bluuebunny/crossref_metadata_2025 huggingface dataset:
6‐Hydroxydopamine Lesions of the Locus Coeruleus Induce a Paradoxical Increase in Growth Hormone Secretion in Male Rats
Authors: Bluet‐Pajot M. T. , Mounier F. , Durand D. , Kordon C. , Llorens‐Cortes C. , Videau C. , Epelbaum J.
Published: September 2006

While the pharmacology of noradrenaline effects on growth hormone (GH) secretion has been extensively studied, the precise localization of noradrenergic neurons involved remains unclear. In the present work, we investigated whether A6 noradrenergic neurons located in the locus coeruleus can play a role in the rhythmic pattern of GH secretion or in the sensitivity of the hormone response to different external challenges. Three weeks after bilateral 6‐hydroxydopamine injections (8μg/3μl) into the locus coeruleus, hypothalamic noradrenaline concentrations were reduced by 60%. Pulsatile GH secretory patterns were observed in unanaesthetized, freely moving control, sham‐operated or locus coeruleus‐lesioned male rats. The amplitude of the pulses and the area under the curves during the 6‐ or 12‐h sampling period were twice as high in locus coeruleus‐lesioned than in control and sham‐operated rats. In contrast, trough levels of GH and intervals between GH peaks were similar in all groups. Prolactin, adrenocorticotrophin, thyroid‐stimulating hormone and luteinizing hormone plasma levels were not affected by the lesion. GH responses to two centrally acting drugs i.e. clonidine (2.5, 5 and 10μg/100g body wt) and morphine (200μg/100g body wt) were also highly amplified in locus coeruleus‐lesioned rats. In contrast, GH responses to two peptides directly acting on somatotrophs i.e. GH‐releasing factor (0.05 and 1.25μg/100g body wt) and vasoactive intestinal peptide (1.5μg/100g body wt) were the same in sham‐operated and lesioned animals.

These data suggest that noradrenergic inputs from the locus coeruleus exert a selective inhibitory influence on GH secretion through centrally mediated mechanisms.
The Distribution and Development of Delta Sleep‐Inducing Peptide‐Like Immunoreactivity in Postnatal and Prepubertal Guinea‐Pig Brain
Authors: Pu L‐P. , Dubois P. M.
Published: September 2006

The distribution and development of delta sleep‐inducing peptide (DSIP) in the guinea‐pig brain were studied in 2‐ to 60‐day‐old animals by using the indirect immunofluorescence method. DSIP‐immunoreactive perikarya were observed in the olfactory bulb and tubercle, diagonal band of Broca, septum, preoptic area, anterior and lateral hypothalamus, arcuate nucleus and hippocampus. In addition to the densest innervation of the median eminence, DSIP‐immunoreactive fibres were widely localized from forebrain to mesencephalon. The field of immunoreactive fibre endings appeared to be in close association with either the blood vessels of brain, ventricles, subarachnoid space or immunolabelled perikarya. Furthermore, throughout development the topographic distribution pattern of immunolabelled neuronal elements seemed to be similar. However, a generalized increase in number, immunofluorescence intensity and varicosities of DSIP fibres was displayed with the growth. The present results provide an anatomical basis for understanding multiple actions of DSIP in the central nervous system and future research for DSIP on development.
Effect of Enhancement of Cholinergic Tone on the Growth Hormone Response to Acute Hyperglycaemia or Thyrotropin‐Releasing Hormone in Dogs
Authors: Arce Victor M. , Cella Silvano G. , Locatelli Vittorio , Müller Eugenio E.
Published: September 2006

The effect of enhancement of cholinergic tone by pyridostigmine on the growth hormone (GH) response to thyrotropin‐releasing hormone (TRH) or glucose‐induced acute hyperglycaemia was tested in six adult unanaesthetized beagle dogs. Both TRH (5μg/ kg iv) and glucose (2 g/kg orally) did not significantly alter baseline GH levels but reduced the GH response to GH‐releasing hormone (GHRH) (2 μg/kg iv), although this effect was more clear‐cut with TRH than with glucose. Pretreatment with pyridostigmine (2 mg/kg orally) counteracted the inhibitory effect of hyperglycaemia on the GHRH‐induced GH release, but had no effect on the inhibition induced by TRH. In summary, these results indicate that: 1) acute hyperglycaemia and TRH play an inhibitory role on GHRH‐stimulated GH secretion in dogs; 2) the inhibitory effect of acute hyperglycaemia is mediated via hypothalamic cholinergic neurotransmission, whereas other neurotransmitter pathways would be. involved in the effect of TRH.
Prostaglandins of the E Series Stimulate Cyclic AMP Accumulation and N‐Acetylation of Serotonin in Chick Pineal Cells
Authors: Van Camp Gilles , Voisin Pierre , Sugden David , Dray Fernand , Coilin Jean‐Pierre
Published: September 2006

We investigated the effects of prostaglandins on cyclic AMP (cAMP) levels and on the activity of the rate‐limiting enzyme of melatonin biosynthesis, arylalkylamine‐N‐acetyltransferase (NAT). The study was performed on primary cultures of dispersed chick pineal cells. Prostaglandin E, (PGE,) increased cAMP levels 2‐fold and this stimulation went up to 4‐fold in the presence of a phosphodiesterase inhibitor. The PGE,‐ evoked increase in cAMP levels did not desensitize over 6 h. The potency order of a series of prostaglandins to increase cAMP levels (PGE1 PGE1>PGA2>PGD2≃PGF2α) agreed with the pharmacological profile of the adenylate cyclase‐coupled prostaglandin receptor. Inhibition of endogenous prostaglandin synthesis by two cyclooxygenase inhibitors (indomethacin and aspirin) caused a 30% decrease in cAMP levels. This effect was completely reversed by the addition of exogenous PGE1 or PGE2. Indomethacin and aspirin also caused a 50% decrease in NAT activity. Prostaglandins of the E series increased NAT activity up to 2‐fold above basal level and restored NAT activity after inhibition by indomethacin or aspirin.

These results are the first illustration of a role for prostaglandins in chick pineal cells. The correlations observed between cAMP levels and NAT activity suggest that the regulation of NAT activity by prostaglandins of the E series might be mediated by changes in cAMP concentration.
Multiplex Solution Hybridization‐Ribonuclease Protection Assay for Quantitation of Different Ribonucleic Acid Transcripts from Snap‐Frozen Neuroendocrine Tissues of Individual Animals
Authors: Jakubowski Moshe , Roberts James L.
Published: September 2006

We have compiled a protocol for simultaneous quantitation of a variety of gene transcripts in multiple individual brain and pituitary gland dissections for studying pretranslational regulation of neuroendocrine systems in vivo, using experimental designs compatible with meaningful statistical power. To facilitate collection of many samples at a time, the tissue was snap‐frozen in chilled liquid Freon and stored at −80 °C until further processing. In this way, as many as five different brain and pituitary gland dissections per rat could be collected from eight rats in about an hour. The snap‐frozen tissue was suitable for isolation of separate cytoplasmic and nuclear RNA fractions using homogenization in the presence of detergents. To facilitate homogenization of many samples at a time, we devised a method in which the tissue was repeatedly expelled through a 22 gauge hypodermic needle attached to a 1‐ml plastic syringe used as a disposable, ready‐to‐use homogenizer. In order to promote dissolution of lipid membrane structures which are prevalent in the brain, the lysis buffer has been optimized to include the detergent sodium deoxycholate in addition to Nonidet P‐40. Specific RNA transcripts were analyzed using a quantitative solution hybridization‐ribonuclease protection assay coupled with polyacrylamide gel electrophoresis. The value of this highly sensitive assay has been expanded by including several molecular probes against a variety of neuroendocrine mRNA sequences simultaneously (e.g. progonadotropin‐releasing hormone, proopiomelanocortin, tyrosine hydroxylase, dopamine D2 receptor, prolactin), thus increasing the amount of information obtained from each sample in one assay. Furthermore, each sample was routinely co‐assayed for cyclophilin mRNA, an abundant, generally non‐regulated mRNA whose levels reflected the individual variability in sample processing, thus serving as an internal reference. Once stored in hybridization solution, as many as 100 samples could be analyzed simultaneously for several different RNA transcripts in one assay. This protocol provides a powerful tool for studying regulation of neuroendocrine systems at the molecular level in vivo, using sample sizes suitable for applying statistical analysis of meaningful statistical power.
Corticosterone Reduces Synaptic Inhibition in Rat Hippocampal and Neocortical Neurons in vitro
Authors: Zeise Marc L. , Teschemacher A. , Arriagada J. , Zieglgänsberger W.
Published: September 2006

We examined the effect of corticosterone (10‐7 to 10‐5 M) on membrane properties and postsynaptic potentials, by means of intracellular recordings from neocortical and hippocampal CA1 pyramidal neurons of the intact adult rat in vitro. Corticosterone reduced both the early and the late components of the orthodromically‐evoked inhibitory postsynaptic potential in both structures. The glucocorticoid receptor antagonist RU 38486 (10‐6 M) prevented this effect in the hippocampus. In hippocampal, but not in neocortical pyramidal neurons, corticosterone reduced a depolarizing membrane transient evoked by a depolarizing current step and increased the threshold for eliciting action potentials evoked by depolarizing current pulses. Corticosterone did not detectably alter the afterhyperpolarization following repetitive neuronal discharges evoked by current injection, in either the neocortex or in the hippocampus. Excitatory postsynaptic potentials, action potentials, membrane potential and membrane input resistance were also unchanged.

The decrease in synaptic inhibition together with the reduction of electrical excitability in the hippocampus, would imply a modulation of response characteristics in pyramidal neurons such that repeated synaptic inputs become more efficient and low frequency input is blunted.
The Maintenance of Normal Parturition in the Rat Requires Neurohypophysial Oxytocin
Authors: Luckman S. M. , Antonijevic I. , Leng G. , Dye S. , Douglas A. J. , Russell J. A. , Bicknell R. J.
Published: September 2006

The neuropeptide oxytocin has long been known as a potent contractor of the uterus. However, it has remained difficult to attribute a definite role for neurohypophysial oxytocin in either the initiation or continuation of labour (1). Most recently, Lefebvre and colleagues (2) have suggested that oxytocin produced in the uterus, rather than in the hypothalamus, may be more important in parturition since at term the uterus of the rat contains 70‐fold more mRNA for oxytocin than the hypothalamus, and this disappears at about the time of parturition. Despite the high levels of mRNA the uterus contains only nanogram quantities of immunoreactive oxytocin per gram wet weight at term (2), compared to microgram quantities present in the pituitary (3,4). Here we show that activation of the neurohypophysial oxytocin system occurs, as reflected by expression of immunoreactivity for Fos in the hypothalamic supraoptic nucleus, and that this activation is indeed critical for normal parturition, since its inhibition results in a significant prolongation of parturition. In addition, we present evidence that pulsatile delivery of oxytocin into the circulation is important for the efficient progress of parturition, indicating that a major role of the neuronal circuits regulating oxytocin secretion for parturition, as is already known for suckling, is to produce an appropriately patterned hormonal output for efficient biological action.
Luteinizing Hormone‐Releasing Hormone and Gamma‐Aminobutyric Acid Neurons in the Medial Preoptic Area are Synaptic Targets of Dopamine Axons Originating in Anterior Periventricular Areas
Authors: Horvath Tamas L. , Naftolin Frederick , Leranth Csaba
Published: September 2006

The aim of this study was to characterize further the transmitter content and the location of the parent cells of tyrosine hydroxylase‐immunoreactive boutons terminating on luteinizing hormone‐releasing hormone‐ and glutamic acid decarboxylase‐immunoreactive neurons in the rat medial preoptic area. Electron microscopic immunostaining for luteinizing hormone‐releasing hormone, tyrosine hydroxylase or glutamic acid decarboxylase was performed on desipramine‐pretreated (to protect norepinephrine and epinephrine axons) rats which received a stereotaxic injection of 6‐hydroxydopamine into the medial preoptic area anteroventral periventricular nucleus 48 h prior to sacrifice. This treatment induced acute degeneration of dopamine axon terminals characterized by the development of autophagous cytolysosomes, an early morphological sign of catecholamine axon degeneration. To further define the cells of origin of these dopamine boutons, the anterograde marker Phaseolus vulgaris leucoagglutinin was iontophoretically applied to the zona incerta. Six days later, rats received a 6‐hydroxydopamine injection into the zona incerta or the lateral ventricle, and 48 h later, double immunostaining was performed for Phaseolus vulgaris leucoagglutinin and tyrosine hydroxylase, luteinizing hormone‐releasing hormone, or glutamic acid decarboxylase on preoptic area vibratome sections.

Following the 6‐hydroxydopamine injection into the anteroventral periventricular nucleus, autophagous cytolysosome‐containing degenerated axons were found in synaptic contact with both luteinizing hormone‐releasing hormone and GABA neurons in the medial preoptic area, confirming that these are dopaminergic connections. Following the double injection treatment, 6‐hydroxydop‐amine‐induced degenerated, Phaseolus vulgaris leucoagglutinin‐labeled dopamine axons originating in the zona incerta were not found to contact luteinizing hormone‐releasing hormone‐containing or GABA cells. Instead, many degenerated, Phaseolus vulgaris leucoagglutinin‐immunopositive boutons were observed in the dorsomedial and paraventricular nuclei of the hypothalamus. These observations indicate that tyrosine hydroxylase‐immunoreactive fibers terminating on the medial preoptic area luteinizing hormone‐releasing hormone and GABA cells are dopaminergic and most probably originate from dopamine neurons located in anterior periventricular areas of the hypothalamus.
Lesions of the Suprachiasmatic Nucleus Indicate the Presence of a Direct Vasoactive Intestinal Polypeptide‐Containing Projection to Gonadotrophin‐Releasing Hormone Neurons in the Female Rat
Authors: van der Beek Eline M. , Wiegant Victor M. , van der Donk Hans A. , van den Hurk Rob , Buijs Ruud M.
Published: September 2006

In non‐seasonal breeders like the rat, the influence of the suprachiasmatic nucleus (SCN) on reproduction is most clearly expressed in the female. Complete lesions of the SCN induce persistent oestrus (anovulation) in intact female rats, whereas oestrogen implantation in ovariectomized rats results in daily luteinizing hormone surges. Vasoactive intestinal polypeptide (VIP), a peptide synthesized in cell bodies of the SCN, inhibits the increase in pulsatile luteinizing hormone release observed in ovariectomized female rats. In search of the anatomical basis for these observations, the present study employs an immunocytochemical double staining for VIP and gonadotrophin‐releasing hormone (GnRH) at the light microscopical level. It was demonstrated that approximately 45% of the GnRH positive neurons in the diagonal band of Broca, the preoptic and anterior hypothalamic area of female rats are innervated by VIP‐containing processes. To investigate whether these VIP‐containing fibres represent a direct projection of the SCN to the GnRH system, unilateral thermic SCN lesions were made.

Lesions that unilaterally destroyed the majority of the VIP synthesizing cells in the SCN resulted in at least a 50% decrease of the VIP innervation of GnRH cell bodies at the lesioned side compared to the intact side. Lesions not affecting the VIP synthesizing cell population in the SCN did not change the percentage of GnRH neurons innervated by VIP‐containing fibres, while partial lesions resulted in intermediate effects.

These results indicate that the majority of the light microscopical VIP‐containing input on GnRH neurons in the hypothalamus is derived from the SCN. It is suggested that the reported effects of VIP on luteinizing hormone release may, at least in part, be induced via a direct effect of VIP on GnRH cell bodies. This direct SCN‐GnRH pathway provides an anatomical basis for diurnal influences on the regulation of the female reproductive cycle.
Endogenous Catecholamines Modulate Growth Hormone Release in the Conscious Rat During Hypoglycaemia but not in the Basal State
Authors: Chapman Ian M. , Kapoor Ranjna , Willoughby John O.
Published: September 2006

To investigate the role of endogenous catecholamines and 5‐hydroxytryptamine in the control of growth hormone (GH) secretion, secretory profiles of GH and prolactin were measured in conscious, male rats following intravenous administration of either 1) α2 antagonist idazoxan 2 mg/kg, a dose that blocked α2 agonist induced GH rise, 2) α1 antagonist prazosin 1 mg/kg, 3) non‐specific β‐blocker propranolol 1.5 mg/kg, a dose that prevented β2 agonist (salbutamol) induced inhibition, 4) serotonin antagonist cyprohep‐tadine 0.5 mg/kg, a dose that inhibited serotonin agonist quipazine induced GH rise, or 5) control. No drug altered mean GH or prolactin levels and pulsatile GH release persisted. Unilateral injections of prazosin, propranolol and idazoxan were made into the medial basal hypothalamus and preoptic‐anterior hypothalamic area and of cyproheptadine into the medial basal hypothalamus, all with no effect on short‐term GH release. GH and prolactin secretory profiles were measured after giving rats 6 units/kg intravenous insulin. Blood glucose levels fell to less than 50% basal. Hypoglycaemia caused a non‐significant 30% fall in mean 2 h GH. Intravenous idazoxan, prazosin, propranolol and cyproheptadine (doses as in first study) did not modify the blood glucose fall, but idazoxan produced a significant reduction of mean GH compared to insulin alone (4 ± 1.1 ng/ml SEM, idazoxan/insulin versus 16 ± 5.6 ng/ml, saline/insulin). The lack of an effect of a‐ and β‐blockers on normal, pulsatile GH release is against a role for endogenous catecholamines in controlling this release. Catecholamines may be released during insulin‐induced hypoglycaemia and preferentially bind to α2 receptors to increase GH secretion, thus attenuating the suppression of GH levels that would otherwise occur.
Galanin‐Induced Growth Hormone Secretion in Conscious Rats: Evidence for a Possible Involvement of Somatostatin
Authors: Tanoh Tsutomu , Shimatsu Akira , Ishikawa Yasuhiro , Ihara Chihiro , Yanaihara Noboru , Imura Hiroo
Published: September 2006

Intracerebroventricular administration of galanin (GAL) potently evoked growth hormone (GH) secretion in conscious male rats. Pretreatments with neostigmine and cysteamine blunted the GAL‐induced GH secretion. Pretreatment of animals with a specific anti‐somatostatin serum significantly inhibited the GAL‐induced GH secretion. On the contrary, GH‐releasing hormone‐induced GH secretion was significantly enhanced with cysteamine and anti‐somatostatin serum. These results suggest that somatostatin is involved in GAL‐induced GH secretion in rats.
In vitro Regulation of Rat Prolactin Messenger Ribonucleic Acid Poly(A) Tail Length: Modulation by Bromocriptine
Authors: Carter David A. , Li‐Jin Chew , Murphy David
Published: September 2006

Recent analysis of endocrine gene transcripts has revealed that several hormone mRNAs exhibit regulated size changes (due to alterations in length of the 3’poly(A) tail) which may function as an additional level of control in the determination of gene expression. We have now shown, through the novel application of an organ culture technique, that prolactin mRNA exhibits a similar regulated change in poly(A) tail length when rat anterior pituitary glands are explanted. The effect is observed in glands of either male or female rats and is specific with respect to growth hormone and α‐tubulin mRNAs. Furthermore, we have also found that the size change in prolactin mRNA is attenuated in the presence of bromocriptine, indicating regulation through a dopaminergic pathway.
Molecular Identification of Two Types of lnterleukin‐1 Receptors in the Murine Pituitary Gland
Authors: Parnet Patricia , Brunke Deborah L. , Goujon Emmanuelle , Mainard Jacques Demotes , Biragyn Arya , Arkins Sean , Dantzer Robert , Kelley Keith W.
Published: September 2006

The present study was carried out to characterize interleukin‐1 (IL‐1) receptors on murine pituitary cells. Receptor autoradiography confirmed the existence of binding sites for IL‐1α in the murine adenphypophysis, but not in the neural or intermediate lobes. Specific binding of IL‐1 to isolated pituitary membranes revealed a Kd of 0.9 nM with a Bmax of 37 fmol/mg protein. To examine the possibility that the adenohypophysis synthesizes a receptor for IL‐1, immunocytochemistry experiments with a specific monoclonal antibody against the type I receptor revealed the existence of this protein in only the adenohypophysis. Identity of the type I IL‐1 receptor was similar to that found on T cells as determined by: 1) amplification of the predicted 619 bp fragment spanning the cytoplasmic, transmembrane and extracellular domains from RNA of pituitary and T cell origin, as well as clonal AtT‐20 pituitary cells, and 2) restriction fragment analysis and sequencing of the amplified cDNAs. The pituitary gland and AtT‐20 cells also expressed transcripts for the newly identified type II receptor for IL‐1 as assessed by amplification of a specific 325 bp fragment, restriction fragment analysis and nucleotide sequencing, and these transcripts were similar to those found on B lymphocytes. These data identify two different forms of the IL‐1 receptor in both normal and transformed pituitary calls and establish that these receptors are similar at the molecular level to those first identified on T and B lymphocytes.
Membrane Properties of Identified Guinea‐Pig Paraventricular Neurons and their Response to an Opioid μ‐Receptor Agonist: Evidence for an Increase in K+ Conductance
Authors: Kasai Masanori , Tasker Jeffrey G. , Wuarin Jean‐Pierre , Dudek F. Edward
Published: September 2006

Intracellular recordings were obtained from neurons in the paraventricular nucleus (PVN) of guinea‐pig hypothalamic slices. Passive and active properties of the neurons were determined, and when possible, recorded neurons were injected with biocytin. The effects of a μ‐receptor opioid agonist [D‐Ala2, Nme‐Phe4, Gly5‐ol]enkephalin (DAGO) were studied in order to determine which types of cells have μ receptors and to test the hypothesis that an increase in K+ conductance causes μ‐receptor‐mediated inhibition in the PVN.

The recorded cells inside the PVN were divided into two groups, primarily on the basis of the presence or absence of a low threshold Ca2+ spike (LTS). In one group of neurons, LTS potentials could not be evoked (non‐LTS cells, n = 42). In another group of neurons (n = 35), LTS potentials with one or two Na+ spikes could be initiated with depolarizing pulses superimposed on steady hyperpolarizing currents; however, these neurons did not fire robust bursts (i.e. non‐bursting LTS cells). The mean time constant of non‐LTS cells (19.9±1.6ms; mean ± SEM) was significantly shorter than that of non‐bursting LTS cells (26.7 ± 2.1 ms). There were no differences in the mean resting membrane potential, spike amplitude, spike duration, input resistance, spike threshold and pattern of synaptic inputs between the two groups. Intracellular labeling with biocytin combined with cresyl violet counter‐staining demonstrated that the two types of cells were located in the PVN. The mean diameters of non‐LTS cells along their long axis (25.4 ± 1.7 μm) and short axis (17.5 ±2.2 μm, n = 7) were larger than those of non‐bursting LTS cells (22.1 ±1.0 μm, 15.2 ± 1.1 μm, respectively, n = 12); this suggests that non‐LTS cells are magnocellular neurons and non‐bursting LTS cells are parvocellular neurons, as previously described in the rat.

Bath application of DAGO at a concentration of 1 μM inhibited 7 of 25 (28%) non‐LTS cells tested, and 4 of 21 (19%) non‐bursting LTS cells. The main effect of DAGO on PVN cells was a hyperpolarization of membrane potential (4 to 15 mV) with a decrease of input resistance (20% to 38%). Quinine (100 μM to 1 mM), a K+ channel blocker, eliminated or reduced inhibitions mediated by DAGO. The apparent reversal potential of the hyperpolarization induced by the μ‐receptor agonist was about −85 mV.

These data in the guinea‐pig PVN suggest that non‐LTS cells are putative magnocellular neurons, and non‐bursting LTS cells are parvocellular neurons. The μ‐receptor agonist, DAGO, directly hyperpolarizes approximately equal numbers of each of these cell types. Experiments with quinine and injected currents support the hypothesis that DAGO activation of μ receptors on neurons in the region of the PVN leads to an increase in K+ conductance.
Seasonal Variation of Neuropeptide Y Actions on Growth Hormone and Gonadotropin‐ll Secretion in the Goldfish: Effects of Sex Steroids
Authors: Peng Chun , Trudeau Vance L. , Peter Richard E.
Published: September 2006

The effects of neuropeptide Y (NPY) on growth hormone (GH) and gonadotropin‐II (GtH‐II) release in different reproductive stages were studied using perfused pituitary fragments of female goldfish. The GH and GtH‐II release responses to 5‐min pulses of NPY were relatively small in sexually regressed fish (July), intermediate in recrudescent fish (December), and maximal in sexually mature (= prespawning) fish (May). To test if sex steroids can modulate NPY action, the effects of in vivo implantation of 17β‐estradiol (E2) and testosterone (T) (both at 100 μg/g dosage) on NPY‐induced GH and GtH‐II secretion were examined. In sexually regressed goldfish, implantation of T significantly enhanced NPY‐induced GH and GtH‐ll release from perfused pituitary fragments; implantation of E2 potentiated the NPY‐induced GtH‐II, but not GH release. However, steroid implantation did not affect responses to NPY when this experiment was repeated using pituitaries from sexually mature fish. To test the hypothesis that steroids may act directly at the level of the pituitary to potentiate NPY action, pituitary fragments taken from sexually regressed goldfish were incubated with 100 nM T for 24 h, and the GH and GtH‐ll responses to 5‐min challenges of NPY assessed in the presence of T. Both GH and GtH‐ll responses to NPY were not affected by treatment with T in vitro, suggesting that T does not act directly at the level of the pituitary. Since we have found that gonadotropin‐releasing hormone (GnRH) in part mediates the effects of NPY on GH and GtH‐ll release, the possibility that steroids may potentiate the actions of NPY on GnRH release were also examined. In sexually regressed fish, NPY did not alter GnRH release either from pituitary fragments or preoptic anterior hypothalamic slices. When fish were pretreated with E2 and T by in vivo implantation, NPY significantly stimulated the release of GnRH. Taken together, these results demonstrate that: 1) there is a seasonal variation of NPY action on GH and GtH‐ll release in the female goldfish; 2) sex steroids, especially T, potentiate the effects of NPY on GH and GtH‐ll release in sexually regressed fish, when the endogenous steroid levels are low; and 3) the seasonality of NPY actions and the potentiation by steroids may be mediated, at least in part, by enhanced stimulation of GnRH release.
Estrogen Receptor Immunostaining in the Preoptic Area and Medial Basal Hypothalamus of Estradiol Benzoate‐ and Prazosin‐Treated Female Guinea‐Pigs
Authors: Malik Karl F. , Feder Harvey H. , Morrell Joan I.
Published: September 2006

Evidence has accumulated showing that the α1‐adrenergic antagonist prazosin decreases nuclear estrogen binding in the hypothalamus of the guinea‐pig. In this study we asked if prazosin treatment alters estrogen receptor (ER) protein content as reflected by changes in ER‐immunoreactivity. The monoclonal rat antibody H222 directed against ER was used to detect ER‐immunoreactive (ER‐ir) cells in eight specific preoptic and hypothalamic brain regions of ovariectomized Hartley strain guinea‐pigs treated with estradiol benzoate and 1.0mg/kg prazosin or vehicle. Immunocytochemical parameters which provided optimum conditions for detection of even modest changes in ER‐immunoreactivity were first established. Then, using these optimum conditions, we compared 1) the mean number of ER‐ir profiles, 2) the mean density of ER‐ir staining, and 3) the distribution of ER‐ir staining density readings, between conditions within each of the eight brain regions. No differences in any of these measures were found between prazosin‐ and vehicle‐treated females. We also compared the percentage of ER‐ir nuclear profiles across the eight cell groups investigated in estradiol benzoate‐ and vehicle‐treated females. The medial preoptic area had by far the highest percentage (48%) of ER‐ir profiles (P<0.05) compared to all seven other brain regions (23% to 32% ER‐ir cells). Our data, showing that ER‐immunoreactivity is not reduced (6 h) after prazosin treatment, suggests that mechanisms other than alterations in ER protein should be considered when interpreting the effects of prazosin on the retention of estradiol by nuclear or cytosolic extracts of hypothalamic lysates.
Sexual Dimorphism in the Distribution of a‐Neoendorphin‐Like Immunoreactivity in the Anterior Pituitary of the Rat
Authors: Bello Aixa R. , Kah Olivier , Croix Dominique , Ciofi Philippe , Tramu Gérard
Published: September 2006

The localization of the opioid peptide α‐neoendorphin (α‐Neo‐E) was studied in the anterior pituitary of normal and castrated male and normal female rats, Immunoreactive (ir) cells were noted in both sexes. These α‐Neo‐E‐ir cells were further characterized using double immunostaining with an elution‐restaining procedure. It was seen that in males, α‐Neo‐E‐ir cells corresponded mainly to luteinizing hormone/follicle‐stimulating hormone cells and a few thyroid‐stimulating hormone (TSH) cells, whereas in females, virtually all α‐Neo‐E‐ir cells corresponded to TSH cells.

Castration of male rats caused, within 3 to 5 days, a dramatic decrease in the number of α‐Neo‐E‐ir gonadotrophs, whereas the number of a‐Neo‐E‐ir TSH cells tended to increase. Two weeks after castration, however, most α‐Neo‐E‐ir cells were also follicle‐stimulating hormone‐ir.

This study demonstrates that in the anterior lobe of the rat, α‐Neo‐E‐ir is located within gonadotrophs and/or thyrotrophs, depending on the sex. In addition, results obtained following castration suggest that the expression of this peptide in the anterior pituitary depends upon the steroid environment, possibly indicating that α‐Neo‐E is implicated in the regulation of the pituitary‐gonadal axis.
Concomitant Changes of Messenger Ribonucleic Acid Levels of Secretogranin II, VGF, Vasopressin and Oxytocin in thé Paraventricular Nucleus of Rats After Adrenalectomy and During Lactation
Authors: Mahata S. K. , Mahata M. , Hörtnagl H. , Fischer‐Colbrie R. , Steiner H.‐J. , Dietze O. , Winkler H.
Published: September 2006

In situ hybridization was used to study the mRNA levels for secretogranin II and VGF in comparison with those of oxytocin and vasopressin in the hypothalamus of rats. VGF is a widespread constituent of large dense core vesicles which is selectively induced in PC12 cells by nerve growth factor. After adrenalectomy the mRNA levels of secretogranin II, VGF and vasopressin were increased 4‐ to 5‐fold in the parvocellular neurons of the paraventricular nuclei. In lactating rats the message for oxytocin and secretogranin II were significantly elevated in the magnocellular neurons of the paraventricular and supraoptic nuclei, whereas for VGF only a smaller non‐significant increase was observed. As shown by immunoelectron microscopy secretoneurin (a peptide derived from secretogranin II) and oxytocin are co‐stored in the large dense core vesicles of the hypothalamo‐neurohypophysial neurons.

These results demonstrate that stimulation of both parvo‐ and magnocellular neurons of the hypothalamus induces a concomitant increase of the messages for secretogranin II and VGF together with those of vasopressin and oxytocin.
Denervation of the Rat Posterior Pituitary Gland: Validation of a Stereotaxic Method
Authors: Makara G. B.
Published: September 2006

A stereotaxic surgical method was developed for interrupting the nerve fibres running through the rat pituitary stalk to the posterior pituitary gland without obliterating the hypothalamo‐pituitary portal circulation. The pituitary stalk was compressed by the blunt tip of an L‐shaped rotating knife. Successful operations produced mild diabetes insipidus, disappearance of arginine vasopressin from the neural lobe, accumulation of arginine vasopressin and neurosecretory material in the pituitary stalk and no infarction in the anterior lobe of the pituitary gland. In female rats, the oestrous cycle was only temporarily disturbed. Plasma prolactin and corticosterone levels were high during the first 24 h after the stalk compression but returned to normal baseline levels from the second day after the operation. One week after the operation plasma adrenocorticotropin and prolactin levels were in the control range while plasma a‐melanocyte‐stimulating hormone was elevated. Denervation of the posterior pituitary gland may help in studying the neural control of intermediate lobe function and the role of the neural lobe in various endocrine conditions, and may serve as a model for lesions of the pituitary stalk and formation of ectopic neurohypophysis in the human.
Immunocytochemistry of Amino‐Acids in the Rodent Pituitary Using Extremely Specific, Very High Titre Antisera
Authors: Pow D. V.
Published: September 2006

The compartmental interrelationships of the metabolically related amino‐acids glutamate, GABA and glutamine and the metabolically unrelated amino‐acids taurine and glycine in the rodent pituitary, were investigated by light microscopic immunocytochemistry using highly specific antisera. Glutamate‐like immunoreactivity was abundant in astrocytes in the posterior pituitary. Glutamine immunoreactivity was present only at low levels in the posterior pituitary, but was abundant in astrocytes within the intermediate lobe. Other glia‐like ceils in the anterior pituitary were also glutamine‐immunoreactive. GABA immunoreactivity was abundant in the intermediate lobe but absent from anterior and posterior lobes. The GABA immunoreactivity mainly took the form of small punctata, the majority of which were in intimate apposition to the glutamine‐immunoreactive glia. Strong taurine immunoreactivity was present in astrocytes in the posterior pituitary but only weak labelling was present in intermediate and anterior lobes of the pituitary. Specific glycine immunoreactivity was not detected in the pituitary. These results suggest that glutamate‐immunoreactive astrocytes in the posterior pituitary, unlike glia in loci such as the retina, do not convert much, if any, of their glutamate content into glutamine (or if they do, it is rapidly further metabolized to another compound), whereas those astrocytes in the intermediate lobe do contain glutamine. The spatial association of GABAergic fibres with glutamine‐positive astrocytes raises the possibility that astrocytes in the intermediate lobe receive a GABAergic innervation. Glutamate, glutamine and taurine (or their metabolites) may have roles as neuroactive substances regulating pituitary secretion.
Androgenic Regulation of Expression of β‐Tubulin Messenger Ribonucleic Acid in Motoneurons of the Spinal Nucleus of the Bulbocavernosus
Authors: Matsumoto Akira , Arai Yasumasa , Hyodo Susumu
Published: September 2006

Expression of β‐tubulin mRNA was examined in androgen‐sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in adult male rats by in situ hybridization histochemistry using cDNA encoding mouse β‐tubulin. Hybridizable β‐tubulin mRNA was localized in the somata and proximal dendrites of SNB motoneurons. Removal of androgen by castration significantly reduced the expression level of β‐tubulin mRNA in the SNB motoneurons, whereas the change was prevented by testosterone treatment. On the contrary, castration or testosterone treatment did not induce any changes in the expression level of β‐tubulin mRNA in the androgen‐insensitive motoneurons of the retrodorsolateral nucleus. These results suggest that androgen regulates the expression of β‐tubulin gene in the SNB motoneurons and may provide evidence for the molecular mechanisms of hormonally‐induced neuronal plasticity in the SNB motoneurons.
The Adenylyl Cyclase‐Cyclic AMP System Modulates Morphological and Functional Development of Hypothalamic β‐Endorphin Neurons in Culture
Authors: Yang Zhiyu , Huang Weiqing , Lee Dan , Copolov David L. , Lim Alan T.
Published: September 2006

In rats, opioidergic β‐endorphin (βEP1–31) is produced and released from neurons of arcuate nuclei in the hypothalamus. Although the neuropeptide has been implicated in sexual maturation and stress‐induced reproductive dysfunction, the intra‐hypothalamic regulation of βEP neurons remains unclear. Employing long‐term monolayer cultures of neonatal rat hypothalamic cells, we report here that 4 days of treatment with 10 μM forskolin increased approximately 3‐fold (P<0.01) the proportion of immunoreactive (ir)‐ βEP positive neurons bearing neurites. In addition, treatment of forskolin also enhanced ir‐βEP release (634 ± 59 pg/well; mean β SE, n = 4, P<0.01) by 14‐fold and ir‐βEP content (119±13 pg/well; P<0.01) by 2‐fold above that of vehicle‐treated cultures; in both instances, the EC50 and the Emax, of forskolin were approximately 10μM and 100 μM, respectively. The forskolin‐stimulated release of ir‐βEP was mimicked by cholera toxin and (Bu)2cAMP treatment in a dose‐related manner, but not by pertussis toxin. Although by itself 3‐isobutyl‐1‐methyl‐xanthine (100μM) only doubled ir‐βEP secretion, it markedly potentiated the stimulatory effect of forskolin. This forskolin‐induced stimulation was reversible and in cultures re‐exposed to the same drug within the first 24 h period, there was a marked increase in the stimulated release of ir‐βEP (P < 0.05); re‐challenge of forskolin at later stages, however, induced a smaller but significant secretion of ir‐βEP (P<0.01) compared to that of vehicle‐treated control cultures. Sephadex G‐50 gel Chromatographie profile of the media prepared from forskolin‐treated cultures revealed a major ir‐βEP peak of 3 K M. Highperformance liquid chromatography analysis showed that ir‐βEP of the 3 K M, species was eluted with a retention time similar to that of synthetic rat βEP1–31. We thus conclude that the adenylyl cyclase‐cAMP system plays an important role in the modulation of βEP1–31 production and release from hypothalamic βEP neurons in culture. Furthermore, the functional responsiveness and the orphological development of these neurons are affected, at least in part, by the intrinsic activity of the adenylyi cyclase‐cAMP system.
Ultrastructural Evidence for Changes in Synaptic Input to the Hypothalamic Luteinizing Hormone‐Releasing Hormone Neurons in Photosensitive and Photorefractory Starlings
Authors: Parry Dilys M. , Goldsmith Arthur R.
Published: September 2006

Neural input to the hypothalamic luteinizing hormone‐releasing hormone (LHRH) neurons was investigated in male starlings (Sturnus vulgaris) using electron microscopy combined with immunocytochemistry. Birds (4 to 6 in each group) were sampled at four stages of a photoperiodically induced reproductive cycle: (a) sexually immature but photosensitive, under short days; (b) during sexual maturation after 7 to 25 long days; (c) during gonadal regression after 35 to 50 long days; and (d) when fully photorefractory after 11 months exposure to long days. The length of the perikaryal membrane, the number and length of axo‐somatic terminals in contact with it and the number and length of synaptic modifications within the terminals were measured for a minimum of six LHRH neurons in each brain. The number of axo‐somatic terminals per neuron and the number per unit length of perikaryal membrane did not differ in birds of groups (a), (b) and (c), but was significantly greater (P<0.05) in the fully refractory birds (group d). Similarly, the number of synaptic modifications was significantly greater (P<0.05) in group (d) than in the other groups. These results are consistent with increased neural input to the LHRH perikarya in photorefractory birds after prolonged exposure to long days, although there was no indication of a change in input at the time of gonadal regression.
Vasostatins, Comprising the N‐terminal Domain of Chromogranin A, Suppress Tension in Isolated Human Blood Vessel Segments
Authors: Aardal Sidsel , Helle Karen B. , Elsayed Said , Reed Rolf K. , Serck‐Hanssen Guldborg
Published: September 2006

Chromogranin A (CGA) belongs to a family of highly acidic proteins which are co‐stored and co‐released with the catecholamines from the mammalian adrenal gland and occur in nmolar concentrations in the human circulation. A vascular function for the adrenomedullary released and circulating CGA has yet to be established. The present study reports on the novel vasoinhibitory effect of the N‐terminal domain of the adrenomedullary CGA in isolated segments of the human internal thoracic artery (ITA) and saphenous vein (SV). The collective term vasostatin(s) refers to N‐terminal fragments (CGA1–76 and CGA1–113) of apparent molecular weights 7 to 22 kD, to indicate their vascular inhibitory effects. The sustained contractions evoked by the potent vasoconstrictor peptide, endothelin‐1 (ET‐1) were suppressed when ITA and SV segments were preincubated for 15 min with vasostatins (72 nM). The vasoinhibitory effects were not dependent on an intact endothelium and suppression of the response to 35 nM ET‐1 was ∼77% and ∼40% in endothelium‐denuded ITA and SV segments, respectively. In endothelium‐denuded SV segments the vasostatins suppressed the maximal sustained tension response but not the potency for ET‐1, indicating that the vasostatin effect did not involve interference with ET‐1 binding to its vascular receptor. Preincubation of endothelium‐denuded SV segments with nifedipine (1 μM) inhibited the sustained response to ET‐1 ≥10 nM by 50%. By the same protocol a 20 to 40% suppression of the contraction evoked by ET‐1 ≥10 nM was obtained in segments preincubated with vasostatins (72 nM), suggesting that inhibition of Ca2 channel opening might indirectly contribute to the effect of vasostatins on the sustained response to ET‐1. The mechanisms for these inhibitory effects of vasostatins on the sustained ET‐1 contractions in human blood vessels remain to be elucidated.
Chromogranin A: Secretion of Processed Products from the Stimulated Retrogradely Perfused Bovine Adrenal Gland
Authors: Helle Karen B. , Marley Philip D. , Angeletti Ruth Hogue , Aunis Dominique , Galindo Estelle , Small David H. , Livett Bruce G.
Published: September 2006

Chromogranin A (CGA) is a member of a family of highly acidic proteins co‐stored and co‐secreted with adrenaline and noradrenaline in the adrenal medulla. A number of biologically active fragments of CGA (CGAFs) have been characterized including a group of small N‐terminal fragments collectively named vasostatins due to their vascular inhibitory activity. In the present study, the release of CGAFs, including CGA N‐terminal fragments, from the isolated, retrogradely perfused bovine adrenal gland, has been studied under basal conditions and during nerve stimulation and perfusion with acetylcholine. The CGAFs were characterized by SDS‐PAGE followed by immunoblotting with antisera to specific sequences within the CGA molecule. Many different CGAFs were released during stimulation of the glands. Antisera to CGA1–40 and CGA44–76 detected a 7 kD protein whose release was increased during stimulation. This component co‐migrated with synthetic CGA1–76, was not immunoreactive to antisera to CGA79–113 or CGA124–143, and was seen whether or not the serine protease inhibitor aprotinin was present in the perfusion medium. The release of an ∼ 18 kD component, which stained with antisera to CGA1–40, CGA44–76 and CGA79–113, but not to chromostatin (CGA124–143), was also increased during stimulation. Components of 22 kD and larger were detected with antisera to chromostatin, but not with antisera to CGA1–40, CGA44–78 and CGA79–113. Two of these components of 22 to 24 kD were enhanced during nerve stimulation in the presence of aprotinin. The results indicate that processed Chromogranin A fragments are secreted from the bovine adrenal medulla during stimulation of chromaffin cells. The major fragments secreted appear to be the N‐terminal fragments of CGA, CGA1–76 and CGA1–113, which would arise as a result of processing of CGA at the first and second pairs of basic amino acids. A number of larger CGAFs, possibly containing the chromostatin sequence CGA124–143 at their N‐terminal, and components similar in size to intact CGA and to proteoglycan forms of CGA, are also secreted from the perfused bovine adrenal gland during stimulation.
Changes in Oxytocin Immunoreactivity and mRNA Expression in the Sheep Brain during Pregnancy, Parturition and Lactation and in Response to Oestrogen and Progesterone
Authors: Broad K. D. , Kendrick K. M. , Sirinathsinghji D. J. S. , Keverne E. B.
Published: September 2006

The effects of pregnancy, parturition and lactation and exogenous treatments with oestradiol and progesterone on oxytocin (OXY) immunoreactivity and gene expression in the sheep brain were investigated. Immunocytochemistry was used to demonstrate that increased OXY‐immunoreactivity occurred in cells of the paraventricular (PVN) and supraoptic nuclei (SON), the bed nucleus of the stria terminalis (BNST), the anterior commissural nuclei (ACN) and the periventricular part of the medial preoptic area (PvMP). Oxytocin immunoreactive terminals were also seen in the accessory olfactory nucleus, the glomerular and peri‐glomerular layers of the olfactory bulb, the lateral septum, the zona incerta and the pars compacta of the substantia nigra. Compared to ovariectomized and late pregnant animals, the intensity of immunoreactivity was increased in all of these oxytocinergic elements at parturition, during lactation and following exogenous treatment with oestradiol. The OXY‐immunoreactivity was also more intense in late pregnant animals compared to ovariectomized ones. Quantitative in situ hybridization histochemistry showed that cells in the PVN, SON, BNST and PvMP all showed significantly increased expression of OXY mRNA in animals at parturition and during lactation compared to late pregnant or ovariectomized animals. Expression levels in late pregnant animals were also significantly higher than in ovariectomized ones. Progesterone treatment significantly increased OXY mRNA in the PVN, SON, BNST and PvMP whereas oestradiol treatment was only effective in the PVN, BNST and PvMP. Combined treatment with these steroids did not significantly increase OXY mRNA levels in comparison with their administration alone.

These results show that OXY‐immunoreactivity and mRNA expression are at their highest in the sheep brain when maternal behaviour is induced. The increased synthesis/storage of the peptide at parturition may be due to changes in circulating concentrations of both progesterone and oestradiol during late pregnancy.