Regional brain contents of serotonin, dopamine and their metabolites in the selectively bred high- and low-alcohol drinking lines of rats

Alcohol, Vol. 6, pp. 317-320. ©Pergamon Press plc, 1989. Printed in the U.S.A.

0741-8329/89 $3.00 + .00

Regional Brain Contents of Serotonin, Dopamine and Their Metabolites in the Selectively Bred Highand Low-Alcohol Drinking Lines of Rats M. A. G O N G W E R , J. M. M U R P H y , t

W. J. M c B R I D E , L. L U M E N G A N D T . - K . LI

Alcohol Research Center, Departments of Psychiatry, Medicine and Biochemistry Institute of Psychiatric Research and Regenstrief Institute Indiana University School of Medicine and R.L. Roudebush Veterans Administration Medical Center, Indianapolis, IN 46202 R e c e i v e d 13 January 1989; A c c e p t e d 27 M a r c h 1989

GONGWER, M. A., J. M. MURPHY, W. J. McBRIDE, L. LUMENG AND T.-K. LI. Regional brain contents of serotonin, dopamine and their metabolites in the selectively bred high- and low-alcohol drinking lines of rats. ALCOHOL 6(4) 317-320, 1989.--The contents of dopamine (DA), serotonin (5-HT) and their primary acid metabolites were assayed in ten brain regions of the selectively bred high-alcohol drinking (HAD) and low-alcohol drinking (LAD) lines of rats. Compared with the LAD line, the contents of 5-HT and/or 5-hydroxyindoleacetic acid were approximately 10-20% (p<0.05) lower in several brain regions of the HAD line (cerebral cortex, striatum, nucleus accumbens, septal nuclei, hippocampus and hypothalamus). The levels of DA, 3,4-dihydroxyphenylacetic acid and homovanillic acid were also 10-20% lower in the nucleus accumbens and anterior striatum (p<0.05) of the HAD animals. These data are in agreement with previous findings that comparatively lower levels in 5-HT and DA systems are associated with high-alcohol drinking in rodents and support the involvement of certain 5-HT and DA pathways in the mediation of alcohol drinking behavior. High-alcohol drinking (HAD) rats Low-alcohol drinking (LAD) rats Regional brain monoamines 3,4-Dihydroxyphenylacetic acid Homovanillic acid Serotonin 5-Hydroxyindoleacetic acid

STUDIES on selectively bred rats of the alcohol-preferring P line have indicated that ethanol-naive P animals consistently have lower contents of serotonin (5-HT) and its primary metabolite, 5-hydroxyindoleacetic acid (5-HIAA), in several brain regions (e.g., cerebral cortex, striatum, nucleus accumbens, hippocampus and hypothalamus), when compared with ethanol-naive rats of the alcohol-nonpreferring NP line (14,18). Research on inbred strains of mice has similarly indicated that relatively low brain 5-HT levels are associated with high alcohol preference (29,30). Compared with rats of the NP line, P rats have also been found to have lower contents of dopamine (DA) and its metabolites, 3,4dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in the nucleus accumbens (18). In agreement with the preceding findings, an inverse relationship has been observed between ethanol drinking scores of rats from the N/Nih heterogeneous stock and the content of 5-HT and DA in some brain regions (17), However, the findings for the N/Nih rats were not as robust as with the P and NP rats and, since the N/Nih rats in that study were not

Dopamine

ethanol naive, it could not be ruled out that the differences in brain monoamines may have arisen from the different quantities of ethanol consumed by the animals that exhibited preference. The P and NP rats were derived from a Wistar stock (11). A breeding program was recently undertaken to select new lines of high-alcohol drinking (HAD) and low-alcohol drinking (LAD) rats based upon the N/Nih rats as the foundation stock (8,10). The purpose of the present study was to determine if, after eight generations of selective breeding, the HAD and LAD rats would exhibit differences in regional brain monoamine contents comparable to those previously observed between the P and NP lines (14,18). Because the HAD and LAD lines are derived from a different foundation stock, agreement with the findings in the P and NP lines would provide considerable validity for the hypothesis that deficits in specific brain 5-HT and DA systems are involved in excessive alcohol drinking behavior (2,13).

~Requests for reprints should be addressed to James M. Murphy, Institute of Psychiatric Research, Indiana University School of Medicine, 791 Union Drive, Indianapolis, IN 46202.

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TABLE 1

TABLE 2

CONTENTS OF 5-HT AND 5-HIAA IN CNS REGIONS OF THE HAD AND LAD LINES OF RATS

CONTENTS OF DA, DOPAC AND HVA IN CNS REGIONS OF THE HAD AND LAD LINES OF RATS

Region

Frontal Cortex LAD HAD Posterior Cortex LAD HAD Anterior Striatum LAD HAD Posterior Striatum LAD HAD Nucleus Accumbens LAD HAD Septal Nuclei LAD HAD Hippocampus LAD HAD Hypothalamus LAD HAD Thalamus LAD HAD Tegmentum LAD HAD

nmol/g Tissue Wet wt. (mean = SEM) 5-HT 5-HIAA

8.0 ± 0.17 7.3 ± 0.07*

2.7 ___ 0.13 2.4 __+ 0.11

4.5 ± 0.17 4.1 ± 0.14

1.8 ± 0.07 1.6 ± 0.07*

2.6 ± 0.11 2.7 ± 0.13

1.9 2 0.10 1.6 --- 0.08

5.6 ± 0.32 4.6 ± 0.24*

4.1 + 0.22 3.3 ± 0.12+

10.0 ± 0.34 9.3 ± 0.16

3.6 ± 0.14 3.1 ± 0.13'

8.i ± 0.42 7.1 ± 0.33

2.8 -* 0.11 2.4 _* 0.06+I.

4.1 + 0.17 3.5 ± 0.17"

2.2 -* 0.09 1.7 _* 0.07+

9.5 _+ 0.28 8.5 ± 0.17+

3.7 ± 0. t6 3.1 ± 0.08t

7.4 ± 0.29 6.8 ± 0.29

3.8 ± 0.18 3.4 ± 0.26

7.8 ± 0.27 7.1 ± 0.23

4.5 -* 0.19 4.0 -~ 0.21

*p<0.05 and +p<0.01 by t-test for HAD compared with LAD. i N - 10-12L

METHOD Adult male rats (approximately 300-500 g) were from the $8 generation of the H A D (N = 12) and L A D (N = 12) lines (10). The rats were housed individually in a colony r o o m on a 12 hour light/dark cycle; lights were on at 0600. Food and water were always available ad lib and the rats used in this study were never exposed to ethanol. In order to assess the genetic predisposition to alcohol preference or nonpreference in these lines after eight generations of selective breeding, other H A D and L A D male rats from the same generation were tested over a four-week period for the free-choice consumption of 10% (v/v) ethanol in the presence of ad lib food and water (9,11). Rats of the H A D line ( N = 3 0 ) consumed 5.6---0.4 g ethanol/kg body wt/day, and L A D rats (N = 31) averaged 1.0 ± 0.2 g/kg/day. All rats were handled daily for at least one week and were killed between 1100-1500 hours by the near-freezing technique (26). The brains were rapidly removed and dissected into ten regions in a cold box at - 20°C or lower. With the ventral surface of the brain up, a coronal cut was made 2 m m anterior to the optic chiasm to obtain the frontal cortex. The nucleus accumbens, septal nuclei and anterior striatum were dissected from a 2 m m coronal section resulting from a second cut at the optic chiasm (4). The posterior cortex, posterior striatum, hippocampus, hypothalamus,

Region

Frontal Cortex LAD 0.48 HAD 0.50 Posterior Cortex LAD 2.3 HAD 2.5 Anterior Striatum LAD 68 HAD 55 Posterior Striatum LAD 42 HAD 42 Nucleus Accumbens LAD 51 HAD 46 Septal Nuclei LAD 15 HAD 14 Hippocampus LAD 0.27 HAD 0.19 Hypothalamus LAD 2.7 HAD 2.6 Thalamus LAD 2.0 HAD 2.1 Tegmentum LAD 1.6 HAD 1.8

nmol/g Tissue Wet wt. (mean ~-_ SEM) DA DOPAC H VA

± 0.06 ± 0.06

0.21 _+ 0.02 0.23 = 0.03

(I.46 ± 0.05 0.51 ± 0.06

-+ 0.3 -+ 0.3

0.22 + 0.02 (I.22 ± 0.02

(I.22 z 0.1)2 0.18 ± 0.02

+ 3 ± 3,

6.5 5.4

~ 0.4 -+ OAt

.3.4 2.7

z 0.2 -~ 0.2*

± 4 ± 4

3.1 3.1

_+ 0.4 ± 0.4

2.8 2.7

± 0.3 _+ 0.3

= 2 -+ 2*

6.0 5.1

-+ 0.1 ± 0.2+

3.0 2.4

-+ 0.1 -+ 0.1+

= 3 -+ 2

1,7 1.3

-+ 0.2 _+ 0.1

1.4 1.2

= 0.2 (8l ± 0.1

± 0.09 ± 0.07

0.01 ± 0.004 (7) 0.03 ± 0.001 (4)

0.12 ± 0.01 0.11 ± 0.01

± 0.1 -+ 0.2

0.27 _+ 0.02 0.24 ± 0.02

0.19 _+ 0.02 0.17 ± 0.01

± 0.3 _* 0.3

0.34 ± 0.04 (9) 0.30 _+ 0.04

0.36 ± 0.03 (6) 0.50 ± 0.06 (31

± 0.2 ± 0.2

0.31 ± 0.05 0.31 _- 0.04

0.50 ± 0.11 (6) 0.65 ± 0.06 (7)

*p<0.05 and tp<0.005 by t-test for HAD compared with LAD. IN = 10-12 unless otherwise noted.)

thalamus and tegmentum were dissected according to their identifiable landmarks form the remaining, caudal portion of the brain. The colliculi, pons, medulla and cerebellum were discarded. The brain parts were frozen at - 70°C until assayed for the contents of DA, D O P A C , H V A , 5-HT and 5 - H I A A by HPLC with electrochemical detection according to published procedures (14,15). Statistical differences between the L A D and H A D groups were determined by Student's t-test. RESULTS The regional brain contents of 5-HT and 5-H1AA for the H A D and L A D rats are given in Table 1. With one exception (5-HT in the anterior striatum), both 5-HT and 5 - H I A A levels tended to be lower in all CNS regions of the H A D compared with the L A D rats. The differences were significant for 5-HT ( 9 - 1 8 % ) in the frontal cortex, posterior striatum, hippocampus and hypothalamus, and for 5 - H I A A (14-23%) in the posterior cortex, posterior striatum, nucleus accumbens, septal nuclei, hippocampus and hypothalmus. Table 2 s h o w s the regional brain contents of DA, D O P A C and H V A for the H A D and L A D rats. Compared with the L A D group, the levels of D A and its metabolites were significantly lower in the anterior striatum (19-21%) and nucleus accumbens (10-20%) of the H A D animals, but the lines did not differ significantly in the

BRAIN MONOAMINES AND ALCOHOL INTAKE

319

TABLE 3 SUMMARYOF DIFFERENCESIN REGIONALBRAIN5-HT AND 5-HIAA CONTENTSOF RATS SELECTIVELYBRED FOR ALCOHOLPREFERENCE

Region Frontal Cortex Posterior Cortex Posterior Striatum Nucleus Accumbens Septal Nuclei Hippocampus Hypothalamus

HAD Line (% LAD Values) 5-HT 5-HIAA 91" 91 82* 93 88 85* 89*

89 89* 80* 86* 86* 77* 84*

TABLE 4 SUMMARYOF DIFFERENCESIN REGIONALBRAINDA, DOPAC AND HVA CONTENTSOF RATS SELECTIVELYBRED FOR ALCOHOLPREFERENCE

P Line (% NP Values) 5-HT 5-HIAA 82* na 73* 79* na 78* 79*

88* na 89 92 na 74* 76*

*p<0.05 for the alcohol-preferring line (HAD or P) compared with the nonpreferring line (LAD or NP) by t-test. na = Data not available. Data for P and NP rats are from published reports (14,18), except previously unpublished data for the posterior striaturn.

other eight brain regions. DISCUSSION The regional brain differences in the contents of 5-HT and 5-HIAA (Table 3) and DA, DOPAC and HVA (Table 4) between the HAD and LAD rats are compared with findings reported previously for P and NP lines (14,18). A considerable degree of concordance is observed. Since the rats in the present study and the P and NP rats in prior studies (14,18) were naive to ethanol, the observed differences in the contents of brain monoamines are not a consequence of ethanol exposure, but are due to the selective breeding for alcohol preference and nonpreference. The genetic basis for the lower contents of the monoamines and metabolites in CNS regions of the P and HAD rats relative to the NP and LAD rats has yet to be identified. The lower contents could result from a slower rate of monoamine metabolism (lower synthesis and/or neuronal activity), or a lower proportion of axon terminals and/or fewer neurons. In the present study with the HAD and LAD lines and in previous studies with the P and NP lines (14,18) and with the N/Nih rats (17), the alcohol-preferring animals tended to have lower contents of 5-HT and 5-HIAA in most forebrain regions. The widespread differences in the contents of 5-HT and 5-HIAA may indicate that any one or several of the 5-HT pathways which innervate the affected brain areas are involved in alcohol preference. However, previous evidence suggests that the 5-HT dorsal ascending pathway of the dorsal raphe nucleus, which sends projections to several brain areas including the nuclueus accumbens and striatum (25), may be specifically activated by ethanol (5), and chronic alcohol consumption by the P line of rats produces ethanol tolerance within the 5-HT pathway projecting to the nucleus accumbens (20). Selective breeding for alcohol preference appears to produce lower contents of DA and its metabolites that are restricted to the nucleus accumbens and perhaps also the anterior striatum (Tables

Region

HAD Line (% LAD Values) DA DOPAC HVA

P Line (% NP Values) DA DOPAC HVA

Anterior Striatum Nucleus Accumbens

81" 90*

98 76*

83* 85*

79* 80*

84 76*

84* 69*

*p<0.05 for the alcohol-preferring line (HAD or P) compared with the nonpreferring line (LAD or NP) by t-test. Data for the P and NP rats are from previously published data (18). 2, 4). The nucleus accumbens receives its DA projections primarily from cell bodies in the ventral tegmental area (VTA) (1), and the lower contents of DA, DOPAC and HVA indicate a relative decrease in the functional capacity and/or number of neurons in this pathway for rats of the HAD compared with the LAD line. The DA pathway from the VTA to the nucleus accumbens is an important component of the brain reward system (24,28) and has been implicated in the reinforcing properties of several drugs of abuse, including ethanol (3,6). The anterior striatum receives its DA projections mostly from the substantia nigra, but the anteromedial portion of the striatum does receive DA innervation from the VTA (1) which may account for the lower contents of DA, DOPAC and HVA observed in this area for the HAD compared with the LAD line. If this is the case, then it appears that the lower DA levels in the alcohol-preferring rats may be limited to specific projections from the VTA since lower contents of DA and its metabolites (Table 2) were not observed in other CNS regions (e.g., frontal cortex and septal nuclei) innervated by the VTA (1). Rats of the LAD and NP lines, like most stock rats, consume low amounts of 10% (v/v) ethanol when given free choice, while rats of the HAD and P lines find alcohol rewarding. Animals from both preferring lines, in the presence of ad lib food and water, voluntarily consume amounts of 10% (v/v) ethanol that exceed 5 g/kg body wt/day [vide methods (10)] and will perform an operant response to obtain alcohol (7, 22, 23). It has also been demonstrated that rats of the P line will self-administer ethanol intragastrically, presumably for the postingestive pharmacological properties (27). If the regionally lower contents of 5-HT, DA and their metabolites of the alcohol-preferring lines reflect a relative decrease in neuronal capacity or functioning associated with alcoholseeking behavior, pharmacological manipulations that increase the synaptic availability of these monoamine transmitters should attentuate alcohol consumption. In agreement with this supposition, 5-HT and DA uptake inhibitors have consistently been found to attenuate alcohol consumption in these lines of rats (12, 16, 19, 21). In summary, the results of the present study corroborate previous findings that relatively lower contents of DA, 5-HT and their metabolites in some CNS regions are associated with high alcohol preference. The present data also provide additional evidence for the involvement of certain 5-HT and DA pathways in regulating alcohol drinking behavior (2,13).

ACKNOWLEDGEMENTS The authors gratefully acknowledge the skillful technical assistance of Mr. Steve Cunningham. Supported by AA-03243, AA-07462 and AA07611.

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(i()NGWER £T,t7

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