Ecotoxicology, 4, 1-4 (1995)
Alteration in the neurotransmitter levels in the brain of the freshwater snakehead fish (Channa punctatus) exposed to carbofuran K R I S H N A G O P A L 1 and M U R A L I D H A R
RAM 2
1Aquatic Toxicology Laboratory, Industrial Toxicology Research Centre, Post Box No. 80, M G Marg, Lucknow-226001, India 2M D Shukla Inter College, Nadan Mahal Road, Lucknow, India
Received 4 August 1993; revised and accepted 31 January 1994
Channa punctatus a freshwater fish exposed to carbofuran (0.6 mg 1-1) for 15 days exhibited alterations in norepinephrine (NE), dopamine (DA) and serotonin (5-HT) levels. NE levels increased in the cerebellum and decreased in the medulla and cerebral cortex relative to the controls. Increased DA levels were observed in the cerebellum and medulla whereas DA levels decreased in the cerebral cortex relative to the controls. Depletion of 5-HT was noticed in the cerebellum, medulla and cerebral cortex relative to controls. The results suggested an overall decrease in the level of neurotransmitters in the cerebral cortex region of the brain. The neurotoxic effect of carbofuran was not uniform in the brain but was localized to the brain regions which regulate motor activity and behaviour in fish. Keywords: carbofuran; neurotransmitters; cholinesterase; behavioural pattern.
Introduction
Carbofuran is a broad spectrum insecticide used extensively in agriculture. In fishes carbamate pesticides like other organophosphates are known to be acetylcholinesterase (AchE) inhibitors and to act as nerve poisons (Jash and Bhattacharya 1983). In teleosts the biogenic amines are found to control varied functions such as behavioural patterns, migration, schooling, circadian rhythms and locomotor activity (Fingerman and Russel 1980) and seasonal reproductive status (Sloley et al. 1986, Holdway et al. 1988). Although behavioural changes are frequently claimed to be a clue of the earliest sign of toxicity, insufficient correlative studies have been reported with particular reference to brain neurotransmitters (Fingerman and Russel, 1980, Sastry and Siddiqui 1982, Saxena and Mani 1985, Holdway et al. 1986). In the present study attempts have been made to investigate the level of neurotransmitters in discrete areas of fish brain to determine the neurotoxic potential of carbofuran.
0963-9292 t~) 1995 Chapman & Hall
2
Gopal and Ram
Materials and methods
Adult freshwater fish Channa punctatus weighing (10 + 2 g) were collected from local resources and acclimatized to standard laboratory conditions. The toxicity tests were conducted in wide-mouthed glass containers with 201 of tap water with temperature 20 + 2°C, pH 6.8-7.0, dissolved oxygen 6.4-7.2 mg 1-1, alkalinity 95-100 mg 1-1 as CaCO3 and hardness l18-120mg 1-1 as CaCO3. Carbofuran dissolved in water was added to the model ecosystem (glass container) containing the test fish to attain the desired nominal concentration (0.6 mg 1-1). A parallel group of control fish was also maintained under identical conditions. Exposures were carried out over a period of 15 days. The brains of six fish from both experimental and control tanks were removed and deep frozen at 0°C. Parts of the brain were dissected out into three regions, the medulla, cerebellum and cerebral cortex, on a chilled ice glass plate and homogenized in N-butanol. Norepinephrine (NE) and dopamine (DA) were extracted in 0.1 M phosphate buffer and 5-hydroxytryptamine (5-HT) was extracted in N-heptane. Fluorescence of NE was measured at 385 nm activation/485 nm emission wavelengths. After 20 min D A was measured at 320 nm activation/385 nm emission and 5-HT was measured at 360 nm activation/470 nm emission (Jacobowitz and Richardson 1978). The fluorescence reading was converted into Hg NE, DA or 5-HT per g of tissue. Eighty per cent recovery was observed. Data was analysed by the Student's t-test (n = 6). Results and discussion
Fish exposed to carbofuran exhibited significant (p < 0.001) increases in NE in the cerebellum whereas a significant decrease (p < 0.001) was observed in the medulla and cerebral cortex. Carbofuran-exposed fish showed a significant (p < 0.001) increase in D A in the cerebellum and medulla followed by a significant decrease (p < 0.001) in the cerebral cortex (Table 1). The 5-HT level decreased (p < 0.001) in the cerebellum, medulla and cerebral cortex (Table 1). Enhanced surfacing activity, distance travelled and frequent opercular movements were observed in the fish exposed to carbofuran as symptoms of an overt toxicity. The biogenic amines are neurotransmitter substances responsible for specific kinds of functions. Catecholamines and 5-HT in the brain have been related to the aetiology of depression (Borsini et al. 1981). It has been reported that changes in the concentrations of biogenic amines may directly affect fish behaviour (Fingerman and Russel 1980). A non-lethal pulse dose of methoxychlor was found to produce an initial alteration in biogenic amine levels (Holdway and Dixon 1986). D D T was found to inhibit NE and DA and elevate the level of 5-HT in goldfish (McDonald 1979). Similarly killifish (Fundulus grandis) exposed to Aroclor 1242 for 24 h demonstrated a significant decrease in the concentration of NE and DA (Fingerman and Russel 1980). It has been reported that the 5-HT level of the cerebellum in mammals depends on the availability of tryptophan and its precursor (Fernstrom and Wurtman 1971). The selective toxicity of carbofuran on the aminergic system in various parts of the brain could have important consequences. The biogenic amine catabolizing enzyme monoamine oxidase in the brain has been associated with the control of pituitary activity in teleosts (Wigham and Ball 1976) and is firmly linked with the control of gonadal development (Change et al. 1983). Environmental changes associated with modulation of gonadotropic activity by
Alteration in neurotransrnitter levels in the brain
3
Table 1. Levels of the neurotransmitters, norepinephrine (NE), dopamine (DA) and 5hydroxytryptamine (5-HT) ~ g per g fresh tissue weight) in different parts of the brain of freshwater snakehead fish Channa punctatus exposed to carbofuran Neurotransmitters Norepinephrine (NE)
Dopamine (DA)
5-Hydroxytryptamine (5-HT)
Treatment
Cerebellum
Medulla
Cortex
Control
0.45 + 0.01
0.6 + 0.01
0.79 + 0.01
Carbofuran (0.6 mg 1-1)
0.88 + 0.01"**
0.4 + 0.01'**
0.31 + 0.003***
Control
0.16 + 0.003
0.16 _+ 0.003
0.79 + 0.004
Carbofuran (0.6 mg 1-1)
0.82 + 0.02***
0.52 + 0.004
0.17 + 0.002**
Control
0.81 + 0.02
0.87 + 0.002
0.64 + 0.004
Carbofuran (0.6 mg 1-1)
0.31 _+ 0.01"**
0.0 _+ 0.004*** 0.38 -+ 0.01"*
Each value represents the mean + SEMfor six observations. ***p < 0.001, **p < 0.01, *p < 0.05 (Student's t-test).
the brain-pituitary complex have been reported (Breton et al. 1988). It is well known that neurotransmitters play an important role in blood pressure and temperature regulation (Anand et al., 1985). Changes in surfacing activity, distance travelled and hanging duration that were observed in pesticide-exposed conditions might be due to the alteration in biogenic amines (Ram and Gopal 1991). It is assumed that the pesticides exerted their effects on these neurotransmitter substances by activating the corresponding metabolizing enzymes involved in the biosynthesis, catabolism and turnover rate. Environmental chemicals such as acrylamide and manganese reported to induce disorders in the central nervous system of mammals by modifying the neurotransmitter functions of the biogenic amines and changes in the concentration of the availability of the neurotransmitter at the synapse could modify the sensitivity of neurotransmitter receptors (Dixit et al. 1981, Agarwal et al. 1983).
Acknowledgements The authors are grateful to the Director, ITRC, Lucknow for his keen interest and guidance. Thanks are also due to Mrs Syamala Das for the typing of this manuscript.
References Agarwal, A.K., Anand, M., Zaidi, N.F. and Seth, P.K. (1983) Involvement of serotonergic receptors in endosulfan neurotoxicity. Biochem. Pharrnacol. 32, 3591-3. Aldegunde, M., Parafita, M. and Duran, R. (1986) Effect of lindane on brain monoamine metabolism. Gen. Pharmacol. 17, 633-5.
4
Gopal and Ram
Anand, M., Mehrotra, S., Gopal, K., Sur, R.N. and Chandra, S.V. (1985) Role of neurotransmitters in endosulfan induced aggressive behaviour in normal and lesioned rats. Toxicol. Lett. 24, 79-84. Borsini, F., Bendotil, C., Welkov, V., Rech, R. and Somairin, R. (1981) Immobility test effects of 5-hydroxytryptaminergic drug and role of catecholamines in the activity of some antidepressants. J. Pharm. Pharmacol. 33, 33-8. Breton, B.E., Sambroni, and Gillet, C. (1988) Gonadotropin releasing hormone (GNRH) and gonadotropin (GTH) variation around and spawning period in a wild population of roach rutiles from Lemon lake. Female Aquat. Living Res. 1, 93-9. Chang, J.P., Cook, A.F. and Peter, R.E. (1983) Influence of catecholamines in gonadotropin secretion in goldfish ( Carassius auratus). Gen. Comp. Endocrinol. 49, 22-32. Dixit, R., Hussain, R., Mukhtar, H. and Seth, P.K. (1981) Effect of acrylamide on biogenic amines levels monoamine oxidase and cathepsin-D activity of rat brain. Environ. Res. 26, 168-173. Fernstrom, J.D. and Wurtman, R.J. (1971) Brain serotonin content physiological dependence on plasma tryptophan level. Science 173, 149-52. Fingerman, S.W. and Russel, L.C. (1980) Effects of the polychlorinated biphenyl Aroclor 1242 on the locomotor activity and on the neurotransmitters dopamine and norepinephrine in the brain of gulf killifish (Fundulus grandis). Bull. Environ. Contain. Toxicol. 25, 682-7. Holdway, D.A. and Dixon, D.G. (1986) Effects of methoxychlor exposure of flagfish eggs, Jordenella floridae on hatchability of juvenile methoxychlor tolerance and whole body levels of tryptophan, serotonin and 5-hydroxyindoleacetic acid (5HIAA) Water Res. 20, 893-7. Holdway, D.A., Sloley, B.D., Munkittrick, K.R., Downer, R.G.H. and Dixon, D.G. (1988) Effect of gender and reproductive status on brain catecholamines and indoleamine levels of flagfish (Jordenella floridae). Fish Physiol. Biochem. 5, 153-8. Jacobowitz, D.M. and Richardson, J.S. (1978) Method for the rapid determination of norepinephrine, dopamine and serotonin in the same brain region. Pharmacol. Biochem. Behav. 8, 515-19. Jash, N.B. and Bhattacharya, S. (1983) Delayed toxicity of carbofuran in fresh water teleost Channa punctatus. Indian J. Exp. Biol. 17, 693-7. McDonald, T.O. (1979) Comparison of the effects of DDT and parathion on the concentration of serotonin, norepinephrine and dopamine in the brain retina/pigment epithelium choroid of the gold fish. Doctoral dissertation, Tulane University, New Orleans. Ram, M.D. and Gopal, K. (1991) Neurobehavioural changes in fresh water fish (Channa punctatus) exposed to fenitrothion. Bull. Environ. Contain. Toxicol. 47, 455-8. Ram, M.D., Verma, P.K. and Gopal, K. (1990) Effect of lindane on the levels of biogenic amines in various parts of brain of fresh water fish Channa punctatus (Bloch). J. Adv. Zool. 11, 79-84. Sastry, K.V. and Siddiqui, A.A. (1982) Chronic toxic effects of carbamate pesticide sevin on carbohydrate metabolism in a fresh water snakehead fish Channa punctatus. Toxicol. Lett. 14, 123-30. Saxena, P.K. and Mani, K. (1985) Quantitative study of testicular recruidescence in fresh water teleost Channa punctatus (Bloch) exposed to pesticides. Bull. Environ. Contain. Toxicol. 34, 597-607. Sloley, B.D. and Downer, R.G.H. (1984) Distribution of 5-hydroxytryptamine and indole alkylamine metabolites in the American cockroaches (Periplaneta americana). J. Comp. Biochem. Physiol. 79, 282-6. Sloley, B.D,, Hickie, B.E., Dixon, D.G., Downer, R.G.H. and Martin, R.J. (1986) The effect of sodium pentachlorophenate diet and sampling procedure on amine and tryptophan concentrations in the brain of rainbow trout (Salmo gairdneri, Richardson). J. Fish Biol. 28,267-77. Wigham, T. and Ball, J.N. (1976) In vivo evidence for catecholaminergic inhibition of prolactin secretion in the teleost Puntius latipinna. J. Comp. Physiol. 110, 135-43.
Alteration in the neurotransmitter levels in the brain of the freshwater snakehead fish (Channa punctatus) exposed to carbofuran K R I S H N A G O P A L 1 and M U R A L I D H A R
RAM 2
1Aquatic Toxicology Laboratory, Industrial Toxicology Research Centre, Post Box No. 80, M G Marg, Lucknow-226001, India 2M D Shukla Inter College, Nadan Mahal Road, Lucknow, India
Received 4 August 1993; revised and accepted 31 January 1994
Channa punctatus a freshwater fish exposed to carbofuran (0.6 mg 1-1) for 15 days exhibited alterations in norepinephrine (NE), dopamine (DA) and serotonin (5-HT) levels. NE levels increased in the cerebellum and decreased in the medulla and cerebral cortex relative to the controls. Increased DA levels were observed in the cerebellum and medulla whereas DA levels decreased in the cerebral cortex relative to the controls. Depletion of 5-HT was noticed in the cerebellum, medulla and cerebral cortex relative to controls. The results suggested an overall decrease in the level of neurotransmitters in the cerebral cortex region of the brain. The neurotoxic effect of carbofuran was not uniform in the brain but was localized to the brain regions which regulate motor activity and behaviour in fish. Keywords: carbofuran; neurotransmitters; cholinesterase; behavioural pattern.
Introduction
Carbofuran is a broad spectrum insecticide used extensively in agriculture. In fishes carbamate pesticides like other organophosphates are known to be acetylcholinesterase (AchE) inhibitors and to act as nerve poisons (Jash and Bhattacharya 1983). In teleosts the biogenic amines are found to control varied functions such as behavioural patterns, migration, schooling, circadian rhythms and locomotor activity (Fingerman and Russel 1980) and seasonal reproductive status (Sloley et al. 1986, Holdway et al. 1988). Although behavioural changes are frequently claimed to be a clue of the earliest sign of toxicity, insufficient correlative studies have been reported with particular reference to brain neurotransmitters (Fingerman and Russel, 1980, Sastry and Siddiqui 1982, Saxena and Mani 1985, Holdway et al. 1986). In the present study attempts have been made to investigate the level of neurotransmitters in discrete areas of fish brain to determine the neurotoxic potential of carbofuran.
0963-9292 t~) 1995 Chapman & Hall
2
Gopal and Ram
Materials and methods
Adult freshwater fish Channa punctatus weighing (10 + 2 g) were collected from local resources and acclimatized to standard laboratory conditions. The toxicity tests were conducted in wide-mouthed glass containers with 201 of tap water with temperature 20 + 2°C, pH 6.8-7.0, dissolved oxygen 6.4-7.2 mg 1-1, alkalinity 95-100 mg 1-1 as CaCO3 and hardness l18-120mg 1-1 as CaCO3. Carbofuran dissolved in water was added to the model ecosystem (glass container) containing the test fish to attain the desired nominal concentration (0.6 mg 1-1). A parallel group of control fish was also maintained under identical conditions. Exposures were carried out over a period of 15 days. The brains of six fish from both experimental and control tanks were removed and deep frozen at 0°C. Parts of the brain were dissected out into three regions, the medulla, cerebellum and cerebral cortex, on a chilled ice glass plate and homogenized in N-butanol. Norepinephrine (NE) and dopamine (DA) were extracted in 0.1 M phosphate buffer and 5-hydroxytryptamine (5-HT) was extracted in N-heptane. Fluorescence of NE was measured at 385 nm activation/485 nm emission wavelengths. After 20 min D A was measured at 320 nm activation/385 nm emission and 5-HT was measured at 360 nm activation/470 nm emission (Jacobowitz and Richardson 1978). The fluorescence reading was converted into Hg NE, DA or 5-HT per g of tissue. Eighty per cent recovery was observed. Data was analysed by the Student's t-test (n = 6). Results and discussion
Fish exposed to carbofuran exhibited significant (p < 0.001) increases in NE in the cerebellum whereas a significant decrease (p < 0.001) was observed in the medulla and cerebral cortex. Carbofuran-exposed fish showed a significant (p < 0.001) increase in D A in the cerebellum and medulla followed by a significant decrease (p < 0.001) in the cerebral cortex (Table 1). The 5-HT level decreased (p < 0.001) in the cerebellum, medulla and cerebral cortex (Table 1). Enhanced surfacing activity, distance travelled and frequent opercular movements were observed in the fish exposed to carbofuran as symptoms of an overt toxicity. The biogenic amines are neurotransmitter substances responsible for specific kinds of functions. Catecholamines and 5-HT in the brain have been related to the aetiology of depression (Borsini et al. 1981). It has been reported that changes in the concentrations of biogenic amines may directly affect fish behaviour (Fingerman and Russel 1980). A non-lethal pulse dose of methoxychlor was found to produce an initial alteration in biogenic amine levels (Holdway and Dixon 1986). D D T was found to inhibit NE and DA and elevate the level of 5-HT in goldfish (McDonald 1979). Similarly killifish (Fundulus grandis) exposed to Aroclor 1242 for 24 h demonstrated a significant decrease in the concentration of NE and DA (Fingerman and Russel 1980). It has been reported that the 5-HT level of the cerebellum in mammals depends on the availability of tryptophan and its precursor (Fernstrom and Wurtman 1971). The selective toxicity of carbofuran on the aminergic system in various parts of the brain could have important consequences. The biogenic amine catabolizing enzyme monoamine oxidase in the brain has been associated with the control of pituitary activity in teleosts (Wigham and Ball 1976) and is firmly linked with the control of gonadal development (Change et al. 1983). Environmental changes associated with modulation of gonadotropic activity by
Alteration in neurotransrnitter levels in the brain
3
Table 1. Levels of the neurotransmitters, norepinephrine (NE), dopamine (DA) and 5hydroxytryptamine (5-HT) ~ g per g fresh tissue weight) in different parts of the brain of freshwater snakehead fish Channa punctatus exposed to carbofuran Neurotransmitters Norepinephrine (NE)
Dopamine (DA)
5-Hydroxytryptamine (5-HT)
Treatment
Cerebellum
Medulla
Cortex
Control
0.45 + 0.01
0.6 + 0.01
0.79 + 0.01
Carbofuran (0.6 mg 1-1)
0.88 + 0.01"**
0.4 + 0.01'**
0.31 + 0.003***
Control
0.16 + 0.003
0.16 _+ 0.003
0.79 + 0.004
Carbofuran (0.6 mg 1-1)
0.82 + 0.02***
0.52 + 0.004
0.17 + 0.002**
Control
0.81 + 0.02
0.87 + 0.002
0.64 + 0.004
Carbofuran (0.6 mg 1-1)
0.31 _+ 0.01"**
0.0 _+ 0.004*** 0.38 -+ 0.01"*
Each value represents the mean + SEMfor six observations. ***p < 0.001, **p < 0.01, *p < 0.05 (Student's t-test).
the brain-pituitary complex have been reported (Breton et al. 1988). It is well known that neurotransmitters play an important role in blood pressure and temperature regulation (Anand et al., 1985). Changes in surfacing activity, distance travelled and hanging duration that were observed in pesticide-exposed conditions might be due to the alteration in biogenic amines (Ram and Gopal 1991). It is assumed that the pesticides exerted their effects on these neurotransmitter substances by activating the corresponding metabolizing enzymes involved in the biosynthesis, catabolism and turnover rate. Environmental chemicals such as acrylamide and manganese reported to induce disorders in the central nervous system of mammals by modifying the neurotransmitter functions of the biogenic amines and changes in the concentration of the availability of the neurotransmitter at the synapse could modify the sensitivity of neurotransmitter receptors (Dixit et al. 1981, Agarwal et al. 1983).
Acknowledgements The authors are grateful to the Director, ITRC, Lucknow for his keen interest and guidance. Thanks are also due to Mrs Syamala Das for the typing of this manuscript.
References Agarwal, A.K., Anand, M., Zaidi, N.F. and Seth, P.K. (1983) Involvement of serotonergic receptors in endosulfan neurotoxicity. Biochem. Pharrnacol. 32, 3591-3. Aldegunde, M., Parafita, M. and Duran, R. (1986) Effect of lindane on brain monoamine metabolism. Gen. Pharmacol. 17, 633-5.
4
Gopal and Ram
Anand, M., Mehrotra, S., Gopal, K., Sur, R.N. and Chandra, S.V. (1985) Role of neurotransmitters in endosulfan induced aggressive behaviour in normal and lesioned rats. Toxicol. Lett. 24, 79-84. Borsini, F., Bendotil, C., Welkov, V., Rech, R. and Somairin, R. (1981) Immobility test effects of 5-hydroxytryptaminergic drug and role of catecholamines in the activity of some antidepressants. J. Pharm. Pharmacol. 33, 33-8. Breton, B.E., Sambroni, and Gillet, C. (1988) Gonadotropin releasing hormone (GNRH) and gonadotropin (GTH) variation around and spawning period in a wild population of roach rutiles from Lemon lake. Female Aquat. Living Res. 1, 93-9. Chang, J.P., Cook, A.F. and Peter, R.E. (1983) Influence of catecholamines in gonadotropin secretion in goldfish ( Carassius auratus). Gen. Comp. Endocrinol. 49, 22-32. Dixit, R., Hussain, R., Mukhtar, H. and Seth, P.K. (1981) Effect of acrylamide on biogenic amines levels monoamine oxidase and cathepsin-D activity of rat brain. Environ. Res. 26, 168-173. Fernstrom, J.D. and Wurtman, R.J. (1971) Brain serotonin content physiological dependence on plasma tryptophan level. Science 173, 149-52. Fingerman, S.W. and Russel, L.C. (1980) Effects of the polychlorinated biphenyl Aroclor 1242 on the locomotor activity and on the neurotransmitters dopamine and norepinephrine in the brain of gulf killifish (Fundulus grandis). Bull. Environ. Contain. Toxicol. 25, 682-7. Holdway, D.A. and Dixon, D.G. (1986) Effects of methoxychlor exposure of flagfish eggs, Jordenella floridae on hatchability of juvenile methoxychlor tolerance and whole body levels of tryptophan, serotonin and 5-hydroxyindoleacetic acid (5HIAA) Water Res. 20, 893-7. Holdway, D.A., Sloley, B.D., Munkittrick, K.R., Downer, R.G.H. and Dixon, D.G. (1988) Effect of gender and reproductive status on brain catecholamines and indoleamine levels of flagfish (Jordenella floridae). Fish Physiol. Biochem. 5, 153-8. Jacobowitz, D.M. and Richardson, J.S. (1978) Method for the rapid determination of norepinephrine, dopamine and serotonin in the same brain region. Pharmacol. Biochem. Behav. 8, 515-19. Jash, N.B. and Bhattacharya, S. (1983) Delayed toxicity of carbofuran in fresh water teleost Channa punctatus. Indian J. Exp. Biol. 17, 693-7. McDonald, T.O. (1979) Comparison of the effects of DDT and parathion on the concentration of serotonin, norepinephrine and dopamine in the brain retina/pigment epithelium choroid of the gold fish. Doctoral dissertation, Tulane University, New Orleans. Ram, M.D. and Gopal, K. (1991) Neurobehavioural changes in fresh water fish (Channa punctatus) exposed to fenitrothion. Bull. Environ. Contain. Toxicol. 47, 455-8. Ram, M.D., Verma, P.K. and Gopal, K. (1990) Effect of lindane on the levels of biogenic amines in various parts of brain of fresh water fish Channa punctatus (Bloch). J. Adv. Zool. 11, 79-84. Sastry, K.V. and Siddiqui, A.A. (1982) Chronic toxic effects of carbamate pesticide sevin on carbohydrate metabolism in a fresh water snakehead fish Channa punctatus. Toxicol. Lett. 14, 123-30. Saxena, P.K. and Mani, K. (1985) Quantitative study of testicular recruidescence in fresh water teleost Channa punctatus (Bloch) exposed to pesticides. Bull. Environ. Contain. Toxicol. 34, 597-607. Sloley, B.D. and Downer, R.G.H. (1984) Distribution of 5-hydroxytryptamine and indole alkylamine metabolites in the American cockroaches (Periplaneta americana). J. Comp. Biochem. Physiol. 79, 282-6. Sloley, B.D,, Hickie, B.E., Dixon, D.G., Downer, R.G.H. and Martin, R.J. (1986) The effect of sodium pentachlorophenate diet and sampling procedure on amine and tryptophan concentrations in the brain of rainbow trout (Salmo gairdneri, Richardson). J. Fish Biol. 28,267-77. Wigham, T. and Ball, J.N. (1976) In vivo evidence for catecholaminergic inhibition of prolactin secretion in the teleost Puntius latipinna. J. Comp. Physiol. 110, 135-43.
