Stimulatory role of prolactin on the development of tuberoinfundibular dopaminergic neurones in prepubertal female rats: studies with cysteamine and somatostatin.

Cysteamine, a potent depletor of prolactin and somatostatin, was used to determine the role of prolactin and somatostatin in the control of central dopamine neurones in prepubertal rats. Cysteamine (100 mg/kg, i.p., twice daily) was injected for 7, 14 or 21 days in 28-day-old Sprague-Dawley female rats in one study and for 3 days in 35-day-old rats in another. In control rats, the 3, 4-dihydroxyphenylacetic acid (DOPAC) levels in the median eminence increased threefold from day 35 to day 49, and serum prolactin concentration increased about 50%. Cysteamine lowered serum prolactin concentrations to 20%, and median eminence DOPAC and dopamine levels to 32-50% of control levels in both studies. The DOPAC levels in the nucleus accumbens and striatum were also lowered, while both DOPAC and dopamine in the paraventricular nucleus and periventricular nucleus (A14) were increased by cysteamine. A single injection of rat prolactin (0.01, 0.1 or 1 mg/kg) significantly increased DOPAC or DOPA levels in the median eminence, nucleus accumbens and striatum, but not in the paraventricular nucleus or A14 at 14 h later in 28-day old female rats or in 40-day-old rats pretreated with cysteamine. In contrast, central injection of somatostatin dose (0.001-1 microg/rat) and time (30-90 min) dependently decreased the DOPAC levels in the median eminence, paraventricular nucleus and A14 and increased those in the nucleus accumbens and striatum of adult female rats. These results indicate that serum prolactin is important for the maturation and maintenance of dopamine systems in the median eminence, nucleus accumbens and striatum, while somatostatin exhibits inhibitory and stimulatory effects on hypothalamic and midbrain dopamine systems, respectively.

Progesterone advances the diurnal rhythm of tuberoinfundibular dopaminergic neuronal activity and the prolactin surge in ovariectomized, estrogen-primed rats and in intact proestrous rats.

A diurnal change of tuberoinfundibular dopaminergic (TIDA) neuronal activity exists in female rats, which is prerequisite for the estrogen-induced afternoon PRL surge. Because progesterone (P4) administered in the morning can advance and amplify the PRL surge, it is of interest to learn whether its action involves the TIDA neuron. In adult ovariectomized and estrogen-primed Sprague-Dawley rats, P4 (2 mg/kg, s.c.), given at 0800 h, exhibited a significant effect in advancing and amplifying the afternoon PRL surge, as determined by both chronic catheterization and decapitation methods of blood sampling. The afternoon decrease of TIDA neuronal activity, as determined by 3,4-dihydroxyphenylacetic acid concentration in the median eminence, was also advanced from 1400 to 1300 h. These effects of P4 on PRL surge and TIDA neuronal activity were shown to be dose- (from 0.5-4 mg/kg) and estrogen-dependent. To determine whether the effect of P4 was indeed acting via specific P4 receptor (PR), we used a PR antagonist, RU486, an antisense oligodeoxynucleotide (ODN) for PR messenger RNA (mRNA), and an antibody against PR in this study, to answer this question. Treatments of RU486 (5 mg x 3, s.c.) for 1-2 days before, and on the sampling day, were effective in antagonizing the effects of P4 on TIDA neuronal activity and on PRL secretion. Intracerebroventricular injection of an antisense ODN (4 nM) for PR mRNA or of an antibody (1:1 and 1:5) against PR for 2 days (24 and 48 h before decapitation) also were effective. Treatments of RU486 on the sampling day only, of sense ODN for PR mRNA, or of diluted PR antibody (1:10) were without significant effect. The involvement of P4 or PR on modulating the TIDA neuronal rhythm and the PRL surge also was shown in proestrous rats. In conclusion, P4 may play a significant modulatory role on rhythmic changes of the TIDA neuronal activity and the PRL surge in the female rats.

Nicotinic control of tuberoinfundibular dopaminergic neuron activity and prolactin secretion: diurnal rhythm and involvement of endogenous opioidergic system.

The possible involvement of cholinergic and opioidergic neurons in the control of diurnal changes of tuberoinfundibular dopaminergic (TIDA) neuronal activity was reported. Adult Sprague-Dawley rats ovariectomized and treated with estrogen were used. All drugs were administered centrally through preimplanted intracerebroventricular cannula, and both TIDA neuronal activity and serum prolactin level were determined. Nicotine (10 ng/3 microl/rat) given at 10:00 h significantly inhibited TIDA neuronal activity from 5 to 30 min and stimulated serum PRL levels at 5 and 15 min. Co-administration of either mecamylamine (1 microg) or naloxone (2.5 microg) prevented both nicotine's effects. A dose-related (0.1-100 ng) effect of nicotine on TIDA neuronal activity and serum PRL level was also observed in the morning when TIDA neuronal activity is high and serum PRL level is low, but not in the afternoon when the former activity is low and the latter is high. When atropine (20 microg), naloxone (25 microg) or Nor-BNI (20 microg) was given at 14:00 h all increased the lowered TIDA neuronal activity in the afternoon. When atropine was co-administered with either naloxone or Nor-BNI, however, no additive effect was observed. Submaximal doses of atropine (0.2 microg), mecamylamine (0.1 microg) or naloxone (0.25 microg) was also effective in stimulating the afternoon levels of TIDA neuronal activity and inhibiting serum PRL, and no additive effect was observed either. Moreover, simultaneous injection of morphine (15 microg) prevented atropine's effect in the afternoon. These results indicate that cholinergic neurons may act through activating the endogenous opioidergic neurons to exhibit an inhibitory effect on TIDA neuronal activity and a stimulatory one on prolactin secretion. A diurnal difference in its endogenous activity between morning and afternoon was also implicated.

Stimulatory effect of central oxytocin on tuberoinfundibular dopaminergic neuron activity and inhibition on prolactin secretion: neurochemical and electrophysiological studies.

The posterior pituitary hormone, oxytocin (OT), has been shown to have either a stimulatory or an inhibitory effect on PRL secretion depending on the route of administration. Whether its central inhibitory effect involves the tuberoinfundibular dopaminergic (TIDA) neuron was the focus of this study. Adult female Sprague-Dawley rats ovariectomized for 1 week, implanted with sc estrogen-containing capsules and intracerebroventricular cannulas for 6 more days were used. TIDA neuron activity was determined by measuring the concentration of 3,4-dihydroxyphenylacetic acid or 3,4-dihydroxyphenylalanine in the median eminence by HPLC with electrochemical detection. Intracerebroventricular injection of OT induced both dose (0.01-1 microgram/rat)- and time (30-90 min)-dependent increases in 3,4-dihydroxyphenylalanine or 3,4-dihydroxyphenylacetic acid levels in the median eminence. Serum PRL levels were also decreased 30 min after the injection. The use of a specific OT antagonist, [d(CH2)5, Tyr(Me)2, Orn8]vasotocin, not only blocked the effect of OT on TIDA neuron activity, it further lowered it to below control levels, indicating the existence of an endogenous OT activity. When 1 microgram OT was administered at 1200 h, it also reversed the diurnal decrease in TIDA neuron activity at 1500 h. The effects of OT on the electrical activities of dorsomedial arcuate neurons were also tested using single unit recording in brain slices. In 33 neurons tested with OT, 66.7% were stimulated by OT in 0.5- to 50-nmol doses, and no inhibitory effect was observed. The rest were not responsive. In conclusion, both neurochemical and electrophysiological studies demonstrated that central OT may play a stimulatory role in regulating the TIDA neurons and, in turn, inhibition of PRL secretion.

Sexual differences in the diurnal changes of tuberoinfundibular dopaminergic neuron activity in the rat: role of cholinergic control.

A diurnal change of the tuberoinfundibular dopaminergic (TIDA) neuron activity in ovariectomized rats treated with or without estrogen was recently reported, and the endogenous cholinergic system may be responsible for its induction. Whether a similar rhythm exists in intact female or male rats was the focus of this study. TIDA neuron activity was assessed by measurement of the precursor or metabolite of dopamine in the median eminence by HPLC with electrochemical detection. In intact female Sprague-Dawley rats, diurnal changes in TIDA neuron activity were observed during all stages of the estrous cycle, i.e., proestrus, estrus, and diestrus 1, and they were nearly identical. No such rhythm, however, was observed in intact male rats. Castration alone decreased and increased basal TIDA neuron activity in female and male rats, respectively, and estrogen treatment increased the activity in both sexes. The diurnal changes in TIDA neuron activity, however, were observed only in the female rats, not the male rats, regardless of castration or estrogen treatment. Serum prolactin levels in the male rats exhibited no diurnal changes either, irrespective of whether the animals had been castrated or treated with estrogen. Central administration of mecamylamine (1 micrograms/3 microliters per rat, i.c.v.), a nicotinic receptor antagonist, in the morning (at 1000 h) or afternoon (at 1500 and 1700 h) induced a differential effect on the TIDA neuron activity of intact female rats, i.e., no effect in the morning and stimulation in the afternoon; it had no effect on the TIDA neuron activity in the male rats regardless of the injection time. In castrated male rats treated with estrogen, however, mecamylamine treatment further increased TIDA neuron activity, but still with no diurnal difference. In summary, a sexual difference in the diurnal changes of TIDA neuron activity was observed, and these changes may be under differential control by the cholinergic system.

An endogenous cholinergic rhythm may be involved in the circadian changes of tuberoinfundibular dopaminergic neuron activity in ovariectomized rats treated with or without estrogen.

We recently reported that a circadian change in the activities of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons exists in ovariectomized (OVX) rats treated with or without estrogen. The involvement of an endogenous cholinergic control mechanism was the focus of this study. Adult female Sprague-Dawley rats OVX for 2 weeks and treated with or without a long-acting estrogen (poly-estradiol phosphate, 0.1 mg/rat, sc) were used in the study. An intracerebroventricular cannula for drug injection was implanted into the lateral cerebroventricle of each rat 6 days before experiment. TIDA neuron activity was determined by measuring the concentrations of 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxyphenylalanine in the median eminence by HPLC plus electrochemical detection. Serum PRL levels were determined by RIA. Neither atropine nor mecamylamine, two cholinergic receptor antagonists, had any effect on TIDA neuron activity in the morning before 1200 h, when endogenous TIDA neuron activity is high. Both drugs, however, exhibited a dose-related stimulating effect on the TIDA neuron activity in the afternoon, when endogenous activity is low. The estrogen-induced afternoon PRL surge was also blocked by a single injection of atropine or mecamylamine at 1300 h. The rhythmic changes in endogenous TIDA neuron activity and their responses to atropine were also observed in OVX rats with no estrogen replacement. In conclusion, a daily change in endogenous cholinergic neuron activity may be responsible for the change in TIDA neuron activity in female rats, which is a prerequisite for the PRL surge.

Circadian changes of serum prolactin levels and tuberoinfundibular dopaminergic neuron activities in ovariectomized rats treated with or without estrogen: the role of the suprachiasmatic nuclei.

Variations of serum prolactin (PRL) levels and activities of tuberoinfundibular dopaminergic (TIDA) neurons during the afternoon of ovariectomized (OVX) rats treated with or without estrogen were determined in this study. Long-term OVX rats treated with or without polyestradiol phosphate (0.1 mg/rat, s.c.) were decapitated every hour from 10.00 to 19.00 h (except 11.00 and 13.00 h). Serum PRL and median eminence (ME) dihydroxyphenylacetic acid (DOPAC) or dihydroxyphenylalanine (DOPA) levels were determined by radioimmunoassay and high performance liquid chromatography plus electrochemical detection, respectively. A prominent PRL surge started and peaked around 14.00-15.00 h, and remained significantly higher than levels of 10.00 and 12.00 h throughout the afternoon. Significant decreases of ME DOPAC and DOPA concentrations were also observed between 14.00 and 19.00 h. In OVX rats with no estrogen replacement, no PRL surge was observed and the changes of ME DOPAC concentrations during the afternoon were not significant except for that at 17.00 h. The ME DOPA accumulation, however, exhibited significantly lower levels from 14.00 to 19.00 h than that at 12.00 h, indicating that an endogenous rhythm for DA synthesis existed in OVX rats. In estrogen-treated OVX rats bearing bilateral lesions of the suprachiasmatic nuclei, both changes in serum PRL level and TIDA neuron activity were abolished. We conclude that an endogenous rhythm of the activities of TIDA neurons may exist in both OVX and OVX plus estrogen-treated rats. The rhythm is regulated by the suprachiasmatic nuclei and may be amplified by estrogen for the induction of PRL surge.

Enhanced tuberoinfundibular dopaminergic neuron activity in thyroidectomized, ovariectomized and estrogen-treated rats with hyperprolactinemia.

The involvement of the tuberoinfundibular dopaminergic (TIDA) neurons in the genesis of hyperprolactinemia in female thyroidectomized rats treated with estrogen was the focus of this study. Rats that were ovariectomized (3 weeks), thyroidectomized (2 weeks) and treated with estrogen for 6 days had a 5- to 10-fold increase in serum prolactin (PRL) levels, while the rats receiving the same treatments but without estrogen had lower PRL levels. The activity of TIDA neurons, using dihydroxyphenylacetic acid (DOPAC) concentration or the DOPAC/dopamine (DA) ratio in the median eminence (ME) as an index, was increased and decreased in estrogen-treated and untreated rats, respectively. The increases in serum PRL level and the activity of TIDA neurons were dependent on the duration of thyroidectomy and could both be lowered by daily injection of thyroid hormone (20 or 100 micrograms/kg b.w. thyroxine, i.p.) for 12 days in a dose-dependent manner. Using dihydroxyphenylalanine (DOPA) accumulation in the ME as another index, we also found an increased DOPA/DA ratio in estrogen-treated hypothyroid rats, which effect could be reversed by replacement of thyroid hormone. Furthermore, treatments with bromocryptine (3 mg/kg b.w./day, s.c.) for 3 days or with cysteamine (100 mg/kg b.w., s.c.) at 26, 15 and 2 h before the rats were sacrificed lowered both serum PRL level and TIDA neuron activity. Since PRL can exert a short-loop feedback control on its own secretion, these results indicate that the increased serum PRL levels in estrogen-treated hypothyroid female rats should be the cause, but not the result, of the increased activity of TIDA neurons.

Extracellular single-unit studies of suprachiasmatic neurons in brain slices. Effects of serotonin, dopamine, carbachol and LHRH.

Suprachiasmatic nucleus (SCN) is the major rhythm generating center in the central nervous system. Extracellular single-unit activities of the SCN neurons were recorded in brain tissue slices either from ovariectomized (OVX) rats or from OVX rats treated with estrogen (polyestradiol phosphate, PEP, 0.1 mg/rat, sc). Neuronal responses to luteinizing hormone-releasing hormone (LHRH) and various neurotransmitters, i.e., serotonin (5-HT), dopamine (DA) and carbachol (Carb, an acetylcholine agonist) were tested. A total of 137 units were recorded from OVX and 81 from OVX + PEP rats. The steady state firing rates of SCN neurons were between 0-9 spikes/sec in OVX and 0-23 in OVX + PEP group. Among them, most fired irregularly (86.9% in OVX and 71.6% in OVX + PEP rats), and a few fired regularly (10.2% and 25.9%, respectively). Few silent units were detected (2.9% and 2.5%, respectively). It seems that estrogen treatment somewhat increased the percentage of regular firing units. As for the agents tested in this study, LHRH by itself had no significant effect on the SCN neuronal activity; 5-HT affected more than 40% of the units, with about equal number of excitation (16.8%) and inhibition (24.4%), while Carb inhibited nearly half of the units tested (49.5%), excited only a few (10.3%). All the actions of these three agents were not significantly affected by in vivo estrogen pretreatment. Dopamine, however, excited more units (50%) in OVX than in OVX + PEP group (27.1%). Although LHRH had no direct effect by itself, it exerted a significant modulatory role on the actions of the other three agents. It either potentiated (42.9%) or inhibited (53.6%) the action of 5-HT, and it significantly potentiated the action of Carb (57.1%). For the action of DA, LHRH had a more significant potentiation effect on OVX + PEP than in OVX rats. (40.9% vs. 12.5%), while the inhibitory effect was the same. In conclusion, 5-HT, DA and Carb all exert significant effects on SCN neuronal activities. LHRH can have a modulatory role upon the action of 5-HT, DA and Carb, and estrogen pretreatment specifically affects that of DA in the SCN.

The influence of raphe lesions, p-chlorophenylalanine, and ketanserin on the estrogen-induced afternoon prolactin surge.

Female Sprague-Dawley rats were ovariectomized and 2 weeks later injected sc with 100 micrograms polyestradiol phosphate, a long-acting estrogen, to induce the afternoon PRL surge. In one series of experiments, the dorsal raphe (DRN), median raphe (MRN), and median raphe-pontine (MRN-RPn) regions were lesioned using a radiofrequency lesion maker set at 56 degrees C for 1 min. Lesions were induced, and atrial catheters were implanted on the day of estrogen injection. Six days later, blood samples were obtained every 2 h from 1100-2100 h to monitor the afternoon PRL surge. The animals were killed, and the hypothalami were quickly frozen on dry ice and stored at -60 degrees C for determination of amine content using HPLC with electrochemical detection. The rest of the brain was fixed and sectioned to verify the location of brain lesions. Complete lesions of the DRN markedly attenuated the PRL surge (P less than 0.001) compared to the effect of sham or incomplete lesions. MRN lesions did not significantly alter the PRL surge; however, MRN lesions that included the RPn significantly (P less than 0.001) attenuated the afternoon PRL surge. Significant decreases in serotonin (5-HT) and 5-hydroxyindoleacetic acid concentrations were observed in the arcuate, ventromedial, suprachiasmatic, and medial preoptic nuclei of the hypothalamus, but not in the median eminence of the lesioned rats. DRN and MRN-RPn lesions decreased the 5-HT concentration in the ARC and MPN compared to sham lesion and DRN incomplete lesion values. Other hypothalamic areas did not show a similar effect of the lesions. The concentrations of norepinephrine, dopamine, or dihydroxyphenylacetic acid were not altered by the lesions. In a second series of experiments one group of animals was injected ip with 10 mg/kg ketanserin, a serotonergic antagonist, at 1200 h on the sampling day. A second group was given 250 mg/kg p-chlorophenylalanine (PCPA) at 1700 h for 2 days before blood sampling was initiated on the third day. Both ketanserin and PCPA completely blocked the PRL surge. In the PCPA-injected animals, the 1100 and 1300 h PRL values were significantly higher (P less than 0.01) than those in vehicle-injected controls. Animals injected with ketanserin at 1200 h had a 1300 h PRL value significantly higher (P less than 0.01) than was observed in vehicle-injected animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrophysiological test of an amphiphilic beta-structure in LHRH action.

Micropipettes were used to record electrical activity from single neurons in hypothalamic tissue slices, in the hypothalamic arcuate nucleus (ARC), and in the periventricular and suprachiasmatic preoptic nuclei (POA). Responses were measured following in vitro application of luteinizing hormone releasing hormone (LHRH), and two analogues: LHRH models 1 and 3. Model 1 (pyroGlu-His-Trp-Ser-Phe-Thr-Ile-Lys-Ile-ThrNH2) had amino acid substitutions in residues 5-10 designed to form an amphiphilic beta-strand structure. Model 3 (pyroGlu-His-Trp-Ser-Phe-Gly-Ile-Lys-Pro-SerNH2) was also designed to possess amphiphilic characteristics, but also more closely to resemble the native peptide. Electrical recording results showed that LHRH was able both to excite or inhibit different hypothalamic neurons, and that it was more effective in the preoptic area than in the arcuate nucleus. Responses to LHRH model 3 were strongly correlated with responses to LHRH, in their occurrence and their direction, for both of the brain regions studied. Moreover, LHRH models 1 and 3 also retained the neuromodulatory effects of LHRH on cellular responses to norepinephrine and serotonin. Thus, LHRH analogues, designed to possess an amphiphilic beta-structure, preserved some of the properties of LHRH when tested electrophysiologically in the central nervous system.