Rat dopaminergic function in the retina during aging

Neurobiology of Aging, Vol. 2, pp. 229,-231, 1981. Printed in the U.S.A.

Rat Dopaminergic Function in the Retina During Aging F. R I C C A R D I , V. C O V E L L I , P. F. S P A N O , S. G O V O N I A N D M. T R A B U C C H I 1

Departments of Pharmacology and Pharmacognosy Universities of Milan and Padua, Via A. Del Sarto 21, 20129 Milan, Italy R e c e i v e d 22 June 1981 RICCARDI, F., V. COVELLI, P. F. SPANO, S. GOVONI AND M. TRABUCCHI. Rat doparainergic function in the retina during aging. NEUROBIOL. AGING 2(3)229-231, 1981.--Parameters of dopaminergic transmission were studied in the retina of mature (3--4 months) and aged (23--24 months) rats. In the retina of senescent rats we found significantly higher dihydroxyphenylacetic acid (DOPAC) levels and a higher number of (3H)-spiroperidol binding sites. We detected also an increase of (aH)-methionine-enkephalin binding sites. The changes in the density of (aH)-spiroperidol and PH)-Metenkephalin binding sites in the retina are opposite to those observed in the brain of aged rats. Aging

Retina

Dopamine

CHEMICAL and histochemical studies have shown that dopamine (DA) is the predominant monoamine in the neural retina in a number of animal species [4,10]. The DA neurons in the retina appear to be part of the amacrine cell population located in the inner nuclear cellular layer, interconnected with other different cell types. Electrophysiological experiments with exogenously applied DA indicate that this amine may function as an inhibitory transmitter in the rabbit retina [1]. At the same time there is evidence that dopaminergic junctional ceils in the retina are capable of storing and accumulating DA. In addition, these cells may release DA following stimulation of the retina with light [15]. Exposure of rats to light increases tyrosine hydroxylase activity and DA formation [12], indicating that the rate of retinal DA formation is modulated by light exposure. In addition, the retina of different mammals, rat included, contains an adenylyl cyclase (AC) which is preferentially stimulated by DA, and which is sensitive to pharmacological and physiological influences [24]. Specific binding sites for (3H)spiroperidol are observed in rat retina homogenates; the pharmacological characterization of these binding sites suggests an association with DA receptors [19]. It is also noteworthy that in different dopaminergic areas of the central nervous system there appear to be two populations of dopamine receptors [14, 25, 26]: the D-1 are those that are associated with the adenylyl cyclase system, whereas the D-2 are not. Watling et al. [28] have reported that D-2 receptors are not present in the retina, suggesting that this area contains a pure population of D-I receptors. Several recent studies have found changes in brain neurotransmitter receptors in aged animals and humans [17]. In particular, a decreased density of dopaminergic receptors associated with adenylyl cyclase (D-1 receptors), has been

observed in various areas of central nervous system. In contrast, D-2 receptors appear to be unchanged [7, 18, 20]. In 24 month old rats the DA sensitive adenylyl cyclase activity (DA-AC) is decreased in striatum, nucleus accumbens, tuberculum offactorium and substantia nigra [6]. Interestingly, in the same group of animals, the retinal DA.AC activity is increased [6]. In our study we measured DA receptor density and affinity using (3H)-spiroperidol and DA metabolism measuring the concentrations of dihydroxyphenylacetic acid (DOPAC) in the retina of adult and old rats. The results indicate that the DOPAC concentration and the number of binding sites for PH)-spiroperidol are increased in aged rats. Furthermore, we also examined opiate receptor binding using (3H)-methionine-enkephalin as a ligand, since others have described the presence of opiate binding sites in the retina of rat, cow, toad and skate [11]. In the central nervous system, opiates interact with dopaminergic transmission [27] and opiate receptors are present on DA neurons [22]. Further, the density of opiate receptors is decreased in various brain areas of aged rats [21 ]. An interesting question, therefore, was whether the pattern of change of opiate receptor was different in retina than in the brain, as observed for DA. In fact, the results indicate an increased number of opiate receptors in old rats' retinas. METHOD

Mature (3--4 months) or senescent (23--24 months) male Sprague-Dawley rats (Charles River, Calco, Italy) were used in our study. The animals were randomly caged to avoid environmental differences, housed at constant temperature and humidity, and exposed to a light cycle of 12 hrs a day. Animals had free access to food and water. Senescent rats

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Copyright © 1981 A N K H O International Inc.m0197-4580/81/030229-03500.80/0

230

RICCARDI E T A I . . •

A 75-

A

......

--~

mature ( S - 4 m o n t h s ) a g e ~ (~1-1~4 months)

~A

B 150 1

o

O,

0-

~ 100-

E

A 25-

B

"x\,

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~

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50

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N

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2's

5'o r tool/m0 o ~ o t / n ~

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FIG. 1. Scatchard analysis of (SH)-spiroperidol (A) and (=H)-methionin¢enkephalin (B) specific bindings in ~ 1 ~m~ p~cpgrations of mature (3-4 months) and aged (23-24 months) rats, A: the apparent Ko a ~ unchanged while maximum specific binding 0B~a,j isincreased by I00CAin retina of aged rats. B: similarly, B~.x values are increased by 35~ in respect to mature whereas Ko values are Unchanged. Values are the mean of four determinations with samples in triplicate.

with patholog/cal affections (tumors, respiratory infections, etc.) were excluded from our study. Dopaminergic receptors were studied with the method of Bun e t al. [3] using (SH)-spiroperidol (Amersham, 23 Ci/mmol) as ligand. Briefly, retinal tissue suspension (about 0.6 nag protein) was incubated at 37"C for 10 rain in the presence of various concentrations of ligand. Samples were filtered under vacuum through Wbatman GF/B filters. Specific (SH)-spiroperidol binding was measured as the difference in binding obtained w/th incubation in the presence or in absence of 10"e M haloperidol. Opiate binding sites were studied with the method of Howells e t ai. [1 I] using (SH)-methionine-enkephalin (New England Nuclear, 23.7 Ci/mmol) as ligand. After 2.5 hrs of incubation at 0~C, 1 ml aliquots (0.3-0.5 mg of protein) of the rat retinal membrane suspension were filtered under vacuum. ~ was assayed in presence and absence of the unla~ ~ at a final concentration of 1 IzM. Dthydroxyphenylacefic acid OX)PAC) levels were measured according to the micromethod described by Argiolas et al. [2]. Protein content was measured according to Lowry et al. [16]. RESULTS

Steady state levels of dihydroxyphenytacetic acid (DOPAC) (a primary marker of DA metabolism) were reduced in striatum ofal~d rats by 30~, while inthe retina the levels are sil~ificantly increased by 240~ (Table 1). Figure I shows the Scatchard plots of (q-l)-spiroperidol binding and PH)-methionine enkephalin binding in retinal membrane preparations of mature (3-4 months) and aged (23-24 months) rats. A 100% increase of (3H)-spiroperidot binding was detected in the retina of aged rats compared with

TABLE 1 STR1ATALAND RETINAL DOPACLEVELS (z~!/!~,~Y~UE), IN MATURE 0~-4 MONTHS) AND AGED (23-24 ~ ; ) RATS

Mature Aged

Striatum

cA

Retina

cA

2.5 _. 0.1 1.6 ± 0.2*

-30~

0.135 ± 0.02 0.455 -,- 0.04*

+240CA

*p<0.01 in respect to mature rats. Values are the means ± S,E. of three experiments run in quadruplicate.

mature rats. This increase in (.3H)-spiroperido|bindingmay be ascribed to an increase in Bmx values from 40.0± 1.7 to 82-+1.5 fmoVmg protein) while KD values gre urtchRnged (0.31±0.01 nM and 0.32"0.02 nM, respectively). (~H)-Methionine enkephalin binding in the getina of aged rats exhibited an increase in Bmax values (from 94±2.4 to 127-+ 3.6 fmol/mg protein) whereas K v values were not modified (3.5-+0.8 nM and 3.4-+0.7 nM, respectively). DISCUSSION Similar to a previously reported increase in DA-AC activity in the retina of aged rats [6] the present study reports an increase of (SH)-spiroperidol binding sites and of DOPAC steady state levels. This increase in dopamine markers in the retina contrasts markedly with the age-related decreases in various dopaminergic parameters (such as DA concentration, turnover, catabolism, uptake and receptor function [5, 13, 23]), observed in different brain areas.

R E T I N A L D O P A M I N E R G I C F U N C T I O N AND A G I N G The concomitant increase in the number of (3H)spiroperidol binding sites and DOPAC levels is difficult to explain. However, it should be mentioned that, although in an opposite direction, the same unusual dissociation of presynaptic activity and receptor function is observed also in the brain (decrease of DA turnover and of the number of binding sites). We may speculate that aging affects those mechanisms regulating the receptor sensitization and desensitization in response to altered presynaptic activity. Along this line, Greenberg and Weiss |8,9] showed an impaired capacity of old rats to increase B-adrenergic receptor density in brain and pineal in response to a decreased adrenergic input. In their study, however, the aged rats retained the

231 ability to decrease the receptor density following repeated desmethylimipramine administration. Perhaps more puzzling than the dopamine alterations is the increase in the n u m b e r of binding sites for (SH)-Metenkephalin observed in the retina of aged rats. Although the immediate causes and functional consequences are unclear, these changes clearly demonstrate that aging does not affect all receptors for a given neurotransmitter in a similar fashion throughout the organism. ACKNOWLEDGMENTS The animals used in this study were kindly supplied by the Italian Study Group on Brain Aging.

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