Neuroscience Letters, 98 (1989) 345-350 345 Elsevier Scientific Publishers Ireland Ltd.

NSL 05976

Striatal D1 dopamine receptor density fluctuates during the rat estrous cycle

Danie l L6vesque, Sophie G a g n o n and Th6r6se Di Pao lo

School of Pharmacy, Laval University, Quebec, Que. (Canada) and Department of Molecular Endocrinology, Laval University Medical Center, Ste-Foy, Que. (Canada)

(Received 15 August 1988; Revised version received 29 November 1988; Accepted 30 November 1988)

Key words: D, dopamine receptor; Rat estrous cycle; Striatum; Antagonist binding site; 17B-Estradiol; Progesterone; Prolactin; SCH 23390

Striatal D~ dopamine (DA) antagonist binding sites were investigated in intact female and male rats, ovariectomized (OVX) animals and during the 4-day estrous cycle. The affinity of the striatal D1 receptor as labelled with [3H]SCH 23390 remains unchanged in intact male, female rats during the estrous cycle and OVX animals. By contrast, ovariectomy decreased striatal Dl receptor density by 179 (P< 0.01) com- pared to intact female rats while a small but significant higher density was observed in intact male com- pared to female rats (10% higher, P < 0.05). The density of striatal Dl DA receptor was higher on the day of diestrus I (DI) and diestrus II (DII) (P< 0.01 vs OVX) and fluctuates throughout the estrous cycle with a maximum on the day of DII (P< 0.05 vs proestrus PM). Our results show for the first time that striatal D, DA receptors fluctuate during the estrous cycle and can be modulated by gonadal steroids.

Studies on dopamine (DA) transmission across the estrous cycle of the rodent

show the importance of the gonads in striatal DA regulation. Striatal DA levels were shown to be decreased on the day of estrus (E) and diestrus I (DI) in rat [9] and mice

[19] while homovanillic acid (HVA) concentrations are increased and DA turnover was highest on the day of E in mice [22]. The rate of DA uptake was shown to be

greatest during diestrus [10]. Male-female differences in striatal DA levels in rats and mice also suggest a modulation by gonads of DA transmission [9, 18]. During the

rat estrous cycle, the striatal D2 DA receptor total density and affinity of the antago-

nist binding site is constant while the agonist high to low affinity site densities peak

in diestrus II (DII) [14]. Dl DA receptors, mostly concentrated in the striatum [11], have not yet been investigated during the estrous cycle. Therefore, the aim of the pre-

sent study was to probe the DA Di receptor during the rat 4-day estrous cycle. Adult Sprague-Dawley intact female and intact male rats were purchased from

Charles River Canada Inc., St-Constant, Que. They were housed 2 per cage and maintained at 22-23°C on a 14:10 h light--dark cycle (lights on from 05.00 to 19.00

Correspondence: T. Di Paolo, Department of Molecular Endocrinology, Laval University Medical Center, 2705, Laurier Boulevard, Ste-Foy, Que. G1V 4G2, Canada.

0304-3940/89/$ 03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd.

346

h) and received rat chow and water ad libitum. Bilaterally ovariectomized (OVX) rats were used for experiments 14 days later and only female rats demonstrating at least two consecutive 4-day estrous cycles were included in the experiment. Vaginal smears were taken daily to monitor the estrous cycle. Rats were sacrificed by decapitation in the afternoon (between 4.00 and 5.00 h) for the afternoon proestrus (PPM) group and in the morning (between 8.30 and 9.30 h) for the other groups. Trunk blood was collected into heparinized tubes and plasma was separated by centrifugation at 4000 g for 10 min and kept at -20°C until assayed for prolactin (PRL), 17fl-estradiol (E2) and progesterone (P). The steroids were measured in duplicate by radioimmunoas- says (RIA) using rat hormone and rabbit antiserum provided by Dr. A. B61anger [5]. PRL was measured in duplicate by double-antibody RIA using rat PRL-I-3 and rab- bit antisera (anti-rat PRL-S-3) kindly provided by National Hormone and Pituitary Program, Baltimore.

The striata were dissected immediately, frozen in dry ice and kept at -70°C until assay. Tissues were prepared according to our previously published procedures [16]. Namely, striata were homogenized in a glass homogenizer in 100 volumes (w/v) of phosphate buffer (81 mM Na2HPO4, 19 mM KH2PO4, 2 mM MgCI2, pH 7.4) and centrifuged at 40000 g for 15 min. This washing procedure was repeated twice and the final pellet resuspended in 100 volumes of the same cold buffer. The D, DA anta- gonist binding sites were investigated in individual animals by saturation isotherms with [N-methyl-3H]SCH 23390 (Amersham, 80-90 Ci/mmol). Binding assays were performed according to Billard et al. [6] with minor modifications. Incubation (for 1 h at 22°C) was initiated by the addition of membrane suspension (0.15 ml contain- ing an average of 0.1 mg of protein measured by the method of Lowry et al. [25]) to tubes containing 1.750 ml of phosphate buffer, 0.05 ml [N-methyl-3H]SCH 23390

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Fig. 1. Equilibrium dissociation constant (Kd) and maximal receptor density (Bmax) of striatal D, DA recep- tor labelled with [3H]SCH 23390 in intact female, intact male and ovariectomized rats. Values represent means ___ S.E.M. obtained from 8 rats determined individually.

347

(8 concentrations, 0.05-0.5 nM) and 0.05 ml SCH 23390 (1/tM, to estimate non-spe- cific binding) or 0.01% ascorbic acid. The assay was done in triplicate. The incubation was terminated by rapid filtration through Whatman GF/C glass fiber filters under vacuum followed by 3 rapid rinses (4 ml) with ice-cold buffer. Bound [N-methyl- 3H]SCH 23390 was determined by liquid scintillation spectrometry at an efficiency of 56%. Equilibrium dissociation constant (Ka) and density of receptor binding sites (Bmax) were estimated by computerized Scatchard transformation of specific binding from isotherm-saturation experiments. Scatchard plots were linear (R 2 ranging from 0.95 to 0.99) indicating saturation to one receptor population. Statistical evaluation of Kd and Bmax as well as hormone data were performed by the Duncan-Kramer mul- tiple range tests [23].

S t r i a t a l D 1 DA receptor density and affinity in intact female rats at random stage of the estrous cycle, OVX female and intact male rats are shown in Fig. 1. Ovariec- tomy decreased (16.8%, P < 0.01 vs intact female), the density of [3H]SCH 23390 binding and a small but significant higher density was observed in intact male com- pared to female rats (9.6% higher, P < 0.05). By contrast, the affinity of [3H]SCH 23390 striatal binding remained unchanged.

Plasma E2, P and PRL concentrations of rats measured during the estrous cycle are shown in Table I. The surge of plasma PRL was seen in PPM and the E2 peak was on the day of proestrus, with the highest level on PPM. P concentrations were more phasic with one peak on the day of DI and DII and one on PPM (Table I). These results are in good agreement with previous reports [7, 14].

The affinity of [3H]SCH 23390 striatal binding remained unchanged throughout the estrous cycle (Fig. 2); by contrast, the density was higher on the day of DI and DII (P < 0.01 vs OVX) and fluctuated significantly throughout the estrous cycle (Fig. 2) with the highest level of receptor on the day of DII (P < 0.05 vs PPM).

Our results show in female rats that density of striatal Dt DA receptors vary during the estrous cycle or after ovariectomy and is lower than in male rats. By contrast, the affinity of the D i receptor remains unchanged in all cases.

TABLE I

RAT P L A S M A P R O L A C T I N , 17~-ESTRADIOL A N D P R O G E S T E R O N E LEVELS D U R I N G T H E 4-DAY E S T R O U S C Y C L E A N D I N O V A R I E C T O M I Z E D R A T S

Values represent the mean 4- S.E.M. of 10-12 rats analyzed individually in duplicate.

G r o u p 17fl-Estradiol Progesterone Prolactin

(pg/ml) (ng/ml) (ng/ml)

Ovariectomized 5.1 4- 0.7 b,c 0.4 + 0. I b,, 3.3 4- 0.4 ¢

Estrus 12.3 _ 1.5 a-~ 5.2 _ 0.7 a.c 3.6 +_0.7 c

Diestrus I 15.3__+ 1.0 a-~ 11.3--+ 1.2 a,b 4.1 +0 .7 c

Diestrus II 15.6 + 1.1 a-~ 12.2 4-1.8 a.b 3.8 4- 0.7 c

Proestrus A M 28.4 4- 2. I a.c 5.0 4- 0.4 a,c 2.1 4- 0.5 c

Proest rus PM 52.1 4- 5. I a,b 11.14-1.9 ~,b 142.7 4-16.8

~P < 0.01 vs ovariectomized; bp < 0.01 VS PAM; ¢P < 0.01 vs PPM.

348

150

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R A T H O R f ' I ~ A L S T A T U S

Fig. 2. Equilibrium dissociation constant (Kd) and maximal receptor density (B,,,) of striatal DI DA recep- tor labelled with [3H]SCH 23390 during the 4-day estrous cycle (E, estrus; DI, diestrus I; DII, diestrus II; PAM, proestrus AM and PPM, proestrus PM) and in ovariectomized rats (OVX). Values represent means + S.E.M. obtained from 10-12 rats determined individually. The 100~ values were 0.233 +_ 0.018 nM for Kd and 943 + 57 fmol/mg of protein for Bmax.

A pool of the D1 densities during the estrous cycle of Fig. 2 is higher than the OVX rats' D 1 receptor density (1108 + 67 and 943 + 57 fmol/mg of protein for estrous cycle female and OVX rats, respectively, P < 0.05). Moreover, this mean of DI densities is similar to the density obtained with intact female rats at random stages of the estrous cycle shown in Fig. I indicating that the value in our first experiment is a good reflection of the average D1 DA receptor density during the estrous cycle.

The density of striatal D2 DA receptors is the same in male and intact or OVX female rats [14, 20]. However, intact male rats display more stereotypy induced by apomorphine than intact female rats [1, 20]. In addition, OVX animals display more apomorphine- [20] and amphetamine- [2] induced stereotypy than intact female rats while striatal D~ DA receptor density is decreased and D2 DA receptor density re- mains unchanged after ovariectomy. Sex differences in Dl DA receptor density and its fluctuations during the estrous cycle may partly explain the behavioral variations occurring in these different hormonal states where D2 DA receptor density and affi- nity for the antagonist binding site remains unchanged. The effect of D] DA receptor changes on behaviors could be direct or via D2 DA receptors through DJD2 DA receptor interactions. Indeed, stimulation of both D~ and D2 DA receptors appears necessary for full expression of postsynaptic effects of DA agonists [8] and respecti- vely opposite and permissive roles of Dt receptor on behavioral responses to DA ago- nists have been reported [21, 24].

During the rat estrous cycle, we have observed [14] that D2 DA striatal receptor

349

agonist binding sites vary with the highest ratio of densities of high to low states on the day of DII while the affinity and total density remains~lanchanged. Gordon [17] has reported a dose-related suppression of apomorphine-irlduced~stereotypy 24 h af- ter E2 treatment and an enhancement 48 h or more after, By anal0gy, ~ the fluctuations of the D1 DA receptor density could be explained by a biphasi¢ effect of estradiol first inhibitory in PPM with the surge of E2 then later stimulatory in DI and II. A biphasic effect of P on DA systems has also been observed. Dluzen and Ramirez [15] have shown, in the corpus striatum of estrogen-primed OVX rats; a stimulating effect 2-12 h after P on spontaneous and amphetamine-evoked DA release while 24 h later, a decrease is observed. Moreover, we have shown that a small dose of P, which repro- duces plasma level observed in the estrous cycle can acutely (within 1 h) increase striatal dopaminergic activity in rats [13]. PRL, LH and FSH which fluctuate during the estrous cycle [7, 14] may also be involved in the variation of the D1 receptor observed.

We have previously shown that tardive dyskinesia in humans can be improved with estrogens [3] and that neuroleptic-induced dopaminergic supersensitivity in rats and monkeys can be suppressed with estrogen injections [4, 12]. Knowledge of the hormo- nal influences on Dl and D2 DA receptors may thus be useful in improving therapeu- tic regimen using dopaminergic agonist and antagonist drugs.

In summary, our results show a sex difference in the total density of D1 DA recep- tors and fluctuations during the estrous cycle while these parameters have previously been shown to remain constant for D2 DA receptors. Our results for the first time demonstrate that physiological hormonal fluctuations as occur during the estrous cy- cle can affect striatal D~ DA receptors.

This work was supported by the Medical Research Council of Canada.

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