bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of N‐Methyl‐D,L‐Aspartate on Beta‐Endorphin and Prolactin Secretion in Rams Exposed to Long or Short Days
Authors: Lincoln G. A. , Wu F. C. W.
Published: September 2006

In a previous study we demonstrated that the injection of the excitatory amino‐acid N‐methyl‐D, L‐aspartate (NMDA) stimulates an acute increase in the peripheral blood concentrations of luteinizing hormone in Soay rams, and this response varies with the photoperiodically‐induced reproductive cycle. To extend these observations, we have now measured the changes in the blood concentrations of β‐endorphin and prolactin in the same animals to establish whether NMDA stimulates the secretion of other pituitary hormones. Groups of adult Soay rams were exposed to alternating 16‐weekly periods of long and short days to induce a long‐term cycle in the endogenous secretion of β‐endorphin (maximum under short days) and prolactin (maximum under long days). NMDA injected intravenously caused a dose‐dependent increase in the blood plasma concentrations of β‐endorphin (over the range 1 to 20 mg/kg NMDA). The initial increase in β‐endorphin occurred within 2 to 4 min with peak levels after 20 to 100 min. The magnitude of the β‐endorphin response was greatest following exposure to long days when the endogenous secretion of β‐endorphin was low, while the duration of the response was greatest following exposure to short days when the endogenous secretion of β‐endorphin was high. NMDA injected intravenously also caused a dose‐dependent increase in the blood plasma concentrations of prolactin but this only occurred following exposure to long days when the endogenous secretion of prolactin was high. At this time the initial increase in prolactin concentrations occurred within 2 to 4 min after the injection of NMDA as for β‐endorphin but the values continued to increase for 2 to 4 h. In a separate experiment, it was shown that pretreatment of the rams with dexamethasone (synthetic glucocorticoid, 133.4 μg/kg iv) blocked the β‐endorphin response to NMDA but had no effect on the prolactin response. This indicates that NMDA stimulates the secretion of β‐endorphin from the corticotrophs probably acting centrally to induce the release of corticotrophin‐releasing hormone and/or arginine vasopressin, while NMDA acts through separate mechanisms to affect the secretion of prolactin.

The overall results show that NMDA can be used as a probe to investigate the neuroendocrine control of β‐endorphin and prolactin in addition to luteinizing hormone as described previously. The multiple responses are likely to represent the effects of NMDA acting on the hypothalamus to induce the acute release of peptides and other hormones into the pituitary portal blood system to affect the corticotrophs, lactotrophs and gonadotrophs. The variation in responsiveness to NMDA related to the photoperiodic cycle may reflect changes in the synthesis and storage of the hypothalamic hormones and the influence of other neural systems. Alternatively, the changes in the secretion of endogenous excitatory amino‐acids and NMDA receptors may constitute part of the neuroendocrine mechanism relaying the effects of photoperiod.
Melatonin Inhibits the Activation of Cyclic AMP‐Dependent Protein Kinase in Cultured Pars Tuberalis Cells from Ovine Pituitary
Authors: Hazlerigg D. G. , Morgan P. J. , Lawson W. , Hastings M. H.
Published: September 2006

The effect of melatonin upon the activation of the intracellular effector enzyme, cyclic AMP (cAMP)‐dependent protein kinase (PKA), was investigated in primary cultures of ovine pars tuberalis cells. Incubation of these cells with forskolin caused a rapid and dose‐dependent activation of PKA (ED50 10∼6M). When cells were incubated with forskolin and melatonin simultaneously, the activation of PKA by forskolin was dramatically inhibited. This inhibitory effect of melatonin was dose‐dependent (ED50 10−10M). Furthermore, treatment with melatonin rapidly deactivated PKA in cells prestimulated with forskolin. When pars tuberalis cell extracts were incubated with 8N3‐[32P]cAMP, an analogue of cAMP used for photoaffinity labelling of native PKA, specific binding was observed in three bands with Mr of 54, 52 and 48 kd, representing the regulatory subunits of PKA II (in phosphorylated and dephosphorylated forms) and PKA I, respectively. These results indicate that melatonin is a potent inhibitory regulator of cAMP‐mediated signal transduction in the ovine pars tuberalis, and suggest that the cellular effects of melatonin in this tissue are mediated by the dephosphorylation of specific substrate proteins.
Cortisol Exerts Site‐, Context‐ and Dose‐Dependent Effects on Agonistic Responding in Hamsters
Authors: Hayden‐Hixson Diane M. , Ferris Craig F.
Published: September 2006

Site‐, context‐ and dose‐dependent actions of intrahypothalamic cortisol administration on the agonistic behaviors of adult male golden hamsters (n = 128 dyads) were examined. When cortisol‐treated animals were tested in paired encounters with aggressive cholesterol‐treated opponents, chronic (≥ 24 h) cortisol treatment (1 mm implants) induced significant (P < 0.05) submission in three medial hypothalamic areas (anterior hypothalamic area > medial preoptic area > ventromedial hypothalamus), but aggression in the paraventricular nucleus or third ventricle. In contrast, chronic cortisol treatment in the anterior hypothalamic area resulted in high levels of aggression during paired encounters with submissive opponents, and during territorial aggression tests with juvenile male intruders. Acute (≥ 20 min) cortisol treatment in the anterior hypothalamic area (100 nl injections) induced significant submission after 10−2 M, but significant aggression after 10−6 M microinjections in paired encounters with aggressive vehicle‐injected opponents. These findings suggest glucocorticoid‐sensitive mechanisms within the anterior hypothalamus modulate aggressive responding during intrasexual social encounters.
Immunccytochemical Localization of Angiotensinogen in Rat Brain: Dependence of Neuronal Immunoreactivity on Method of Tissue Processing
Authors: Campbell Duncan J. , Sernia Conrad , Thomas Walter G. , Oldfield Brian J.
Published: September 2006

There is disagreement between laboratories on the presence and location of angiotensinogen immunostaining in neuronal cells. We examined this issue by using different antisera and histological procedures to stain for angiotensinogen in brains from normal, colchicine‐treated and nephrectomized rats. Five different antisera from three laboratories were used to stain sections of paraffin‐embedded tissue, frozen sections and Vibratome sections of cerebral cortex, thalamus, hypothalamus, brainstem and cerebellum. All five antisera and all three tissue treatments were effective in showing angiotensinogen staining in glial cells, with the most intense staining being achieved in Vibratome sections. All five antisera gave identical results. Neuronal staining was also found with all antisera but mostly in paraffin‐embedded sections, with occasional light staining in frozen sections. No neuronal staining was observed in Vibratome sections. Neuronal staining was frequently perivascular, tended to have a more variable anatomical localization, and to occasionally lack bilateral symmetry in coronal sections. These results provide an explanation for the disagreement between laboratories on the presence and location of angiotensinogen immunostaining in neuronal cells. Taken together with the limited concordance between published sites of angiotensinogen and angiotensin II staining, and the recent demonstration by hybridization in situ of a specifically glial cell localization of angiotensinogen mRNA, our own results suggest a need for caution in the interpretation of neuronal staining with anti‐angiotensinogen antisera.
Effect of Hypophysectomy and Growth Hormone Replacement on Hypothalamic Growth Hormone‐Releasing Factor Messenger Ribonucleic Acid Levels
Authors: Eccleston Lynne M. , Powell John F. , Clayton Richard N.
Published: September 2006

The mechanisms by which the pituitary gland, and growth hormone (GH) in particular, affect growth hormone‐releasing factor (GRF) gene expression have been addressed using the technique of in situ hybridization. Anatomically matched sections through the mediobasal hypothalamus of control and hypophysectomized male rats, with or without GH hormone replacement, were analysed to obtain information on GRF mRNA levels within the arcuate nucleus and around the ventromedial hypothalamus. Hypophysectomy resulted in a 70% increase in the amount of GRF mRNA per cell (P<0.001), within neurons in the arcuate nucleus. GH replacement and T4 replacement separately partially attenuated this increase (GH replacement P< 0.001 versus hypophysectomy, T4 replacement P<0.05 versus hypophysectomy). Additionally, after hypophysectomy there was an 80% increase in the number of cells expressing the GRF gene in neurons around the ventromedial hypothalamus, when compared to shamoperated controls (P<0.01). Both GH and T4 replacement separately partially attenuated this phenomenon (P<0.01 versus hypophysectomized animals). Hypothyroidism alone did not affect GRF mRNA levels in either the arcuate nucleus or in the area surrounding the ventromedial hypothalamus. These results show that hypophysectomy increases GRF mRNA levels in two separate ways: by increasing the amount of mRNA produced per cell within the arcuate nucleus, and by increasing the number of cells expressing the gene in the area surrounding the ventromedial hypothalamus. This increase in the number of GRF mRNA‐containing cells after hypophysectomy could result from the recruitment of neurons which previously did not express the GRF gene, and may reflect the plasticity of the adult central nervous system in response to a changing endocrine environment. This could represent part of a sensor mechanism to drive the production of GRF in the arcuate nucleus in response to extreme disruption of the GRF/ GH feedback loop.
Regulation of Chromogranin A and Chromogranin B (Secretogranin I) Synthesis in Bovine Cultured Chromaffin Cells
Authors: Galindo Estelle , Bader Marie‐France , Aunis Dominique
Published: September 2006

In the present study we investigated the regulation of Chromogranin A (CGA) and Chromogranin B (CGB) biosynthesis in bovine chromaffin cells maintained in primary culture. Cellular proteins were labelled with [35S]methionine and the incorporated radioactivity was used as an index of the synthesis rate. The radioactivity incorporated into CGA was determined by immunoprecipitation, and that into CGB was quantified by a dot immunobinding assay using specific antibodies. Incubation of cells with carbamylcholine, nigh K+ or histamine, three potent stimulators of catecholamine secretion in chromaffin cells, increased the rate of CGA and CGB synthesis. On the other hand bradykinin, angiotensin II and prostaglandin E2, which cause little secretion, also produced an increase in both CGA and CGB synthesis. These results suggest that in chromaffin cells, the biosynthesis of chromogranins is not closely linked to the secretory activity. Inhibition of protein kinase C by sphingosine or by long‐term treatment with phorbol esters, completely abolished the synthesis of CGA and CGB induced by carbamylcholine, bradykinin and prostaglandin E2 but decreased only partially the stimulating effect of histamine. Thus, protein kinase C may not be the sole effector involved in the secretagogue‐induced modulation of Chromogranin synthesis. Forskolin, an activator of adenylate cyclase had no effect on CGA synthesis, but significantly enhanced the incorporation of radioactivity into CGB. The effect of forskolin was not modified by protein kinase C inhibitors and was additive to that induced by phorbol esters indicating that cyclic AMP did not stimulate CGB synthesis through a protein kinase C‐dependent pathway. These observations suggest that the biosynthesis of CGA and CGB in chromaffin granules is independently regulated.
Androgen‐Dependent and ‐Independent Aromatase Activity Coexists with Androgen Receptors in Male Guinea‐Pig Brain
Authors: Connolly Peter B. , Roselli Charles E. , Resko John A.
Published: September 2006

Using a microdissection technique we localized androgen receptors and aromatase activity (AA) in the brain of male guinea‐pigs. In addition, we evaluated the effects of castration and androgen replacement on androgen receptor dynamics and induction of AA. In the castrate animal, cytosolic androgen receptor content was highest in the basal hypothalamus, specifically in the median eminence‐arcuate nucleus (> 15 fmol mg protein 1), while lesser levels were found in the preoptic regions and amygdala. Nuclear receptor content was highest (> 150 fmol mg DNA −1) in the median eminence‐arcuate nucleus, periventricular region of the preoptic area and cortical amygdala. All regions investigated showed a significant decrease in nuclear receptors following castration and an increase with androgen replacement. However, reciprocal changes in cytosolic androgen receptors were not always evident. Aromatase activity was high in the cortical amygdala, medial amygdala, periventricular region of the preoptic area and bed nucleus of the stria terminalis. Castration and androgen replacement had significant stimulatory effects on AA in the ventral medial hypothalamus, median eminence‐arcuate nucleus, cortical amygdala and periventricular regions of the preoptic area and anterior hypothalamus. Thus, androgen receptors and AA are unevenly distributed throughout the subcortical regions of the male guinea‐pig brain and respond differently to endocrine stimuli. Our data demonstrate that AA is androgen‐dependent in some subcortical regions which contain androgen receptors. Even though nuclear receptors in all brain regions were affected by castration and dihydrotestosterone treatment, the events were not always linked to AA regulation. Due to this difference in regulation, AA may serve divergent functions in guinea‐pig brain.
Electrical Stimulation of the Basolateral Amygdala Elicits Only Growth Hormone Secretion Among Six Anterior Pituitary Hormones in the Pentobarbital‐Anesthetized Male Rat
Authors: Koibuchi Noriyuki , Kakegawa Tadao , Suzuki Mitsuo
Published: September 2006

In order to investigate the role of the basolateral amygdala on secretion of the adenohypophyseal hormones, the basolateral amygdala was electrically stimulated for 10 min through a chronically implanted electrode in the pentobarbital‐anesthetized adult male rat. Blood samples were withdrawn through a Silastic cannula implanted in the right atrium. Electrical stimulation of the basolateral amygdala increased plasma growth hormone, decreased plasma follicle‐stimulating hormone and suppressed the increase in plasma adrenocorticotropin. Plasma prolactin, thyroid‐stimulating hormone and luteinizing hormone were not significantly changed. These results indicate that electrical stimulation of the basolateral amygdala in the pentobarbital‐anesthetized adult male rat causes a simultaneous change in secretion of several adenohypophyseal hormones.
Neurohormonal Factors Involved in the Control of the Nocturnal Prolactin Surge that Precedes Parturition in the Rat
Authors: Grattan David R. , Averill Robert L. W.
Published: September 2006

A nocturnal surge of prolactin (PRL) occurs in the dark period preceding parturition in the rat. The roles of oxytocin, vasoactive intestinal peptide (VIP), serotonin and the opioids in controlling the antepartum PRL surge were investigated by examining PRL secretion over the last 2 days of pregnancy in the presence of antagonists to these neurohormonal factors. Serial blood samples were collected from unanesthetized, freely moving rats via indwelling jugular cannulae, and plasma PRL was measured by radioimmunoassay. In control rats PRL levels rose in a nocturnal surge peaking at 223 ± 34 ng/ml (n = 6) at 0500 h on day 21 of pregnancy, the day of parturition. Intra‐arterial infusion of the oxytocin antagonist desGly‐NH2d(CH2)5[Tyr(Me)2, Thr4]‐OVT at a dose sufficient to completely block milk ejection (10 μg/h) had no effect on this PRL surge. Infusion of the VIP antagonist [4Cl‐D‐Phe6,Leu17]‐VIP at 2 μg/h from 2200 h on day 20 until 0500 h on day 21 significantly attenuated the antepartum PRL surge, reducing the peak to 76 ± 28 ng/ml at 0500 h on day 21 (n = 6; P<0.001). Naloxone, the opiate receptor antagonist, inhibited the antepartum PRL surge in a dose‐dependent manner. Infused at 2 mg/h naloxone partially reduced the magnitude of the PRL surge, which peaked at 128 ± 24 ng/ml at 0300 h on day 21 (n = 4; P<0.05), while at 10 mg/h naloxone totally abolished the PRL surge (n = 6; P<0.001). Injection of the serotonin synthesis inhibitor ρ‐chlorophenylalanine (250 mg/kg, sc at 1700 h on days 19 and 20 of pregnancy) increased the magnitude of the antepartum PRL surge to a peak of 327 ± 48 ng/ml at 0500 h on day 21 (n = 5), compared with 244 ± 24 ng/ml at the same time in vehicle‐injected controls (P<0.05; n = 5). The results demonstrate that the antepartum PRL surge is stimulated by an opioid mechanism, and also by VIP. Oxytocin and serotonin have no role in stimulating PRL secretion during late pregnancy.
Luteinizing Hormone Surge in Adult Female Rats After Androgen Priming
Authors: Loke D. F. M. , Ratnam S. S. , Goh H. H.
Published: September 2006

The effects of androgens alone and in combination with oestrogens on luteinizing hormone secretion in adult female rats were studied after 1, 2 and 4 months of priming. Each adult female rat was implanted with a Silastic capsule filled with testosterone or dihydrotestosterone alone or together with another similar capsule of oestradiol. An oestrogen challenge test was carried out in each rat after priming. Blood samples were collected before androgen priming. During the oestrogen challenge test, blood samples were collected before (0 h or day 0) and at 12, 24, 36 and 48 h after the intramuscular injection of 50 μg oestradiol vaierate for rats primed for 1 month, or daily for 5 days (days 1 to 5) for other rats. An oestrogen challenge test was also performed in two groups of untreated male and female rats which served as controls.

The results indicated that long‐term androgen priming had resulted in the attenuation or total blockade of the luteinizing hormone surge which normally occurs during an oestrogen challenge test in adult female rats. This suggests that androgens may be important modulators of the luteinizing hormone surge mechanism in rats. Furthermore, the fact that the luteinizing hormone surge can be blocked by androgen priming in adult female rats suggests that the sexual dimorphic response to oestrogen feedback is not immutably imprinted in the hypothalamic‐pituitary axis as was originally proposed.
Characterization and Ontogenesis of N‐Methyl‐D‐Aspartate‐Evoked Luteinizing Hormone Secretion in Immature Female Rats
Authors: MacDonald Michael C. , Wilkinson Michael
Published: September 2006

The excitatory amino‐acid L‐glutamate appears to be involved in the neural regulation of puberty. We have now characterized the stimulatory effect of the glutamate agonist N‐methyl‐D‐aspartic acid (NMDA) on the secretion of luteinizing hormone (LH) in immature female rats. NMDA injections (subcutaneously) rapidly induce LH secretion (maximum at 8 min) with an ED50 of 3.25 mg/kg determined at postnatal day 30. The LH response to NMDA develops between 10 and 15 days after birth, reaches a maximum at approximately day 27 and disappears again by day 32, an age at which 80% of the rats are still prepubertal. In confirmation of work by others, we also showed that postpubertal male rats are unresponsive to NMDA. The effect of NMDA on LH release is blocked by prior injection of the glutamate antagonist MK‐801 (0.1 mg/kg). MK‐801 alone is able to reduce LH secretion in acutely ovariectomized rats but has little effect in intact, prepubertal rats. NMDA may not act exclusively on the gonadotropin‐releasing hormone neuron since inhibition of norepinephrine biosynthesis by the drug U‐14,624 largely prevents NMDA‐induced LH secretion. Interestingly, rats treated neonatally with the neurotoxin monosodium glutamate responded normally to NMDA when tested at postnatal day 25. Finally, removal of the ovaries 48 h previously reduced the LH response to NMDA. Full responsiveness was restored following estrogen priming. In summary, hypothalamic glutamate receptors of the NMDA subtype comprise a significant regulatory component of LH secretion in prepubertal female rats. LH responsiveness appears to be age‐, dose‐, estrogen‐ and norepinephrine‐dependent.
Corticotropin‐Releasing Factor Binding Sites Undergo Axonal Transport in the Rat Vagus Nerve
Authors: Mercer J. G. , Lawrence C. B. , Copeland P. A.
Published: September 2006

Corticotropin‐releasing factor (CRF) binding sites were found to be present in the rat vagus nerve and underwent axonal transport. Binding sites accumulated on both sides of ligatures placed on the nerve and at similar rates following ligation of right or left cervical vagal trunks of either male or female rats. CRF binding sites also accumulated proximal and distal to ligatures on subdiaphragmatic vagal trunks. Binding was specific, reversible and inhibited by the CRF receptor antagonist α‐helical‐CRF(9–41). [125l]Tyr0‐ovine‐CRF binding to rat vagus nerve was not guanine nucleotide‐sensitive. CRF and cholecystokinin binding sites were transported at a similar rate in the cervical vagus, although turnover of CRF binding sites was more rapid. No differences in CRF binding site transport were observed between Zucker rats of lean or obese genotype.
Further Evidence that Most Luteinizing Hormone‐Releasing Hormone Neurons are not Directly Estrogen‐Responsive: Simultaneous Localization of Luteinizing Hormone‐Releasing Hormone and Estrogen Receptor Immunoreactivity in the Guinea‐Pig Brain
Authors: Watson Robert E. , Langub M. Chris , Landis John W.
Published: September 2006

Gonadotropin secretion from the pituitary is regulated in large part by steroid action on the brain. An important question concerns whether luteinizing hormone‐releasing hormone (LHRH) neurons themselves transduce steroid signals, or whether, alternatively, steroid‐sensitive interneuronal populations regulate their activity. A previous study in the rat employing steroid autoradiography combined with LHRH immunocytochemistry revealed that only an exceedingly small percentage of LHRH‐immunoreactive (ir) neurons was estrogen concentrating. This study has examined the relationship of estrogen receptive and LHRH‐ir cells in the male and female guinea‐pig brain with double label immunocytochemistry. Since estrogen receptor‐ir, as demonstrated with antibody H222, is known to be confined predominantly to the cell nucleus, whereas LHRH‐ir is localized mainly in the cytoplasm, single cells can be double‐labeled. Diaminobenzidine tetrahydrochloride was used for localization of LHRH‐ir while nickel‐enhanced diaminobenzidine tetrahydrochloride was used for localization of estrogen receptor‐ir. The results revealed that there were many brain nuclei that contained both LHRH and estrogen receptor‐positive cells, including the preventricular periventricular nucleus, the anterior subcompact nucleus of the medial preoptic nucleus (MPNa), the remainder of the medial preoptic nucleus, the retrochiasmatic area, and the anterior, dorsomedial, ventrolateral and arcuate nuclei. However, of a total of 2,604 LHRH‐ir cells that were examined, we observed only 5 double‐labeled cells (<0.2%). The double‐labeled cells were not restricted to a single nucleus; they were present in the MPNa, the retrochiasmatic area and the arcuate nucleus. Moreover, at the light microscopic level, LHRH cells quite frequently appeared to be apposed to estrogen receptor‐positive cells (8.8% in the female), especially in the MPNa. These results lend further support to the notion that estrogen‐mediated regulation of the LHRH system is achieved primarily through estrogen receptive interneurons. However, due to the existence of LHRH‐LHRH synaptic interactions, the possibility also exists that a small population of estrogen‐sensitive LHRH neurons could contribute to generalized activation of the LHRH system.
Differential Regulation of Luteinizing Hormone‐Releasing Hormone and Galanin Messenger Ribonucleic Acid Levels by Alpha1Adrenergic Agents in the Ovariectomized Rat
Authors: Weesner Gary D. , Bergen Hugo T. , Pfaff Donald W.
Published: September 2006

α1‐Adrenergic control of luteinizing hormone‐releasing hormone (LHRH) and galanin mRNA levels was examined in ovariectomized rats. Each rat was ovariectomized and a permanent bilateral cannula was implanted 1 mm dorsal to the preoptic area. Eleven to 14 days later, each rat received one of three treatments: prazosin (α1 antagonist, n = 8), methoxamine (α1 agonist, n = 5) or control (no drug, n = 7). Each drug was suspended in a polyacrylamide gel matrix and delivered to the preoptic area via the bilateral cannula. After 24 h of continuous exposure to the adrenergic agents (or control), rats were anesthetized, decapitated and brains were stored in liquid nitrogen until sectioned (7 μm) on a cryostat. In situ hybridization was performed using a [32P]‐end‐labelled 59mer complementary to LHRH mRNA. Reduced silver grains, proportional to LHRH mRNA content, were quantified for number of LHRH cells per section, number of grains per labelled cell and total number of grains in labelled cells. Compared to the controls, prazosin caused a 32% decrease (P<0.05) in the number of cells expressing the LHRH gene. The LHRH cells from untreated animals had a median of 53 grains/cell with a smooth distribution above and below the median. Treatment with prazosin reduced the median number of grains/cell to 36 (P<0.05). When the number of grains in labelled cells were totalled, prazosin decreased (P<0.01) the number of grains by 47%. Surprisingly, methoxamine caused no quantitative changes in any of the parameters examined. This might be explained if LHRH transcription in control animals was proceeding at near‐maximal rates supported, in part, by an endogenous α1 ligand. Alternatively, continuous exposure to this agonist may have resulted in desensitization to its stimulatory effects. When anatomically matched brain tissue sections from these animals were examined for galanin mRNA, no differences among experimental groups were detected. In conclusion, administration of an α1‐adrenergic antagonist into the preoptic area suppressed levels of LHRH mRNA but not galanin mRNA. Therefore, the data suggest that an endogenous α1 ligand, such as norepinephrine (or epinephrine), is required to maintain a high level of LHRH gene expression in the ovariectomized rat.
NADPH‐Diaphorase (Nitric Oxide Synthase) Staining in the Rat Supraoptic Nucleus is Activity‐Dependent: Possible Functional Implications
Authors: Pow David V.
Published: September 2006

NADPH‐diaphorase has recently been shown to be the enzyme nitric oxide (NO) synthase, and to be present in the rat supraoptic nucleus (SON) and posterior pituitary. Investigations were carried out to assess whether there is any difference in the extent to which this enzyme is present, as assessed by light‐microscopic histochemistry, in SON of normal and dehydrated male Wistar rats. In normal rats there was clear cellular heterogeneity; cells located in the ventral and caudal areas of the SON stained only weakly or not at all, while cells in the rostro‐dorsal areas of the nucleus stained strongly. Dehydration of rats for 12 h caused a large and rapid increase in staining intensity of the nucleus, particularly of cells in its ventral and caudal parts. On the basis of its known biological actions, and the kinetics of its induction, it is suggested that NO would be a strong candidate as a modulator of SON and posterior pituitary morphology and function, with the potential to rapidly modulate blood flow, neuronal activity, and possibly astrocyte morphology, in response to changes in neuronal activity.
Glucocorticoid Effects on the Molecular Forms of Adrenocorticotrophic Hormone Secreted by Cultures of Rat Anterior Pituitary Cell Monolayers
Authors: Norman James C. , Gurney Alison M. , Gillham Brian
Published: September 2006

The effects of glucocorticoids and corticotrophin‐releasing factor (CRF) on the release of various molecular forms of adrenocorticotrophic hormone (ACTH) have been investigated in primary cultures of rat anterior pituitary. The rat cells responded to a 30 min challenge with CRF by secreting increased amounts of ACTH, as assessed both by bioassay, using rat adrenocortical cells, and by radioimmunoassay. Inclusion of a synthetic glucocorticoid, such as dexamethasone (DEX), in the incubation for 5 min prior to, and during the CRF challenge, had no effect on the response as measured by radioimmunoassay. Bioassay, however, indicated profound suppression of the response to CRF. This discrepancy between ACTH immuno‐ and bioactivity was investigated by fractionating the immunoreactive ACTH species using high‐performance liquid chromatography. The lower molecular weight (<15kd) forms (ACTH1–39, phosphorylated ACTH1–39 and glycosylated ACTH1±39) were separated from higher molecular weight (>15kd) forms (i.e. ACTH biosynthetic intermediate and proopiomelanocortin) using C‐18 Sep‐Pak. The lower molecular weight molecules were further resolved into glycosylated and non‐glycosylated ACTH, using an acetonitrile gradient high‐performance liquid chromatography with trifluoroacetic acid as an ion‐pairer. Neither the proportion of low molecular weight forms of ACTH, nor that of glycosylated ACTH1–39 secreted in response to CRF, were affected by DEX. Further fractionation of non‐glycosylated ACTH, also using acetonitrile gradient high‐performance liquid chromatography but with heptafluorobutyric acid as the ion‐pairer, yielded peaks corresponding to phosphorylated and non‐phosphorylated ACTH1–39. DEX significantly increased the proportion of phosphorylated ACTH secreted in response to CRF by 18%. An additional effect of DEX was revealed when Sep‐Pak extracts were treated with alkaline phosphatase, prior to analysis. After dephosphorylation, it became clear that the peptides released by CRF‐stimulated cells were different if DEX was present in the medium. The peptide released in the presence of DEX (ACTH‐S) had a slightly, but consistently, different retention time on high‐performance liquid chromatography and very little biological activity. Antibody cross‐reactivity studies suggested that ACTH‐S was modified in the 1–24 region of the peptide. It is concluded that challenge of anterior pituitary cells in primary culture with CRF, following 5 min previous exposure to DEX, results in a molecular change. The consequence of this is that ACTH immunoreactivity is released, but the molecule has reduced biological activity. This may be part of the mechanism by which fast feedback inhibition of ACTH secretion is effected.
Hemorrhage‐Induced Activations of Adrenocorticotropin Release and Catecholamine Metabolism in the Ventrolateral Medulla are Differently Affected by Glucocorticoid Feedback
Authors: Ponec J. , Lachuer J. , Suaud‐Chagny M. F. , Tappaz M.
Published: September 2006

We have compared the effects of increasing doses of dexamethasone on the hemorrhage‐induced stimulation of the corticotropic axis and the metabolism of the catecholamines of the A1 group in the ventrolateral medulla. Adrenocorticotropin was measured in sequential samples of plasma while the metabolism of the catecholamines was recorded by in vivo electrochemistry in urethane‐anesthetized rats. Combined intracerebroventricular injection of specific adrenergic blockers (α1‐antagonist, prazosin and ß‐antagonist, propranolol) prevented the stimulation of the adrenocorticotropin release by hemorrhage. Pretreatment with dexamethasone (1 mg/kg sc) fully blocked the hemorrhage‐induced adrenocorticotropin release but did not affect the concomitant stimulation of the catecholamine metabolism in A1 cells. The latter was partially decreased only with the highest dose (10 mg/kg sc). While a central catecholaminergic input appears to be necessary for the hemorrhage‐induced stimulation of the corticotropic axis, it does not seem to play a significant role in the feedback regulation by glucocorticoids.
Central Somatostatin Inhibits Cholecystokinin‐lnduced Oxytocin Secretion in Conscious Rats
Authors: Blackburn Ruth E. , Frederick Stephen E. , Strieker Edward M. , Verbalis Joseph G.
Published: September 2006

Recent studies have demonstrated the presence of fibres immunoreactive for somatostatin‐28 (SS‐28), which originate in the brainstem and selectively innervate the magnocellular oxytocin (OT) cells of the supraoptic nucleus. The potential physiological relevance of this pathway was investigated in the present study by measuring plasma OT levels in response to intraperitoneal administration of cholecystokinin in conscious male rats pretreated with intracerebroventricular injections of either SS‐28 or artificial cerebrospinal fluid. Cholecystokinin treatment produced the expected marked rise in plasma OT levels in control rats pretreated intracerebroventricularly with artificial cerebrospinal fluid. However, this response was markedly blunted by prior intracerebroventricular administration of SS‐28, even though SS‐28 itself had no effect on basal plasma OT levels, nor did it impair OT release in response to hypertonic saline injection. These results demonstrate that centrally injected SS‐28 can selectively block cholecystokinin‐stimulated release of OT in rats, and support an inhibitory role for this peptide in brainstem‐mediated neurohypophysial hormone secretion.

Central SS‐28 administration also elicited up to 3‐fold increases in the amount of food ingested in 1 h by previously sated rats. These observations suggest the possibility that endogenous SS‐28 may be involved in stimulating food intake in rats, and establish the basis for future studies to clarify the role of this neuropeptide in controlling ingestive behaviours.
Effect of Constant Light, Pinealectomy and Guanosine Triphosphate Gamma‐S on the Density of Melatonin Receptors in the Rat Suprachiasmatic Nucleus: A Possible Implication of Melatonin Action
Authors: Gauer F. , Masson‐Pévet M. , Pévet P.
Published: September 2006

We report here the effects of pinealectomy and exposition to constant light on the density of melatonin receptors in the suprachiasmatic nuclei of the rat using quantitative autoradiography. The Bmaxvalues were significantly increased when the animals were maintained in constant light for 3 days (8.22 ± 0.95 fmol/mg protein versus 4.55±0.14 fmol/mg protein in control group, 12 h light/12 h dark cycle (12L/12D), n = 6). A similar increase was also observed in rats pinealectomized 3 days before sacrifice and then maintained either under 12L/12D (Bmax7.56±0.80 fmol/mg protein) or in constant light (Bmax7.85±1.02 fmol/mg protein), while Kdvalues failed to show any variations after constant light and/or pinealectomy.

The effect of GTPγS on the density of rnelatonin binding sites was also investigated in control animals and after 3 days of constant light. In 12L/12D animals, the Bmax shifted from 5.94 ± 0.14 fmol/mg protein in the absence of GTPγS to 3.97±0.22 fmol/ mg protein in the presence of 50μiM GTPγS. In animals maintained for 3 days in constant light, a similar decrease in the Bmax value was observed (8.95 ± 0.25 fmol/mg protein in absence and 5.95 ± 0.22 fmol/mg protein in presence of 50 μ GTPγS). In both cases, Kd values were not affected by GTPγS.

Pinealectomy and constant light exposition are known to induce a suppression of the nocturnal peak of plasma rnelatonin and to keep plasma rnelatonin concentrations at a very low level. These results could suggest a regulatory effect of rnelatonin on the density of its own receptors which are shown here to be also coupled with a G‐protein.
Characterization of the Parabrachial Nucleus Input to the Hypothalamic Paraventricular Nucleus in the Rat
Authors: Jhamandas Jack H. , Harris Kim H. , Petrov Theodor , Krukoff Teresa L.
Published: September 2006

The brainstem parabrachial nucleus (PBN) is viewed as an increasingly important site for the transfer of autonomic‐related information to more rostral structures in the forebrain including the hypothalamus. In this study, we examined electrophysiologically in vivo and anatomically the nature of PBN input to the hypothalamic paraventricular nucleus (PVN) and particularly to the vasopressin‐and oxytocin‐secreting magnocellular neurosecretory cells within this nucleus.

In urethane‐anaesthetized rats, extracellular recordings from 108 antidromically identified neurosecretory PVN cells revealed an excitatory (37/43 cells) and less frequently an inhibitory (6/43 cells) response consequent to electrical stimulation in the PBN. Both vasopressin (12/37 cells)‐and oxytocin (9/37 cells)‐secreting neurons appear to respond to the PBN stimulus. Four cells projecting to the neurohypophysis could also be antidromically activated from PBN, and this observation may be indicative of collateral branching in some PVN neurosecretory neurons. In addition, recordings from 60 non‐magnocellular (i.e. non‐neurohypophysially‐projecting) PVN cells revealed a facilitatory response (43/60 cells) following PBN stimulation, Iontophoretic injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA‐L) were made within the rat lateral PBN and brains prepared for immunocytochemical examination of projections to the PVN region. PHA‐L‐labelled fibres and terminals were visualized within both the parvocellular and magnocellular divisions of the PVN. In addition, labelled fibres were also seen in a region immediately dorsal to the PVN. PHA‐L‐labelled fibres with axonal varicosities and boutons were visualized over immunocyto‐chemically‐identified vasopressin and oxytocin neurons within the magnocellular PVN. These convergent electrophysiological and anatomical data provide evidence for a PBN projection to the PVN that is predominantly excitatory to both magnocellular neurosecretory and non‐magnocellular cells. Moreover, with respect to vasopressin‐and oxytocin‐secreting cells, the PBN input appears to be directed at both populations of peptidergic neurons.
Electrical Activity of Dorsal Horn Neurons During the Suckling‐Induced Milk Ejection Reflex in the Lactating Rat
Authors: Fénelon Valérie S. , Poulain Dominique A.
Published: September 2006

In lactating rats, suckling elicits the milk ejection reflex which consists of an intermittent synchronous activation of hypothalamic oxytocin neurons which releases oxytocin into the bloodstream. We here investigated the electrophysiological behaviour of spinal cord neurons linked to mammary innervation in relation to suckling and the suckling‐induced milk ejection reflex. Experiments were carried out on 58 urethane‐anaesthetized rats, paralysed with gallamine triethiodide and artificially ventilated. Fixation of the spinal cord and laminectomy significantly slowed down the reflex, which occurred in only 27% of the animals. In these rats, 31 dorsal horn neurons at the thoraco‐lumbar level were found to be excited by nipple stimulation. During suckling by a litter of at least nine pups, they displayed an irregular pattern of brief bursts of activity (peak firing rate 22.0 ± 3.2 Hz, mean ± SD) correlated to the bouts of suckling of the pups. Seventeen out of 19 cells tested by stimulation of at least 2 adjacent nipples received convergent input from different ipsilateral nipples. Out of 11 cells tested, 8 were also activated by stimulation of a contralateral nipple. Fourteen out of 30 units were recorded through at least one reflex milk ejection. Their firing rate was significantly higher than the firing rate of cells recorded in animals which failed to milk eject (4.4 Hz ± 2.8 versus 1.5 Hz ± 0.7). At the moment of the high frequency discharge of action potentials, occurring in oxytocinergic cells 10 to 15 s before each milk ejection, spinal neurons showed no systematic change in electrical activity. In contrast, the stretch reaction of the pups, which corresponds to an intense period of suckling when milk ejection has started, induced, in 12 cells, a considerable increase in electrical activity. One unit was found to be inhibited by suckling and during the stretch reaction. Ten more units, which were not activated by stimulation of the nipples but responded to stimulation of excitatory receptive fields near the last three pairs of nipples, were recorded through reflex milk ejections: 8 remained silent during reflex milk ejections but 2 were activated when the pups stimulated their excitatory receptive field. We conclude that some dorsal horn neurons, able to respond readily to the suckling movements of pups, appear to receive an ungated input from the nipples. At the time of the activation of oxytocin neurons, they present no particular pattern of activation or inhibition which could account in a simple manner for the intermittence of the high frequency discharge in oxytocinergic cells. However, in so far as these dorsal horn neurons may be part of the milk ejection reflex pathway, their activity, showing convergence and summation of input, and being facilitated in milk ejecting animals, indicates that the reflex does undergo a certain degree of processing at the spinal cord level.
C‐Type Natriuretic Peptide Stimulates Prolactin Secretion by a Hypothalamic Site of Action
Authors: Huang Fong‐Lee S. , Skala Karl D. , Samson Willis K.
Published: September 2006

The most recently discovered member of the family of natriuretic peptides, C‐type natriuretic peptide (CNP), exerts many pharmacologic actions similar to its structural homolog A‐type natriuretic peptide (ANP). Like ANP it failed to significantly alter prolactin release from dispersed, rat anterior pituitary cells incubated under static or dynamic conditions. Unlike ANP, however, which inhibits prolactin secretion in vivo by a hypothalamic action, CNP injection into the third cerebroventricle significantly stimulated prolactin secretion in ovariectomized, conscious rats. The effect was highly significant 15 min after injection and transient, lasting 30 min in animals injected with 2 nmole CNP. In a companion group of rats, significant inhibition of plasma prolactin levels was observed after central administration of similar doses of ANP. These results suggest differing hypothalamic actions of the CNP and ANP perhaps mediated by multiple natriuretic peptide receptors present in the tissue. Further, they provide additional support for unique roles exerted within the central nervous system by these structural homologs.
Central Administration of Serotonin Decreases Tyrosine Hydroxylase Catalytic Activity and Messenger Ribonucleic Acid Signal Levels in the Hypothalamus of Female Rats
Authors: Mathiasen Joanne R. , Arbogast Lydia A. , Voogt James L.
Published: September 2006

We investigated the effect of central serotonin (5‐hydroxytryptamine, 5‐HT) administration on hypothalamic tuberoinfundibular dopamine neurons and related changes in neuronal activity to circulating prolactin (PRL) levels. Ovariectomized rats were treated with either vehicle or 5‐HT through a lateral ventricular cannula in one of two dose paradigms: 1) a bolus of 20 μg, with tissues taken at 30 min, or 2) the same bolus immediately followed by 20 μg/30 min via a syringe pump for 120 min, and tissues taken at 120 min. Blood samples were taken throughout experiments and plasma PRL determined by radioimmunoassay. Under both paradigms, NSD 1015, a dihydroxyphenylalanine (DOPA) decarboxylase inhibitor (25 mg/kg intraarterially) was injected 10 min before decapitation and brain excision followed by stalk‐median eminence dissection. The rate of DOPA accumulation, determined by measuring DOPA levels in the stalk‐median eminence by high‐performance liquid chromatography with electrochemical detection was used as a measure of tyrosine hydroxylase (TH) catalytic activity. Stalk‐median eminence DOPA accumulation in control rats was 29.9 ± 4.2 and 28.8 ± 4,4 ng/mg protein (30 and 120 min experiments, respectively). DOPA accumulation in 5‐HT‐treated rats was significantly reduced (P<0.05) after 30 min to 17.8 ± 1.2 ng/mg protein, but it was similar (21.7 ± 3.9) to controls after 120 min of 5‐HT infusion. 5‐HT levels in the stalk‐median eminence of rats treated with 5‐HT were 13‐ to 17‐fold greater than controls (16.9 to 18.5 ng/mg protein). Plasma PRL levels in both groups increased 10‐fold after 5‐HT treatment with a peak at 5 min, returning to baseline by 120 min. TH mRNA levels were determined by in situ hybridization in a second group of rats which were treated with the 20μg bolus and subsequent 120 min infusion of 5‐HT. TH mRNA signal levels in the arcuate nucleus of control rats averaged 144 ± 21 grains/cell. After treatment with 5‐HT, TH mRNA levels in the arcuate nucleus were significantly lower (P<0.0001) with 69±14 grains/cell. In a third group of rats, the effects of the 30 min 5‐HT treatment on TH catalytic activity and circulating PRL levels was challenged with two 5‐HT2 receptor antagonists, LY53857 (5 mg/kg intraperitoneally) or ketanserin (10 mg/kg intraperitoneally). Neither the 5‐HT‐induced decrease in TH catalytic activity nor the increase in PRL was altered by pretreatment (120 min) with 5‐HT2 antagonists. These data suggest that central 5‐HT is capable of decreasing TH activity and TH mRNA levels in the tuberoinfundibular dopamine neurons and that the decrease in dopaminergic neuronal activity may contribute to the 5‐HT‐induced PRL rise. The changes in TH catalytic activity and PRL after intracerebroventricular administration of 5‐HT do not appear to be mediated by 5‐HT2 receptors.
Neuropeptide Y‐lmmunoreactive Nerve Fibres in the Pineal Gland of the Macaque (Macaca fascicularis)
Authors: Mikkelsen Jens D. , Mick Gérard
Published: September 2006

The distribution of neuropeptide Y (NPY)‐ and the C‐fianking peptide of NPY (CPON)‐immunoreactive elements in the pineal gland of the macaque was investigated by means of immunohistochemistry. NPY‐ and CPON‐immunoreactive nerve fibres were located in the precommissural nucleus, around the stria medullaris, and in the posterior commissure. NPY‐ and CPON‐immunoreactive nerve fibres endowed with bulbous varicosities, were traced from the brain via the pineal stalk into the rostral part of the pineal gland. Furthermore, CPON‐immunoreactive, and to a lesser extent NPY‐immunoreactive nerve fibres, were distributed in the méninges, the choroid plexus and the vasculature related to the pineal organ. Nerve fibres located in the pineal capsule penetrated into the pineal parenchyma, where groups of individual fibres were found most often in an interlobular position. Occasionally, individual nerve fibres dispersed between the pinealocytes were observed. In contrast to the nerve fibres originating from the brain, those originating from the periphery were endowed with smaller immunoreactive nerve terminals. Another apparent difference was that the peripheral nerve fibres innervated only the caudal two‐thirds of the gland, whereas the central fibres were found exclusively in the rostral part of the pineal organ. Rarely, positive neuronal‐like cells were found in the pineal parenchyma. These results show the presence of a moderate number of NPY‐ and CPON‐immunoreactive nerve fibres within the primate pineal organ and strongly indicate that the primate pineal gland is innervated by NPYergic nerve fibres originating from both a peripheral and a central source.