Ovarian steroids influence the activity of neuroendocrine dopaminergic neurons

Brain Research 879 (2000) 139–147 www.elsevier.com / locate / bres

Research report

Ovarian steroids influence the activity of neuroendocrine dopaminergic neurons Jamie E. DeMaria, John D. Livingstone, Marc E. Freeman* Program in Neuroscience, Department of Biological Science, Florida State University, Tallahassee, FL 32306 -4340, USA Accepted 25 July 2000

Abstract The secretion of prolactin (PRL) from the anterior lobe (AL) of the pituitary gland is tonically inhibited by dopamine (DA) of hypothalamic origin. While ovarian steroids play a role in the regulation of the secretion of PRL, their effect on all three populations of hypothalamic neuroendocrine dopaminergic neurons is not fully understood. In this study we describe the effects of ovarian steroids on regulation of the release of DA from tuberoinfundibular dopaminergic (TIDA), tuberohypophyseal dopaminergic (THDA) and periventricular-hypophyseal dopaminergic (PHDA) neurons. Adult female rats were bilaterally ovariectomized (OVX) and, 10 days following ovariectomy (day 0), injected with corn oil (vehicle), estrogen, or estrogen plus progesterone (day 1). Animals were sacrificed every 2 h from 09.00 to 21.00 h by rapid decapitation. Trunk blood was collected and the concentration of PRL in serum was determined by radioimmunoassay. The median eminence (ME) and the AL, intermediate (IL) and neural (NL) lobes of the pituitary gland were dissected and the concentration of DA and DOPAC in each was measured by HPLC-EC. OVX rats presented small but significant increases in the secretion of PRL at 15.00 and 17.00 h. Replacement of estrogen or estrogen plus progesterone increased the basal concentration of PRL. Moreover, injection of estrogen only, or estrogen plus progesterone increased the concentration of PRL in serum at 15.00 h through 19.00 h, respectively, followed by a decrease to baseline thereafter. The turnover of DA in the ME and NL of OVX rats increased at 13.00 and returned to low levels. Turnover of DA in the IL of OVX rats increased in the morning by 11.00 h and remained elevated before decreasing by 17.00 h. The turnover of DA in the ME, IL and NL of OVX rats increased by 19.00 h. Injection of estrogen advanced the increase of TIDA activity by 2 h in the ME compared to OVX rats. Moreover, administration of estrogen suppressed the activity of THDA and PHDA neurons in the afternoon compared to OVX rats. In estrogen plus progesterone-treated rats, the activity of hypothalamic neuroendocrine dopaminergic neurons terminating in the ME, IL, and NL was inhibited prior to the increase in the secretion of PRL. The concentration of DA in the AL diminished prior to the estrogen-induced increase of PRL. Administration of progesterone, in concert with estrogen, delayed the increase of PRL in serum and the decrease of DA in the AL, compared to estrogen-treated rats, by 4 h. These data suggest a major role for ovarian steroids in controlling increases in the secretion of PRL by not only stimulating PRL release from lactotrophs, but also by inhibiting the activity of all three populations of hypothalamic neuroendocrine DAergic neurons.  2000 Elsevier Science B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Neuroendocrine: other Keywords: Estrogen; Prolactin; Pituitary gland; Median eminence; Arcuate nucleus; Periventricular nucleus

1. Introduction With the exception of prolactin (PRL), the secretion of hormones from the anterior lobe (AL) of the pituitary *Corresponding author. Tel.: 11-850-644-3896; fax: 11-850-6444583. E-mail address: [email protected] (M.E. Freeman).

gland is stimulated by hormone-specific releasing factors originating within the hypothalamus [25,46–48]. While a single PRL releasing factor, analogous to those found for other pituitary hormones, has yet to be identified, several candidates, including ovarian steroids, have been considered [41]. The secretion of PRL is tonically inhibited by dopamine (DA) [4]. DA arrives at the AL through three distinct

0006-8993 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02763-3

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dopaminergic (DAergic) pathways: (1) Tuberoinfundibular DAergic (TIDA) neurons arise from throughout the arcuate nucleus and terminate on the primary capillary loops of the long portal vessels within the external zone of the median eminence (ME) [22]. Long portal vessels transport DA released from TIDA neurons to the AL. (2) Tuberohypophyseal DAergic (THDA) neuron cell bodies are located in the rostral portion of the arcuate nucleus and terminate in both the intermediate (IL) and neural (NL) lobes of the pituitary gland [6]. (III) Periventricular-hypophyseal DAergic (PHDA) neurons originate in the periventricular nucleus of the hypothalamus and terminate exclusively in the IL [23,24]. DA released from THDA and PHDA neurons is transported to the AL through short portal vessels. The increasing concentration of estradiol in serum on the afternoon of diestrus-2 of the estrous cycle of the rat stimulates the proestrous increase of PRL in serum [40]. Immunoneutralization of endogenous estrogen during the afternoon of diestrus-2 prevents the increase of PRL on the afternoon of proestrus [20,40]. In addition to stimulating the secretion of PRL [41], estrogen diminishes and / or reverses the inhibitory response of lactotrophs to DA [8,12,19,34], recruits cells within the pituitary gland to produce [31] and secrete [7,18,26] PRL and increases transcription of the PRL gene [30–32,36,38,50–53]. Unlike estrogen, the role of progesterone in the regulation of the secretion of PRL is not as thoroughly defined. However, when administered in concert with estrogen, progesterone is a potent stimulator of the secretion of PRL [10]. It has previously been shown that the activity of dopaminergic neurons terminating in the ME [42] and the concentration of DA in portal vessels is decreased [5] prior to the peak of PRL on the afternoon of proestrus. In addition, we previously reported that the concentration of DA and DOPAC decreases in the AL and IL, but not the NL prior to increased secretion of PRL on the afternoon of proestrus [16]. Moreover, it has been shown that ovarian steroids regulate biosynthesis [2,3,28,49,55] and release [13,14] of DA from TIDA neurons. In this study we have described the effects of ovarian steroids on the activity of all three populations of hypothalamic neuroendocrine dopaminergic neurons. Changes in the concentrations of DA and DOPAC in the ME, AL, IL, and NL and the concentration of PRL in serum were monitored following administration of estrogen and estrogen plus progesterone in ovariectomized rats.

2. Materials and methods

2.1. Animals and in vivo steroid treatments Female Sprague–Dawley rats (200–250 g; Charles River, NC) were housed under 12 h of illumination (initiated at 06.00 h) with water and rat chow available ad

libitum. Animal procedures were approved by the Florida State University Animal Care and Use Committee. Rats were bilaterally ovariectomized (OVX) under Halothane anesthesia and subsequently divided into three groups. Ten days following OVX, rats in group one (estrogen-treated) received an injection of estrogen (20 mg, i.p., Sigma, St Louis, MO) at 10.00 h on day zero and corn oil at 12.50 h on day one. Rats in group two received an injection of estrogen (20 mg, i.p., Sigma) at 10.00 h on day zero and an injection of progesterone (5 mg, i.p., Sigma) at 12.50 h on day one. Rats in group three received an injection of corn oil (200 ml, i.p., vehicle) at 10.00 h on day zero and 12.50 h on day one. Five rats from each treatment group were sacrificed by rapid decapitation every 2 h from 09.00 to 21.00 h on day one. Rats decapitated at 13.00 h on day 1 had received corn oil or progesterone 10 min before decapitation. The ME, AL, IL, and NL from each animal were dissected and individually stored in cryogenic vials with 250 ml of homogenization buffer [0.2 N perchloric acid, 26 mM EGTA, 700 pM dihydroxybenzylamine (DHBA, internal standard)] at 2408C until day of assay. Trunk blood was collected, allowed to clot, centrifuged, and serum was removed and stored at 2408C until the concentration of PRL was measured by RIA.

2.2. HPLC-EC ME, AL, IL, and NL samples were thawed, homogenized, and sonicated. Twenty microliters of homogenate were removed for protein assay. The remaining sample was centrifuged (10 min @ 10003g). The supernatant was filtered through a 0.2 mm nylon microfiltration unit (Osmonics, Livermore, CA), and then placed into autosampler vials. The concentration of DA and DOPAC in each sample was measured using high performance liquid chromatography coupled to electrochemical detection (HPLC-EC), as previously described [15–17,39]. Twenty microliters of each sample was injected by an autosampler (WISP 710 Autosampler, Waters Corp., Milford, MA). Mobile phase consisting of 75 mM sodium dihydrogen phosphate monohydrate (EM Science, Gibbstown, NJ), 1.7 mM 1-octane sulfonic acid (Fisher Scientific), 100 ml / l triethylamine (Aldrich, Milwaukee, WI), 25 mM EDTA (Fisher Scientific), 6% acetonitrile (EMScience), titrated to pH 3.0 with phosphoric acid (Fisher Scientific), was delivered by a dual piston pump (Kratos Analytical Instruments, Ramsey, NJ) at 600 ml / min. Water was purified on a Milli-Q system (Millipore, Bedford, MA) to 18 MV and polished with a C 18 Sep-Pak (Millipore). Catecholamines were separated on a reverse phase C 18 column (MD-150, Dimensions 15033 mm, particle size 3 mm, ESA inc., Chelmsford, MA), oxidized on a conditioning cell (E: 1300 mV, ESA 5010 Conditioning Cell, ESA inc.) and then reduced on a dual channel analytical

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cell (E 1 : 255 mV, E 2 : 2185 mV, ESA 5011 High Sensitivity Analytical Cell, ESA inc.). The change in current on the second analytical electrode was measured by a coulometric detector (ESA Coulochem II, ESA Inc.) and recorded using Baseline 810 software (Waters Corp.). DA and DOPAC were identified on the basis of their peak retention times (RT511.0 and 6.5 min, respectively). The amount of catecholamine in each sample was estimated by comparison to the area under each peak generated by known amounts of each catecholamine. The amount of DHBA (RT57.5 min) recovered was compared to the amount of DHBA added as internal standard and corrected for any loss of sample (usually ,5%). The sensitivity of the assay was 30 pg of DA and 33 pg of DOPAC.

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3. Results

3.1. Concentration of PRL in serum The concentration of PRL in serum is shown in Fig. 1.

2.3. Protein assay The amount of protein in each sample was measured using a modified form of the Pierce BCA Protein Assay Kit (Pierce, Rockford, IL). Homogenate (10 ml) from sonicated tissue samples was aliquoted into 96-well plates (Corning, Corning, NY) in duplicate and 200 ml of BCA solution was added to each well. The plate was incubated for 30 min at 608C, and the absorbance of each well was measured at 562 nm by a plate spectrophotometer (Molecular Devices, Palo Alto, CA). Unknowns were compared against standards of bovine serum albumin.

2.4. Radioimmunoassay ( RIA) The concentration of PRL in serum was determined by RIA as previously described [21] with materials supplied by Dr Albert F. Parlow and the National Hormone and Pituitary Program. Serum concentrations of PRL were expressed as ng / ml in terms of the rat PRL RP-3 standard in assays whose sensitivity averaged 1 ng / ml. The interassay coefficient of variation was 10% and the intra-assay coefficient of variation was 5% for both assays.

2.5. Data analysis Differences in the DOPAC / DA ratio, the concentration of DA in the AL, and the concentration of PRL in serum within individual treatment groups were compared statistically using a one-way ANOVA. Tukey’s honestly significant difference test was used as a post-hoc test for all data sets. Values are considered significant at P,0.05. Differences in the DOPAC / DA ratio, the concentration of DA in the AL, and the concentration of PRL in serum between treatment groups were compared statistically using a two-way ANOVA. Tukey’s honestly significant difference test was used as a post-hoc test for all data sets. Values are considered significant at P,0.05.

Fig. 1. The concentration of PRL in the serum of OVX (A), estrogen(B), and estrogen plus progesterone- (C) treated rats. Each point represents the mean6S.E.M. (n55). Points with dissimilar letters are significantly different. P,0.05.

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The concentration of PRL in serum of OVX rats is low and unchanging for most of the day with the exception of a 25% increase from baseline (P,0.05) occurring between 15.00 and 17.00 h (Fig. 1A). Unlike the steady and relatively unchanging levels of PRL in OVX rats, replacement of ovarian steroids significantly elevates baseline secretion of PRL. The concentration of PRL in serum of estrogen-treated rats is significantly increased (P,0.05) by 15.00 h, peaks by 17.00 h at a concentration three-fold greater than baseline, and returns to low levels by 21.00 h (Fig. 1B). The concentration of PRL in serum of estrogen plus progesterone-treated rats significantly increases by 19.00 h at a concentration five-fold greater than baseline, and diminishes to low levels thereafter (Figs. 1C).

3.2. TIDA neuronal activity: DOPAC /DA turnover in the ME The turnover of DA in the ME of OVX (A), estrogen(B) and estrogen plus progesterone- (C) treated rats is shown in Fig. 2 as the mean DOPAC / DA ratio6S.E.M. In this and subsequent figures the serum PRL concentrations shown in Fig. 1 are duplicated for comparison. DA turnover is significantly increased (P,0.05) by 13.00 h in OVX rats (Fig. 2A), diminishes to low levels by 15.00 h, returns to baseline by 17.00 h and subsequently increases by 21.00 h (Fig. 2A). Injection of estrogen to OVX rats decreases the turnover of DA in the ME between 09.00 and 11.00 h (P,0.05), delays the increase in DA turnover seen in OVX rats until 15.00 h concomitant with the initial increase in the secretion of PRL, and prevents the late evening increase in turnover of DA observed in OVX rats by 21.00 h (Fig. 2B). The DOPAC / DA ratio in estrogen plus progesterone-treated rats increases significantly (P, 0.05) by 13.00 h similar to OVX rats, but unlike OVX rats is depressed through the late afternoon coincident with an increase in serum PRL (Fig. 2C).

3.3. PHDA neuronal activity: DOPAC /DA turnover in the IL The turnover of DA in the IL of OVX rats increases by 11.00 h (P,0.05), returns to low levels by 17.00 h, coincident with the increase of serum PRL, and again increases (P,0.05) through 21.00 h (Fig. 3A). In contrast to the turnover of DA in the IL of OVX rats, the turnover of DA in the IL of estrogen-treated rats is unchanged between 09.00 and 15.00 h (Fig. 3B). The turnover of DA in the IL of estrogen-treated rats subsequently increases (P,0.05) by 17.00 h, coincident with the increase of serum PRL, and returns to low levels concomitant with the levels of PRL in serum (Fig. 3B). This is nearly a mirrorimage of the pattern of PRL secretion in OVX rats. Estrogen plus progesterone treatment significantly increases (P,0.05) DA turnover by 13.00 h, which then decreases thereafter through 17.00 h concomitant with the

Fig. 2. The concentration of PRL in serum and the DOPAC / DA ratio in the median eminence of OVX (A), estrogen- (B), and estrogen plus progesterone- (C) treated rats. Circles indicate mean concentration of PRL in serum6S.E.M. Significant differences in the concentration of PRL are as indicated in Fig. 1. Squares are mean DOPAC / DA ratio6S.E.M. Points with different letters are significantly different (P, 0.05).

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initiation of the increase of PRL in serum (Fig. 3C). This pattern is similar in timing, but differs in magnitude from that of OVX rats. However, unlike OVX rats, the turnover of DA in estrogen plus progesterone-treated rats remains low and unchanging in the IL thereafter (Fig. 3C).

3.4. THDA neuronal activity: DOPAC /DA turnover in the NL The turnover of DA in the NL of OVX rats increases (P,0.05) twice; first by 13.00 h, prior to the increase of PRL in serum, and the second by 19.00 h, during the waning phase of the peak of serum PRL (Fig. 4A). Unlike OVX rats, estrogen treatment causes a decrease (P,0.05) in the activity of THDA neurons by 11.00 h. However, similar to changes in OVX rats, the turnover of DA in the NL of estrogen plus progesterone-treated rats returns to high levels by 13.00 h (Fig. 4B). The turnover of DA in the NL of estrogen-treated rats decreases (P,0.05) steadily thereafter to low levels at 21.00 h coincident with the increase of PRL in serum (Fig. 4B). During the morning, estrogen plus progesterone-treated rats present a DA turnover profile similar to that of the estrogen-treated rats (Figs. 4B, C). However, following injection of progesterone at 13.00 h, DA turnover is significantly (P,0.05) depressed and remains low through 21.00 h (Fig. 4C), prior to and coincident with the increase of PRL in serum.

3.5. Concentration of DA in the anterior lobe

Fig. 3. The concentration of PRL in serum and the DOPAC / DA ratio in the intermediate lobe of OVX (A), estrogen- (B), and estrogen plus progesterone- (C) treated rats. Circles indicate mean concentration of PRL in serum6S.E.M. Significant differences in the concentration of PRL are as indicated in Fig. 1. Squares are mean DOPAC / DA ratio6S.E.M. Points with different letters are significantly different (P, 0.05).

The concentration of DA in the AL of OVX rats increases slightly from 09.00 to 15.00 h (Fig. 5A). Coincident with the slight increase of PRL in serum at 17.00 h, the concentration of DA in the AL decreases to morning levels (P,0.05) and remains low until it increases significantly (P,0.05) again at 21.00 h (Fig. 5A). Although of greater magnitude in estrogen-treated rats, the patterns of DA in the AL are similar between 09.00 h and 13.00 h in the OVX and estrogen-treated groups (Fig. 5A, B). In both groups, the concentration of DA in the AL significantly decreases (P,0.05) coincident with the increase in the concentration of PRL in serum at 15.00 h and remains low through 19.00 h (Fig. 5B). Concomitant with the return of PRL to baseline levels, the concentration of DA in the AL increases to levels similar to those of the morning (Fig. 5B). Similar to the concentration of DA in the AL of OVX rats, the concentration of DA in the AL of estrogen plus progesterone-treated rats is unchanged through 17.00 h and then subsequently decreases through 21.00 h (P,0.05). However, in estrogen plus progesterone-treated rats, the rate of decrease is greater than that seen in the AL of OVX rats. The concentration of DA in the AL of estrogen and progesterone-treated rats remains low through 21.00 h while the secretion of PRL increases (Fig. 5C; P,0.05), unlike the concentration of DA in the AL of estrogen-treated rats, which increased at 21.00 h.

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4. Discussion

Fig. 4. The concentration of PRL in serum and the DOPAC / DA ratio in the neural lobe of OVX (A), estrogen- (B), and estrogen plus progesterone- (C) treated rats. Circles indicate mean concentration of PRL in serum6S.E.M. Significant differences in the concentration of PRL are as indicated in Fig. 1. Squares are mean DOPAC / DA ratio6S.E.M. Points with different letters are significantly different (P,0.05).

While ovarian steroids play a critical role in the direct regulation of PRL secretion [41], they are not the only ´ participants in what is rapidly becoming a melange of regulatory factors. DA is the physiological inhibitor of the secretion of PRL [4]. However, the interplay between ovarian steroids and DA in regulating PRL secretion has yet to be elucidated. In these experiments we report the effects ovarian steroids have on the activity of three populations of hypothalamic neuroendocrine DAergic neurons involved in the control of PRL secretion. In the absence of ovarian steroids the secretion of PRL is low and relatively stable throughout the day [9,10,27]. Administration of exogenous estrogen stimulates the secretion of PRL in OVX rats [9,10]. Immunoneutralization of endogenous estrogen prevents spontaneous secretion of PRL on proestrus [40]. Moreover, administration of progesterone to estrogen primed rats, advances the timing [56] and enhances the secretion of PRL [9,10,27]. The DOPAC / DA ratio in the ME is an effective tool for monitoring the activity of TIDA neurons [35], whereas the DOPAC / DA ratio in the posterior pituitary is an effective tool for measuring the change in activity of THDA and PHDA neurons terminating in the IL and NL [33]. Using DOPAC / DA ratios, we have measured the activity of neuroendocrine dopaminergic neurons terminating in the ME, IL and NL in response to estrogen and estrogen plus progesterone. Additionally, we have measured the concentration of DA in the AL as a measure of DA arriving at the target tissue. It has previously been reported that estrogen inhibits the expression of tyrosine hydroxylase in the arcuate nucleus [2,28] and the release of DA into long portal vessels [13]. The data presented here reflect estrogen inhibition of neuroendocrine dopaminergic neurons. TIDA and THDA neuronal activity in estrogen-treated rats is inhibited in the morning between 09.00 and 11.00 h (Fig. 2B and 4B). Subsequently, the turnover of DA in TIDA neurons in the ME of estrogen-treated rats increases coincident with the initiation of the increase of PRL and returns to low levels at the peak concentrations of PRL (Fig. 2B). The turnover of DA in THDA neurons in the NL of estrogen-treated rats decreases steadily through the initiation, peak, and termination of the increased secretion of PRL (Fig. 4B). The turnover of DA in PHDA neurons of the IL of estrogentreated rats is relatively constant throughout the morning (09.00–15.00 h), increases coincident with the peak of PRL and returns to baseline thereafter (Fig. 3B). These data indicate that estrogen plays a role in the regulation of neuroendocrine dopaminergic neurons by inhibiting the activity of these neurons prior to the initiation of the increase of PRL. Indeed it has been hypothesized that estrogen down regulates the activity of TIDA neurons by diminishing the activity of tyrosine hydroxylase [28]. In addition to acting at the hypothalamus to alter dopa-

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minergic neuronal activity, estrogen serves as a major stimulator of lactotroph recruitment [26,31], PRL synthesis [30–32,37,50,51,53,54] and PRL secretion [9,10]. Progesterone alone does not have a substantial effect on the secretion of PRL [10,45]. However, when administered in concert with estrogen, progesterone is a potent stimulator of the secretion of PRL [10] and has been shown to reverse the estradiol-induced inhibition of tyrosine hydroxylase mRNA in the hypothalamus [2,3]. Administration of progesterone to estrogen-treated animals caused an increase in the activity of all three populations of neuroendocrine dopaminergic neurons at 13.00 h (Figs 2–4C) compared to estrogen-treated rats. The activity of TIDA and THDA neurons returns to low levels immediately following the increase of their activity at 13.00 h, whereas the activity of PHDA neurons decreases over a four hour period. The activity of all three populations of neuroendocrine dopaminergic neurons remains low throughout the initiation of the increase in PRL secretion (Figs. 2–4C). It has been shown that long-term progesterone treatment will stimulate the release of DA into portal vessels [14]. Following progesterone induced increases in the activity of these neurons, the activity of all three populations of neuroendocrine dopaminergic neurons declines rapidly and remains low through the increase of PRL secretion. Progesterone may be acting to deplete neurons of DA so that, concomitant with the increase of PRL secretion, the activity of neuroendocrine dopaminergic neurons is low and unchanging. The sum of the activity of these three populations of neurons is reflected by the concentration of DA in the AL of the pituitary gland (Fig. 5A–C). While the concentration of DA in the AL is not an accurate indicator of hypothalamic neuroendocrine dopaminergic activity [33,35], it reveals the amount of DA arriving at the target cells from all three populations. The relative contribution of DA from each neuron population to the AL cannot be ascertained from these data. However, based on our previous work indicating that all three populations of neuroendocrine dopaminergic neurons contribute DA to the AL [17], it is likely that each population contributes DA to the regulation of the secretion of PRL. In each treatment group the concentration of DA in the AL decreases prior to the increase in the secretion of PRL (Fig. 5). Moreover, the degree and timing of the decline appears to be mediated by the presence of ovarian steroids. The abundance of DA in the AL of OVX rats is low throughout most of the morning. The concentration of DA in the AL of OVX rats increases slightly (P,0.05) by 13.00 h, then decreases by 17.00 h, coincident with the slight increase in the concentration of PRL in serum (Fig. 5A). Estrogen treatment of OVX rats doubles the concentration of DA in the AL compared to oil-treated OVX rats. The concentration of DA in the AL of estrogen-treated rats increases by 13.00 h and subsequently declines concomitant with the initiation of the increase of PRL. The concentration of DA in the AL

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Fig. 5. The concentration of PRL in serum and the concentration of DA in the AL of OVX (A), estrogen- (B), and estrogen plus progesterone- (C) treated rats. Circles indicate mean concentration of PRL in serum6S.E.M. Significant differences in the concentration of PRL are as indicated in Fig. 1. Squares are mean DA concentration6S.E.M. Points with different letters are significantly different (P,0.05).

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of estrogen-treated rats remains low through 19.00 h, then increases back to values similar to those at 09.00 h (Fig. 5B). The decrease of DA in the AL at 15.00 h in estrogentreated rats is two-fold) greater than that of OVX rats. The apparent decrease of DAergic tone on the lactotroph, in concert with the lactotroph-priming action of estrogen [7,8,12,18,19,26,30–32,34,36,38,41,50–53], initiates an increase in the secretion of PRL. Administration of progesterone to estrogen-treated rats magnifies the inhibitory effects of estrogen only on the concentration of DA in the AL. No decrease in the concentration of DA in the AL occurs during the morning. However, the decrease of DA in the AL of estrogen plus progesterone-treated rats during the afternoon which is coincident with the increase of PRL secretion, is almost three fold greater and more abrupt than that of estrogentreated animals (Fig. 5C). The delay in the decrease of the concentration of DA in the AL of estrogen and progesterone-treated rats is expected given the delaying effects progesterone has on the activity of neuroendocrine dopaminergic neurons [56]. These changes initiate an increase in the secretion of PRL that is two-fold greater and occurs several hours later than that seen in the estrogen-treated rat. We have demonstrated that PRL plays a role in activating all three populations of hypothalamic neuroendocrine DAergic neurons [15]. It has been shown that the activity of TIDA, THDA, and PHDA neurons decreases prior to an increase in endogenous PRL [15]. However, the mechanism for inactivation of these neurons is still unclear. These data support the notion that ovarian steroids, especially estrogen, are responsible for relieving the dopaminergic inhibition of lactotrophs. Indeed, estrogen decreases the number of dopamine receptors in the AL [1,43] and decreases tyrosine hydroxylase production [2] and activity [28] in the arcuate nucleus. In addition to directly inhibiting the activity of TIDA, THDA, and PHDA neurons, estrogen uncouples inhibitory subunits of G-proteins coupled to inhibitory DA D 2 receptors on lactotrophs [34], increases calcium influx into lactotrophs [11,44], and upregulates PRL-R number on neuroendocrine DAergic neurons [29]. These data, taken together with other reports, suggest that while PRL is responsible for activation of all three populations of hypothalamic neuroendocrine DAergic neurons, estrogen is responsible for inhibiting their activity prior to an endogenous increase in PRL secretion. Moreover, administration of progesterone to estrogen-treated animals intensifies and delays the inhibitory effects of estrogen to produce a later and greater secretion of PRL.

Acknowledgements The authors wish to thank DeAnn Scarborough for her technical assistance. Dr Albert F. Parlow and the National

Hormone Pituitary Program are thanked for the PRL RIA reagents. This work was supported by NIH DK 43,200 to MEF.

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