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Activation of dopamine-containing amacrine cells of retina: light-induced increase of acidic dopamine metabolites
125
Brain Research, 260 (1983) 125-127 Elsevier Biomedical Press
Activation of dopamine-containing amacrine cells of retina: light-induced increase of acidic dopamine metabolites JOSEPH COHEN, MARIA H A D J I C O N S T A N T I N O U and N O R T O N H. NEFF*
Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, DC 20032 (U.S.A.) (Accepted October 18th 1982)
Key words: retina - amacrine cell - dopamine turnover - apomorphine - haloperidol - homovanillic acid 3,4-dihydroxyphenylacetic acid
The acidic metabolites of dopamine, homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC), are present in rat retina. DOPAC is the most abundant metabolite. Both metabolites increase in parallel when rats are taken from a dark to a lighted environment. Haloperidol treatment also increases the metabolites in both dark and light while apomorphine decreases both metabolites in dark and light and partially antagonizes the increase induced by haloperidol.
The dopamine-containing amacrine cells of the rat retina are found in the inner plexiform layer9. Exposing dark-adapted rats to light results in an increase of dopamine synthesis4,5 and release5 from these neurons. Concomitant with the increase ofdopamine synthesis is an increase of the affinity of tyrosine hydroxylase for its pteridine cofactor6. The administration of neuroleptic drugs also stimulates dopamine synthesis and increases the affinity of tyrosine hydroxylase for the pteridine cofactora. Activation of the nigrostriatal pathway after administering neuroleptic drugs has been ascribed to the presence of a negative neuronal feedback loop which induces activation of dopaminergic neurons when dopamine receptors are blocked2. A similar activation mechanism has been proposed for the retinal dopaminergic neurons3. In contrast to neuroleptic drugs, the administration of dopamine agonists results in reduced dopamine synthesis in the striatum8 and retina 4. We now report that apomorphine and haloperidol are capable of modifying the retinal content of dopamine metabolites in the dark when the tyrosine hydroxylase of the amacrine cell system appears * To whom correspondence should be addressed. 0006-8993/83/0000-0000/$ 03.00 © 1983 Elsevier Biomedical Press
to be at a basal state and in the light when the system appears to be activated. Male Sprague-Dawley rats, 150-200 g, (ZivicMiller Labs, Allison Park, PA) were decapitated under light or dark conditions, and retinas removed as described previously4 for the subsequent analysis of dopamine, homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC). Drug treatments are described in the Table I. Dopamine and its metabolites were analyzed by HPLC with electrochemical detection. For HVA determinations retinas from one animal were homogenized in 0.2 N HCIO4, 200 ~1, containing acid ascorbic (0.002% w/v). After centrifugation at 28,000 x g for 10 min the supernatant (100/xl) was injected directly into the HPLC system. For dopamine and DOPAC estimation, retinas were homogenized in HCIO4 as described and extracted through alumina ~. The HPLC system consisted ofa Bio-Sil ODS$5 15 cm column (Bio-Rad, Richmond, CA), with a mobile phase of citric acid, 0.05 M, titrated with sodium acetate, 0.1 M, to pH 4.8, containing sodium octyl sulfate, 100 mg/1. The
126 TABLEI Dopamine, H VA and DOPA C content in rat retina in a light and dark environment andJbllowing treatment with haloperidol and apomorphine
Treatments: rats were maintained on a 12 h light:dark cycle for at least 3 days before experimentation. At 11.5 h of darkness the rats were injected intraperitoneally with saline, apomorphine 10 mg/kg and/or haloperidol 5 mg/kg, In light-exposed animals, the lights were turned on 30 min after administration of drugs and the rats killed after 30 min of exposure to light. Apomorphine was administered 2 min before haloperidol when both drugs were administered to the same animals. Dark-adapted animals were sacrificed 60 min after saline, apomorphine and/or haloperidol while still in the dark. Data presented as the mean + S.EM. l?eatment
pmol/mgprotein + S.E.M. (n = 5 10) l,ight
Saline Haloperidol Apomorphine APO + HALO
Dark
Dopamine
t t VA
DOPA C
Dopamine
H VA
DOPA C
16 _+ 1 13 -+ 1* 15 + 1 It + 0.4*
4.4 _ 0.2*** 5.8 + 0.4* 1.3 + 0.6* 3.5 _+ 0.2**
9.8 14 + 4.0 _ 7.0 _
16 +_ 2 14 _+ 0.2 9.4 +_ t* 13 __ 2*
1.9 _+ 0,4 2.7 4" 0,1" 0.80 -- 0.07* 2.5 -+ 0.2**
3,7 __ 0.3 7.7 + 0.8* 2,1 _+ 0.2* 5.2 _+ 0.5**
0.5*** 1" 0.6* 0.2**
* P ~ 0.05 when compared with saline-treated animals. ** P ~Z0.05 when compared with apomorphine-treated animals. *** P <~ 0.05 when compared to dark-adapted saline-treated animals. buffer was filtered, degased and 10% v / v methanol added. T h e flow rate was 1. l m l / m i n . The electrochemical detector (BAS, W. Lafayette, IN) was set to a potential o f 0.50 V. The retention times for D O P A C , HVA and d o p a m i n e were 4.25, 10.15, 15.30 min. respectively. T h e addition o f the alumina step for the analysis o f d o p a m i n e and D O P A C eliminated some interfering materials and concentrated the catechols. The retinal content of e n d o g e n o u s substances was calculated from standard curves or from added 3 , 4 - d i h y d r o x y b e n z y l a m i n e (10 pmol) as an internal standard. As a pharmacological identification o f the substances asssayed, we administered either reserpine or the m o n o a m i n e oxidase inhibitor, pargyline. As expected, reserpine decreased d o p a m i n e whereas pargyline increased d o p a m i n e and decreased the metabolites (data not shown). Protein was d e t e r m i n e d by the p r o c e d u r e o f Lowry et alT. The d o p a m i n e content in the retina o f rats killled in the dark was about 16 p m o l / m g protein (Table I). Exposure to light for 30 min did not change the d o p a m i n e content. Exposure to light, however, produced significant increases in the content of d o p a m i n e metabolites. D O P A C increased 2.6-fold and H V A increased 2.3-fold, During both light and dark periods, D O P A C was the major metabolite o f d o p a m i n e found in the retina, The ratio o f D O P A C to HVA remai-
ned essentially the same when rats were taken from dark to light, a value o f a b o u t 2. H a l o p e r i d o l was given to animals in the dark and 60 min later they were killed while still in the dark (Table I). F o r comparison, a second group o f rats was treated with haloperidol and killed 60 min later with the last 30 rain in the light. H a l o p e r i d o l caused a significant increase o f retinal d o p a m i n e metabolites in rats kept in the dark, and also in rats placed in the light where metabolites were already elevated by light exposure (Table I). By subtraction o f metabolite content, light exposure increased H V A by a b o u t 2.5 and D O P A C by a b o u t 6.1 p m o l / m g protein i n d e p e n d e n t l y o f the increase induced by t r e a t m e n t with haloperidol. T h a t is, the response to light was minimally altered by treatment with haloperidol. T h e r e was a fall o f d o p a m i n e in the light-exposed, haloperidoltreated rats. In contrast to haloperidol, a p o m o r p h i n e administration resulted in a fall o f the retinal content o f H V A and D O P A C in the dark and in the light (Table I). Morever, it also partially antagonized the rise o f metabolites following treatment with haloperidol. D o p a m i n e content generally decreased following treatment with a p o m o r p h i De. As a consequence of exposure to light, the content o f retinal D O P A C and H V A increased
127 significantly above that f o u n d in the dark, while d o p a m i n e content remained unchanged. The increase of D O P A C and H V A content is an indication that the end result o f exposure to light is an e n h a n c e m e n t o f d o p a m i n e synthesis, utilization and metabolism, and parallels the results of Iuvone et al? .6, who demonstrated that exposure to light results in the activation of tyrosine hydroxylase and the increase of retinal d o p a m i n e turnover. The administration of haloperidol has been shown to activate retinal tyrosine hydroxylase of rats kept in the dark or placed in the light3, and as we have now shown it increases the acidic metabolites of d o p a m i n e in both environments as well. Apparently, activation of dopaminergic neurons by light and haloperidol takes place in the retina by i n d e p e n d e n t mechanisms. A study of the activation o f tyrosine hydroxylase by light and haloperidol led to a similar conclusion 3. The results are also consistent with the absence ofcli1 Anton, A. H. and Sayre, D. F., A study of the factors affecting the aluminum oxide trihydroxyindole procedure for the analysis of catecholamines, J. Pharmacol. exp. Ther., 138 (1962) 360-375. 2 Carlsson, A. and Lindqvist, M., Effect ofchlorpromazine or haloperidol on formation of 3-methoxytyramine and normetanephrine in mouse brain, A cta pharmacol, toxicol., 20(1963) 140-144. 3 Cohen, J., Iuvone, P. M. and Neff, N. H., Neuroleptic drugs activate tyrosine hydroxylase of retinal amacrine cells,J. Pharmacol. Exp. Ther., 218 (1981) 390-394. 4 Da Prada,M., Dopamine content and synthesis in retina and n. accumbens septi: pharmacologicaland light-induced modifications. In E. Costa and G. L. Gessa (Eds.),
nical reports that haloperidol significantly alters vision. A p o m o r p h i n e decreased the content o f retinal HVA and D O P A C in both dark and light environments, and it also reduces the increase in these metabolites as a consequence of haloperidol treatment. Two mechanisms m a y be responsible for this action: direct inhibition of tyrosine hydroxylase a n d / o r interaction with dopaminergic receptors. In summary, the major metabolite of dopamine found in the rat retina is DOPAC. Exposure to light causes a parallel independent increase o f the H V A and D O P A C content. Haloperidol also increased H V A and D O P A C but this increase appears to be i n d e p e n d e n t of the increase induced by exposure to light. The rise of HVA and D O P A C induced by light and haloperidol can be partially inhibited by treatment with apomorphine.
6
7 8
Advances in Biochemical Psychopharmacology, Vol.16,
Raven Press, New York, 1977,pp. 311-319. 5 luvone, P. M., Galli, C. L., Garrison-Gund, C. K. and Neff, N. H., Light stimulates tyrosine hydroxylase activ-
9
ity and dopamine synthesis in retinal amacrine neurons, Science, 202 (! 978) 901-902. Iuvone, P. M., Galli, C. L. and Neff, N. H., Retinal tyrosine hydroxylase: comparison of short-term and longterm stimulation by light, Molec. Pharmacol., 14 (1978) 1212-1219. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (195l) 265-275. Roth, R. H., Waiters, J. R., Murrin, L. C. and Morgenroth, V. H. III, Dopamine neurons: role of impulse flow and presynaptic receptors in the regulating tyrosine hydroxylase. In E. Usdin and W. E. Bunney (Eds.), Pre-and Postsynaptic Receptors, Marcel Dekker, New York, 1975, pp. 5-48. Schmitt, F. O., Dev, P. and Smith, B. H., Electrotonic processing of information in brain cells, Science, 193 (1976) 114-120.
Brain Research, 260 (1983) 125-127 Elsevier Biomedical Press
Activation of dopamine-containing amacrine cells of retina: light-induced increase of acidic dopamine metabolites JOSEPH COHEN, MARIA H A D J I C O N S T A N T I N O U and N O R T O N H. NEFF*
Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, DC 20032 (U.S.A.) (Accepted October 18th 1982)
Key words: retina - amacrine cell - dopamine turnover - apomorphine - haloperidol - homovanillic acid 3,4-dihydroxyphenylacetic acid
The acidic metabolites of dopamine, homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC), are present in rat retina. DOPAC is the most abundant metabolite. Both metabolites increase in parallel when rats are taken from a dark to a lighted environment. Haloperidol treatment also increases the metabolites in both dark and light while apomorphine decreases both metabolites in dark and light and partially antagonizes the increase induced by haloperidol.
The dopamine-containing amacrine cells of the rat retina are found in the inner plexiform layer9. Exposing dark-adapted rats to light results in an increase of dopamine synthesis4,5 and release5 from these neurons. Concomitant with the increase ofdopamine synthesis is an increase of the affinity of tyrosine hydroxylase for its pteridine cofactor6. The administration of neuroleptic drugs also stimulates dopamine synthesis and increases the affinity of tyrosine hydroxylase for the pteridine cofactora. Activation of the nigrostriatal pathway after administering neuroleptic drugs has been ascribed to the presence of a negative neuronal feedback loop which induces activation of dopaminergic neurons when dopamine receptors are blocked2. A similar activation mechanism has been proposed for the retinal dopaminergic neurons3. In contrast to neuroleptic drugs, the administration of dopamine agonists results in reduced dopamine synthesis in the striatum8 and retina 4. We now report that apomorphine and haloperidol are capable of modifying the retinal content of dopamine metabolites in the dark when the tyrosine hydroxylase of the amacrine cell system appears * To whom correspondence should be addressed. 0006-8993/83/0000-0000/$ 03.00 © 1983 Elsevier Biomedical Press
to be at a basal state and in the light when the system appears to be activated. Male Sprague-Dawley rats, 150-200 g, (ZivicMiller Labs, Allison Park, PA) were decapitated under light or dark conditions, and retinas removed as described previously4 for the subsequent analysis of dopamine, homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC). Drug treatments are described in the Table I. Dopamine and its metabolites were analyzed by HPLC with electrochemical detection. For HVA determinations retinas from one animal were homogenized in 0.2 N HCIO4, 200 ~1, containing acid ascorbic (0.002% w/v). After centrifugation at 28,000 x g for 10 min the supernatant (100/xl) was injected directly into the HPLC system. For dopamine and DOPAC estimation, retinas were homogenized in HCIO4 as described and extracted through alumina ~. The HPLC system consisted ofa Bio-Sil ODS$5 15 cm column (Bio-Rad, Richmond, CA), with a mobile phase of citric acid, 0.05 M, titrated with sodium acetate, 0.1 M, to pH 4.8, containing sodium octyl sulfate, 100 mg/1. The
126 TABLEI Dopamine, H VA and DOPA C content in rat retina in a light and dark environment andJbllowing treatment with haloperidol and apomorphine
Treatments: rats were maintained on a 12 h light:dark cycle for at least 3 days before experimentation. At 11.5 h of darkness the rats were injected intraperitoneally with saline, apomorphine 10 mg/kg and/or haloperidol 5 mg/kg, In light-exposed animals, the lights were turned on 30 min after administration of drugs and the rats killed after 30 min of exposure to light. Apomorphine was administered 2 min before haloperidol when both drugs were administered to the same animals. Dark-adapted animals were sacrificed 60 min after saline, apomorphine and/or haloperidol while still in the dark. Data presented as the mean + S.EM. l?eatment
pmol/mgprotein + S.E.M. (n = 5 10) l,ight
Saline Haloperidol Apomorphine APO + HALO
Dark
Dopamine
t t VA
DOPA C
Dopamine
H VA
DOPA C
16 _+ 1 13 -+ 1* 15 + 1 It + 0.4*
4.4 _ 0.2*** 5.8 + 0.4* 1.3 + 0.6* 3.5 _+ 0.2**
9.8 14 + 4.0 _ 7.0 _
16 +_ 2 14 _+ 0.2 9.4 +_ t* 13 __ 2*
1.9 _+ 0,4 2.7 4" 0,1" 0.80 -- 0.07* 2.5 -+ 0.2**
3,7 __ 0.3 7.7 + 0.8* 2,1 _+ 0.2* 5.2 _+ 0.5**
0.5*** 1" 0.6* 0.2**
* P ~ 0.05 when compared with saline-treated animals. ** P ~Z0.05 when compared with apomorphine-treated animals. *** P <~ 0.05 when compared to dark-adapted saline-treated animals. buffer was filtered, degased and 10% v / v methanol added. T h e flow rate was 1. l m l / m i n . The electrochemical detector (BAS, W. Lafayette, IN) was set to a potential o f 0.50 V. The retention times for D O P A C , HVA and d o p a m i n e were 4.25, 10.15, 15.30 min. respectively. T h e addition o f the alumina step for the analysis o f d o p a m i n e and D O P A C eliminated some interfering materials and concentrated the catechols. The retinal content of e n d o g e n o u s substances was calculated from standard curves or from added 3 , 4 - d i h y d r o x y b e n z y l a m i n e (10 pmol) as an internal standard. As a pharmacological identification o f the substances asssayed, we administered either reserpine or the m o n o a m i n e oxidase inhibitor, pargyline. As expected, reserpine decreased d o p a m i n e whereas pargyline increased d o p a m i n e and decreased the metabolites (data not shown). Protein was d e t e r m i n e d by the p r o c e d u r e o f Lowry et alT. The d o p a m i n e content in the retina o f rats killled in the dark was about 16 p m o l / m g protein (Table I). Exposure to light for 30 min did not change the d o p a m i n e content. Exposure to light, however, produced significant increases in the content of d o p a m i n e metabolites. D O P A C increased 2.6-fold and H V A increased 2.3-fold, During both light and dark periods, D O P A C was the major metabolite o f d o p a m i n e found in the retina, The ratio o f D O P A C to HVA remai-
ned essentially the same when rats were taken from dark to light, a value o f a b o u t 2. H a l o p e r i d o l was given to animals in the dark and 60 min later they were killed while still in the dark (Table I). F o r comparison, a second group o f rats was treated with haloperidol and killed 60 min later with the last 30 rain in the light. H a l o p e r i d o l caused a significant increase o f retinal d o p a m i n e metabolites in rats kept in the dark, and also in rats placed in the light where metabolites were already elevated by light exposure (Table I). By subtraction o f metabolite content, light exposure increased H V A by a b o u t 2.5 and D O P A C by a b o u t 6.1 p m o l / m g protein i n d e p e n d e n t l y o f the increase induced by t r e a t m e n t with haloperidol. T h a t is, the response to light was minimally altered by treatment with haloperidol. T h e r e was a fall o f d o p a m i n e in the light-exposed, haloperidoltreated rats. In contrast to haloperidol, a p o m o r p h i n e administration resulted in a fall o f the retinal content o f H V A and D O P A C in the dark and in the light (Table I). Morever, it also partially antagonized the rise o f metabolites following treatment with haloperidol. D o p a m i n e content generally decreased following treatment with a p o m o r p h i De. As a consequence of exposure to light, the content o f retinal D O P A C and H V A increased
127 significantly above that f o u n d in the dark, while d o p a m i n e content remained unchanged. The increase of D O P A C and H V A content is an indication that the end result o f exposure to light is an e n h a n c e m e n t o f d o p a m i n e synthesis, utilization and metabolism, and parallels the results of Iuvone et al? .6, who demonstrated that exposure to light results in the activation of tyrosine hydroxylase and the increase of retinal d o p a m i n e turnover. The administration of haloperidol has been shown to activate retinal tyrosine hydroxylase of rats kept in the dark or placed in the light3, and as we have now shown it increases the acidic metabolites of d o p a m i n e in both environments as well. Apparently, activation of dopaminergic neurons by light and haloperidol takes place in the retina by i n d e p e n d e n t mechanisms. A study of the activation o f tyrosine hydroxylase by light and haloperidol led to a similar conclusion 3. The results are also consistent with the absence ofcli1 Anton, A. H. and Sayre, D. F., A study of the factors affecting the aluminum oxide trihydroxyindole procedure for the analysis of catecholamines, J. Pharmacol. exp. Ther., 138 (1962) 360-375. 2 Carlsson, A. and Lindqvist, M., Effect ofchlorpromazine or haloperidol on formation of 3-methoxytyramine and normetanephrine in mouse brain, A cta pharmacol, toxicol., 20(1963) 140-144. 3 Cohen, J., Iuvone, P. M. and Neff, N. H., Neuroleptic drugs activate tyrosine hydroxylase of retinal amacrine cells,J. Pharmacol. Exp. Ther., 218 (1981) 390-394. 4 Da Prada,M., Dopamine content and synthesis in retina and n. accumbens septi: pharmacologicaland light-induced modifications. In E. Costa and G. L. Gessa (Eds.),
nical reports that haloperidol significantly alters vision. A p o m o r p h i n e decreased the content o f retinal HVA and D O P A C in both dark and light environments, and it also reduces the increase in these metabolites as a consequence of haloperidol treatment. Two mechanisms m a y be responsible for this action: direct inhibition of tyrosine hydroxylase a n d / o r interaction with dopaminergic receptors. In summary, the major metabolite of dopamine found in the rat retina is DOPAC. Exposure to light causes a parallel independent increase o f the H V A and D O P A C content. Haloperidol also increased H V A and D O P A C but this increase appears to be i n d e p e n d e n t of the increase induced by exposure to light. The rise of HVA and D O P A C induced by light and haloperidol can be partially inhibited by treatment with apomorphine.
6
7 8
Advances in Biochemical Psychopharmacology, Vol.16,
Raven Press, New York, 1977,pp. 311-319. 5 luvone, P. M., Galli, C. L., Garrison-Gund, C. K. and Neff, N. H., Light stimulates tyrosine hydroxylase activ-
9
ity and dopamine synthesis in retinal amacrine neurons, Science, 202 (! 978) 901-902. Iuvone, P. M., Galli, C. L. and Neff, N. H., Retinal tyrosine hydroxylase: comparison of short-term and longterm stimulation by light, Molec. Pharmacol., 14 (1978) 1212-1219. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (195l) 265-275. Roth, R. H., Waiters, J. R., Murrin, L. C. and Morgenroth, V. H. III, Dopamine neurons: role of impulse flow and presynaptic receptors in the regulating tyrosine hydroxylase. In E. Usdin and W. E. Bunney (Eds.), Pre-and Postsynaptic Receptors, Marcel Dekker, New York, 1975, pp. 5-48. Schmitt, F. O., Dev, P. and Smith, B. H., Electrotonic processing of information in brain cells, Science, 193 (1976) 114-120.