bluuebunny/crossref_metadata_2025 huggingface dataset:
Pineal Photoreceptor Cells in Culture: Fine Structure, and Light/ Dark Control of Cyclic Nucleotide Levels and Melatonin Secretion
Authors: Falcoón Jack , Bégay Valérie , Besse Colette , Ravault Jean‐Paul , Collin Jean‐Pierre
Published: September 2006

Trout pineal photoreceptor cells were dissociated by trypsin‐DNase digestion and further purified by a Percoll gradient centrifugation. Total cells or purified photoreceptor cells were then embedded in a collagen gel, or layered on culture‐treated polycarbonate membranes, or maintained in suspension, with RPMI 1640 medium or BGjb medium. It has been shown that cells maintain a rhythmic production of melatonin for at least seven 24 h light/dark cycles under these conditions. In this complementary study, the morphofunctional state of the photoreceptor cells was examined 1) by electron (transmission, scanning) microscopy, and 2) by pharmacological tests under different lighting conditions. Using polycarbonate membranes together with RPMI 1640 medium appeared the most suitable. The segmented organization of photoreceptor cells was well preserved when using the culture‐treated membranes. It tended to disappear in cells embedded in the collagen gel and was lost after passage through the Percoll gradient. However, this one allowed obtention of an homogeneous population of photoreceptors, as recognized by their intracellular components. Intracellular organelles were rather well preserved in the embedded photoreceptors. The study also provides novel information on the nature of second messengers involved in the photoperiodic control of melatonin production in photoreceptor cells. From the effects of an adenylyl cyclase activator and a phosphodiesterase inhibitor it appeared that 1) total cells and Percoll‐selected cells behaved similarly, 2) the nocturnal rise in melatonin secretion was associated with an increase in cAMP content, and 3) a fall in cAMP may be a mechanism through which light reduces melatonin secretion by photoreceptor cells. Cyclic GMP, the metabolism of which also appeared to be controlled by light, did not seem involved in the photoperiodic control of melatonin production. The method proposed herein offers interesting perspectives for the study of the photoneuroendocrine properties of isolated photoreceptor cells.
Immunocytochemical Identification of Oestrogen Receptors in the Ovine Pars Tuberalis: Localization Within Gonadotrophs
Authors: Skinner Donal C. , Herbison Allan E. , Robinson Jane E.
Published: September 2006

The pars tuberalis (PT) of the pituitary gland shows dense binding of melatonin and consequently this region may be involved in modulating seasonal reproduction. Oestrogen is well established as a critical gonadal steroid in controlling seasonally via its ability to alter luteinizing hormone (LH) release. Using immunocytochemistry techniques with antibodies specific for the oestrogen receptor (ER) and the ovine βLH (oβLH) subunit, we have identified large populations of ER‐immunoreactive (‐IR) and LH‐IR cells in the anteroventral region of the ovine PT. In contrast, few ER‐ or LH‐IR cells were identified in the anterodorsal or posterior regions of the PT. Double‐labelling experiments revealed that all ER‐IR cells in the PT are also immunoreactive for LH. These results show that cells immunoreactive for the ER are concentrated in the anteroventral aspect of the PT and that these receptors are located in the nuclei of the PT gonadotrophs. These results suggest that the anteroventral PT, a region which also expresses high melatonin binding, may be a site of integrated oestrogen and melatonin action on LH secretion from the PT.
Adenosine Inhibits Alpha‐Melanocyte‐Stimulating Hormone Release from Frog Pituitary Melanotrophs. Evidence for the Involvement of A1 Adenosine Receptors Negatively Coupled to Adenylate Cyclase
Authors: Chartrel Nicolas , Tonon Marie‐Christine , Lamacz Marek , Vaudry Hubert
Published: September 2006

Adenosine is recognized as an important modulator of cell activity. In particular, adenosine regulates the secretion of adrenocorticotropin from anterior pituitary cells. However, the possible role of adenosine on the pars intermedia has never been investigated. In the present study, we have examined the effect of adenosine on α‐melanotropin (α‐MSH) secretion from the intermediate lobe of the pituitary of the frog (Rana ridibunda), using the perifusion technique. When whole neurointermediate lobes were exposed to graded doses of adenosine (10−9 to 10−4 M), a dose‐dependent inhibition of a‐MSH release was observed. Repeated pulses of adenosine (5 ± 10−5 M) induced a reproducible inhibition of α‐MSH secretion without any desensitization phenomenon. The effect of adenosine was mimicked by the non‐selective agonist 5′‐N‐ethylcarboxamide‐adenosine and the highly specific adenosine A, receptor agonist N6‐[R‐phenylisopropyl]‐adenosine (R‐PIA). In contrast the selective adenosine A2 receptor agonist, CGS 21680, induced a slight stimulation of α‐MSH release. Adenosine‐induced inhibition of α‐MSH secretion was blocked by the non‐selective adenosine antagonist, 8‐(p‐sulfophenyl)‐theophyline. Adenosine and R‐PIA also inhibited α‐MSH secretion from acutely dispersed pars intermedia cells. Adenosine did not block thyrotropin‐releasing hormone‐induced α‐MSH release from perifused neurointermediate lobes. In contrast, adenosine inhibited both acetylcholine‐evoked and muscarine‐evoked α‐MSH secretion. Finally, R‐PIA induced a significant inhibition of basal and forskolin‐stimulated cyclic AMP levels in whole neurointermediate lobes. The present results demonstrate that adenosine exerts a direct inhibitory effect on α‐MSH release from melanotrope cells through activation of the A1 receptor subtype, negatively coupled to adenylate cyclase. These data suggest that adenosine may play a physiological role in the regulation of hormone release from the intermediate lobe of the pituitary.
Dysregulation of the Hypothalamus‐Pituitary‐Adrenal Axis in Male and Female, Genetically Obese (ob/ob) Mice
Authors: McGinnis Ralph , Walker Joseph , Margules David , Aird Fraser , Redei Eva
Published: September 2006

The autosomal, recessive obesity of ob/ob mice is associated with hypercorticosteronemia and amelioration of most symptoms of obesity following adrenalectomy. Increased adrenocorticotropic hormone (ACTH) secretion has been hypothesized on the basis of several reports of higher pituitary ACTH content in ob/ob mice compared to lean littermates. However, the only measurement of ACTH blood concentration found lower levels in ob/ob mice than in leans suggesting that hypercorticosteronemia might result solely from an enhanced adrenal response to ACTH and also suggesting that the ob/ob's elevated pituitary ACTH content might be due to decreased ACTH secretion rather than increased ACTH synthesis. In our study, basal serum ACTH levels were higher in ob/ob males and females compared to sex‐matched lean littermates. Anterior pituitary ACTH synthesis was also elevated as indicated by increased content of ACTH and proopiomelanocortin mRNA in obese mice of both sexes; however hypothalamic corticotropin‐releasing factor content was not different in lean and obese mice. Basal serum ACTH and corticosterone (CS) levels showed normal circadian rhythm in both phenotypes and sexes, but the circadian increase in CS level was much greater in obese mice than in leans despite equal serum ACTH increases in the two phenotypes. Ether stress at both peak and trough of the circadian rhythm also stimulated much larger serum CS increases in obese mice even though ACTH increases were again equal in the two phenotypes. Taken together, these results strongly indicate that ob/ob mice have increased synthesis and secretion of pituitary ACTH despite the presence of chronically elevated serum CS. This hyperactivity of the hypothalamo‐pituitary‐adrenal axis appears to be most pronounced in ob/ob females since pituitary ACTH content was equal in obese males and females despite much higher circulating CS levels in the females. Furthermore, the results also indicate an enhanced response to ACTH by the adrenal cortex of the obese mouse. Thus, ob/ob mice exhibit abnormal hypothalamo‐pituitary‐adrenal axis function with hyperactivity occurring at the level of pituitary ACTH synthesis/secretion as well as at the level of adrenocortical response to ACTH.
Diencephalic Noradrenaline Depletion Impairs the Corticosterone Response to Footshock but does not Affect Conditioned Fear
Authors: Selden Nathan R. W. , Robbins Trevor W. , Everitt Barry J.
Published: September 2006

This study tested the hypothesis that hypothalamic noradrenaline (NA) depletion induced by 6‐hydroxydopamine alters neuroendocrine, but not behavioural, responses to aversive stimuli. Sham‐operated and NA depleted rats were exposed to pairings of an auditory (clicker) CS and (footshock) US in a distinctive environment. Subjects were tested for preference of a ‘safe’ environment over the one in which they were shocked, as a measure of effective conditioning to the contextual stimuli present in the distinctive environment. Subjects were also tested, in a separate operant chamber, for the suppression of drinking in the presence of the auditory stimulus, as a measure of effective conditioning to the explicit auditory CS. Blood samples were collected immediately following each phase of the behavioural experiment and were later analysed for plasma Corticosterone concentration.

Behavioural and Corticosterone responses of individual control animals to the CS were positively correlated, consistent with previous results. This correlation was not present in the NA depleted group. The lesioned rats also showed a severely attenuated Corticosterone response to the footshock US. By contrast, NA depletion had no effect on any behavioural measure of CS or contextual conditioning. Together with previous experiments, these results suggest that diencephalic NA projections are more likely to mediate neuroendocrine, and coeruleo‐cortical NA projections are more likely to mediate behavioural responses to conditioned and unconditioned aversive stimuli.
Changes in Proopiomelanocortin Primary Transcript Levels in the Anterior Pituitary Accompany Increased Adrenocorticotropin Secretion During the Diurnal Surge
Authors: Kwak Seung P. , Young Elizabeth A. , Akil Huda , Watson Stanley J.
Published: September 2006

Proopiomelanocortin (POMC) gene transcription in the anterior pituitary varies during stress and glucocorticoid feedback. These changes appear to parallel alterations in peptide release. The diurnal rhythm of the hypothalamo‐pituitary‐adrenal axis also involves the periodic excursion of adrenocorticotropin (ACTH) levels in plasma, but it is not clear whether the diurnal release is accompanied by changes at the transcriptional level. In the present study, we have initially characterized the heteronuclear species of POMC (hnPOMC) RNA found in the anterior pituitary by a Northern blot analysis and subsequently used this method to quantitate relative changes in the levels of heteronuclear transcript during diurnal stimulation. Two species of RNA migrating at 6.0 kb and 4.1 kb were found in the nuclear fraction of the anterior pituitary. Successive probing by various POMC cRNAs indicated that the 6.0 kb fragment was the primary transcript and the 4.1 kb fragment corresponded to the intron A‐containing processing intermediate of POMC.

The nuclear species were quantitated after acute swim stress and during the diurnal ACTH secretion. Acute swim increased plasma ACTH levels by 243% after 30 min. This was paralleled by a 214% increase in the primary transcript RNA levels. Endogenous circadian stimulation in the evening produced a smaller rise of plasma ACTH (79%), and was accompanied by a 34% increase in POMC hnRNA levels. Nuclear processing intermediate (4.1 kb) and the mRNA levels did not vary during the evening. These results suggest that the diurnal mechanism transiently increases ACTH release as well as POMC gene transcription in the anterior pituitary. Release and transcription appear to be tightly coupled during circadian activation as well as during stress.
Vasoactive Intestinal Polypeptide/Peptide Histidine Isoleucine‐Immunoreactive Neuron Systems in the Basal Hypothalamus of a Rat Strain with Deficient Prolactin Release in Response to Stress
Authors: Ceccatelli S. , Fahrenkrug J. , Eneroth P. , Hökfelt T.
Published: September 2006

There is evidence for involvement of vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) in control of prolactin secretion. In fact VIP‐ and PHI‐like immunoreactivities have been demonstrated in the hypotha/amic paraventricular nucleus and at the median eminence level. Using immunohistochemistry we have compared the distribution of immunoreactive VIP and PHI in the hypothalamus of male Sprague‐Dawley rats and BS rats, a rat strain which has a deficient release of prolactin after stressful stimuli. Quantitative information was obtained by radioimmunoassay for VIP. VIP‐ and PHI‐positive cell bodies were found in the parvocellular part of the paraventricular nucleus in colchicine‐treated rats and in nerve fibres within the median eminence of untreated rats to the same extent in both strains. Furthermore, intravenous injection of VIP caused a significant increase in serum prolactin tevets in both strains. However, at the median eminence level in BS rats, the blood vessels located in the lateral aspects of the median eminence did not show the dense VIP/PHI innervation seen in Sprague‐Dawley rats. Also, a thick VIP/PHI‐positive nerve bundle present on the surface of the median eminence of Sprague‐Dawley rats could not be seen in BS rats. Radioimmunoassay analysis revealed that VIP levels in the median eminence were twice as high in Sprague‐Dawley as compared to BS rats. Taken together, these results suggest that the defect in the prolactin release mechanism present in BS rats is not confined to the paraventricular system or the pituitary, but could be due to a deficit in VIP/PHI in fibres associated with portal vessels at the median eminence level.
Ontogeny of Expression and Glucocorticoid Regulation of the Arginine Vasopressin Gene in the Rat Hypothalamic Paraventricular Nucleus
Authors: Grino Michel , Burgunderf Jean‐Marc
Published: September 2006

Corticotropin‐releasing factor and arginine vasopressin (AVP) are the two major hypothalamic factors that regulate anterior pituitary adrenocorticotropin secretion during stress. We have previously reported that the expression of the corticotröpin‐releasing factor gene in the hypothalamus and its regulation by glucocorticoids were not mature during the first week of life in the rat, i.e. during the stress non‐responsive period. In this report, we studied the ontogeny of expression of the AVP gene in the hypothalamic paraventricular nucleus in rats using in situ hybridization. AVP mRNA was detected as early as day 20 of gestation (E20) both in the parvocellular and the magnocellular portion of the paraventricular nucleus. The levels of expression of the AVP gene increased steadily from E20 to the third day after birth (P3) and remained stable from P3 to P14. Bilateral surgical adrenalectomy induced an increase in AVP mRNA levels in the parvocellular portion of the paraventricular nucleus, but not in the magnocellular portion, in both 7‐day‐old and 14‐day‐old rats, suggesting that the glucocorticoid regulation of the AVP synthesizing neurons of the paraventricular nucleus is mature in the developing postnatal rat.
Long‐Term Treatments with Morphine and Naloxone have Sex‐Differentiated Effects on Luteinizing Hormone Secretion in Chronically Catheterized Fetal Pigs
Authors: Behrens‐Herrler Sadie , Parvizi Nahid
Published: September 2006

Short‐ (one bolus injection) and long‐term (repeated injections over a period of at least 7 days) effects of drugs on pituitary function in pig fetuses were studied to investigate the influence of morphine and naloxone on luteinizing hormone secretion in the chronically catheterized pig fetus between days 102 and 110 of gestation (term: 113±1 SD day). Both substances were intravenously administered in two doses: 0.1 mg and 1 mg/fetus. Repeated injections at 2‐day intervals enabled us to study short‐ as well as long‐term effects. Morphine acutely inhibited luteinizing hormone secretion both in male and female fetuses. Long‐term treatment with morphine at both doses caused an inhibition of basal luteinizing hormone (levels before treatment on each day) in females (0.1 mg: r=−0.60, P<0.001; 1 mg: r=−0.45, P<0.05) while male fetuses were unaffected. Naloxone did not have any short‐term effects, either in females or in males. In the long‐term study, however, naloxone at 1 mg dose decreased basal luteinizing hormone levels in male fetuses (r=−0.55, P<0.01), whereas in females no effect was evident. Co‐administration of 0.1 mg naloxone +0.1 mg morphine abolished the long‐term inhibitory effect observed in females when 0.1 mg morphine alone was given. These results indicate that the link between opioids and the luteinizing hormone system is functional in the pig from at least 2 weeks before birth. Furthermore, there is a sex difference in the long‐term effects of both morphine and naloxone. The origin of the apparently paradoxical long‐term effect of naloxone in male fetuses remains unclear.
Sex‐Related Differences in Chromogranin A, Chromogranin B and Secretogranin II Gene Expression in Rat Pituitary
Authors: Fischer‐Colbrie R. , Schmid K. W. , Mahata S. K. , Mahata M. , Laslop A. , Bauer J. W.
Published: September 2006

Chromogranin A, an acidic secretory protein, is widely distributed throughout diverse endocrine cells and the central and peripheral nervous systems. Chromogranin A is co‐stored and co‐secreted from secretory vesicles together with the endogenous hormones or neurotransmitters. Recently, two peptides derived from the Chromogranin A precursor have been shown to inhibit secretion from endocrine cells. In the present study, we investigated the regulation of the biosynthesis of Chromogranin A by estrogen in various tissues. In the pituitary, steady‐state levels of Chromogranin A mRNA were markedly reduced by 64% in estrogen‐treated male rats. At the protein level, a comparable decrease was found. Chromogranin B and secretogranin II, two other secretory proteins co‐stored with Chromogranin A, were slightly increased by estrogen. In pituitaries of female rats Chromogranin A mRNA and protein levels were significantly lower than in males. For Chromogranin B on the other hand, a 2‐fold increase of mRNA levels was found. Our observations demonstrate that physiologic concentrations of estrogen strongly affect Chromogranin A levels in the pituitary resulting in a sex‐related difference in Chromogranin A gene expression. Based on these and previous results demonstrating increased biosynthesis of Chromogranin A by glucocorticoids and calciferol, we suggest that a typical and characteristic feature of the Chromogranin A gene is its regulation by at least three different classes of steroid hormones.
Radial Glia‐Like Cells in the Supraoptic Nucleus of the Adult Rat
Authors: Bonfanti Luca , Poulain Dominique A. , Theodosis Dionysia T.
Published: September 2006

Conventional light and confocal microscopy of thick vibratome sections of the hypothalamus of adult male and female rats immunostained for the astrocytic marker glial fibrillary acidic protein (GFAP) revealed that the supraoptic nucleus (SON) contains two morphologically distinct types of astrocytes. One has a stellate form, similar to that of most astrocytes in the adult CMS. The other has a morphology reminiscent of radial glia in the developing CNS: from their cell bodies, located along the ventral glia lamina (VGL), arise one long thick process that spans the SON in the coronal plane, several horizontally‐oriented processes that form a dense network in the VGL, and a short process oriented towards the pia. The latter astrocytes are immunoreactive for vimentin, an intermediate filament protein of immature glial cells and a marker for radial glia. The stellate astrocytes showed no vimentin immunoreactivity. The functional significance of each type of supraoptic astrocyte is at present unknown but the presence of radial glia‐like cells in this hypothalamic region suggests that the SON retains a certain degree of immaturity during adulthood, that may be linked to its well known capacity to undergo neuronal‐glial plasticity under physiological and experimental stimulation.
The Role of Oxytocin Release in the Mediobasal Hypothalamus of the Sheep in Relation to Female Sexual Receptivity
Authors: Kendrick K. M. , Fabre‐Nys C. , Blache D. , Goode J. A. , Broad K. D.
Published: September 2006

In vivo microdialysis and retrodialysis were used to investigate the role of oxytocin (OXY) release in the mediobasal hypothalamus (MBH) of the ewe in the control of sexual receptivity. Initial experiments showed that OXY release was significantly increased in ovariectomized animals treated with progesterone and oestradiol when they were sexually receptive towards males and received intromissions. No such increases were seen during tests where the ewes were receptive but the males were prevented from achieving intromission. By contrast, OXY release was significantly reduced in tests where the ewes were not receptive to the male. In a second experiment artificial vaginocervical stimulation (VCS) was found to significantly increase OXY release when the animals were treated with oestradiol and this effect was potentiated by progesterone priming. OXY release in the MBH was not significantly altered by VCS in the presence of progesterone priming alone. Plasma OXY concentrations were significantly increased by VCS following all three hormone treatments but no one treatment was significantly more effective than another. Noradrenaline release in the MBH was only significantly increased following VCS when progesterone priming was given before oestradiol treatment. No effects of VCS on release of GABA, glutamate or dopamine were seen but their basal concentrations were significantly increased by the combined steroid treatment compared to oestradiol alone. In a third experiment it was found that OXY (10 μM) infused bilaterally into the MBH of receptive ewes, by retrodialysis, significantly decreased sexual receptivity and increased the release of noradrenaline and GABA. Finally, in a fourth experiment is was shown that multiple intromissions significantly reduced sexual receptivity.

These results show that OXY release in the MBH is increased by natural or artificial VCS and that this is modulated by sex steroids. The inhibitory action of OXY infusions into the MBH on sexual receptivity suggests that a functional role of this peptide within this brain region might be to mediate decreases in sexual receptivity which we observed following multiple intromissions. It is probable that the behavioural effects of this peptide are primarily mediated directly rather than through a modulation of noradrenaline and GABA release.
Oxytocin Pretreatment Enhances Arginine Vasopressin‐lnduced Motor Disturbances and Arginine Vasopressin‐lnduced Phosphoinositol Hydrolysis in Rat Septum: A Cross‐Sensitization Phenomenon
Authors: Poulin Paule , Pittman Quentin J.
Published: September 2006

The recent observation that the central oxytocin (OT) receptor has high affinity for both OT and arginine vasopressin (AVP) raises the possibility that it may be involved in some of the central actions of AVP. Repeated intracerebroventricular (icv) injections of AVP in rats evoke an unusual sensitization phenomenon in that a first exposure to the peptide enhances the sensitivity (sensitization) of the brain to a second exposure. This report investigates the possibility that the OT receptor may be involved in the mediation of the phenomenon of sensitization, using OT, a specific OT receptor agonist, [Thr4, Gly7]OT, and a specific OT receptor antagonist, d(CH2)5, [Tyr(Me)2, Thr4, Tyr‐NH29]OVT (compound 6; cpd 6), as well as a V1 AVP receptor antagonist, d(CH2)5Tyr(Me)AVP. Peptides were injected icv in conscious, adult male Sprague‐Dawley rats. The data showed that: 1) a first icv AVP injection (10 pmol/5μl) enhanced the sensitivity of the rat brain to the motor response of a second AVP injection (10 pmol/5 μl) given 24 h later; 2) injection of d(CH2)5Tyr(Me)AVP (100 pmol/5 μl icv) but not cpd 6, (100 pmol/5 μl icv) 2 min prior to the first AVP injection, blocked AVP‐induced sensitization; 3) a first injection of OT or [Thr4, Gly7]OT (10 pmol/5 μl) enhanced the sensitivity of the brain to the motor actions of a subsequent AVP injection given 24 h later; 4) the magnitude of this cross‐sensitization induced by OT pretreatment varied with dose and appeared to be ten times more potent than the sensitization induced by a first AVP injection; 5) injection of cpd 6 (100 pmol/5 μl) but not d(CH2)5Tyr(Me)AVP (100 pmol/5 μl icv) 2 min prior to the first OT injection (1 pmol/5 μl) blocked the cross‐sensitization induced by OT; 6) an injection of OT (100 to 1,000 pmol/5 μl) or [Thr4, Gly7]OT (10 pmol/5 μl) in rats that had been cross‐sensitized with OT or [Thr4, Gly7]OT pretreatment did not evoke enhanced motor responses; 7) OT injected 2 min prior to the second AVP injection in AVP‐sensitized rats did not block the enhanced AVP‐induced motor responses; 8) AVP‐induced [3H]inositol monophosphate accumulation in septal slices was also enhanced in rats cross‐sensitized by OT pretreatment.

These results suggest that while pre‐exposure of the rat brain to both AVP and OT alters the responsiveness of the rat brain to subsequent AVP exposures, AVP sensitization appears to be mediated via the V1 AVP receptor, whereas cross‐sensitization by OT may be mediated via the OT receptor. The ability of OT to alter the responsiveness of the rat brain to subsequent AVP injection suggests a role for this peptide in modulating central AVP actions.
Colchicine‐lnduced Accumulation of Estrogen Receptor and Progestin Receptor Immunoreactivity in Atypical Areas in Guinea‐Pig Brain
Authors: Blaustein Jeffrey D. , Olster Deborah H.
Published: September 2006

Using immunocytochemical techniques, cells containing estrogen and progestin receptors have been observed in many discrete regions of the guinea‐pig forebrain, including the mediobasal hypothalamus and preoptic area. While most reaction product is located within cell nuclei, we have reported abundant reaction product in perikaryal cytoplasm and neuronal processes as well. Ultrastructural analysis has revealed the presence of estrogen and progestin receptors in atypical subcellular sites within the hypothalamus, including dendrites and axon terminals. In order to determine if microtubule‐dependent intracellular transport is involved in intraneuronal transport of steroid hormone receptors, ovariectomized guinea‐pigs were injected intracerebroventricularly with the microtubule inhibitor, colchicine, and brain sections at the level of the hypothalamus were immunostained for estrogen receptors. This treatment resulted in the appearance of estrogen receptor immunoreactivity in the paraventricular and mediodorsal thalamic region, areas typically devoid of estrogen receptor‐immunoreactive cells in guinea‐pigs. In a second study on progestin receptors, we observed the colchicine‐induced accumulation of progestin receptor immunoreactivity in the paraventricular thalamic, mediodorsal thalamic and lateral dorsal thalamic areas as well as in the medial amygdala, all areas typically devoid of progestin receptor immunoreactivity. While estradiol injection induced progestin receptor immunoreactivity in the hypothalamus and preoptic area as described previously, it had no effect on the colchicine‐induced accumulation in the thalamus and amygdala. These results provide evidence that in some neurons, progestin receptors and estrogen receptors are transported intracellularly, apparently at a rapid enough rate that they do not ordinarily accumulate within the perikaryon.
Potential Corticotropin‐Releasing Factor Pathways in the Rat Brain as Determined by Bilateral Electrolytic Lesions of the Central Amygdaloid Nucleus and the Paraventricular Nucleus of the Hypothalamus
Authors: Koegler‐Muly Susan M. , Owens Michael J. , Ervin Gregory N. , Kilts Clinton D. , Nemeroff Charles B.
Published: September 2006

The projection fields of corticotropin‐releasing factor (CRF)‐containing perikarya in the rat central nervous system were studied using a combination of electrolytic lesions, microdissection and radioimmunoassay. The effects of bilateral electrolytic lesions of the central nucleus of the amygdala (Ce) or the paraventricular nucleus (PVN) of the hypothalamus were measured by a sensitive and specific radioimmunoassay. Following lesions of the Ce, CRF concentrations in the locus ceruleus (LC) were significantly decreased. Following lesions of the PVN, CRF concentrations in the median eminence were markedly decreased (>85%), with smaller but consistent reductions of CRF in the hippocampus as well. In contrast to the Ce lesions, PVN lesions resulted in increases in CRF concentratixhtmlons in the LC.

These results further confirm the projection of CRF‐containing cells from the PVN to the median eminence, provide evidence for a PVN‐hippocampal CRF pathway, and suggest that the PVN modulates CRF neurons innervating the LC. Moreover, the existence of a CRF‐containing pathway from the Ce to the LC appears likely, and such a circuit may play a role in the behavioral and physiological responses to stress.
Substance P Inhibits the Release of Anterior Pituitary Adrenocorticotrophin via a Central Mechanism Involving Corticotrophin‐Releasing Factor‐Containing Neurons in the Hypothalamic Paraventricular Nucleus
Authors: Larsen Philip J. , Jessop David , Patel Hemant , Lightman Stafford L. , Chowdrey Hardial S.
Published: September 2006

Chronic osmotic stimulation influences the hypothalamo‐adenohypophysial axis by inhibiting the synthesis of hypothalamic corticotrophin‐releasing factor (CRF‐41) and subsequently the secretion of basal and adrenalectomy‐elevated adrenocorticotrophin from the adenohypophysis. In the present study, we used a substance P antagonist to test the hypothesis that this inhibition is mediated centrally by substance P or other tachykinins. In control rats and rats given 2% saline to drink for 12 days, intracerebroventricular administration of a substance P antagonist elevated plasma adrenocorticotrophin and corticosterone levels. Using quantitative in situ hybridization histochemistry, it was also demonstrated that CRF mRNA increased in the medial parvocellular division of the paraventricular nucleus of saline‐treated as well as control rats 6 h after intracerebroventricular administration of the antagonist, while vasopressin mRNA in the medial parvocellular division of the paraventricular nucleus was increased in the control animals only. These results provide evidence that central endogenous substance P has an inhibitory influence over the synthesis and release of CRF‐41 both under normal conditions and during a chronic osmotic stimulus.
Altered Ratios of Beta‐Endorphin : Beta‐Lipotropin Released from Anterior Lobe Corticotropes with Increased Secretory Drive. I. Effects of Diminished Glucocorticoid Secretion
Authors: Young Elizabeth A. , Przewlocki Ryszard , Patel Paresh , Watson Stanley J. , Akil Huda
Published: September 2006

Previous studies have demonstrated that acute stress or ovine corticotropin‐releasing hormone (oCRH) in vivo, or oCRH in vitro, stimulates release of β‐endorphin over β‐lipotropin from anterior pituitary corticotropes. This occurs despite the predominance of β‐lipotropin in corticotrope peptide stores. In vitro studies with primary anterior pituitary cultures suggested that chronic exposure to oCRH results in a shift towards more β‐lipotropin secretion into the media than with short‐term exposure. The current studies explored whether increased secretory drive in vivo results in a similar shift towards more β‐lipotropin. We used removal of glucocorticoids by adrenalectomy or metyrapone blockade of corticosterone synthesis, to stimulate endogenous secretion of CRH and vasopressin. Both treatments resulted in shifts of the ratio of β‐endorphin: β‐lipotropin in plasma of experimental animals in comparison to the sham‐treated control rats. In vitro testing with oCRH of anterior lobe cultures from adrenalectomized or metyrapone‐treated rats demonstrated similar effects of these treatments on the ratio of β‐endorphin : β‐lipotropin. These changes occurred despite similar ratios of β‐endorphin : β‐lipotropin in anterior pituitary peptide stores.
Testosterone Enhances GABA and Taurine but not N‐Methyl‐D,L‐Aspartate Stimulation of Gonadotropin Secretion in the Goldfish: Possible Sex Steroid Feedback Mechanisms
Authors: Trudeau V. L. , Sloley B. D. , Peter R. E.
Published: September 2006

The effects of gonadal steroids on GABA‐, taurine (TAU)‐ and N‐methyl‐D, L‐aspartate (NMA)‐induced gonadotropin‐II (GTH‐II) release were investigated in male and female goldfish in vivo. In sexually regressed goldfish (both sexes mixed), intraperitoneal implantation for 5 to 10 days with solid Silastic pellets containing testosterone (100 μg/g), oestradiol (100 μg/g) or progesterone (100 μg/g) was previously shown to elevate serum sex steroid levels to values comparable to those in sexually mature animals, and to potentiate gonadotropin‐releasing hormone‐stimulated GTH‐II release. In the present study, testosterone but not oestradiol or progesterone enhanced the stimulatory effects of exogenous GABA (100 μg/g) on GTH‐II release in vivo. TAU (1 mg/g) stimulated GTH‐II release in sexually regressed mixed sex and sexually recrudescent male goldfish, and both testosterone and oestradiol implantation enhanced GTH‐II release induced by TAU. The glutamate agonist NMA (25 to 50 μg/g) was also found to stimulate GTH‐II release; however it was relatively less effective in elevating serum GTH‐II levels than GABA and TAU, and its effects were not modulated by sex steroid treatments. Pretreatment of goldfish with α‐methyl‐p‐tyrosine to deplete brain and pituitary catecholam‐ines did not affect NMA action on GTH‐II release. Our results indicate that GABA, TAU and NMA are involved in the neuroendocrine regulation of GTH‐II release in goldfish, and support the idea that testosterone participates in the positive feedback regulation of pituitary gonadotropin release in a non‐mammalian vertebrate by enhancing GABA‐ and TAU‐stimulated GTH release in vivo.
Development of Gonadotrophs and Thyrotrophs in the Female Foetal Sheep Pituitary: Immunocytochemical Localization Studies
Authors: Thomas Gregory B. , McNeilly Alan S. , Brooks A. Nigel
Published: September 2006

In order to investigate the ontogenesis of cell types in the pituitary gland, anterior pituitaries were collected from female foetal sheep at days 70, 100 and 130 of gestation (term = 145 days). Cells containing the common α‐subunit and the specific β‐subunits of luteinizing hormone (LH), follicle‐stimulating hormone (FSH) and thyroid‐stimulating hormone (TSH) were immunolocalized using the avidin‐biotin immunoperoxidase technique. LHβ‐containing cells were first detected in the foetal pituitary by day 70 of gestation. The number and intensity of staining of these LHβ cells increased by day 100 but had declined again by day 130. Immunopositive a‐subunit and FSHβ cells appeared by day 100 of gestation and had further increased in number and staining intensity by day 130. Cells containing TSHβ were present at day 70 and progressively increased in abundance and intensity through gestation. These data indicate that the development of LH‐ and FSH‐containing cells in the female foetal sheep pituitary is differentially regulated during foetal life, and that in the sheep free a‐subunit is not produced in significant amounts before the specific β‐subunits.
Direct Stimulation of the Pituitary by Transfer of Activated Leucocytes
Authors: Knight Richard A. , Stephanou Anastasis , Sarlis Nicholas J. , Chowdrey Hardial S. , Lightman Stafford L.
Published: September 2006

Some strains of rat develop arthritis, and have profoundly elevated adrenocorticotropin (ACTH) and corticosterone, following intradermal injection of an adjuvant containing heat‐killed mycobacteria. Transfer to syngeneic recipients of immune spleen (IS) cells taken from arthritic rats 14 days after injection of the adjuvant, but not of non‐immune cells, causes increased circulating ACTH and increased pituitary proopiomelanocortin (POMC) mRNA. Transfer of immune cells does not transfer the disease, but does protect recipients from subsequent challenge with the adjuvant. In these immune‐protected rats, the secondary immune response raises ACTH and POMC to levels similar to those seen in arthritic rats. These data show that endogenous inflammatory mediators have direct actions on the neuroendocrine system.
Application of a Percoll Density Gradient to Separate and Enrich Porcine Pituitary Cell Types
Authors: Torronteras R. , Castaño J. P. , Ruiz‐Navarro A. , Gracia‐Navarro F.
Published: September 2006

The application of a Percoll density gradient cell separation procedure to the pituitary gland of neonatal, prepubertal and mature pigs is described. After enzymatic dispersion, cell viability was 90% to 98% as determined by uptake of Trypan Blue. Recovery of dissociated cells after application of the gradient ranged from 80% to 95%. The dissociated cells were separated in several fractions that were characterized immunocytochemically using different antisera. We obtained highly enriched fractions of gonadotrope (gonadotropins), somatotrope (growth hormone) and lactotrope (prolactin) cells for each age. Concentration of the cell types (purity) was higher than 60% in the following fractions: 1) gonadotropin cells, fraction 15 (1.033 g/cc of density) from mature animals; 2) growth hormone cells, fraction 3 (1.121 g/cc of density) from neonatal animals and fraction 9 (1.087 g/cc) from prepubertal animals; and 3) prolactin cells, fraction 7 (1.094 g/cc) and 10 (1.082 g/cc) from neonatal animals and fraction 14 (1.051 g/cc) from mature pigs. In thyrotrope (thyroid‐stimulating hormone) and corticotrope (adrenocorticotropin) cells, enriched fractions were also obtained, although the values of purity were lower (20% to 58%). In conclusion, the proposed cell separation and enrichment technique is suitable for the isolation, purification and examination of porcine pituitary cell types and subpopulations, and offers major advantages such as simplicity, rapidity, efficiency and reproducible results.
Neuroanatomical and Physiological Evidence for the Involvement of Pituitary Adenylate Cyclase‐Activating Polypeptide in the Regulation of the Distal Lobe of the Frog Pituitary
Authors: Yon Laurent , Jeandel Lydie , Chartrel Nicolas , Feuilloley M. , Conlon J. Michael , Arimura Akira , Fournier Alain , Vaudry Hubert
Published: September 2006

Pituitary adenylate cyclase‐activating polypeptide (PACAP) is a 38 amino‐acid peptide which belongs to the glucagon/secretin/ vasoactive intestinal peptide superfamily. The sequence of PACAP is identical in all mammalian species studied so far but frog PACAP differs by one amino‐acid from mammalian PACAP. The aim of the present study was to investigate the presence of PACAP in the hypothalamo‐pituitary complex of the frog Rana ridibunda and to determine the biological activity of frog PACAP on homologous pituitary cells. The distribution of PACAP‐containing neurons and fibers was examined by the indirect immunofluores‐cence method using an antiserum raised against the N‐terminal region of the peptide. In the hypothalamus, PACAP‐immunoreactive perikarya were localized in the preoptic nucleus and the dorsal and ventral infundibular nuclei. Beaded nerve fibers were observed coursing from the ventral infundibular nucleus to the external vascular layer of the median eminence. A dense network of immunoreactive axons terminated in the vicinity of the capillaries of the hypophysial portal system. The neurointermediate lobe and the distal lobe of the pituitary were devoid of immunoreactive elements. The amount of PACAP‐like immunoreactive material in hypothalamus extracts was measured by radioimmunoassay; the apparent concentration of PACAP was 4.5 ng/mg protein. Synthetic frog PACAP38 and PACAP27 induced a similar dose‐dependent stimulation of cAMP production in isolated frog distal lobe pituitary fragments (ED50= 2 × 10−8 M). At the maximum dose tested (5 × 10−6 M), both frog PACAP38 and PACAP27 produced a 4‐fold increase in cAMP production. In contrast, the truncated form [Des‐His1frog PACAP38 did not affect adenylate cyclase activity demonstrating therefore that the N‐terminal histidyl residue is essential for the biological activity of the peptide. [Des‐His1]frog PACAP38 did not antagonize the stimulatory effect of frog PACAP38 or PACAP27 on cAMP production. Taken together, these data support the concept that, in amphibians as in mammals, PACAP may act as a hypophysiotropic neuropeptide.
Local Tetrodotoxin Blocks Chronic Stress Effects on Corticotropin‐Releasing Factor and Vasopressin Messenger Ribonucleic Acids in Hypophysiotropic Neurons
Authors: Sawchenko P. E. , Arias C. A. , Mortrud M. T.
Published: September 2006

To test the hypothesis that synaptic inputs to the paraventricular nucleus mediate stress‐induced increases in corticotropin‐releasing peptide expression in the paraventricular nucleus of the hypothalamus (PVH), relative levels of the mRNAs encodingv corticotropin‐releasing factor (CRF) and arginine vasopressin (AVP) were followed, in situ, in animals subjected to chronic footshock stress and concurrent local administration of tetrodotoxin or vehicle. Consistent with previous findings, a 7‐day exposure to chronic footshock resulted in a 2.1‐fold increase in CRF mRNA levels in the parvocellular division of the PVH. The footshock paradigm also resulted in at least a 41% increase in AVP transcripts in this same region; this effect was localized predominantly to CRF‐immunoreactive neurons. The Stressor did not significantly alter AVP mRNA levels in the magnocellular division of the PVH. Tetrodotoxin, administered to the PVH via osmotic minipump, blocked the stress‐induced rise in CRF and AVP mRNAs, but had no significant effect on basal levels of these transcripts. The results support the view that maintenance of the enhanced central drive on pituitary‐adrenal activity seen in response to chronic stress is mediated via neural inputs to the PVH.
Involvement of Central Corticotropin‐Releasing Factor (CRF) in Suckling‐Induced Inhibition of Luteinizing Hormone Secretion in Lactating Rats
Authors: Walker Claire‐Dominique , Dallman Mary F. , Palmer Abraham A. , Steele Marianne K.
Published: September 2006

The lack of ovulation and the inhibition of reproductive functions observed in many species during lactation is closely related to the intensity of the suckling stimulus. However, the mechanisms by which suckling inhibits hypothalamic GnRH and pituitary LH secretion in rats are still unclear. Since we recently demonstrated that suckling is a persistent stimulus to the adrenococortical system of the rat, we tested the hypothesis that suckling‐induced activation of central CRF release may mediate the associated inhibition of GnRH secretion. Lactating females were ovariectomized (OVX) on day 2 of lactation, and equipped with icv guide cannula on day 2 and indwelling jugular catheters on day 5 before testing on day 7. Lactating females were separated from their pups for 24 h prior to the suckling test with the following pretreatments: 1) icv injection with artificial CSF (aCSF) or a specific CRF antagonist, alpha‐helical CRF (9–41), (25μg/rat, CRF‐AX) 15 min prior to pup reunion or 2) iv injection of normal sheep serum (NSS) or CRF antiserum (CRF‐AB) 4 h prior to pup reunion. Plasma ACTH, LH and PRL concentrations were determined prior to and at various intervals after pup reunion. After 3 h of suckling, LH and PRL responses to a bolus injection of GnRH (10 ng/rat) were measured; a bolus injection of Angiotensin II (All, 5 μg/rat) was administered after 4 h to test for ACTH responses. Non‐lactating females injected with GnRH and All were used as controls.

In animals treated icv with aCSF or iv with NSS, the suckling stimulus resulted in increased ACTH and PRL secretion with a decrease in LH release. Iv CRF‐AB abolished the increase in ACTH, but did not affect the increase in PRL nor the decrease in LH secretion. In contrast, and in support of our hypothesis, icv CRF‐AX abolished the suckling‐induced decline in LH secretion without altering the increase in PRL and ACTH secretion. Finally, we did not observe lactation‐related changes in LH (or PRL) responses to a bolus GnRH injection or in ACTH responses to All stimulation.

Our results suggest that: 1) central CRF release is involved in the decrease in LH secretion which occurs following suckling in lactating females, and 2) the changes in LH secretion during suckling are not mediated by an effect of CRF at the median eminence or by acute changes in pituitary responsiveness to GnRH.
Effect of a Single Intravenous Injection of N‐Methyl‐D,L‐Aspartic Acid on Secretion of Luteinizing Hormone and Growth Hormone in Holstein Bull Calves
Authors: Shahab M. , Nusser K. D. , Griel L. C. , Deaver D. R.
Published: September 2006

The role of N‐methyl‐D‐aspartate (NMDA) receptor activation in the central regulation of luteinizing hormone (LH) and growth hormone (GH) was tested by administering a bolus intravenous dose of N‐methyl‐D,L‐aspartic acid (NMA), a NMDA receptor agonist, to 24‐week‐old intact (n = 5), estradiol‐treated intact (n = 3) and castrated (n = 3) Holstein bull calves. The calves were bled for 12 h pre‐ and 100 min post‐NMA injection (1.75 mg/kgBW) periods at 10min intervals. Concentrations of LH and GH in plasma were measured by specific RlA. Prior to administration of NMA, the average concentration of LH, but not GH, differed significantly among the 3 groups. As expected, administration of estradiol prevented the normal ontogeny of pulsatile LH secretion, while castration resulted in an increased frequency of LH discharges. Injection of NMA resulted in an acute (P<0.001) release of LH in 3 of 5 intact and 3 of 3 estradiol‐treated intact calves with the peak response being observed at 20 min (3.18 ±1.3 and 5.58 ± 1.3 ng/ml, respectively) following the challenge. Treatment with NMA did not alter the release of LH in castrate calves. Concentrations of GH in plasma increased (P<0.001) within 20 to 30 min after administration of NMA in intact, estradiol‐treated intact and castrate calves with a similar response being observed in each group. Based on these findings, we suggest an involvement of glutamatergic neurotransmission in the hypothalamic or supra‐hypothalamic control of LH and GH secretion, and that the excitatory effects of NMDA receptor activation on LH release are overtly influenced by gonadal steroids in bull calves.
Immunocytochemical Localization of Progesterone Receptors in Galanin Neurons in the Guinea Pig Hypothalamus
Authors: Warembourg Maryvonne , Jolivet Andre
Published: September 2006

A double‐label immunocytochemical technique was used to determine whether progesterone receptor‐containing neurons in the female guinea‐pig hypothalamus also contained galanin. Adult ovariectomized guinea‐pigs were primed by estradiol to induce progesterone receptors and injected intracerebroventricularly with colchicine to visualize galanin‐immunopositive neurons. A small proportion of progesterone receptor‐containing perikarya in the medial preoptic area and the mediobasal hypothalamus were found to be immunoreactive for galanin. The medial preoptic, periventricular and arcuate nuclei showed the greatest concentration of double‐labelled cells. Galanin varicosities appeared in close proximity to neurons with progesterone receptor‐containing nuclei. These results provide neuroanatomical evidence that a subset of hypothalamic galanin‐immunoreactive neurons is directly regulated by progesterone.