Unique responses to mitochondrial complex I inhibition in tuberoinfundibular dopamine neurons may impart resistance to toxic insult.

Tuberoinfundibular dopamine (TIDA) neurons are spared in Parkinson's disease (PD), a disorder that causes degeneration of midbrain nigrostriatal dopamine (NSDA) and mesolimbic dopamine (MLDA) neurons. This pattern of susceptibility has been demonstrated in acute complex I inhibitor-induced models of PD, and extrinsic factors such as toxin distribution, bioactivation, entry into the cell and sequestration into vesicles are postulated to underlie the resistance of TIDA neurons. In the present experiments, direct exposure to rotenone or 1-methyl-4-phenylpyridinium (MPP+) had no effect on mediobasal hypothalamic TIDA neurons, but significantly increased the percentage of apoptag immunoreactive neurons in midbrain primary NSDA and MLDA cultures. In vivo 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) exposure caused an initial decrease (by 4 h) in dopamine (DA) in brain regions containing axon terminals of TIDA (median eminence [ME]), NSDA (striatum [ST]) and MLDA (nucleus accumbens [NA]) neurons. By 16 h after MPTP treatment, DA concentrations in ME returned to control levels, while ST and NA DA levels remained low up to 32 h after treatment with MPTP. When mice and rats were chronically treated with MPTP and rotenone, respectively, the same pattern of susceptibility emerged. TIDA neurons were unaffected while NSDA neurons suffered loss of cell bodies and axon terminal DA. These experiments demonstrate that the resistance of hypothalamic TIDA neurons is not likely to be due to extrinsic factors, and that further examination of the intrinsic properties of these neurons may elucidate mechanisms that can be translated into neuroprotective strategies in PD.

Thyrotropin-releasing hormone mediates serotonin-induced secretion of GH in cattle.

Serotonin stimulates secretion of growth hormone (GH) in cattle, but the mechanism is unknown. In rats, thyrotropin-releasing hormone (TRH) mediates serotonin-induced secretion of GH. We hypothesized that the same is true in cattle. Cattle were fed for 2h daily to synchronize secretion of GH, such that concentrations of GH were high before and low after feeding. Our first objective was to determine whether or not feeding suppresses serotonin receptor agonist (quipazine) induced secretion of GH. Holstein steers were injected with quipazine (0.2 mg/kg BW) either 1 h before or 1 h after feeding. Quipazine-induced secretion of GH which did not differ in magnitude before and after feeding. If TRH mediates serotonin-induced secretion of GH, then magnitude of TRH-induced secretion of GH should not be different before and after feeding (our second objective). Sixteen meal-fed Holstein steers were injected with 0.3 microg TRH/kg BW either 1 h before or 1 h after feeding. Indeed, magnitude of TRH-induced secretion of GH before and after feeding was not different. Our third objective was to inhibit endogenous TRH with 3,5,3'-triiodothyronine (T(3)) and examine basal, GH-releasing hormone (GHRH)-, TRH- and quipazine-induced secretion of GH. Sixteen Holstein steers were injected daily with either T(3) (3 or 6 microg/kg BW) or vehicle for 20 days and then challenged sequentially with vehicle or GHRH, TRH, or quipazine. T(3) did not affect basal, GHRH- or TRH-induced secretion of GH, but reduced basal secretion of thyroxine. T(3) reduced but did not completely block quipazine-induced secretion of GH. In conclusion, TRH mediates, in part, serotonin-induced secretion of GH in cattle.

Pituitary adenylate cyclase-activating polypeptide induces secretion of growth hormone in cattle.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a hypothalamic neuropeptide that stimulates release of growth hormone (GH) from cultured bovine anterior pituitary gland cells, but the role of PACAP on the regulation of in vivo secretion of GH in cattle is not known. To test the hypothesis that PACAP induces secretion of GH in cattle, meal-fed Holstein steers were injected with incremental doses of PACAP (0, 0.1, 0.3, 1, 3, and 10 microg/kg BW) before feeding and concentrations of GH in serum were quantified. Compared with saline, injection of 3 and 10 microg PACAP/kg BW increased peak concentrations of GH in serum from 11.2 ng/ml to 23.7 and 21.8 ng/ml, respectively (P < 0.01). Peak concentrations of GH in serum were similar in steers injected with 3 or 10 microg PACAP/kg BW. Meal-fed Holstein steers were then injected with 3 microg/PACAP/kg BW either 1 hr before feeding or 1 hr after feeding to determine if PACAP-induced secretion of GH was suppressed after feeding. Feeding suppressed basal concentrations of GH in serum. Injection of PACAP before feeding induced greater peak concentrations of GH in serum (19.2 +/- 2.6 vs. 11.7 +/- 2.6 ng/ml) and area under the response curve (391 +/- 47 vs. 255 +/- 52 ng. ml(-1) min) than injection of PACAP after feeding, suggesting somatotropes become refractory to PACAP after feeding similar to that observed by us and others with growth hormone-releasing hormone (GHRH). We concluded that PACAP induces secretion of GH and could play a role in regulating endogenous secretion of GH in cattle, perhaps in concert with GHRH.

GH-releasing peptide-6 overcomes refractoriness of somatotropes to GHRH after feeding.

After a meal, somatotropes are temporarily refractory to growth hormone-releasing hormone (GHRH), the principal hormone that stimulates secretion of growth hormone (GH). Refractoriness is particularly evident when free access to feed is restricted to a 2-h period each day. GH-releasing peptide-6 (GHRP-6), a synthetic peptide, also stimulates secretion of GH from somatotropes. Because GHRH and GHRP-6 act via different receptors, we hypothesized that GHRP-6 would increase GHRH-induced secretion of GH after feeding. Initially, we determined that intravenous injection of GHRP-6 at 1, 3 and 10 microg/kg body weight (BW) stimulated secretion of GH in a dose-dependent manner. Next, we determined that GHRP-6- and GHRH-induced secretion of GH was lower 1 h after feeding (22.5 and 20 ng/ml respectively) than 1 h before feeding (53.5 and 64.5 ng/ml respectively; pooleds.e.m.=8.5). However, a combination of GHRP-6 at 3 microg/kg BW and GHRH at 0.2 microg/kg BW synergistically induced an equal and massive release of GH before and after feeding that was fivefold greater than GHRH-induced release of GH after feeding. Furthermore, the combination of GHRP-6 and GHRH synergistically increased release of GH from somatotropes cultured in vitro. However, it was not clear if GHRP-6 acted only on somatotropes or also acted at the hypothalamus. Therefore, we wanted to determine if GHRP-6 stimulated secretion of GHRH or inhibited secretion of somatostatin, or both. GHRP-6 stimulated secretion of GHRH from bovine hypothalamic slices, but did not alter secretion of somatostatin. We conclude that GHRP-6 acts at the hypothalamus to stimulate secretion of GHRH, and at somatotropes to restore and enhance the responsiveness of somatotropes to GHRH.

Somatostatin inhibits alpha-2-adrenergic-induced secretion of growth hormone-releasing hormone.

The purpose of this experiment was to determine the role of growth hormone-releasing hormone (GHRH) and somatostatin (SRIH) neurons in mediating alpha(2)-adrenergic receptor-induced stimulation of growth hormone (GH) secretion in cattle. Our first objective was to determine if stimulation of alpha(2)-adrenergic receptors increases activity of GHRH neurons in the arcuate nucleus (ARC) and/or decreases activity of SRIH neurons in periventricular (PeVN) and ARC nuclei. Clonidine (an alpha(2)-adrenergic agonist) or vehicle (saline) were injected i.v. into steers and dual-label immunohistochemistry was performed to quantify the number of GHRH and SRIH neurons expressing Fos and Fos-related antigens (Fos/FRA) as markers of neuronal activity. Clonidine increased concentrations of GH in serum and decreased activity of SRIH neurons in the PeVN, but not in the ARC. Clonidine did not alter activity of GHRH neurons in the ARC. Our second objective was to determine if clonidine decreases secretion of SRIH from perifused slices of hypothalami, which contain perikarya and terminals of GHRH and SRIH neurons, and from explants of hypophysial stalk alone, which contain only terminals of GHRH and SRIH neurons. Clonidine failed to alter release of GHRH or SRIH from hypothalamic slices, but stimulated release of GHRH from explants of hypophysial stalk. Blockade of SRIH receptors enabled clonidine to stimulate release of GHRH from slices of hypothalami, but also stimulated release of SRIH. These results suggest that alpha(2)-adrenergic-induced secretion of GH occurs via a dual mechanism involving inhibition of SRIH neurons in the PeVN and direct stimulation of GHRH release from axon terminals in the median eminence.

Neuroregulation of growth hormone secretion in domestic animals.

Growth hormone (GH) is essential for postnatal somatic growth, maintenance of lean tissue at maturity in domestic animals and milk production in cows. This review focuses on neuroregulation of GH secretion in domestic animals. Two hormones principally regulate the secretion of GH: growth hormone-releasing hormone (GHRH) stimulates, while somatostatin (SS) inhibits the secretion of GH. A long-standing hypothesis proposes that alternate secretion of GHRH and SS regulate episodic secretion of GH. However, measurement of GHRH and SS in hypophysial-portal blood of unanesthetized sheep and swine shows that episodic secretion of GHRH and SS do not account for all episodes of GH secreted. Furthermore, the activity of GHRH and SS neurons decreases after steers have eaten a meal offered for a 2-h period each day (meal-feeding) and this corresponds with reduced secretion of GH. Together, these data suggest that other factors also regulate the secretion of GH. Several neurotransmitters have been implicated in this regard. Thyrotropin-releasing hormone, serotonin and gamma-aminobutyric acid stimulate the secretion of GH at somatotropes. Growth hormone releasing peptide-6 overcomes feeding-induced refractoriness of somatotropes to GHRH and stimulates the secretion of GHRH. Norepinephrine reduces the activity of SS neurons and stimulates the secretion of GHRH via alpha(2)-adrenergic receptors. N-methyl-D,L-aspartate and leptin stimulate the secretion of GHRH, while neuropeptide Y stimulates the secretion of GHRH and SS. Activation of muscarinic receptors decreases the secretion of SS. Dopamine stimulates the secretion of SS via D1 receptors and inhibits the secretion of GH from somatotropes via D2 receptors. Thus, many neuroendocrine factors regulate the secretion of GH in livestock via altering secretion of GHRH and/or SS, communicating between GHRH and SS neurons, or acting independently at somatotropes to coordinate the secretion of GH.

Degeneration of capsaicin-sensitive sensory nerves leads to increased salt sensitivity through enhancement of sympathoexcitatory response.

We have previously shown that neonatal degeneration of capsaicin-sensitive sensory nerves renders a rat responsive to a salt load with an increase in blood pressure and a decrease in natriuretic response. To test the hypothesis that the enhanced sympathoexcitatory response to a high salt intake contributes to the development of hypertension in this model, newborn Wistar rats were given 50 mg/kg capsaicin and/or 80 mg/kg guanethidine subcutaneously. Control rats were treated with vehicle. After the weaning period, male rats were grouped as the following and given a high sodium diet (4%) for 2 weeks: capsaicin and guanethidine coadministration (CAP-GUA), capsaicin only (CAP), guanethidine only (GUA), and vehicle control (CON). Norepinephrine concentrations in the atrium were significantly lower in CAP-GUA and GUA than in CON rats (P<0.05). Twenty-four-hour urine and sodium excretions were significantly lower in CAP than in CAP-GUA, GUA, and CON rats (P<0.05). Mean arterial pressure (mm Hg) was significantly higher in CAP (180+/-10) than in CAP-GUA (106+/-1), GUA (133+/-5), and CON (122+/-3) rats (P<0.05). Thus, sympathectomy restores the natriuretic response to a high salt intake and prevents the development of salt-sensitive hypertension induced by sensory denervation. These data indicate that sensory nerves counterbalance the prohypertensive effect of the sympathetic nerves to maintain blood pressure within normal range during salt loading.

Effects of prolactin on expression of Fos-related antigens in tyrosine hydroxylase-immunoreactive neurons in subdivisions of the arcuate nucleus.

Dual immunohistochemistry was employed to determine the effects of prolactin on expression of Fos and its related antigens (FRA) in tuberoinfundibular dopamine (TIDA) neurons located in the dorsomedial (DM) and ventrolateral (VL) subdivisions of the arcuate nucleus (ARC) in the male rat. Systemic administration of the DA receptor antagonist haloperidol caused a sustained (up to 12 h) increase in plasma prolactin concentrations that was accompanied by a transient increase (at 3 h) in the percentage of tyrosine hydroxylase (TH)-immunoreactive (IR) neurons containing FRA-IR nuclei in the DM-ARC. In contrast, haloperidol caused a prolonged (1. 5 to 12 h) decrease in the percentage of TH-IR neurons with FRA-IR nuclei in the VL-ARC. Haloperidol had no effect, however, on the overall number of TH-IR neurons in either of these regions. Co-administration of prolactin antisera (PRL-AB) blocked haloperidol-induced increases in both plasma prolactin concentrations and the percentage of TH-IR neurons expressing FRA in the DM-ARC, but had no effect on haloperidol-induced inhibition of FRA expression in TH-IR neurons in the VL-ARC. Intracerebroventricular (i.c.v.) administration of prolactin also increased the percentage of TH-IR neurons containing FRA-IR nuclei in the DM-ARC, but this effect was of longer duration (up to 6 h) than that of haloperidol in all but the most caudal portion of the DM-ARC. In the VL-ARC, prolactin caused a transient increase (at 1.5 h) in the percentage of TH-IR containing FRA-IR nuclei. These results demonstrate that prolactin regulates immediate early gene expression in TIDA neurons in male rats, and reveal that there are temporal differences in the responsiveness of discrete subpopulations of these neurons to prolactin. Prolactin causes a short-lived increase in FRA expression in TIDA neurons in the VL-ARC which is followed by a more prolonged activation of FRA expression in TIDA neurons in the DM-ARC.

Prolactin regulation of tuberoinfundibular dopaminergic neurons: immunoneutralization studies.

The purpose of this study was to determine the effects of acute hypoprolactinemia on tuberoinfundibular dopamine (DA) neurons using a rabbit anti-rat prolactin antiserum (PRL-AB) to immunoneutralize circulating prolactin under basal conditions and at various times after haloperidol-induced hyperprolactinemia. The specificity of PRL-AB for prolactin was determined by examining the ability of unlabelled hormone to displace binding of 125I-labelled prolactin to PRL-AB. Tuberoinfundibular DA neuronal activity was estimated by measuring the concentrations of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the median eminence which contains terminals of these neurons. Systemic (i.v.) administration of 200 microl of PRL-AB decreased plasma prolactin concentrations below detectable levels for at least 4 h, and this was accompanied by a pronounced decrease in DOPAC concentrations in the median eminence of females, but not males. Central (i.c.v.) administration of 2 microl PRL-AB diluted up to 1:100 mimicked the inhibitory effect of systemic administration of PRL-AB on median eminence DOPAC concentrations suggesting that the tonic stimulatory effect of prolactin on the basal activity of tuberoinfundibular DA neurons in females occurs via a central site of action. In male rats, blockade of anterior pituitary DA receptors with haloperidol (1 mg/kg; s.c.) caused an prompt (by 1 h) increase in plasma prolactin concentrations which was maintained for at least 12 h. Haloperidol-induced hyperprolactinemia also caused a delayed (at 6 and 12 h) increase in median eminence DOPAC concentrations in these animals which was blocked by PRL-AB. Exposure of rats to initial priming periods of endogenous hyperprolactinemia of up to 6 h duration (followed by 6 h or more of PRL-AB-induced hypoprolactinemia) failed to alter median eminence DOPAC concentrations unless prolactin exposure was reinstated by an i.c.v. injection of prolactin. These results confirm that prolactin mediates the stimulatory effects of haloperidol on tuberoinfundibular DA neurons, and reveal that delayed induced activation of these neurons by prolactin is dependent upon a priming period of sustained hyperprolactinemia longer than 3 h for initiation and maintenance of this response.

Feeding reduces activity of growth hormone-releasing hormone and somatostatin neurons.

Secretion of growth hormone (GH) is synchronized among castrate male cattle (steers) around feeding when access to feed is restricted to a 2-hr period each day. Typically, concentrations of GH increase before and decrease after feeding. Our objectives were to determine whether i) concentrations of GH decrease in blood after start of feeding; ii) activity of immunoreactive growth hormone-releasing hormone (GHRH-ir) neurons decreases in the arcuate nucleus (ARC) after feeding; iii) activity of immunoreactive somatostatin (SS-ir) neurons in the periventricular nucleus (PeVN) and ARC increase after feeding; and iv) GHRH stimulates release of GH to a similar magnitude at 0900 and at 1300 hr, in steers fed between 1000 and 1200 hr. Blood samples were collected at 20-min intervals from 0700 to 1300 hr. Groups of steers were euthanized at 0700, 0900, 1100, and 1300 hr (n = 5 per group). Dual-label immunohistochemistry was performed on free-floating sections of hypothalami using antibodies directed against Fos and Fos-related antigens (Fos/FRA) as a marker of neuronal activity in immunoreactive GHRH and SS neurons. Concentrations of GH were high before and decreased after feeding. The percentage of SS-ir neurons containing Fos/FRA-ir in the PeVN was 50% lower (P<0.01) at 1100 hr and 36% lower (P<0.05) at 1300 hr than at 0900 hr. There was no change in percentage of SS-ir neurons containing Fos/FRA-ir in the ARC. The percentage of GHRH-ir neurons containing Fos/FRA-ir in the ARC was 66% lower (P<0.05) at 1100 hr and 65% lower (P<0.05) at 1300 hr than at 0700 hr. In contrast, the number of GHRH-ir neurons increased from 0700 to 1300 hr. GHRH-induced release of GH was suppressed at 1300 hr compared with 0900 hr. In conclusion, reduced basal and GHRH-induced secretion of GH after feeding was associated with decreased activity of GHRH neurons in the ARC and decreased activity of SS neurons in the PeVN.

Feeding-induced increases in insulin do not suppress secretion of growth hormone.

Secretion of growth hormone (GH) is reduced for several hours after feeding when access to feed is restricted to a 2-hr period each day. We hypothesized that increased secretion of insulin after feeding inhibits release of GH from the anterior pituitary gland. Our objectives were to determine whether: 1) alloxan prevents concentrations of insulin from increasing after feeding steers; 2) concentrations of GH remain high after feeding alloxan-treated steers; and 3) GH-releasing hormone (GHRH) stimulates greater release of GH in alloxan-treated, than in control, steers after feeding. Steers were injected iv with either saline (control) or with alloxan (110 mg/kg) (n = 4 per group). Concentrations of insulin were not different (P = 0.61) between control and alloxan-treated steers before feeding (87.5 +/- 33.6 pmol/l). However, alloxan prevented insulin from increasing (P < 0.001) after feeding (131.8 pmol/1) compared with control steers (442.0 pmol/l) (pooled SEM = 47.5). Overall, GH was higher (P < 0.05) in alloxan-treated (6.4 ng/ml) than in control steers (3.7 ng/ml) (pooled SEM = 0.7), but GH decreased (P < 0.001) after feeding in both groups. Iv injection of GHRH stimulated release of GH 1 hr before, but not when injected 1 hr after feeding (P < 0.001). In addition, net areas under the GH curve were not significantly different between control and alloxan-treated groups. We conclude that increased concentrations of insulin after feeding do not mediate feeding-induced suppression of GH secretion in steers.

Contribution of dopamine neurons in the medial zona incerta to the innervation of the central nucleus of the amygdala, horizontal diagonal band of Broca and hypothalamic paraventricular nucleus.

Results of previous studies suggested that incertohypothalamic dopamine (IHDA) neurons located in the medial zona incerta (MZI) project to the central nucleus of the amygdala (cAMY), horizontal diagonal band of Broca (HDB), and paraventricular nucleus (PVN). The overall goal of the present study was to determine the relative contribution of IHDA neurons to the DA innervation of these brain regions. A combined fluorescent and in situ hybridization histochemical procedure was employed to localize the retrograde tracer fluoro-gold (FG) in cells expressing tyrosine hydroxylase (TH) mRNA in the MZI following its iontophoretic injection into either the cAMY, HDB or PVN. For comparison, the numbers of dual labeled FG/TH mRNA neurons in the midbrain were also determined. One week after unilateral injection of FG into the cAMY, cells containing FG+TH mRNA were found in the ipsilateral MZI, substantia nigra zona compacta (SNC) and ventral tegmental area (VTA). The total numbers of cells labeled with FG varied with the size of the injection site, but the ratio of dual labeling in the MZI to that of the SNC-VTA remained constant across animals at approximately 1:6. FG injections into the HDB resulted in a ratio of dual labeled cells in the ipsilateral MZI and VTA of approximately 1:2, but no dual labeled cells were found in the SNC. Dual labeled cells were only found in the ipsilateral MZI in animals receiving FG injections in the PVN. Thus, DA terminals in the PVN originate exclusively from IHDA neurons in the MZI, whereas these neurons provide only a portion of the DA innervation of the cAMY and HDB. The similar distribution of dual labeled cells in the MZI following FG injections into the cAMY, HDB and PVN suggests that perikarya of IHDA neurons projecting to these regions are not organized into distinct groups.

Opposing roles for dopamine D1 and D2 receptors in the regulation of hypothalamic tuberoinfundibular dopamine neurons.

The purpose of the present study was to characterize pharmacologically dopamine D1 receptor-mediated inhibition of tuberoinfundibular dopamine neurons in males rats, and to determine if inhibitory dopamine D1 receptors oppose stimulatory dopamine D2 receptors and account for the inability of mixed dopamine receptor agonists to alter the activity of these neurons. Tuberoinfundibular dopamine neuronal activity was estimated by measuring the concentrations of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the median eminence, the region of the hypothalamus containing terminals of these neurons. Administration of the dopamine D1 receptor agonist (+/-)-1 phenyl-2,3,4,5-tetrahydro-(1 H)-3-benzazepine-7,8-diol (SKF38393) decreased median eminence DOPAC and increased plasma prolactin concentrations, whereas administration of the dopamine D1 receptor antagonist ((-)-trans,6,7,7a,8,9,13b-hexahydro-3-chloro-2-hydroxy-N-methyl-5H -benzo[d]naphtho-[2,1 b]azepine (SCH39166) increased median eminence DOPAC concentrations but had not effect on plasma prolactin. The inhibitory effect of SKF38393 on median eminence DOPAC concentrations was blocked by SCH39166. These results demonstrate that acute activation of dopamine D1 receptors inhibits the activity of tuberoinfundibular dopamine neurons and thereby increases prolactin secretion, and that under basal conditions dopamine D1 receptor-mediated inhibition of tuberoinfundibular dopamine neurons is tonically active. Administration of the dopamine D2 receptor agonist (5aR-trans)-5,5a,6,7,8,9,9a,10-octahydro-6-propyl-pyridol[2, 3-g]quinazolin-2-amine (quinelorane) increased median eminence DOPAC concentrations, and SKF38393 caused a dose-dependent reversal of this effect. Administration of the mixed dopamine D1/D2 receptor agonist R(-)-10,11-dihydroxy-apomorphine (apomorphine) had no effect per se, but blocked quinelorane-induced increases in DOPAC concentrations in the median eminence. These results reveal that concurrent activation of dopamine D1 and D2 receptors nullifies the actions of each of these receptors on tuberoinfundibular dopamine neurons, which likely accounts for the lack of an acute effect of mixed dopamine D1/D2 receptor agonists on these hypothalamic dopamine neurons.

Pharmacological evidence that neurotensin mediates prolactin-induced activation of tuberoinfundibular dopamine neurons.

The purpose of this study was to investigate the role of neurotensin (NT) receptors in mediating the stimulatory effects of prolactin on the activity of tuberoinfundibular dopamine (TIDA) neurons in male and female rats. TIDA neuronal activity was estimated by measuring concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) in terminals of these neurons in the median eminence (ME). Haloperidol activates TIDA neurons indirectly by blocking D2 receptors on pituitary lactotropes, thereby increasing secretion of prolactin. Twelve hours after administration of haloperidol (1 mg/kg, s.c.), DOPAC concentrations in the ME were increased. Blockade of NT receptors with the selective antagonist SR-48692 had no effect per se on basal DOPAC concentrations in the ME but produced a dose-related (10-1,000 microg/kg, i.p.; 1 h) reversal of haloperidol-induced increases in ME DOPAC concentrations. In contrast, SR-48692 had no effect on either basal or haloperidol-induced increases in plasma prolactin. SR-48692 also blocked the stimulatory effects of prolactin (10 microg/rat, i.c.v.; 12 h) on ME DOPAC concentrations. SR-48692 was equally effective in blocking the stimulatory effects of haloperidol and prolactin on TIDA neurons in male and female rats. These results suggest that NT mediates the induced stimulatory effect of hyperprolactinemia on the activity of TIDA neurons in both males and females, whereas the tonic regulation of these neurons by prolactin in females occurs via an NT-independent mechanism.

Stimulation of dopamine D1 receptors increases activity of periventricular somatostatin neurons and suppress concentrations of growth hormone.

The selective dopamine D1 receptor agonist, SKF38393, stimulates release of somatostatin (SS) from perifused bovine hypothalamic slices. Therefore, we hypothesized that SKF38393 activates SS neurons, which, via release of SS, would suppress concentrations of growth hormone (GH) in serum in calves. Our objectives were to determine whether SKF38393: (1) increases the percent of immunoreactive c-Fos protein and Fos-related antigens (Fos/FRA) detected in somatostatin neurons in periventricular (PeVN) and arcuate (ARC) hypothalamic nuclei; (2) reduces concentrations of GH in serum; (3) suppresses growth hormone-releasing hormone (GHRH)-induced release of GH. Meal-fed steers were used to perform these objectives because a synchronous pulse of GH occurs 1-2 hr before feeding in steers allowed access to feed for 2 hr each day. In Experiment 1, two groups of four Holstein steers were injected s.c. with either vehicle (sterile water) or SKF38393 (5 mg/kg BW). Steers were injected i.v. with a lethal dose of sodium pentobarbital 100 min later and their brains were fixed with 4% paraformaldehyde. Dual-label immunohistochemistry was performed on 40 microns free-floating sections using antiserum to SS and to Fos/FRA on sections containing PeVN and ARC nuclei. More SS neurons were detected in the PeVN than in the ARC. The percent of SS neurons with immunoreactive Fos/FRA present was 2.9-fold higher in SKF38393-treated compared with vehicle-injected steers in the PeVN, but was unchanged in the ARC. In Experiment 2, eight Holstein steers were injected s.c. with either vehicle (sterile water) or SKF38393 (5 mg/kg BW) 140 min before meal-feeding. In contrast to controls, concentrations of GH in serum of SKF38393-treated steers did not increase during 140 min before meal-feeding. In Experiment 3, eight Holstein steers were injected s.c. with either vehicle (sterile water) or SKF38393 (5 mg/kg BW), then 100 min later, each steer was injected i.v. with [Leu27,Hse45] bGHRH1-45 lactone (0.2 micrograms/kg BW). Bovine GHRH stimulated release GH into serum in both groups, but concentrations of GH were lower in SKF38393-treated steers. These results show that stimulation of D1 receptors selectively increases activity of SS neurons in the PeVN, and this increased activity is associated with suppressed basal- and GHRH-induced release of GH in serum of meal-fed steers.

Regulation of growth hormone-releasing hormone and somatostatin from perifused, bovine hypothalamic slices. II. Dopamine receptor regulation.

An in vitro perifusion system for bovine hypothalamic tissue was utilized to examine the role of D1 and D2 dopamine receptors in the regulation of somatostatin (SRIF) and growth hormone-releasing hormone (GHRH) release. Up to three sagittal slices (600 microns) of bovine hypothalamus, immediately parallel to the midline, were cut in an oxygenated balanced salt solution at 4 degrees C, placed in 5 cc syringes, and perifused at 37 degrees C with oxygenated minimum essential medium-alpha at a flow rate of 0.15 ml/min. Five experiments were conducted, and medium effluent was collected every 20 min before (two samples), during (one or three samples), and after (six samples) treatment. Areas under SRIF and GHRH response curves (AUC), adjusted by covariance for pretreatment values, were calculated from samples collected during the treatment/post-treatment period. Activation of D1 receptor with 10(-8) M and 10(-6) M SKF 38393 increased AUC for SRIF from 5.6 (control) to 420 and 500 +/- 57.8 ng.ml-1 min, but 10(-10) M SKF 38393 was ineffective. Relative to controls, release of GHRH was decreased 50% in the 10(-6) M SKF 38393 group. Blockade of D1 receptors with SCH 23390 had no effect on basal release of either SRIF or GHRH, but prevented SKF 38393-induced release of SRIF and SKF 38393-induced suppression of GHRH. In contrast, quinelorane, a D2 receptor agonist, and haloperidol, which blocks D2 receptors, did not affect release of SRIF or GHRH. We concluded that activation of D1 dopamine receptors, but not D2 dopamine receptors, stimulates release of SRIF and inhibits release of GHRH from the bovine hypothalamus.

Regulation of growth hormone-releasing hormone and somatostatin from perifused, bovine hypothalamic slices. I. Alpha 2-adrenergic receptor regulation.

An in vitro perifusion system was developed for bovine hypothalamic tissue to examine the role of alpha 2-adrenergic receptors in the regulation of growth hormone-releasing hormone (GHRH) and somatostatin (SRIF) release. Up to three sagittal slices (600 microns) of hypothalamus, immediately parallel to the midline, were cut in an oxygenated balanced salt solution at 4 degrees C, placed in 5 cc syringes, and perifused at 37 degrees C with oxygenated minimum essential medium-alpha at a flow rate of 0.15 ml/min. Three experiments were conducted, and medium effluent was collected every 20 min before (two samples), during (one or three samples), and after (six samples) treatment. Areas under GHRH and SRIF response curves (AUC), adjusted by covariance for pretreatment values, were calculated from samples collected during the treatment/post-treatment period. Location from which slices were cut, relative to the sagittal midline, had no effect on basal release of GHRH and SRIF, but variation in basal release of GHRH and SRIF differed among animals. Medium containing 60 mM KCI increased AUC for GHRH 39% and 161% for SRIF when compared with perifusion of medium alone, thereby verifying that tissue remained viable for at least 14 hr. Activation of alpha 2-adrenergic receptor with 10(-6) and 10(-4) M clonidine increased AUC for GHRH from 54.8 (control) to 79.1 and 108.7 +/- 2.5 ng.ml-1 min for 10(-6) M and 10(-4) M clonidine, respectively. Guanabenz, another alpha 2-adrenergic receptor agonist, at 10(-8), 10(-6), and 10(-4) M also increased GHRH release from 45.5 (control) to 52.8, 66.2, and 86.7 +/- 1.6 ng.ml-1 min, respectively. Clonidine and guanabenz did not affect release of SRIF. An alpha 2-adrenergic receptor antagonist, idazoxan, blocked clonidine-induced release of GHRH without affecting release of SRIF. We concluded that alpha 2-adrenergic receptor stimulation of in vivo growth hormone secretion in cattle is mediated via an increase in release of GHRH and not a change in release of SRIF.

Regulation of growth hormone-releasing hormone and somatostatin from perifused, bovine hypothalamic slices. III. Reciprocal feedback between growth hormone-releasing hormone and somatostatin.

An in vitro perifusion system for bovine hypothalamic tissue was used to determine if growth hormone-releasing hormone (GHRH) and somatostatin (SRIF) modulate each other's release, and whether SRIF mediates D1-agonist-induced suppression of GHRH in cattle. Up to three sagittal slices (600 microns) of bovine hypothalamus, immediately parallel++ to the midline, were cut in an oxygenated balanced salt solution at 4 degrees C, placed in 5 cc syringe barrels, and perifused at 37 degrees C with oxygenated minimum essential medium-alpha at a flow rate of 0.15 ml/min. Three experiments were conducted, and medium effluent was collected every 20 min before (two samples), during (one or three samples), and after (six samples) treatment. Areas under GHRH and SRIF response curves (AUC), adjusted by covariance for pretreatment values, were calculated from samples collected during the treatment/post-treatment period. Perifusion of SRIF at 10(-6) M and 10(-4) M decreased AUC for GHRH from 86.3 (control) to 65.4 and 59.5 +/- 6.3 ng.ml-1 min, but 10(-8) M SRIF was ineffective. Relative to controls, 10(-8).10(-6), and 10(-4) M GHRH increased release of SRIF 190, 675, and 1,135%, respectively. Activation of D1 receptors with 10(-6) M SKF 38393 increased AUC for SRIF from 12.5 ng.ml-1 min (control) to 484.9 ng.ml-1 min and decreased AUC for GHRH from 36.4 ng.ml-1 min (control) to 18.2 ng.ml-1 min. Blockade of SRIF action with a SRIF antagonist, cyclo-[7-aminoheptanoyl-phe-D-trp-lys-thr(bzl)], increased release of GHRH 1.9-fold. In addition, the SRIF antagonist blocked SKF 38393-induced suppression of GHRH. We concluded that GHRH and SRIF interact within the bovine hypothalamus/pituitary stalk to modulate the release of the other. Moreover, SRIF mediates the inhibitory effects of activation of D1 receptors on release of GHRH in cattle.

Effects of selective activation of dopamine D2 and D3 receptors on prolactin secretion and the activity of tuberoinfundibular dopamine neurons.

Dopamine agonists with activity at both dopamine D2 and D3 receptor subtypes stimulate tuberoinfundibular dopamine neurons and inhibit prolactin secretion from the anterior pituitary. The purpose of the present study was to identify the dopamine receptor subtypes mediating these effects using recently developed selective agonists for dopamine D2 (PNU-95,666) and D3 (PD128907) receptors. The activity of tuberoinfundibular dopamine neurons was estimated by measuring either the synthesis (accumulation of 3,4-dihydroxyphenyl-alanine [DOPA] following inhibition of decarboxylase activity) or metabolism (3,4-dihydroxyphenylacetic acid [DOPAC] concentrations) of dopamine in the median eminence, the region of the hypothalamus containing axon terminals of these neurons. In one experiment, the activity of mesolimbic dopamine neurons was also determined by measuring DOPA accumulation in terminals of these neurons in the nucleus accumbens. Activation of dopamine D2 receptors with PNU-95,666 caused dose- and time-related increases in DOPAC concentrations in median eminence which were temporally correlated with decreases in plasma prolactin concentrations. Activation of dopamine D3 receptors with PD128907 decreased DOPA concentrations in the nucleus accumbens, but had no effect on concentrations of DOPAC or DOPA in the median eminence or prolactin in plasma. These results reveal that tuberoinfundibular dopamine neurons are regulated by dopamine D2 rather than D3 receptors, and suggest that the ability of mixed dopamine D2/D3 receptor agonists to increase the activity of these neurons is mediated by an action at dopamine D2 receptors. Furthermore, these results confirm that tuberoinfundibular dopamine neurons are not regulated by inhibitory dopamine D2 or D3 autoreceptors.

Role of gonadal steroids in determining sexual differences in expression of Fos-related antigens in tyrosine hydroxylase-immunoreactive neurons in subdivisions of the hypothalamic arcuate nucleus.

Dual immunohistochemistry was employed to examine the role of gonadal steroids in determining sexual differences in the expression of Fos and its related antigens (FRA) in tuberoinfundibular dopaminergic (TIDA) neurons located in the dorsomedial (DM-) and ventrolateral (VL-) subdivisions of the arcuate nucleus (ARC). In the DM-ARC, there was no sexual difference in the number of tyrosine hydroxylase (TH)-immunoreactive (-IR) perikarya, but the number of these containing FRA-IR was greater in females than in males in all but the most caudal region. In the VL-ARC, there were more TH-IR perikarya in males than in females, but there was no sexual difference in the numbers of those containing FRA-IR throughout the entire rostrocaudal extent of this nucleus. Ovariectomy decreased the number of TH-IR perikarya containing FRA-IR in the DM-ARC, but not in the VL-ARC, whereas orchidectomy increased the number of TH-IR perikayra containing FRA-IR in both the DM-ARC and VL-ARC. These gonadectomy-induced effects were reversed by estrogen and testosterone, respectively. These results reveal gonadal steroid-dependent sexual differences in the regulation of immediate early gene expression in anatomically discrete subpopulations of TIDA neurons. In females, estrogen stimulates FRA expression in TIDA neurons in the DM-ARC, whereas in males, testosterone inhibits FRA expression in TIDA neurons in both the DM-ARC and the VL-ARC.