Tuberoinfundibular Dopaminergic Neurons Research Articles

Prolactin replacement must be continuous and initiated prior to 21 d of age to maintain hypothalamic dopaminergic neurons in hypopituitary mice.

The prolactin (PRL) deficit in mice homozygous for the spontaneous Ames dwarf (df) mutation coincides with a marked reduction in the number of PRL-regulating tuberoinfundibular dopaminergic (TIDA) neurons. The TIDA deficit develops after 14 21 d postnatally and may be prevented by PRL replacement initiated at 12, but not at 60, d of age. The present study was designed to define further the developmental period during which PRL can prevent the deficit in the number of TIDA neurons in df/df mice, as well as to evaluate whether exposure to PRL neonatally affects the response to PRL by TIDA neurons in later development. To address the first aim, litters of df/df and normal (DF/df) mice were treated daily with ovine PRL (50 microg intraperitoneally), starting at 12, 21, or 30 d of age. To address the second aim, DF/df and df/df animals treated with PRL for 30 d starting at 12 d of age were subjected to PRL withdrawal (15 d of saline vehicle treatment), followed by PRL retreatment. All brains were evaluated using both catecholamine histofluorescence and tyrosine hydroxylase (TH) immunocytochemistry. Total numbers of TH-immunostained cells were counted in area A12 (TIDA neurons) and in A13 (medial zona incerta). Qualitatively, catecholamine fluorescence in A12 perikarya and terminals in df/df mice was enhanced by PRL treatment initiated at 12 or 21, but not at 30, d of age. TH immunostaining intensity was enhanced in all df/df PRL-treated groups, compared with saline treatment. However, total numbers of TH-positive TIDA neurons were reduced significantly in df/df mice treated with PRL beginning at 21 or 30 d, as well as with saline at 12 d, compared with similarly treated DF/df groups and with df/df animals treated with PRL beginning at 12 d (p < 0.01 for all comparisons). Among dwarf mice treated with PRL beginning at 12 d, followed by PRL withdrawal, the numbers of TH-positive TIDA neurons were greater than those of saline-treated dwarfs, but less than those in DF/df mice (p < 0.05 for both comparisons). In dwarfs retreated with PRL after withdrawal, the TIDA population was also smaller than that in normal animals (p < 0.05), although it was larger than in vehicle-treated dwarfs of the same age (p < 0.05).

Fasting stimulates tuberoinfundibular dopaminergic neuronal activity and inhibits prolactin secretion in oestrogen-primed ovariectomized rats: involvement of orexin A and neuropeptide Y.

Fasting up-regulates central orexigenic systems including orexin A and neuropeptide Y (NPY) and it also inhibits the secretion of prolactin. We hypothesized that fasting may act through orexin A and NPY to influence tuberoinfundibular dopaminergic (TIDA) neurones, the major regulator of prolactin secretion. The effects of orexin A and NPY on TIDA neuronal activity and prolactin secretion were determined in oestrogen-primed ovariectomized rats, and the effects of fasting and the involvement of orexin A and NPY were tested. Orexin A, NPY and its analogs were administered through preimplanted intracerebroventricular (i.c.v.) cannulae. TIDA neuronal activity was determined by measuring concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) or 3,4-dihydroxyphenylalanine in the median eminence. i.c.v. injection of NPY (10 microg) or orexin A (1 microg) concomitantly increased median eminence DOPAC and decreased serum prolactin concentrations. The effect of NPY was mimicked by a Y1 receptor agonist at lower doses (0.1 and 1 microg) and no additive effect was observed when orexin A and the Y1 agaonist were coadministered. Moreover, a Y1 receptor antagonist, BIBP, not only blocked the effect of Y1 agaonist, but also that of orexin A. Treatment with BIBP alone decreased median eminence DOPAC and increased serum prolactin concentrations, indicating that endogenous NPY may play a role. Moreover, fasting for 48 h significantly increased TIDA neuronal activity, both in the morning and afternoon, and the effect was reversed by treatment with either BIBP or an antibody against orexin A. The findings support our hypothesis that fasting stimulates TIDA neuronal activity and inhibits prolactin secretion through up-regulated central orexin A and NPY systems.

Prostaglandins play an important role in diurnal changes of tuberoinfundibular dopaminergic neuronal activity and prolactin secretion in ovariectomized, estrogen-treated rats

Intracerebroventricular injection of prostaglandins (PGs) has long been shown to stimulate the secretion of prolactin (PRL). The action of specific PG and the underlying mechanism, however, remain inconclusive. In this study, we evaluated the involvement of PGs in the diurnal changes of the tuberoinfundibular dopaminergic (TIDA) neurons and PRL secretion in ovariectomized (OVX) rats treated with estrogen (E 2). Prior treatment of indomethacin (50 mg/kg, subcutaneous) for 24 h significantly prevented the afternoon decrease of TIDA neuronal activity and blocked the PRL surge. Intracerebroventricular injection of PGE 1 (5 μg/3 μl/rat) at 1000 h significantly lowered the activity of TIDA neurons, while similar treatment of PGE 2 was without effect. In OVX + E 2 rats pretreated with indomethacin, PGE 1 given at 15, but not 30 or 180 min before decapitation at 1500 h significantly reversed the effects of indomethacin on TIDA neuronal activity, while the blocked PRL surge was not reversed. The PGs also exhibited a stimulatory effect on nigrostriatal dopamine (DA) neurons with PGE 2 being the most effective agent. These results indicate that endogenous PGs play a significant role in the control of central DA neurons, especially the diurnal changes of TIDA neuronal activity and PRL secretion in OVX + E 2 rats.

Calmodulin and a cyclic nucleotide-dependent protein kinase facilitate the prolactin-induced increase in tyrosine hydroxylase activity in tuberoinfundibular dopaminergic neurons.

Many aspects of tuberoinfundibular dopaminergic neuronal function are increased by elevated prolactin (PRL) levels, including the activity of tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of dopamine. This study evaluated the roles of calmodulin, cyclic nucleotide-dependent protein kinase, and calcium/calmodulin-dependent protein kinase II in the PRL-induced increase in tyrosine hydroxylase activity. Ovariectomized rats were treated with haloperidol or ovine PRL (oPRL) for 20-30 h before the experiment, respectively. Treatment with haloperidol increased circulating PRL levels 8-fold and tyrosine hydroxylase activity in the stalk-median eminence 1.8-fold. Treatment with oPRL increased tyrosine hydroxylase activity 1.9-fold. W-7, a calmodulin antagonist, reversed both the haloperidol- and oPRL-induced increase in tyrosine hydroxylase activity to control levels. H-8, a cyclic nucleotide-dependent protein kinase inhibitor, also reversed the haloperidol induced increase in tyrosine hydroxylase activity. KN62, a selective calcium/calmodulin-dependent protein kinase II inhibitor, attenuated the haloperidol-induced increase in tyrosine hydroxylase activity, but KNO4, a structurally related control compound, had no effect. By contrast, the oPRL- and haloperidol-induced increases in tyrosine hydroxylase activity were not altered by KN93, a selective calcium/calmodulin-dependent protein kinase II inhibitor. These data indicate that calmodulin and a cyclic nucleotide-dependent protein kinase contribute to the PRL-induced increase in tyrosine hydroxylase activity, but the role of calcium/calmodulin-dependent protein kinase II is still unclear.

Decreased expression of fos-related antigens (FRAs) in the hypothalamic dopaminergic neurons after immunoneutralization of endogenous prolactin.

In our previous studies we found that administration of exogenous prolactin increased dopamine turnover in the terminal areas of the hypothalamic dopaminergic neurons controlling prolactin secretion from pituitary lactotrophs. In this study we investigated the effect of immunoneutralization of endogenous prolactin on the expression of FRAs in the tuberoinfundibular dopaminergic (TIDA), tuberohypophysial dopaminergic (THDA), and periventricular hypothalamic dopaminergic (PHDA) subpopulations of the hypothalamic dopaminergic neurons. Female rats were ovariectomized on d 0 of the experiment. At 1000 h of d 10, all animals were injected with 20 microg of 17-beta-estradiol sc to induce a proestrous-like surge of prolactin at 1700 h the next day. At 1000 h on d 11, half of the animals were injected with 200 microL of rabbit anti-rat prolactin antiserum ip, while the controls received normal rabbit serum. Groups of animals were sacrificed for immunocytochemistry in 2 h intervals between 1300 and 2100 h. Double-label immunocytochemistry for FRAs and tyrosine hydroxylase (TH) was performed and the results are presented as percentage of TH-immunoreactive neurons expressing FRAs. In the control animals, expression of FRAs decreased at 1500 h, gradually increased by 1900 h, but was lower than the basal levels by 2100 h. Expression of FRAs was significantly lower at 1900 h in the PHDA, THDA and TIDA neurons of prolactin antiserum treated rats than in the controls. These results indicate that elimination of endogenous prolactin from the circulation lowers the activity and/or prevents the reactivation of neuroendocrine dopaminergic neurons at the beginning of the dark phase.

Ovarian steroids influence the activity of neuroendocrine dopaminergic neurons

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.

Dopamine and 7-OH-DPAT may act on D 3 receptors to inhibit tuberoinfundibular dopaminergic neurons

Whether the tuberoinfundibular dopaminergic (TIDA) neurons resided in the dorsomedial arcuate nucleus (dmARN) can respond to dopamine and a dopamine D 3 receptor agonist, 7-hydroxydipropylaminotetralin (7-OH-DPAT), was the focus of this study. In studies using extracellular single-unit recording of dmARN neurons in brain slices obtained from ovariectomized rats, dopamine and 7-OH-DPAT inhibited 60.1% ( n = 141) and 80.9% ( n = 47) of recorded dmARN neurons, respectively. Other dopamine D 1 or D 2 receptor agonists were not as effective. Intracerebroventricular injection of 7-OH-DPAT (10 −9 mol/3 μl) in ovariectomized, estrogen-primed rats significantly lowered the TIDA neuronal activity as determined by 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the median eminence. Co-administration of a putative D 3 receptor antagonist, U-99194A, could prevent the effect of 7-OH-DPAT. Unilateral microinjection of 7-OH-DPAT or dopamine itself (10 −11–10 −9 mol/0.2 μl) into the right dmARN exhibited the same inhibitory effect on TIDA neurons. In all, dopamine may act on D 3 receptors to exhibit an inhibitory effect on its own release from the TIDA neurons.

An Endogenous Serotonergic Rhythm Acting on 5-HT2A Receptors May Be Involved in the Diurnal Changes in Tuberoinfundibular Dopaminergic Neuronal Activity and Prolactin Secretion in Female Rats

The central serotonergic system has long been known to have a stimulatory role on the secretion of prolactin (PRL). The integrity of serotonergic neurotransmission is essential for the expression of the estrogen-induced afternoon PRL surge. Whether its effect on PRL involves change in the activity of tuberoinfundibular dopaminergic (TIDA) neurons has not been ascertained. In adult ovariectomized rats treated with estrogen, depletion of central serotonin (5-HT) by 5,7-dihydroxytryptamine (5,7-DHT, 200 µg/rat, i.c.v.) effectively prevented the afternoon fall in TIDA neuronal activity (using the levels of 3,4-dihydroxyphenylalanine and 3,4-dihydroxyphenylacetic acid (DOPAC), and the ratio of DOPAC/dopamine in the median eminence as indices), and blunted the afternoon PRL surge. A single injection of a 5-HT_2A receptor antagonist, ketanserin (5 mg/kg, i.p. at 12.00 h), also had the same effects on the diurnal changes in TIDA neuronal activity and PRL secretion as the treatment with 5,7-DHT did. Intracerebroventricular injection of a 5-HT₂ receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) in the morning inhibited the TIDA neuronal activity and stimulated PRL secretion in a dose-dependent manner; while injection of a 5-HT₁ agonist, 8-hydroxy-dipropylaminotetralin, was without effect. Injection of DOI in 5,7-DHT-pretreated rats at 14.30 h also lowered the TIDA neuronal activity and reinstated the PRL surge. In all, endogenous 5-HT, acting through the 5-HT_2A receptor, appears to exhibit an inhibitory effect on TIDA neuronal activity during the afternoon, which is essential for the PRL surge.

Stimulatory and entraining effect of melatonin on tuberoinfundibular dopaminergic neuron activity and inhibition on prolactin secretion.

The aims of the present study were to determine if melatonin exerts an effect on prolactin (PRL) secretion via the tuberoinfundibular dopaminergic (TIDA) neurons and if endogenous or exogenous melatonin has an entraining effect on the rhythmic changes of TIDA neuronal activity and PRL secretion. Melatonin given in the morning (10:00 h), dose- (0.01-1 mg/kg, ip) and time- (at 15 and 60 min, but not at 30 min) dependently stimulated TIDA neuronal activity in ovariectomized (OVX), estrogen-treated rats as determined by 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the median eminence (ME). Serum PRL was concurrently inhibited by the injection. Melatonin administered in the afternoon (15:00 h) was even more effective in stimulating the lowered TIDA neuronal activity and inhibiting the increased PRL level than that given in the morning (10:00 h). S-20098, a melatonin agonist was also effective in stimulating the TIDA neurons. In contrast, S-20928, a putative melatonin antagonist, while it had no effect by itself, blocked the effect of S-20098. Although S-20928 failed to prevent melatonin's effect on ME DOPAC levels, six interspaced injections of S-20928, from 18:00 to 01:30 h, significantly blocked the increase of ME DOPAC levels at 03:00 h, indicating that the endogenous melatonin may play a role. We further used rats that received daily injection of melatonin (1 mg/kg, ip) at 18:00 h for 10 days and found that the injection augmented basal TIDA neuronal activity at 11:00 h and blunted the afternoon PRL surge. In all, melatonin can have an inhibitory effect on PRL secretion by stimulating the TIDA neurons, and it may help to entrain the circadian rhythms of both TIDA neuronal activity and PRL secretion.

Immunoneutralization of Endogenous Opioid Peptides Prevents the Suckling-Induced Prolactin Increase and the Inhibition of Tuberoinfundibular Dopaminergic Neurons

Previous studies have shown that the endogenous opioid peptides, acting at specific opiate receptor subtypes, are involved in the suckling-induced prolactin secretory response. The prolactin increase elicited by suckling is due, at least in part, to an inhibition of tuberoinfundibular dopaminergic (TIDA) neurons in the hypothalamus. We investigated the effects of immunoneutralization of dynorphin, leu-enkephalin and met-enkephalin on the suckling-induced prolactin increase and on the activity of the TIDA neurons in lactating female rats between days 7 and 12 postpartum. Rats were injected into the right lateral ventricle with antiserum specific for one of these three peptides. Control rats were administered equal amounts of immunoglobulin proteins. Suckling produced a profound and significant increase in prolactin levels, as well as a decrease in DOPA accumulation in the median eminence of lactating rats. Administration of immunoglobulin concentrations of up to 3.6 µg did not inhibit the prolactin secretory response to the suckling stimulus and did not prevent the suckling-induced inhibition of TIDA neurons. Antisera to all three endogenous opioid peptides abolished the suckling-induced prolactin increase and prevented the inhibition in DOPA accumulation in the median eminence. Thus, the endogenous opioid peptides, dynorphin, leu-enkephalin and met-enkephalin, are essential for the prolactin secretory response to suckling and inhibition of TIDA neuronal activity is at least one of the mechanisms of action utilized by these peptides.

Estradiol Attenuates the Forskolin-Induced Increase in HypothalamicTyrosine Hydroxylase Activity

The purpose of this study was to evaluate interactions between estradiol and the 3′,5′ cyclic adenosine monophosphate (cAMP) signaling pathway to regulate tyrosine hydroxylase (TH) activity in hypothalamic dopaminergic neurons. The first experiment examined the ability of forskolin to activate TH in the tuberoinfundibular dopaminergic neurons of adult ovariectomized rats with or without estradiol treatment. Estradiol treatment reduced both basal and forskolin-stimulated TH activity in the median eminence. The second group of experiments examined the effect of estradiol on the forskolin-induced activation of TH in fetal hypothalamic cells cultures. Estradiol decreased basal TH activity in the hypothalamic cell cultures to 80% of control levels. Forskolin treatment for 1 h increased TH activity in a concentration-dependent manner in control and estradiol-treated cells, but estradiol attenuated the stimulatory response to 0.01–10 µM forskolin. The suppressive effect of estradiol on cAMP-dependent activation of TH was evident with 1–12 h of forskolin treatment. The responses to other activators of the cAMP- protein kinase A pathway, including dibutyryl cAMP and 8-bromo-cAMP, and to a depolarizing stimulus were blunted in estradiol-treated cultures. Forskolin treatment for 1 h increased radiolabeled phosphate incorporation into TH protein in control but not estradiol-treated cells, suggesting that estradiol interferes with the ability of the cAMP pathway to phosphorylate TH. Forskolin caused a time-dependent increase in TH mRNA signal levels in control cultures. The magnitude of the forskolin-induced increase in TH mRNA levels was less in the estradiol-treated cells after 6 h of forskolin treatment, indicating that estradiol hinders cAMP-regulated TH gene expression. These data indicate that estradiol attenuates the ability of hypothalamic dopaminergic neurons to respond to cAMP-dependent stimulation by interfering with phosphorylation mechanisms in the short term and control of TH mRNA levels in the long term.

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.

Neuroendocrine responses to levodopa in multiple system atrophy (MSA).

Hypothalamic dopaminergic pathways are involved in the regulation of growth hormone and prolactin release from the anterior pituitary. Neuroendocrine studies in patients with multiple system atrophy (MSA), in whom there is a reported loss of hypothalamic dopamine, are few and contradictory. We therefore studied the neuroendocrine responses to 250 mg levodopa (plus 25 mg carbidopa) in subjects with MSA (n = 15), and compared them with age- and sex-matched healthy control subjects (n = 8). There were no significant differences in basal or post-levodopa levels of growth hormone (GH), growth hormone-releasing hormone (GHRH), glucose, insulin-like growth factor (IGF-1), or thyroid-stimulating hormone (TSH) between the groups. In patients with MSA, basal levels of prolactin were elevated (21.1 +/- 5.2 ng/mL [mean +/-standard error]) compared with control subjects (12.1 +/- 1.7, p <0.05), and after L-dopa there was increased variability in prolactin response with less suppression compared with control subjects. In conclusion, in patients with MSA, the GHRH and GH responses to L-dopa were preserved and were similar to responses in age-matched control subjects. In contrast, there was impaired dopaminergic suppression of prolactin secretion. In patients with MSA this may represent a selective dysfunction, rather than generalized loss, of tubero-infundibular dopaminergic neurones.

Prolactin activates all three populations of hypothalamic neuroendocrine dopaminergic neurons in ovariectomized rats

Prior studies suggest that prolactin (PRL) stimulates release of dopamine (DA) from tuberoinfundibular dopaminergic (TIDA) neurons. In the present study, the time course over which PRL exerts its effects on all three populations of neuroendocrine dopaminergic (DAergic) neuron populations [TIDA, tuberohypophyseal (THDA) and periventricular-hypophyseal (PHDA)] was determined. Ten days following ovariectomy (OVX), groups of female rats were injected either with 15 μg of ovine PRL (oPRL) or saline at 0900 h. Rats were decapitated every 30 min from 0830 h–1100 h and hourly from 1200 h–1500 h. Trunk blood was assayed for rat PRL (rPRL) and oPRL using species-specific radioimmunoassays (RIAs). The concentration of DA and 3,4-dihydroxyphenylacetic acid (DOPAC) in the median eminence (ME), as well as the anterior (AL), intermediate (IL) and neural (NL) lobes of the pituitary gland were determined by HPLC-EC. The concentration of rPRL in oPRL-treated animals, compared to saline-treated animals, was diminished by 1000 h and again between 1200 h–1500 h. DOPAC/DA ratio, an indicator of dopaminergic neuronal activity, increased spontaneously in the ME, IL, and NL during the afternoon in OVX rats. In animals injected with oPRL at 0900 h, the DOPAC/DA ratio increased in the ME, IL and NL within 1 h. Moreover, a secondary increase in the DOPAC/DA ratio in the IL and NL occurred during the afternoon in oPRL-treated rats. However, the second increase of DA turnover present in the ME of control animals never occurred in oPRL-treated animals. Furthermore, there were two increases in the concentration of DA in the AL: the first coincided with the increased turnover of DA in all three terminal areas and the second with increased DA turnover in the IL and NL. These data suggest that all three populations of hypothalamic neuroendocrine DAergic neurons are activated by PRL and that PHDA/THDA neurons have a second `delayed' activation.

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.

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

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

Differential regulation of hypothalamic tuberoinfundibular dopamine neurones in two dwarf rat models with contrasting changes in pituitary prolactin.

In transgenic growth-retarded (Tgr) rats, expression of human growth hormone (hGH) is targeted to hypothalamic GH-releasing hormone (GHRH) neurones. In these rats, GHRH is reduced and somatostatin expression is increased, resulting in secondary GH deficiency and dwarfism. Tgr rats also show reduced pituitary prolactin (PRL), which may reflect an additional lactogenic feedback action of the hGH transgene, analogous to that in mice transgenic for peripheral hGH which show enhanced dopamine (DA) and tyrosine hydroxylase (TH) expression in the hypothalamic tuberoinfundibular dopaminergic (TIDA) neurones that inhibit PRL secretion. The present study examined DA histofluorescence and TH immunoreactivity in Tgr rats, and also in dw/dw rats, a dwarf strain with primary pituitary GH but not PRL deficiency. Radioimmunoassay confirmed a significant decrease in total pituitary PRL content in Tgr rats, but showed a marked increase in total pituitary PRL in dw/dw rats. Despite their PRL deficiency, Tgr rats showed qualitatively increased TIDA histofluorescence and TH immunoreactivity compared with AS control rats, though the total number of detectable TH-positive TIDA neurones was similar for Tgr and AS. In contrast, dw/dw rats showed increased numbers of TH-immunoreactive TIDA neurones whilst TIDA fluorescence was unchanged, and these findings were not affected in dw/dw rats given bovine GH (200 microg/d s.c. for 7 d). These results suggest that reduced PRL in Tgr rats is due to a local lactogenic feedback effect of hGH to stimulate TIDA neurones. The complex changes in TIDA neurones probably reflect a combination of increased lactogenic feedback in Tgr rats, with an increased (Tgr) or decreased (dw/dw) somatogenic feedback on GHRH neurones, some of which coexpress TH. Thus, the unchanged number of TIDA neurones in Tgr rats may result from hGH stimulation of TH and DA, but a reduction in GHRH-producing cells, whereas increased TIDA neurones in dw/dw rats suggests a stimulation by endogenous PRL with an increased GHRH cell complement due to GH deficiency. These findings therefore indicate that differences in lactogenic feedback in these dwarf rat models are reflected in marked differences in their hypothalamic TIDA neurones.

Single-unit activity of dorsomedial arcuate neurons and diurnal changes of tuberoinfundibular dopaminergic neuron activity in female rats with neonatal monosodium glutamate treatment

Neonatal monosodium glutamate (MSG)-treated rats were used in this study to answer two questions: (1) whether or not the dopamine-responsive dorsomedial arcuate (dm-ARN) neurons are tuberoinfundibular dopaminergic (TIDA) neurons, and (2) whether or not the remaining TIDA neurons in MSG-treated rats are functioning normally. MSG (4 mg/g b. wt., subcutaneously [s.c.]) or saline was given to neonatal Sprague-Dawley rats on days 1, 3, 5, 7, and 9 after birth. The female rats were ovariectomized at 50 days of age and treated with estrogen for 1 week before they were used between 65–90 days of age. The tyrosine hydroxylase-immunoreactive (TH-ir) neurons located in the dm and ventrolateral (vl) parts of the ARN were significantly reduced in MSG-treated rats, as determined by immunohistochemical method. Some TH-ir cells, however, were visible along the border of the third ventricle. Using single-unit recording in brain slices, we found that dopamine inhibited significantly fewer percentage of dm-ARN neurons in MSG-treated (28.2%, n = 39) than in saline-treated rats (73.3%, n = 15). In contrast, bombesin exhibited similar effects (over 70% excitation) in both groups. Using neurochemical means, neonatal MSG treatment produced significant decreases of both 3,4-dihydroxyphenylacetic acid and dopamine levels, but not their ratios, in the median eminence. Moreover, the diurnal change of TIDA neuronal activity persisted in the MSG-treated rats; so did the estrogen-induced afternoon prolactin surge. All these results indicate that neonatal MSG-treatment reduced the number and altered the location of TIDA and dopamine-responsive dm-ARN neurons. The remaining TIDA neurons seemed to be able to maintain their basal activities and diurnal rhythm.

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.

Nitric oxide plays an important role in the diurnal change of tuberoinfundibular dopaminergic neuronal activity and prolactin secretion in ovariectomized, estrogen/progesterone-treated rats.

A significant diurnal change of tuberoinfundibular dopaminergic (TIDA) neuronal activity coincident with the estrogen (E2)-induced afternoon PRL surge has been reported in ovariectomized, E2-primed (OVX+E2) rats. Systemic injection of a nitric oxide (NO) synthase (NOS) inhibitor, N(G)-nitro-L-arginine (L-NA, 50 mg/kg, i.p. at 1000 and 1200 h), significantly blocked the diurnal changes of TIDA neuronal activity and PRL secretion at 1500 and 1700 h in OVX+E2 rats. Coadministration of L-arginine (300 mg/kg, i.p.) with L-NA completely prevented the effects of L-NA. Total nitrite/nitrate levels in the serum of L-NA- and L-NA+L-arginine-treated rats substantiated the effects of L-NA and L-arginine on NO production. Pretreatment of antisense oligodeoxynucleotide (ODN; 1 microg/3 microl; intracerebroventricularly at 48, 24, and 7 h before sacrifice) against the messenger RNA (mRNA) of constitutive NOS, i.e. neuronal NOS or endothelial NOS, was also effective in preventing the diurnal changes of TIDA neuronal activity and PRL surge at 1500 h. The same treatment of antisense ODN against the mRNA of inducible NOS, i.e. macrophage NOS, had no effect. Progesterone (P4) has been reported to advance and augment the diurnal changes of TIDA neuronal activity and the afternoon PRL surge, by 1 h, in both proestrous and OVX+E2 rats. We further showed that L-NA dose dependently (50 but not 5 mg/kg, i.p. at 1000 and 1200 h) blocked the effect of P4 on TIDA neurons and serum PRL at 1300 h, which effect could be negated by simultaneous administration of L-arginine (300 mg/kg, i.p.). Pretreatment with antisense ODNs against the mRNA of neuronal NOS or endothelial NOS, but not macrophage NOS, was also effective in preventing the P4's effect on TIDA neuronal activity and PRL secretion at 1300 h. In summary, NO may play a physiological role in the E2- and P4-regulated diurnal changes of TIDA neuronal activity and PRL secretion.