose cocaine self-administration by naive rats: Effects of body weight and a fixed time one minute food delivery schedule, Pharmacol. Biochem. Behav., 15 (1981) 485-488. 27 Riggin, R.M. and Kissinger, P.T., Determination of tetrahydroisoquinoline alkaloids in biological materials with high performance liquid chromatography, Anal. Chem., 49 (1977) 530-533. 28 Singer, G., Oei, T.P.S. and Wallace, M., Schedule-induced self-injection of drugs, Neurosci. Biobehav. Rev., 6 (1982) 77'--83. 29 Singer, G., Simpson, F. and Lang, W.J., Schedule-induced self-injection of nicotine with recovered body weight, Pharmacol. Biochem, Behav., 9 (1978)387-389. 30 Sjoquist, B., Liljequist, S. and Engel, J., Increased salsolinol levels in rat striatum and limbic forebrain following chronic ethanol treatment, J. Neurochem., 39 (1982) 259-262. 31 Sjoquist, B. and Magnusson, E., Analysis of salsolinol and
128 salsoline in biological samples using deuterium-labelled internal standards and gas chromatography-mass spectrometry, J. Chromatogr., 183 (1980) 17-24. 32 Smith, D., Oei, T.P.S., Ng, K.T. and Armstrong, S., Rat self-administration of ethanol: enhancement by darkness and exogenous melatonin, Physiol. Behav., 25 (1980) 449-455. 33 Smythe, G.A., Bradshaw, J.E. and Vining, R.F., Hypothalamic monoamine control of stress-induced adrenocorticotropin release in the rat, Endocrinology, 113 (1983) 1062-1071.
34 Smythe, G.A., Duncan, M.W. and Bradshaw, .I.E., Salsolinol is a potent stimulus for prolactin secretion in the rat: evidence for hypothalamic dopamine blockade, IRCS Med. Sci. (Biochem.), 9 (1981) 472-473. 35 Takahashi, R.N. and Singer, G., Effects of body weight levels on cannabis self-injection, Pharmacol. Biochem. Behay., 13 (1980) 877-881. 36 von Wartburg, J.P., Acetaldehyde. In M. Sandier (Ed.), Psychopharmacology of Alcohol, Raven Press, New York, 1980, pp. 137-147.