Regulation of prolactin secretion during pregnancy and lactation.

Prolactin plays major roles in maintaining the corpora lutea of pregnancy and in the synthesis of milk during lactation. The hypothalamic mechanisms involved in these functions have been investigated. Mating leads to a surge of prolactin and programs daily surges during early pregnancy. The expression of Fos-immunoreactivity shows that mating activates several hypothalamic nuclei, particularly the arcuate nucleus and medial preoptic area. In the arcuate nucleus, mating is associated with Fos expression in beta-endorphin neurons, and infusion of naloxone blocks both mating-induced and diurnal prolactin surges. Tyrosine hydroxylase-immunoreactive dopamine neurons appear not to participate in surge generation. However, after day 10 of gestation the secretion of placental lactogens suppresses prolactin secretion via activation of dopamine neurons without involvement of beta-endorphin neurons. Intracerebroventricular implantation of placental lactogen-secreting cells will block pregnancy prolactin surges, increase Fos expression in dopamine neurons, and increase tyrosine hydroxylase activity. During lactation the mechanisms regulating dopamine and beta-endorphin neurons are further modified. In early lactation a prolactin-induced increase in tyrosine hydroxylase activity leads to negative feedback, but this effect is lost by mid-lactation. Overriding this negative feedback is the inhibitory effect that suckling has on dopaminergic activity. This may involve beta-endorphin-mediated inhibition of dopamine neurons, as naloxone causes a marked increase in tyrosine hydroxylase activity and suppression of circulating prolactin. However, removal of tonic dopamine inhibition is not sufficient to account for the high levels of prolactin attained during lactation, and additional releasing factors are probably involved. In situ hybrization histochemistry for the most recent candidate, prolactin-releasing peptide, suggests that this may involve brain stem neurons that co-localize noradrenaline. Thus, prolactin secretion during pregnancy and lactation involve complex interactions of regulatory factors and plasticity of neuronal responsiveness.

Mating-activated brainstem catecholaminergic neurons in the female rat.

Central catecholaminergic systems play an important role in the control of reproductive activities including sexual behavior, luteinizing hormone (LH) and prolactin secretion. It has been reported that catecholaminergic neurons in the locus coeruleus (A6) are activated by mating in rabbits and ferrets, animals known as reflex ovulators. This study used Fos as a marker of neuronal activity to examine whether brainstem catecholaminergic neurons are activated by mating in the spontaneous ovulator, the female rat. Proestrous rats receiving intromissions (mated group) from males or mounts-without-intromission (mounted group) were sacrificed along with rats taken directly from their home cage (control group) 90 min after the beginning of mating or mounting. Double-label immunocytochemistry was used to examine the expression of c-Fos in catecholaminergic neurons labeled by tyrosine hydroxylase (TH) antibody, or adrenergic neurons labeled by phenylethanolamine-N-methyl transferase (PNMT) antibody. Double label immunofluorescent immunohistochemistry was used to determine the number of neurons containing the estrogen receptor (ERalpha) that were activated by mating in these brain areas. The results showed that mating-with-intromissions induced a significant increase in the percentage of TH/Fos colabeled neurons in both A1 and A2 cells compared to mounting-without-intromission or control. In both these areas, over 50% ERalpha-ir neurons were activated after mating while mounting-without-intromission did not affect the percentage of colabeled Fos/ERalpha neurons. In A6 region, neither the expression of Fos nor the percentage of TH/Fos colabeled cells was influenced by either mating or mounting compared to controls. The percentage of PNMT-containing neurons colabeled with Fos was not different in C1 and C2 among the three experimental groups. The results indicate that catecholaminergic neurons were activated by mating in A1 and A2 but not in adjoining adrenergic C1 and C2 cells. In contrast to the findings that catecholaminergic neurons in A6 are activated by mating in induced ovulators, mating did not affect neuronal activity in A6 neurons in the female rat. In A1 and A2 areas, a high percentage of neurons containing ERalpha were activated by mating suggesting both tactile and hormonal information may converge on these populations of neurons. The activated catecholaminergic neurons in A1 and A2 may be an important pathway by which sensory information generated during sexual interaction modulates both behavior and pituitary function.

Effects of chronic bromocriptine treatment on tyrosine hydroxylase (TH) mRNA expression, TH activity and median eminence dopamine concentrations in ageing rats.

The purpose of this study was to investigate the age-related changes in the responsiveness of tuberoinfundibular dopamine (TIDA) neurones to chronic hypoprolactinemia induced by treatment with bromocriptine, a dopamine receptor agonist. In one experiment, TIDA neuronal activity after acute hypoprolactinemia or exogenous prolactin was monitored by measuring tyrosine hydroxylase (TH) activity in the stalk median eminence of middle-aged cycling female rats (10-12 months), old constant oestrous rats (18-20 months) and old pseudopregnant rats (22-24 months). In another experiment, middle-aged cycling (10-12 months) rats were treated with bromocriptine for 6 or 12 months. TH activity was measured in the stalk median eminence, TH mRNA levels were measured in the arcuate nucleus and dopamine concentrations were measured in the arcuate nucleus and median eminence. Responsiveness of TIDA neurones to exogenous prolactin and to the withdrawal of bromocriptine in these rats was also tested. While the TIDA neurones in all three age groups responded to acute hypoprolactinemia by showing a reduction in TH activity, older rats failed to respond to exogenous prolactin administration. In contrast, chronic hypoprolactinemia for 12 months enabled the rats to retain TIDA neuronal responsiveness to exogenous prolactin. It also decreased TIDA neuronal function as measured by dopamine concentrations in the median eminence, TH activity in the stalk median eminence and TH mRNA in the arcuate nucleus of ageing rats. The restoration of the responsiveness of these neurones to prolactin stimulation in older rats demonstrates for the first time that hypoprolactinemia produced by chronic bromocriptine treatment indeed provides a neuroprotective effect on TIDA neurones. These results indicate that maintaining a low level of neuronal activity by lowering prolactin levels may be a contributing factor in retaining the plasticity of TIDA neurones.

Mechanisms for suckling-induced changes in expression of prolactin receptor in the hypothalamus of the lactating rat.

The present study aimed to investigate whether increased expression of prolactin receptor (PRL-R) during lactation is caused by suckling-induced hyperprolactinemia or the suckling stimulus itself. Three groups (n=7) of mid-lactating rats were used. Each rat received 3 days of s.c. injection of vehicle or drug before sacrifice on lactation day 10. Rats in the control group received vehicle only and were suckled by pups. The second group received bromocriptine to suppress PRL levels and were suckled by pups. The third group of rats received haloperidol (high PRL) and were deprived of pups. Plasma PRL levels were measured. Animals were perfused with 2% paraformaldehyde for immunofluorescent study. Results showed that PRL-R immunoreactivity in the ventrolateral preoptic, ventromedial preoptic, and ventromedial hypothalamic nuclei was significantly increased in the bromocriptine-treated group compared to the control group, indicating PRL-R expression in these areas may be inhibited by hyperprolactinemia in the presence of the suckling stimulus. The PRL-R in the lateroanterior, ventrolateral and paraventricular nuclei was significantly decreased in the haloperidol-treated group compared to the control group, suggesting that the PRL-R in these areas is most likely regulated by the suckling stimulus itself. The PRL-R in the arcuate nucleus was significantly increased in bromocriptine-treated rats and decreased in haloperidol-treated rats, suggesting that the PRL-R in this nucleus is regulated by mechanisms related to both the stimulus of suckling itself and suckling-induced hyperprolactinemia. These results support the hypothesis that expression of PRL-R in discrete hypothalamic nuclei is differentially regulated by either PRL and/or suckling.

Involvement of endogenous opioidergic neurons in modulation of prolactin secretion in response to mating in the female rat.

Mating in female rats induces an acute prolactin (PRL) release within 60 min and twice-daily surges of PRL throughout the first 10 days of pregnancy to maintain luteal function. Little is known about the brain mechanism whereby the vaginocervical stimulation is processed to induce PRL release. Our recent results revealed an increase in Fos expression in the arcuate nucleus (ARC) following mating in the intact estrous rat, suggesting that a neuronal network in the brain area may participate in conveying and integrating the genitosensory stimulation. To further investigate the phenotype of activated neurons in the ARC, the present study examined whether beta-endorphin (beta-END) and/or dopamine (DA) neurons are activated by mating, and if so, whether activation is involved in the mating-induced acute release of PRL and the establishment of the twice-daily surges of PRL. In experiment 1, proestrous rats receiving intromissions (mated group) from males or mounts without intromission (mounted group) were sacrificed along with rats taken directly from their home cage (control group) 60 min after the beginning of mating or mounting. Expression of Fos in beta-END neurons and expression of fos-related antigen (FRA) in DA neurons, which were labeled by tyrosine hydroxylase (TH) antibody in the ARC were examined by double-label immunocytochemistry. In experiment 2, proestrous females with indwelling atrial catheters were mated with males. Naloxone (10 microl/min, 2 mg/10 min), an opiate antagonist, or saline was infused before, during and after mating. Blood samples were collected during the mating session and also at several times 3 days after mating. The results showed that mating induced a significant increase in the percentage of beta-END/Fos colabeled neurons and a significant decrease in the number of beta-END cells in all subdivisions of the ARC. In contrast, neither the percentage of FRA/TH colabeled cells nor the number of TH cells was influenced by mating. Mating induced an acute increase in PRL release in saline-treated control animals within 30 min and a subsequent diurnal surge (18.00 h) and a nocturnal surge of PRL (2.00 h) 3 days after mating. Naloxone infusion during mating blocked the mating-induced acute PRL response and the diurnal surge of PRL 3 days after mating, but affected neither the nocturnal surge of PRL nor the incidence of pregnancy. These results demonstrate that (1) beta-END neurons but not DA neurons in the ARC are activated in response to mating in proestrous rats, and (2) the mating-induced activation of beta-END neurons may participate in the acute response of PRL release to mating and the memory mechanism for the establishment of the diurnal PRL surge, but not the nocturnal PRL surge in early pregnancy. These results lead to a conclusion that endogenous opioid peptides may be involved in the neuronal transmission of genitosensory stimulation to induce PRL secretion.

Hypothyroidism increases prolactin secretion and decreases the intromission threshold for induction of pseudopregnancy in adult female rats.

In order to understand the mechanism by which thyroid hormones alter prolactin (PRL) secretion, we investigated the role of tuberoinfundibular dopamine (TIDA) neurons and pituitary and hypothalamus vasoactive intestinal peptide (VIP) in thiouracil- (0. 03% in drinking water for 16 days) induced-hypothyroid adult female rats. The intromission threshold for induction of pseudopregnancy also was examined to evaluate the PRL response to coital stimulation in hypothyroid rats. Hypothyroidism in adult female rats did not affect TIDA neuronal activity as measured by tyrosine hydroxylase activity (DOPA accumulation 30 min after administration of m-hydroxybenzylhydrazine dihydrochloride, 100 mg/kg, i.p.) in the stalk-median eminence compared with that in euthyroid rats, whereas pituitary concentration of VIP was dramatically increased. Plasma concentration of PRL was higher at 1100 h of proestrus and estrus in hypothyroid rats as compared with that of euthyroid rats. The proportion of female rats exhibiting pseudopregnancy was higher in hypothyroid animals (100%) receiving seven intromissions than in euthyroid animals (43%). Administration of L-thyroxine in hypothyroid rats decreased the proportion of pseudopregnancy (40%) to the level of euthyroid animals. These results indicate that the increased level of pituitary VIP probably affects PRL secretion in a paracrine or autocrine manner and account for the hyperprolactinemia induced in hypothyroid female rats. No role for TIDA neurons in PRL elevation can be ascribed. A decrease in the intromission threshold for induction of pseudopregnancy might be due to increased levels of PRL in hypothyroid female rats.

Effect of suckling on prolactin receptor immunoreactivity in the hypothalamus of the rat.

Previous studies showed that expression of prolactin (PRL) receptor is increased in numerous hypothalamic nuclei in mid-lactating rats. The increase in PRL receptor expression could be initated by neurohormonal changes during proestrus or pregnancy, or by the suckling stimulus during lactation. The present study investigated whether the PRL receptor expression in numerous hypothalamic nuclei is altered by the suckling stimulus. Three groups (n = 4) of rats on lactation day 10 were used: a continuously suckled group, a nonsuckled group (pups removed for 12 h) and a resuckled group (pups removed for 12 and then resuckled for 9 h). Animals were perfused with 2% paraformaldehyde and brains were sectioned (20 microm) for the immunofluorescence study. Immunoreactivity was semiquantitatively analyzed by counting the immunoreactive cells and measuring the immunostaining intensity in a specific area. Neurons expressing PRL receptors were observed in numerous hypothalamic areas with the highest number being in the arcuate, paraventricular and supraoptic nuclei. The PRL receptor immunofluorescence in several nuclei was significantly decreased in the nonsuckled group, and recovered in the resuckled group. These areas included the ventromedial preoptic, ventrolateral preoptic, lateroanterior hypothalamic, ventrolateral hypothalamic and ventromedial hypothalamic nuclei. PRL receptor immunoreactivity in other areas was not significantly altered by the suckling stimulus. These results demonstrate that expression of PRL receptor in hypothalamic nuclei was differentially affected by the suckling stimulus. PRL receptors in those nuclei which were significantly altered by suckling stimulus may play more critical roles during lactation than those areas which were less sensitive to the suckling stimulus.

Calcitonin inhibition of prolactin secretion in lactating rats: mechanism of action.

The effects of intracerebroventricular (10 ng/rat) or intravenous (10 or 40 microg/15 min/rat) administration of salmon calcitonin (sCT) on the prolactin (PRL) response to suckling and the activity of tyrosine hydroxylase (TH) were examined in lactating rats. Plasma concentration of PRL increased dramatically in control rats after the onset of the suckling stimulus, while administration of sCT resulted in inhibition of PRL response to suckling. The action of sCT was much more effective with intracerebroventricular administration, which totally blocked PRL release, compared to intravenous administration. The intracerebroventricular administration of sCT increased TH activity of tuberoinfundibular dopamine neuron (TIDA) in the stalk-median eminence, as measured by DOPA accumulation, while completely suppressing the PRL response to suckling. Injection of alpha-methyl-p-tyrosine (alpha-MT; 50 mg/kg), an inhibitor of TH and thus dopamine synthesis, increased PRL levels, and suckling caused a further increase in plasma concentrations of PRL. Injection of sCT (intracerebroventricularly) did not inhibit the PRL response to suckling in the presence of a depletion of dopamine. These results suggest that sCT inhibition of PRL secretion in lactating rats is mediated mainly by TIDA neurons without involvement of other neuroendocrine mechanisms.

Distribution of prolactin-releasing peptide mRNA in the rat brain.

In order to identify the distribution of prolactin-releasing peptide (PrRP) mRNA in the rat brain, we independently cloned cDNA of PrRP. Brains were removed from three adult males, and brains from three females each at 0200 and 1400 h on day 7 of pregnancy were obtained. By the nonradioactive in situ hybridization method, the location of PrRP mRNA was detected in very restricted brain areas. The distribution of PrRP mRNA signals was very similar in both sexes. In the hypothalamus, only the ventral part of the caudal dorsomedial nucleus had PrRP mRNA signals. Other forebrain areas did not show any positive signals. In the medulla oblongata, two discrete areas contained PrRP mRNA signals. No positive signal was found in the rostral part of the medulla oblongata extending to the anterior part of the area postrema. The caudal part of the nucleus of the solitary tract (NTS) had neurons with very strong signals of PrRP mRNA. The reticular nucleus showed a few PrRP mRNA positive neurons. The number of PrRP mRNA positive cells in the NTS was not different between experimental groups, although plasma prolactin levels in these animals were different. This anatomical information on the location of PrRP mRNA in the brain provides the framework to understand the physiological functions of PrRP in vivo.

Pituitary hormone gene expression and secretion in human growth hormone-releasing hormone transgenic mice: focus on lactotroph function.

The human GH-releasing hormone (hGHRH) transgenic mouse has a hyperplastic anterior pituitary gland that eventually develops into an adenoma. We showed previously that the number of lactotrophs in the male hGHRH transgenic mouse is increased 2-fold, yet there is no concomitant increase in plasma levels of PRL. To further elucidate underlying changes in lactotroph function in the hGHRH transgenic mouse, the objectives of this study were to 1) examine the relative differences in PRL gene expression in transgenic mice and their siblings, 2) quantify PRL secretion at the level of the individual cell, 3) determine whether tyrosine hydroxylase gene expression and/or activity are altered in the hypothalamus of transgenic mice, and 4) assess dopamine receptor gene expression and functional sensitivity in lactotrophs of transgenic mice. Total PRL messenger RNA (mRNA) levels were increased nearly 5-fold in the hGHRH transgenic mouse, whereas the concentrations of PRL mRNA (PRL mRNA per microg total RNA) were unchanged. In contrast, total PRL contents were unchanged, whereas the concentrations of PRL (micrograms of PRL per mg total protein) were decreased 3-fold. Hypothalamic tyrosine hydroxylase steady state mRNA levels were not altered in the hGHRH transgenic mice, but hypothalamic tyrosine hydroxylase activity was increased 2-fold in transgenic mice. Dopamine D2 receptor mRNA concentrations in the anterior pituitary were increased 2.5-fold in hGHRH transgenic mice, and total pituitary D2 receptor mRNA levels were increased nearly 10-fold. Furthermore, the basal secretory capacity of lactotrophs from transgenic mice was increased significantly at the level of the single cell, and dopamine inhibited the secretion of PRL to a greater extent in hGHRH transgenic mice. Thus, although the total number of lactotrophs is increased 2-fold in hGHRH transgenic mice, the present data are consistent with the hypothesis that increased hypothalamic dopamine synthesis and release coupled with an increase in D2 dopamine receptor gene expression and functional sensitivity in the pituitary result in normal plasma levels of PRL.

Rhythmicity of beta-endorphinergic neuronal activity in the mediobasal hypothalamus during pregnancy in the rat.

During the first half of gestation in the rat, prolactin (PRL) from the anterior pituitary gland exerts its luteotropic function on the ovary to stimulate progesterone secretion. During this period, beta-endorphin stimulates PRL secretion by regulation of dopaminergic neurons in the hypothalamus. During the second half, placental lactogens (PLs) take the place of PRL in maintenance of pregnancy, and initiate a negative feedback to suppress PRL secretion. However, the effect of PLs on beta-endorphinergic neurons is not known. The aim of this study was to examine the possibility that PLs suppress PRL secretion by inhibiting beta-endorphinergic neuronal activity. To accomplish this aim, we examined the changes in the neuronal activity of beta-endorphinergic neurons in the mediobasal hypothalamus, as measured by Fos immunoreactivity, after manipulating the levels of PRL and PLs during pregnancy. On day 4 of pregnancy, animals received either Rcho-1 cells in the lateral ventricle that secrete PLs or HRP-1 cells as controls. In a separate experiment on day 12, hysterectomy was performed to remove the intrinsic source of PLs. These rats received Rcho-1 cells, HRP-1 cells, or nothing. Intracerebroventricular (i.c.v.) injection of Rcho-1 into hysterectomized rats was done to examine the effect of PL replacement. Sham-hysterectomy was also performed as a control. Animals were sacrificed 2 days after each treatment at 0200 h, 1400 h, and 1800 h. Brains were used for dual immunocytochemistry of Fos/beta-endorphin. The neuronal activity of beta-endorphinergic neurons of HRP-1 i.c.v. injected animals showed a daily rhythm, with high levels at 0200 h and 1800 h, and a low level at 1400 h. These animals also exhibited two surges of PRL secretion on day 6 of pregnancy. This rhythmicity of beta-endorphinergic neurons was also observed in Rcho-1 i.c.v. injected animals, which showed very low and unchanging PRL levels. However, the magnitude of neuronal activity was reduced. On day 14 of pregnancy, all four experimental groups showed diurnal rhythms of beta-endorphinergic neurons. This rhythmicity occurred even though PRL was elevated at all three time points in the hysterectomized rats and very low in the Rcho-1 i.c.v. injected hysterectomized and sham-hysterectomized rats. Our results demonstrate that there is a diurnal rhythm of beta-endorphinergic neuronal activity in the mediobasal hypothalamus during pregnancy in the rat. PLs might reduce the neuronal activity of beta-endorphinergic neurons, but only during the first half of pregnancy, partially explaining the suppression of PRL surges.

Effects of naloxone infusion on nocturnal prolactin secretion and Fos/FRA expression in pregnant rats.

Dopamine (DA) secreted by tuberoinfundibular dopaminergic (TIDA) neurons in the hypothalamus is the major inhibitory factor controlling prolactin (PRL) secretion from the anterior pituitary. Endogenous opioid peptides (EOPs), mainly the neuropeptide beta-endorphin, have a stimulatory influence on PRL secretion. During the first half of pregnancy in rats, PRL secretion is characterized by two daily surges, the nocturnal surge and the diurnal surge. We tested the hypothesis that EOPs are critical stimulatory factors in regulating the nocturnal PRL surge, possibly via inhibition of TIDA neuronal activity. Naloxone (NAL), an opioid antagonist, was continuously infused (0.2 mg/10 microL/min i.v.) during the expected time of the nocturnal PRL surge in day 8 pregnant rats. Radioimmunoassay (RIA) was used to measure plasma PRL levels, and the immunocytochemical (ICC) staining of Fos/FRA was performed to detect changes in transcriptional activity of neurons in the hypothalamus. ICC of tyrosine hydroxylase (TH), the rate-limiting enzyme for DA synthesis, was performed to visualize TIDA neurons in the arcuate nucleus. The results showed that the nocturnal surge of PRL was markedly delayed and dampened in NAL-treated rats (p < 0.01). The numbers of both Fos/FRA and (Fos/FRA)/TH dual-staining neurons increased in the arcuate nucleus following NAL treatment (p < 0.05 for both comparisons). These data indicate that EOPs are critical stimulatory factors for the nocturnal PRL surge and that the actions of EOPs are partially mediated via decreasing TIDA neuronal activity.

3,5 cyclic adenosine monophosphate mediates the salmon calcitonin-induced increase in hypothalamic tyrosine hydroxylase activity.

This study examined the effect of salmon calcitonin (sCT) on hypothalamic tyrosine hydroxylase (TH) activity and evaluated the cellular signaling mechanisms involved in the response. Fetal hypothalamic cells were cultured in a defined medium and treated with sCT and/or specific protein kinase inhibitors on day 14 in vitro. sCT (0.1-10 nM) increased both TH activity and cellular cAMP content in a concentration-dependent manner. sCT (10 nM) increased TH activity to 150-175% of control values and resulted in a 10-fold increase in cellular cAMP content. Both the C1a and C1b CT receptor isoforms were present in the cultures, as assessed by RT-PCR. Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), a cAMP antagonist, and H-8, a cyclic nucleotide kinase inhibitor, blocked the sCT-induced increase in TH activity, with complete abolition of the response observed at concentrations of 1 mM and 5 microM, respectively. sCT (10 nM) increased radiolabeled phosphate incorporation into TH protein to 169% of control values and 1 mM Rp-cAMPS completely blocked this effect. In contrast, neither Calphostin C, a protein kinase C inhibitor, nor U-73122, a phospholipase C inhibitor, significantly altered the ability of sCT to increase TH activity. Likewise, the sCT-induced increase in TH activity was observed after pretreating the cells with either BAPTA/AM, an intracellular calcium chelator, or thapsigargin, an inhibitor of the endoplasmic reticulum calcium pump. These data indicate that sCT has a profound stimulatory effect on TH activity in fetal hypothalamic cells and that enhanced phosphorylation of TH coincides with the sCT-induced increase in enzyme activity. Moreover, CT receptors, which are linked to cAMP production, are expressed in the hypothalamic cells and a cAMP-dependent mechanism mediates the sCT-induced activation and phosphorylation of TH.

Feedback effects of placental lactogens on prolactin levels and Fos-related antigen immunoreactivity of tuberoinfundibular dopaminergic neurons in the arcuate nucleus during pregnancy in the rat.

PRL in the rat exerts its luteotropic action during the first half of pregnancy. After midpregnancy, placental lactogens (PLs) take the place of PRL to stimulate progesterone secretion from the corpus luteum. Simultaneously, PLs trigger a negative feedback on PRL secretion. However, the brain mechanisms for the negative feedback induced by PLs are not fully understood. Here, we report changes in plasma PRL levels, tuberoinfundibular dopaminergic (TIDA) neuronal activity as measured by Fos-related antigen (FRA)/tyrosine hydroxylase (TH) immunoreactivity, and TH catalytic activity as measured by dihydroxyphenylalanine (DOPA) accumulation in the stalk-median eminence (SME) after experimental manipulation of PL levels. On day 4 of pregnancy, animals received Rcho-1 cells intracerebroventricularly (i.c.v.) to increase the level of PLs in the brain or HRP-1 cells as controls. On day 12 of pregnancy, hysterectomy alone or i.c.v. HRP-1 injection plus hysterectomy were performed to remove the source of PLs. Rcho-1 i.c.v. injection plus hysterectomy were performed to examine the effect of replacement of the PL source. Sham-hysterectomized animals were used as a control group. Animals were killed 2 days after each treatment at 0200 and 1800 h, which represent the peak times of PRL surges, and at 1400 h, which represents the intersurge time, by either transcardial perfusion for FRA/TH immunocytochemistry or decapitation 30 min after NSD 1015 injection to assess DOPA accumulation with HPLC-electrochemical detection. Rcho-1 cells completely abolished PRL surges on day 6 of pregnancy and increased the percentage of FRA/TH immunoreactivity in the dorsomedial, ventrolateral, and caudal subdivisions of the arcuate nucleus. This change in neuronal activity reflected the amount of DOPA accumulation in the SME, which was high at all time points. On day 14 of pregnancy, removal of the PL source by hysterectomy resulted in increased PRL levels and decreased neuronal activity of TIDA neurons at all three time points. Similar profiles were observed in animals that received i.c.v. HRP-1 injection plus hysterectomy. Replacement of the source of PL with Rcho-1 cells in hysterectomized rats resulted in low PRL secretion, high neuronal activity of TIDA neurons, and high TH catalytic activity. These patterns were the same as those in sham-operated animals. Our results demonstrate that PLs induce an increase in the neuronal activity of dopaminergic neurons, as measured by FRA/TH immunoreactivity and TH catalytic activity in the SME. Removal of the PL source elevates plasma PRL levels at all times during the second half of pregnancy and does not restore PRL surges.

Fos expression in the female rat brain during the proestrous prolactin surge and following mating.

A prolactin (PRL) surge occurs in the female rat during proestrus in response to elevated estradiol levels. The elevated release of ovarian steroids on the day of proestrus is also associated with sexual receptivity. Mating triggers twice-daily PRL surges that supplant the proestrous PRL surge and are responsible for maintaining luteal function during the first half of pregnancy. In order to understand the neuronal mechanisms controlling the proestrous- and mating-induced PRL surges, we examined patterns of Fos expression by immunocytochemistry in specific brain regions as a measure of neuronal activity. Intact female rats were sacrificed at 09.00, 15.00, and 18.00 h on the day of proestrus and the day of diestrus. Brain tissues were also collected at 21.00 h on the day of proestrus from rats receiving intromissions or mounts from males or taken directly from their homecage. On the day of proestrus, the number of neurons expressing Fos in the medial preoptic area (mPOA), medial amygdaloid nucleus (mAMYG), and ventromedial nucleus of the hypothalamus (VMH) was few and was associated with low plasma PRL levels at 09.00 h; however, the number of Fos-positive cells in these brain regions significantly increased at 15.00 and 18.00 h when the proestrous PRL surge occurred. Mating during the evening of proestrus resulted in a dramatic increase in the number of Fos-positive cells in the mPOA, mAMYG, and VMH as well as in the arcuate nucleus (ARC). Nonmated animals in diestrus showed low and unchanged PRL levels and Fos expression in all the brain areas throughout the day. These results suggest that the mPOA, mAMYG, VMH and ARC may be important brain sites for the integration of stimuli associated with proestrous- and mating-induced PRL surges.

Responsiveness of tuberoinfundibular dopaminergic neurons to 5-hydroxytryptophan: effects of aging.

Serotonin is known to stimulate prolactin secretion by decreasing tyrosine hydroxylase (TH) activity in the tuberoinfundibular dopaminergic (TIDA) neurons. However, the effects of aging on the responsiveness of TIDA neurons to serotonin are not known. An effective way to increase serotonergic activity is to administer 5-hydroxytryptophan (5-HTP), a serotonin precursor. The present study was done to investigate the effects of 5-HTP on TIDA neuronal activity in aging animals. Middle-aged (10-12 mo), old (18-20 mo), and very old (22-24 mo) female Sprague-Dawley rats were bilaterally ovariectomized. Ten days later, they were injected iv with 50 mg/kg body wt of 5-HTP or the vehicle for 5-HTP (PBS-HCI). Twenty minutes later, m-hydroxybenzylhydrazine (NSD), a DOPA decarboxylase inhibitor, was administered. Ten minutes later, the animals were killed, and tyrosine hydroxylase (TH) activity was determined by measuring L-DOPA accumulation in the stalk median eminence by HPLC-EC. In all three groups, administration of 5-HTP increased serum prolactin levels significantly. In control middle-aged rats, TH activity (L-DOPA pg/ microg protein) was 33.0+/-5.6. Treatment with 5-HTP decreased TH activity by 60%. Similarly, 5-HTP treatment decreased TH activity by 52 and 56% in 18- to 20- and 22- to 24-mo-old rats, respectively, compared to the control rats. The magnitudes of the 5-HTP-induced decreases in TH activities in middle-aged, old, and very old rats were not different from each other. These results indicate that TIDA neuronal responsiveness to serotonin does not change with age and that 5-HTP is capable of stimulating PRL release even in very old rats.

Endogenous opioid peptides contribute to suckling-induced prolactin release by suppressing tyrosine hydroxylase activity and messenger ribonucleic acid levels in tuberoinfundibular dopaminergic neurons.

The endogenous opioid peptides have been implicated in the control of the suckling-induced PRL rise during lactation. This study examined the role of the endogenous opioid peptides in suppressing tuberoinfundibular dopaminergic neuronal activity during lactation. In the first experiment, lactating rats were constantly exposed to pups. Naloxone (NAL; 60 mg/kg x h; i.v.), an opioid receptor antagonist, or saline was infused for 12 h. Blood was collected before and at 2-h intervals during the infusion. NAL suppressed circulating PRL levels to less than 36% of control values at 4, 6, 8, and 12 h after the onset of the infusion. Tyrosine hydroxylase (TH) activity in the stalk-median eminence and TH messenger RNA signal levels in the arcuate nucleus were determined at the end of the NAL infusion. TH activity and TH messenger RNA signal levels were increased 2.5- and 2.7-fold, respectively, after the 12-h NAL infusion. Even though the time spent with their pups was similar between the two groups, the pups in the NAL-treated group failed to gain weight during the 12-h NAL infusion period, whereas the control litters (8 pups) gained 5 g. In a second experiment, pups were removed from the dams before the 12-h NAL infusion and were returned after 11 h. Blood was collected before the infusion, at 3-h intervals during the pup separation period, and at 15-min intervals after reunion with the pups. Plasma PRL in control and NAL-treated rats was low (1-15 ng/ml) and similar during the separation period. The suckling-induced PRL surge in NAL-treated rats was markedly attenuated to 9-25% of control levels (350-650 ng/ml). After a 1-h suckling episode, TH activity in the stalk-median eminence of NAL-treated rats was 4.5-fold greater than controls. Litter weight gains were significantly less in NAL-treated rats during the 1-h suckling episode. These data indicate that the endogenous opioid peptides are an integral component for increasing PRL release in response to suckling and they act to decrease tuberoinfundibular dopaminergic neuronal activity during lactation, in part, by suppressing TH gene expression.

Effects of chronic hyperprolactinemia on tuberoinfundibular dopaminergic neurons.

Prolactin (PRL) secretion is under the inhibitory regulation of the tuberoinfundibular dopaminergic (TIDA) system. Short-term elevation in PRL levels has been shown to increase the activity of TIDA neurons, however, the responsiveness of TIDA neurons to chronically elevated serum PRL levels is controversial. The purpose of this study was to investigate the effects of prolonged elevations of serum PRL on TIDA neuronal activity. Female Sprague-Dawley rats (2-3 months old) were ovariectomized and implanted (s.c.) with haloperidol (HAL), a dopamine receptor antagonist for 6 or 9 months to produce hyperprolactinemia. Ovariectomized, sham-implanted rats were used as controls. Other groups of intact rats were implanted with HAL or sham-implanted for 9 months and then were implanted with PRL-producing MMQ cells for 6 weeks to further increase circulating PRL levels. TIDA neuronal activity was measured in terms of tyrosine hydroxylase (TH) activity in the stalk-median eminence and was correlated with changes in serum PRL levels. After 6 months of treatment, TH activity in HAL-treated rats was 130% higher than that in the control rats. After 9 months of treatment, TH activity in HAL-treated rats was 81% higher than that in control rats. This increase was significantly less than the increase that occurred after 6 months of treatment. Nine months of HAL-induced hyperprolactinemia followed by implantation of PRL-producing MMQ cells, which resulted in very high levels of PRL, did not increase TH activity in the stalk-median eminence. These results demonstrate that hyperprolactinemia over a prolonged period reduces the responsiveness of TIDA neurons, and these effects vary depending on the duration and intensity of hyperprolactinemia.

Semicircadian rhythms of c-Fos expression in several hypothalamic areas during pregnancy in the rat: relationship to prolactin secretion.

Prolactin (PRL) serves an important luteotrophic function in the rat during early pregnancy, expressed as a nocturnal surge in the early morning and a diurnal surge in the late afternoon. Several areas of the hypothalamus, including the preoptic area (POA), the suprachiasmatic nucleus (SCN) and the ventromedial and dorsomedial nuclei (VM-DM) have been implicated in PRL surges. We investigated the temporal relationship between neuronal activity as measured by c-Fos immunocytochemistry in these areas and PRL secretion during early and late pregnancy. Brains were collected at nine time points (24:00, 02:00, 04:00, 06:00, 10:00, 14:00, 16:00, 18:00 and 20:00 h) on days 6-7 and three time points (02:00, 14:00 and 18:00 h) on days 14-15 of pregnancy. Plasma PRL levels determined by radioimmunoassay revealed two surges with peaks at 02:00 and 18:00 h and a trough at 14:00 h on days 6-7, which were absent on days 14-15 of pregnancy. The number of neurons expressing c-Fos in the anterior medial preoptic nucleus, the medial preoptic area and the medial preoptic nucleus, but not the anteroventral preoptic nucleus of the POA, and the VM-DM, showed a semicircadian rhythm which was maximal at 02:00 h or/and 04:00 and 18:00 h and reached the lowest value at 14:00 h, in parallel with the PRL surges in early pregnancy. However, the temporal pattern of c-Fos in these areas was reversed during late pregnancy, with a peak at 14:00 h and low levels at 02:00 and 18:00 h. PRL surges were absent and levels were uniformly low during these times. Neuronal activity in the SCN did not show any correlation with PRL surges. The dorsomedial subdivision of the SCN showed high neuronal activity during the daytime in both stages of pregnancy. Neuronal activity in the ventrolateral subdivision of the SCN was high during the nighttime in early pregnancy, however it exhibited high levels during the daytime in late pregnancy. These results suggest that the two daily surges of PRL secretion during the first half of pregnancy might be related to the temporal rhythm of neuronal activity in the POA and the VM-DM, and a major change in the pattern of neuronal activity in these hypothalamic areas might result in termination of the PRL surges at midpregnancy.

Prolactin (PRL) receptors are colocalized in dopaminergic neurons in fetal hypothalamic cell cultures: effect of PRL on tyrosine hydroxylase activity.

This study examined the responsiveness of dopaminergic neurons to PRL and the expression of PRL receptors in fetal hypothalamic cells. Hypothalamic cells were cultured in medium containing 5 or 25 mM potassium (K+) with or without 5% FBS. Rat PRL (rPRL) treatment (10-1000 ng/ml) for 10 days increased tyrosine hydroxylase (TH) activity 1.6- to 1.8-fold in dopaminergic neurons cultured in serum-containing medium with 25 mM K+, but not in defined medium or any medium with 5 mM K+. The rPRL-induced increase in TH activity was observed at 10-1000 ng/ml after both 1 and 10 days of rPRL treatment, whereas 1 ng/ml was not effective. TH activity was not altered after 1-12 h of rPRL treatment (100 ng/ml), but was increased 1.4-fold after 1-3 days and 1.8-fold after 5-10 days. The colocalization of PRL receptors and TH was evaluated by double labeled immunocytochemistry. PRL receptor immunostaining was observed in most TH-immunoreactive cells cultured in either defined or serum-containing medium with or without 10 days of rPRL treatment (100 ng/ml). As assessed by reverse transcriptase-PCR, the long form, but not the short form, of the PRL receptor was expressed in the hypothalamic cells regardless of medium composition, similar to the expression pattern in adult mediobasal hypothalamus from ovariectomized rats. These data indicate that a factor present in FBS imparts PRL responsiveness to hypothalamic dopaminergic neurons in vitro. The effective PRL concentrations and the time course for PRL's action in vitro are within the physiological range in vivo. The colocalization of PRL receptor in dopaminergic neurons provides anatomical evidence for a direct effect of PRL, with the long form of the PRL receptor being the predominant form in the hypothalamic cells.