bluuebunny/crossref_metadata_2025 huggingface dataset:
Melatonin Regulates the Phosphorylation of CREB in Ovine Pars Tuberalis*
Authors: McNulty S. , Rosst A. W. , Barrettt P. , Hastings M. H. , Morgan P. J.
Published: September 2006

This study investigated whether melatonin could modulate the phosphorylation of the calcium/cyclic AMP response‐element binding‐protein (CREB) within primary cell cultures of ovine pars tuberalis (oPT) and pars distalis (oPD). Gel shift assays confirmed the presence of nuclear factors able to alter the electrophoretic mobility of a 32P‐labelled CRE oligonucleotide. Two shifted bands were observed probably due to monomer and dimer binding to the CRE. Each band was supershifted by antisera directed against both CREB and the phosphorylated form of CREB (P‐CREB), consistent with a specific role of CREB proteins in transcriptional regulation. To study the physiological role of CREB, the nuclear immunoreactivity for P‐CREB was followed in primary cultures of oPT given different pharmacological treatments. Cells stimulated with forskolin responded with a robust time‐ and dose‐dependent increase in nuclear phospho‐CREB immunoreactivity (P‐CREB‐ir), confirming that activation of this transcription factor occurred through the cyclic AMP‐PKA pathway. Maximal stimulation was achieved within 15 min and persisted for up to 1 h. Treatment with melatonin alone did not alter basal P‐CREB‐ir levels, yet melatonin inhibited the forskolin‐induced increase in P‐CREB‐ir in a dose‐dependent manner (IC50 of between 10−10M and 10−8 M melatonin when tested against 1 μM forskolin). In contrast, in primary cultures of oPD, melatonin failed to block forskolin‐stimulated increases in either the content of cyclic AMP or the intensity of nuclear P‐CREB‐ir, confirming that the action of melatonin upon P‐CREB‐ir is tissue specific. These results demonstrate that, consistent with its inhibitory effect on the activation of PKA within oPT, melatonin prevents or reverses the phosphorylation of CREB induced by activation of the cyclic AMP signal transduction pathway. Therefore melatonin has the potential to regulate gene expression in the oPT by acting upon the CREB transcription factor. However, this paper also shows that 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) which activates PKC also leads to the phosphorylation of CREB in oPT cells, suggesting the potential involvement of other signal transduction pathways in the transcriptional regulation of these cells.
Hypothalamic Estrogen Receptor‐lmmunoreactivity in Prepubertal vs Adult Female Guinea Pigs
Authors: Olster Deborah H.
Published: September 2006

Juvenile guinea pigs (18–20 days old) rarely display lordosis in response to estradiol and progesterone treatments that elicit sexual behavior in adult females. Nor do immature animals release a preovulatory‐like surge of luteinizing hormone in response to estradiol. In vitro radioligand binding assays have revealed similar concentrations of estrogen receptors in thehypothalamus and preoptic area of prepubertal and adult guinea pigs. The aim of the present study was to compare estrogen receptor‐immunoreactivity in a variety of forebrain regions of immature and adult guinea pigs, to determine whether age differences in estrogen receptor levels inmore discrete portions of the hypothalamus and preoptic area exist. Forebrain tissue from juvenile (17 days) and adult females (> 6 weeks), ovariectomized 6 days previously, was processed forestrogen receptor‐immunoreactivity, using Abbott Laboratories' H222 anti‐human estrogen receptor antibody. Juveniles had estrogen receptor‐immunoreactive cells in all of the same regions as adults: medial preoptic area, medial preoptic nucleus, bed nucleus of the stria terminalis, periventricular, paraventricular, dorsomedial and arcuate nuclei, ventrolateral and anterior hypothalamic regions, and amygdala. Among the areas in which estrogen receptor‐immunoreactivity was quantified (medial preoptic area, medial preoptic nucleus, anterior periventricular nucleus, arcuate nucleus and ventrolateral hypothalamus), the only region in which an age difference in estrogen receptor‐immunostaining was observed was the rostral portion of the ventrolateral hypothalamus. Juvenile females had, on average, 30% fewer estrogen receptor‐immunoreactive cells in asample of this region than adults (440 ± 25 vs 626 ± 25, P = 0.001). These data are consistent with the hypothesis that insufficient estrogen receptors in the rostral ventrolateral hypothalamus may underlie, in part, the markedly deficient responses of juvenile female guinea pigs to estradiol.
Down‐Regulation of Estrogen Receptor Immunoreactivity by 17β‐estradiol in the Guinea Pig Forebrain
Authors: Meredith John M. , Auger Catherine J. , Blaustein Jeffrey D.
Published: September 2006

Low amplitude pulses of estradiol‐17β (E2‐17β) are more effective than large single bolus injections or constant exposure to E2‐17β in inducing progesterone‐facilitated sex behavior in female rats and guinea pigs. The present study examined whether the increased responsiveness to E2‐17β is due to an increase in the number of estrogen receptors in the estrogen receptor rich areas of the hypothalamus and amygdala.

Initial studies examined the rapid effects (20 min) of a high dose of E2‐17β (50 μg) on estrogen receptor immunostaining using either the H222 antibody or the ER 21 antiserum. ER 21 immunostaining was not affected by the E2‐17β treatment suggesting that it binds to both occupied and unoccupied estrogen receptors. Therefore the ER 21 antiserum was used to characterize the regulation of estrogen receptor immunoreactivity (ER‐IR) by E2‐17β.

ER‐IR was examined for 48 h and serum E2‐17β for 24 h following a 2 μg s.c. injection of E2‐17β (a dose similar to that used in multiple pulse paradigms). Serum E2‐17β peaked 15 to 30 min following the injection and returned to baseline values by 1 h. In all but one area maximal suppression of ER‐IR occurred at 12 h.

In summary, 1) decreases in estrogen receptor immunoreactivity following E2‐17β are consistent with studies in which estrogen receptors were assayed by binding assays and estrogen receptor mRNA was determined by in situ hybridization; 2) the ER 21 antiserum is able to detect both occupied and unoccupied estrogen receptors and 3) H222 immunoreactivity is influenced by the presence of E2‐17β, so that the level of H222‐IR is a reflection of ligand/receptor binding dynamics.

The data suggest that up‐regulation of estrogen receptors does not account for the increase in behavioral sensitivity which is observed following multiple pulses of E2‐17β.
Activation of Vasopressin Neurons in Aging and Alzheimer's Disease
Authors: Lucassen P. J. , Salehi A. , Pool C. W. , Gonatas N. K. , Swaab D. F.
Published: September 2006

The supraoptic (SON) and paraventricular nuclei (PVN) of the human hypothalamus are production sites of vasopressin (AVP) and oxytocin (OXT). Although the hypothalamus is affected in Alzheimer's disease (AD), previous work has not only shown that in these two nuclei no neurons are lost, neither during aging nor in AD, but that the number of AVP‐expressing neurons and their nucleolar size had even increased with age. These observations indicated that the peptide synthesis of the AVP neurons was activated in the oldest age‐groups. Recently published, qualitative observations, using the area of the Golgi Apparatus (GA) as a sensitive parameter for neurosecretory activity, confirmed the activation of SON and PVN neurons with age in human; however, in this report the neurons were not identified according to their neuropeptide content. In the present quantitative study we determined whether the AVP neurons were indeed activated as a result of the aging process in controls and AD patients. We applied a polyclonal antiserum directed against the medial cisternae of the GA on formalin‐fixed, paraffin‐embedded tissue sections taken from the dorsolateral SON (dl‐SON) of 10 controls and 10 AD patients, and performed our measurements in this area that is known to be predominantly occupied (90–95%) by AVP neurons. In addition, the sparse OXT cells present in the area of study, were excluded from the measurements on the basis of alternative sections stained for OXT. In the dl‐SON, the area occupied by the GA and the cellular profile area per patient were quantified by means of image analysis. The results show a significant increase in GA area with age in controls and in AD, demonstrating an activation of the AVP neurons in the dl‐SON of the human hypothalamus in these two conditions. No changes were observed in the cellular profile areas with age, neither in the controls nor in AD, suggesting that the GA area is a much more sensitive parameter for monitoring activity changes in post‐mortem material than neuronal size. It is proposed that this activation of AVP cells with age, which has been suggested to be a compensatory response to the age‐related loss of AVP receptors in the kidney, might be the basis of the stability of these neurons in aging and AD.
Pituitary Adenylate Cyclase Activating Peptide‐38 (PACAP‐38), PACAP‐27, and PACAP Related Peptide (PRP) in the Rat Median Eminence and Pituitary
Authors: Mikkelsen Jens D. , Hannibalt Jens , Fahrenkrugt Jan , Larsen Philip J. , Olcese James , McArdle Craig
Published: September 2006

Pituitary adenylate cyclase activating peptide (PACAP) is a member of the vasoactive intestinal peptide‐like peptide family. It is found in the hypothalamus, where the PACAP precursor is processed to form PACAP‐38, the C‐terminal truncated PACAP‐27 and PACAP related peptide (PRP). Both PACAPs are potent stimulators of anterior pituitary adenylate cyclase activity, but the physiologically relevant anatomical sources of PACAP and possible importance of PRP in this regard are poorly understood. Using immunocytochemistry with epitope‐specific antisera, we now show that PACAPS38, PACAP27‐ and PRP‐positive nerve fibres are all present in the rat median eminence. The major immunoreactive species present was PACAP38. Numerous PACAP38‐immmunoreactive nerve fibres were observed in the internal layer and a few were present in the posterior pituitary lobe. The external layer of the median eminence contained a few PACAP‐38‐immunoreactive fibres and PACAP‐38‐positive nerve terminals were rarely seen in the perivascular portal spaces. Surprisingly, delicate PACAP‐38‐positive nerve fibres were identified in the anterior pituitary lobe intermingled between the pituitary cells although none of the secretory pituitary cells contained immunoreactive PACAP38, PACAP27 or PRP and preproPACAP mRNA was not detected in the gland by Northern blotting or in situ hybridization. PACAP‐27‐ and PRP‐immunoreactive nerve fibres and terminals were found in the same locations as PACAP‐38 although generally in lower numbers. Specific radioimmunoassays and HPLC revealed that PACAP‐38 accounts for the vast majority of the adenohypophyseal PACAP‐immunoreactivity, whereas PACAP‐27 and PRP were found in low to undetectable concentrations. In primary cultures of rat pituitary cells and in the clonal gonadotrope‐derived aT3‐1 cell line, PACAP‐27 and PACAP‐38 were equipotent stimulators of cAMP accumulation, whereas PRP was ineffective. We conclude that the distribution of PACAP‐imrnunoreactive nerve fibres in the hypothalamus of the adult male rat is not that expected for a classic releasing factor suggesting that other sources of PACAP are relevant for stimulation of anterior pituitary cells or that the hypothalamic PACAP system is activated under specific endocrine or developmental conditions.
Vasoactive Intestinal Polypeptide in the Suprachiasmatic Nucleus of the Mink (Mustela vison) Could Play a Key Role in Photic Induction
Authors: Martinet Lise , Bonnefond Catherine , Peytevin Jocelyne , Monnerie Régine , Marcilloux Jean Claude
Published: September 2006

The present study was conducted to visualize neuropeptides in the SCN of a mustelid, the American mink in which seasonal cycles of reproduction rely totally on the annual changes in day length. At this time, data in mustelids are lacking. Results were obtained with in situ hybridization (ISH) using synthetic oligonucleotide vasopressin (AVP) and somatostatin (SOM) and with single and dual immunohistochemistry (IHC) performed with antisera against AVP, SOM, vasoactive intestinal polypeptide (VIP), gastrin releasing peptide (GRP) and met‐enkephalin (Met‐ENK) in untreated (AVP and VIP) or colchicine (SOM, Met‐ENK and GRP) treated adult male and female mink. The most striking result, evidenced by ISH as well as IHC was the lack of AVP, SOM and Met‐ENK immunoreactive (ir)‐neurons in the SCN. In contrast, strongly VIP ir‐perikarya were widely distributed within the SCN and gave rise to a dense network of fibres extending within the periventricular (peVA) and subparaventricular (subPVA) areas. Weakly GRP ir‐perikarya were also observed in the median part of the SCN. Dual IHC revealed that the magnocellular neurons located just dorsal to the SCN, in the peVA and subPVA co‐stored AVP with VIP, SOM or Met‐ENK. The lack of SCN AVP and SOM ir‐neurons, reported for the first time in a mammalian species, raises the question of their implication in the functions of the circadian pacemaker and its entrainment by the light/dark cycle in other species. The significance of the large neurons co‐storing peptides in the terminal field of VlPergic fibres originating in the SCN has also to be determined. These results suggest that VIP could be of major importance in processing photic information mediating circadian entrainment and consequently annual rhythms.
Opposing Neuroendocrine Actions of the Natriuretic Peptides: C‐Type and A‐Type Natriuretic Peptides do not Interact with the same Hypothalamic Cells Controlling Prolactin Secretion
Authors: Samson Willis K. , Huang Fong‐Lee S. , Fulton R. Jerrold
Published: September 2006

The two major members of the family of natriuretic peptides (NPs) in brain, A‐type natriuretic peptide (ANP) and C‐type natriuretic peptide (CNP) exert opposing actions on the neuroendocrine regulation of prolactin (PRL) secretion. We have targeted for compromise and destruction cells within the diencephalon which bear receptors for ANP (NPR‐A receptors), CNP (NPR‐B receptors), or both peptides (NPR‐C receptors) using novel cytotoxin cell targeting methodology in order to determine if the neuroendocrine effects of these two peptides are exerted on similar cell systems. In animals pretreated with ANP conjugated to the cytotoxic A chain of ricin, central administration of a dose of ANP which is known to inhibit PRL secretion did not alter PRL levels in plasma; however, subsequent administration of CNP elicited the stimulation of PRL secretion. In rats pretreated with CNP‐ricin A chain conjugate, a treatment we hypothesize targets for destruction CNP responsive cells, ANP injection did inhibit PRL secretion, while the stimulatory effect of CNP was absent. These results suggest that the neuroendocrine effects of these two natriuretic peptides on PRL secretion are expressed on different cellular elements of the hypothalamo‐pituitary axis. Furthermore, they reveal that neither peptide acts directly on the tuberoinfundibular dopamine system since pretreatment with either cytotoxin conjugate failed to alter basal PRL levels. Thus ANP and CNP do not appear to express opposing actions on the same cell systems, suggesting the recruitment of each peptide individually by differing, unique stimuli for PRL release.
Central Glucopenia Induced by 2‐Deoxy‐D‐Glucose Stimulates Somatostatin Secretion in the Rat
Authors: Sato Makoto , Murao Koji , Matsubara Shuji , Niimi Michio , Chihara Kazuo , Takahara Jiro
Published: September 2006

The mechanisms involved in 2‐deoxy‐D‐glucose (2‐DG)‐induced growth hormone (GH) suppression in the rat were examined. Conscious male rats were given 2‐DG by intracerebroventricular (icv) injection and the pulsatile GH secretion was monitored for 6 h. The single icv injection of 2‐DG (8 mg/rat) eliminated pulsatile GH secretion in conscious rats. Pretreatment with somatostatin (SS) antiserum completely restored the suppressed GH secretion in the 2‐DG treated rats. Hypothalamic GH‐releasing hormone (GRH) and SS mRNA levels were not altered by single and multiple icv injections of 2‐DG. These findings suggest that 2‐DG‐induced GH suppression is primarily due to hypersecretion of SS without a significant change at the transcription level in the rat.
Long‐Term Antidepressant Treatment Reduces Behavioural Deficits in Transgenic Mice with Impaired Glucocorticoid Receptor Function
Authors: Montkowski Alexandra , Barden Nicholas , Wotjak Carsten , Stec Ingemar , Ganster Julia , Meaney Michael , Engelmann Mario , Reul Johannes M. H. M. , Landgraf Rainer , Holsboer Florian
Published: September 2006

Impaired cognitive function and enhanced activity of the hypothalamic‐pituitary‐adrenocortical system are among the cardinal symptoms of major depression in humans that resolve after successful antidepressant treatment. We used a transgenic mouse model expressing antisense RNA complementary to that of glucocorticoid receptor (GR) mRNA to test the hypothesis that reduced GR function can cause these clinical disturbances. The transgenic mice show profound behavioural changes in a number of animal tests that are indicative of cognitive impairment. These mice also have elevated plasma corticotropin concentrations in response to stress. After long‐term treatment with moclobemide, a reversible inhibitor of monoamine oxidase type A that acts clinically as an antidepressant, both the behavioural deficits and the hormonal alterations disappeared. These observations suggest that a transgenic mouse with GR dysfunction may be a useful model for investigation of drug effects on the cognitive and neuroendocrine aspects of depression.
LHRH Pulse Generator is Stimulated by Naloxone in the Pentobarbital‐Blocked Proestrous Rat
Authors: Kimura Fukuko , Jinnai Kayoko , Sano Akane
Published: September 2006

Previous studies by us and others led us to hypothesize that there are separate LHRH pulse and surge generators in the rat brain. The present study was designed to detect the activity of LHRH pulse generator by checking changes in LH secretion and the multiunit activity (MUA) of the arcuate‐median eminence region of the hypothalamus during infusions of naloxone (NAL, 2 mg/h) in the proestrous rat in which the LHRH surge generator activity was blocked by pentobarbital sodium (PB, 32 mg/kg bw, ip). The animals were subjected to blood sampling in the morning (1000–1300 h) or afternoon (1400–1700), and injected with PB at 09.45 or 13.45, respectively. During saline infusions in the rat given PB injection at either 09.45 or 13.45, serum LH levels were low but fluctuated significantly, suggesting a pulsatile secretion in either the morning or the afternoon period. The pulse intervals were an average 28.2 min in the morning and 42.2 min in the afternoon. NAL infusions decreased the pulse interval significantly, to 22.0 min in the morning and to 27.0 min in the afternoon. In the electrophysiological experiment, characteristic increases in the MUA (volleys), which occur in association with the initiation of an LH pulse and therefore are considered to represent an increased activity of the LHRH pulse generator, appeared during NAL (5 mg/h) infusions in either the morning or the afternoon. These results strongly suggest that separate LHRH pulse and surge generators exist in the brain, and that, even during the critical period of proestrus, the activity of LHRH pulse generator is disclosed by PB, which, on the other hand, arrests the surge generator.
The Effect of NO‐Donors in Bovine and Rat Pineal Cells: Stimulation of cGMP and cGMP‐Independent Inhibition of Melatonin Synthesis
Authors: Maronde E. , Middendorff R. , Mayer B. , Olcese J.
Published: September 2006

The presence of soluble guanylate cyclase in the pineal and its regulation by adrenergic pathways has been well documented. Recent evidence points to adrenergically stimulated nitric oxide generation as a mechanism for coupling this pathway. To what extent nitric oxide (NO) signalling can influence adrenergically stimulated melatonin synthesis has not been investigated. Cyclic guanosine 3′,5′‐monophospate (cGMP) signal transduction in the bovine pineal has also received little attention. We describe in the present report: 1) a dose‐dependent elevation of cGMP in response to the nitrovasodilators, sodium nitroprusside (SNP) and 3‐morpholino‐sydnonimine (SIN‐l), 2) a dose‐dependent inhibition of melatonin synthesis by SNP and SIN‐1, but not by 8‐Br‐cGMP in both bovine and rat pineal cell cultures, which is not due to cytotoxicity as judged by two different approaches, and 3) immunohistochemical evidence for the presence of nitric oxide synthase (NOS) (EC 1.14.23.‐) in the intact bovine pineal gland and in cultured bovine pinealocytes. These data support the view that NOS is a component of the cGMP‐generating system in mammalian pinealocytes. Although NO‐donor molecules are also potent activators of cGMP accumulation, they may have other important actions in the pineal, namely the inhibition of adrenergic‐stimulated melatonin synthesis. As SNP and SIN‐1 exerted this inhibitory effect on cells regardless of whether they were stimulated by isoproterenol, forskolin or 8‐Br‐CAMP it would appear that NO‐donors can act ‘downstream’ from the receptor/adenylate cyclase level.
Expression of Biologically Active Procorticotrophin‐Releasing Hormone (proCRH) in Stably Transfected CHO‐K1 Cells: Characterization of Nuclear proCRH
Authors: Morrison Ewan , Tomasec Peter , Linton Elizabeth A. , Lowry Philip J. , Lowenstein Pedro R. , Castro Maria G.
Published: September 2006

Corticotrophin‐releasing hormone (CRH) is a 41 amino acid neuropeptide which is cleaved at a pair of dibasic amino acids from a larger precursor molecule (pre‐proCRH) by the action of endopeptidases. In cells possessing a regulated secretory pathway, sorting of proneuropeptides and prohormones occurs within the trans‐Golgi network, where they are finally packaged into secretory vesicles to be released in response to an external stimulus. Such cells also possess a constitutive secretory pathway, and neuropeptides are also translocated into this subcellular compartment. We have recently established stably transfected CHO‐K1 cells expressing the rat pre‐proCRH cDNA, and shown that proCRH was localized within the secretory pathway and the nucleus of transfected cells. Both the cytoplasmic and nuclear species of IR‐CRH displayed an apparent molecular weight of approximately 19 kDa, consistent with the size of the uncleaved CRH precursor molecule.

In this paper, we further characterized the bitopological, i.e. nuclear and cytoplasmic localization of proCRH within transfected CHO‐K1 cells. Immunoreactive nuclear CRH was not extractable using detergents (Triton X‐100 and CHAPS), 10 mM salt washes or RNase digestion but could be abolished by digestion with DNase I. These results therefore suggest that nuclear proCRH is in close association with DNA/chromatin. Treatment of transfected cells with inhibitors of protein and RNA synthesis for up to 24 h had no effect upon immunoreactive nuclear CRH, indicating that it is very stable with a long half life. Brefeldin A treatment had no effect upon the nuclear translocation of newly synthesized proCRH, suggesting that late stages of the secretory pathway (i.e. post rough endoplasmic reticulum compartments) of the transfected cells do not play a role in proCRH nuclear transport. We also demonstrate that proCRH synthesized within stably transfected CHO‐K1 cells is capable of stimulating ACTH release from primary cultures of anterior pituitary cells, therefore showing for the first time that the intact precursor is also biologically active and could act as an ACTH secretagogue in‐vivo.
Antisense Vasopressin Oligonucleotides: Uptake, Turnover, Distribution, Toxicity and Behavioral Effects
Authors: Meeker Rick , LeGrand Gordon , Ramirez Julio , Smith Tracy , Shih Ying‐Hsien
Published: September 2006

The uptake, turnover, distribution, toxicity and behavioral effects of antisense vasopressin oligonucleotides were investigated to define how these compounds interact with neural tissue to inhibit translation of a target mRNA. Both phosphorothioate modified and unmodified oligonucleotides are rapidly taken up by mammalian neural tissue. Turnover of the unmodified oligonucleotide was found to be fast (t1/2 < 1 h) relative to the phosphorothioate modified oligonucleotide (t1/2= 12 h). The phosphorothioate vasopressin antisense oligonucleotide suppressed vasopressin synthesis in vitro at concentrations below the toxic threshold of approximately 5 μM. Intracranial injections of phosphorothioate antisense oligonucleotide into the region of the SON in vivo, resulted in a small decrease in vasopressin mRNA and a compensatory drinking response within the first 24 h, consistent with a deficit in vasopressin translation with kinetics similar to those observed in vitro. Water intake returned to normal by the second day indicating relatively rapid clearance of the oligonucleotide and minimal side effects. Although the mechanisms of accumulation and details of the molecular interactions are still unknown, our observation of preferential uptake and/or retention of oligonucleotide within a subset of neurons in vitro suggests some process of selective targeting. Thus, low concentrations of oligonucleotides targeted to the untranslated 5′ end of vasopressin mRNA can be effective for the acute and reversible control of vasopressin synthesis in mammalian CNS with relatively rapid onset of behavioral effects and minimal side effects.
Glutaminyl‐Cyclase Expression in the Bovine/Porcine Hypothalamus and Pituitary
Authors: Böckers Tobias M. , Kreutz Michael R. , Pohl Thomas
Published: September 2006

The expression of the glutaminyl cyclase (QC), an enzyme responsible for the post‐translational modification of N‐terminal pyroglutamyl residues of neuropeptide precursors, was examined in bovine/porcine hypothalamic and pituitary tissue by means of immunocytochemistry and in situ hybridization. In the anterior pituitary a distinct pattern of QC immunoreactivity and mRNA expression was found associated exclusively with somatotrophs. In corticotrophs of the pars intermedia QC expression was undetectable, whereas a small portion of putative pars tuberalis cells in the rostra1 part of the pars distalis were heavily labelled. The neurointermediate lobe was devoid of signals for QC mRNA, but showed significant QC‐immunoreactivity on secretory granules of axonal nerve endings. Also nuclei of the hypothalamus were found to be positive for QC‐immunoreactivity. Intense labelling was observed in the nucleus supraopticus and nucleus paraventricularis. Staining of the nucleus periventricularis was found to be moderate, whereas no labelling of perikarya in the nucleus arcuatus, the preoptic area and the nucleus suprachiasmaticus was detectable. Moreover, varicose fibers stained positive for QC‐immunoreactivity and could be identified in the main transport route from the hypothalamus to the pars neuronalis (tractus hypothalamo hypophysialis). These results suggest that the enzyme is transported via the same routes as its substrate/product to the median eminence or the neural lobe. Furthermore, the mapping of the cellular QC distribution reveals a strikingly distinctive expression pattern, that should be useful for the identification of yet undiscovered places of peptide synthesis and processing.
Contribution of the Ventral Subiculum to Inhibitory Regulation of the Hypothalamo‐Pituitary‐Adrenocortical Axis
Authors: Herman James P. , Cullinan William E. , Morano Maria Ines , Akil Huda , Watson Stanley J.
Published: September 2006

Anatomical studies indicate that the ventral subiculum is in a prime position to mediate hippocampal inhibition of the hypothalamo‐pituitary‐adrenocortical (HPA) axis. The present study evaluated this hypothesis by assessing HPA function following ibotenic acid lesion of the ventral subiculum region. Rats with lesions of the ventral subiculum (vSUB) or ventral hippocampus (vHIPPO) did not show changes in basal corticosterone (CORT) secretion at either circadian peak or nadir time points when compared to sham‐lesion rats (SHAM) or unoperated controls. However, rats with vSUB lesions exhibited a prolonged glucocorticoid stress response relative to all other groups. Baseline CRH mRNA levels were significantly increased in the medial parvocellular paraventricular nucleus (PVN) of the vSUB group relative to controls. CRH mRNA differences were particularly pronounced at caudal levels of the nucleus, suggesting topographic organization of vSUB interactions with PVN neurons. Notably, the vHIPPO group, which received large lesions of ventral CA1, CA3 and dentate gyrus without significant subicular damage, showed no change in stress‐induced CORT secretion, suggesting that the ventral subiculum proper is principally responsible for ventral hippocampal actions on the HPA stress response. No differences in medial parvocellular PVN AVP mRNA expression were seen in either the vSUB or vHIPPO groups. The results indicate a specific inhibitory action of the ventral subiculum on HPA activation. The increase in CRH biosynthesis and stress‐induced CORT secretion in the absence of changes in baseline CORT secretion or AVP mRNA expression suggests that the inhibitory actions of ventral subicular neurons affect the response capacity of the HPA axis.
Morphological Evidence for a Galanin‐Opiate Interaction in the Rat Mediobasal Hypothalamus
Authors: Horvath Tamas L. , Kalra Satya P. , Naftolin Frederick , Leranth Csaba
Published: September 2006

It is well established that hypothalamic galanin‐ and β‐endorphin‐containing circuits play important roles in the neuroendocrine regulation of pituitary hormone secretion and sexual behaviors, as well as in feeding. Recent experimental evidence suggests that an opiate‐galanin interaction may be involved in these neuroendocrine responses. In particular, galanin and β‐endorphin have been shown to stimulate prolactin release from the pituitary, and concurrently, evoke feeding in the rat. The present study was designed to elucidate the morphological component underlying these responses in the hypothalamus. Sections of the mediobasal hypothalamus of colchicine‐pretreated female rats were double immunostained for galanin and β‐endorphin. A dark blue nickel ammonium sulfate‐intensified diaminobenzidine reaction was used to visualize galanin profiles, while b‐endorphin neurons were labeled with a light brown diaminobenzidine reaction. Light microscopy revealed putative connections between galanin boutons and β‐endorphin cells. Electron microscopic examination showed that galanin boutons form axo‐somatic and axo‐dendritic synaptic connections with β‐endorphin neurons. The vast majority (89.6%) of the β‐endorphin‐immunoreactive neurons were found to be contacted by galanin‐immunopositive fibers in the hypothalamus. To determine the origin of the galanin fibers innervating this region, the arcuate nuclei of additional rats were isolated unilaterally using a Halász‐knife. After a ten day survival period, immunostaining was carried out for galanin. The relative surface occupied by galanin immunoreactive profiles on the ipsi‐ and contralateral sides were compared using an image analyzer. This analysis revealed that deafferentation of the arcuate nucleus did not decrease the density of galanin immunoreactive profiles on the isolated side of the arcuate nucleus compared to the control side, thus, indicating that the galanin boutons contacting β‐endorphin cells are most probably of local origin.

These studies support the proposal that galanin‐evoked prolactin secretion and feeding behavior may, in part, be mediated by enhanced β‐endorphin release and raises the possibility that a hypothalamic galanin‐β‐endorphin axis may operate in the control of other pituitary hormones.
The 24‐hour Rhythms in Plasma Growth Hormone, Prolactin and Thyroid Stimulating Hormone: Effect of Sleep Deprivation
Authors: Sadamatsu Miyuki , Kato Nobumasa , Iida Hideharu , Takahashi Saburo , Sakaue Kyoko , Takahashi Kiyohisa , Hashida Seiichi , Ishikawa Eiji
Published: September 2006

Twenty‐four hour secretory rhythms of growth hormone (GH), prolactin (PRL) and thyroid stimulating hormone (TSH) were investigated in 9 normal adult men by means of serial blood sampling at 30 min intervals. The profiles of pituitary hormones were compared in 6 subjects between in normal nocturnal sleep condition and in delayed sleep condition. Plasma GH was measured with use of highly sensitive enzyme immunoassay (EIA) recently developed. Plasma TSH was also evaluated by highly sensitive time‐resolved fluorometric immunoassay (TR‐FIA). Time series analysis of plasma GH and PRL was performed by auto‐ and cross‐ correlation and spectral analysis.

The detection limit of EIA for GH was 0.3 pg/ml and all plasma GH levels were within the detectable range of this EIA. Cross‐correlation and spectral analysis suggested the presence of approximately 2–3 h rhythmicity of plasma GH. Plasma PRL appeared to have some 24‐hour rhythmicity besides its sleep‐dependent component. Sleep deprivation caused marked elevation of plasma TSH during night time. It is suggested that there appears two mechanisms regulating GH secretion: one has a sleep‐independent and ultradian rhythm and another has a sleep‐dependent rhythm.
Daily Melatonin Injections Induce Cytological Changes in Pars Tuberalis‐Specific Cells Similar to Short Photoperiod
Authors: Böckers T. M. , Niklowitz P. , Bockmann J. , Fauteck J.‐D. , Wittkowski W. , Kreutz M. R.
Published: September 2006

Hypophyseal pars tuberalis (PT)‐specific cells are known to exhibit remarkable seasonal changes in morphology especially in photoperiodic animals like the Djungarian hamster Phodopus sungorus. Their high density of melatonin‐receptors leads to the supposition that fluctuations in circulating melatonin levels are a crucial factor for the morphological alterations induced by photoperiodic signals. To prove this hypothesis the nocturnal elevation of melatonin in long photoperiods was prolonged by late afternoon administration of melatonin. We investigated whether this treatment induces cytological changes usually observable under short photoperiod. Electron microscopy revealed that in contrast to hamsters maintained in long photoperiods PT‐specific cells of hamsters injected with melatonin or those kept in short photoperiods appear inactive, containing a relatively high number of secretory granules, sparse endoplasmatic reticulum, irregularly outlined and invaginated cell nuclei and a high amount of glycogen. Furthermore immunoreactivity for the common α‐chain of glycoprotein hormones and β‐TSH was significantly weaker in hamsters kept in short photoperiods or daily injected with melatonin than untreated or vehicle injected controls in long photoperiod. These results demonstrate that an exogenous prolongation of the elevated nocturnal melatonin levels causes a similar morphological appearance of PT‐specific cells as observed in short photoperiods. It is tempting to speculate that the melatonin signal is a direct ‘Zeitgeber’ for the transduction of photoperiodic information to the secretory activity in this cell type.
Evidence that Melatonin Acts in the Pituitary Gland through a Dopamine‐independent Mechanism to Mediate Effects of Daylength on the Secretion of Prolactin in the Ram
Authors: Lincoln G. A. , Clarke I. J.
Published: September 2006

A previous study provided evidence that melatonin acts in the pituitary gland to mediate the effects of daylength on the secretion of prolactin in sheep. This was based on the observation that hypothalamo‐pituitary disconnected (HPD) Soay rams showed normal patterns in the changes in the peripheral blood concentrations of prolactin in response to alterations in photoperiod (10‐fold higher concentrations under long than short days), and in response to exogenous melatonin (rapid decline following the administration of a constant‐release implant of melatonin). The purpose of this study was to establish whether dopamine (DA) might be involved in mediating the effects of melatonin on the secretion of prolactin. Groups of HPD (n = 7) and control Soay rams (n = 8) were treated with vehicle (control, 2.0 ml 0.1 M tartaric acid/saline sc), bromocriptine (DA agonist, 0.06 mg/kg sc) or sulpiride (DA antagonist, 0.6 mg/kg sc), and the acute prolactin responses were measured over the next 4 h. Treatments were carried out under short days (8L:16D, low prolactin), long days (16L:8D, high prolactin), and under long days in the presence of a constant‐release implant of melatonin (low prolactin). The prolactin response to TRH (1.25μg/kg iv) was also measured. Bromocriptine caused a decrease in the plasma concentrations of prolactin in both HPD and control rams under short and long days. Sulpiride had no effect in the HPD rams on any occasion, but caused a very marked increase in the plasma concentrations of prolactin in the control rams under short days, long days, and under long days + melatonin. TRH caused an acute increase in the plasma concentrations of prolactin in the HPD rams under both long and short days although the responses were notably reduced compared with the controls especially under long days + melatonin. Overall, the inhibitory response to the DA agonist in HPD rams indicates the presence of DA D2 receptors linked to functional lactotrophs in the isolated pituitary gland. However, the total lack of a response to the DA antagonist indicates the absence of endogenous DA mechanisms regulating the secretion of prolactin in the HPD rams. The conclusion is that melatonin acts directly on the pituitary gland to mediate effects of photoperiod through a DA‐independent mechanism.
Effects of Chronic Octreotide Treatment on GH Secretory Dynamics and Tumor Growth in Rats Bearing an Ectopic Somatotroph (GC) Tumor
Authors: Mounier Françoise , Bluet‐Pajot Marie Thérèse , Viollet Cécile , Bertherat Jérôme , Timsit José , Tannenbaum Gloria S. , Epelbaum Jacques
Published: September 2006

The effects of octreotide, a long‐acting somatostatin agonist selective of the sstr2/sstr3/sstr5 receptor subtypes, on ectopic GH secretion and tumor growth were investigated in Wistar‐Furth female rats implanted with GH secreting (GC) cells which express mostly somatostatin receptors of the sstr1 and sstr2 subtypes. Octreotide dose dependently inhibited thymidine incorporation (−57%) and GH secretion (−41%) from GC cells in culture. In vivo, 6 weeks after GC cell implantation, plasma GH, IGF‐1 and insulin levels were highly elevated. Cluster analysis of GH secretory dynamics revealed that GH secretion was less pulsatile in GC‐implanted than in control animals. Furthermore, in GC‐implanted animals, passive immunization either with SRIH or GHRH antisera did not affect GH plasma levels. Three weeks after GC cell implantation, when tumors became palpable, octreotide (1 μg/h/kg BW) or saline was infused constantly for three weeks by osmotic minipumps. In octreotide treated rats, GH, IGF‐1 and insulin levels were not different from sham‐implanted animals and tumors weight were reduced by 80%. High affinity somatostatin binding sites were found in equivalent amounts on tumors from octreotide‐treated or saline‐treated animals. These findings indicate that GH secretion in GC‐rats is mainly derived from the tumors and independent of hypothalamic control and that octreotide reduces both GH secretion and tumor growth. We conclude that the GC‐implanted rat represents a good animal model to test the antisecretory and antitrophic properties of somatostatin analogs in vivo.
Sexual Differentiation of the Surge Mode of Gonadotropin Secretion: Prenatal Androgens Abolish the Gonadotropin‐Releasing Hormone Surge in the Sheep
Authors: Herbosa Cristina G. , Dahl Geoffrey E. , Evans Neil P. , Pelt Juanita , Wood Ruth I. , Foster Douglas L.
Published: October 2006

In sheep, the surge mode of gonadotropin secretion is sexually differentiated, i.e. the LH surge is present in the female, but not in the male. The present study tested the hypothesis that sexual differentiation of the LH surge mechanism reflects a sex difference in the pattern of GnRH, and that prenatal androgens abolish the surge mode of GnRH secretion. We monitored the pattern of GnRH secretion in pituitary portal blood after acute treatment with estradiol in gonadectomized postpubertal males (n=6), females (n=6), and androgenized females (exposed prenatally to testosterone from day 30–90 in gestation, n=7). Four capsules, each containing a 30‐mm column of estradiol were implanted s.c. into each lamb to produce high physiologic concentrations of the hormone. Beginning 7 h later, portal and peripheral blood samples were collected hourly for 48 h for measurement of GnRH and LH, respectively. All females exhibited a GnRH surge beginning 13.0±0.4 h after estradiol treatment; this was accompanied by an LH surge. By contrast, only one male produced a small surge in GnRH (1.7 pg/min) with a latency of 32 h; a corresponding increase in LH occurred in this male. Likewise, among the androgenized females, only one exhibited GnRH and LH surges which began at about 22 h after estradiol treatment. Some of the androgenized females had sporadic increases in GnRH which were of lower amplitude than in the control females, and were unaccompanied by rises in LH. These findings provide the first direct evidence that the sex difference in the surge mode of LH secretion results from the sexual differentiation of the pattern of GnRH release. The study also suggests that androgens during prenatal development abolish the GnRH surge and subsequently, the generation of the LH surge.
Calcium Checks cyclic AMP — Corticosteroid Feedback in Adenohypophysial Corticotrophs
Authors: Antoni Ferenc A.
Published: October 2006

This paper summarizes a particular aspect of the stress response — the negative feedback control of anterior pituitary adrenocorticotrophin secretion with special focus on the mechanism of action of protein(s) rapidly induced by glucocorticoids. The main thesis is that the principal intracellular mechanism underlying corticosteroid inhibition of corticotroph secretory function is the opposition of cAMP‐mediated activation by calcium ions. An increase of intracellular cAMP levels in corticotrophs produces a rise in intracellular free Ca2+ known to be essential for triggering hormone secretion. In parallel, calcium regulates agonist‐induced cAMP accumulation through inhibition of adenylyl cyclase and the stimulation of cAMP‐degrading phosphodiesterase. Furthermore, a key action of cAMP is the inhibition of a slow, sustained potassium current which is activated by calcium ions. Collectively, the actions of calcium constitute a powerful intracellular feedback inhibition of cAMP‐induced cellular activation. Analysis of corticosteroid action in mouse corticotroph tumour (AtT20) cells indicates that the essence of corticosteroid feedback inhibition is the amplification of intracellular calcium feedback. A common mediator of the inhibitory actions of calcium may be the calcium receptor protein calmodulin the de novo synthesis of which is rapidly stimulated by glucocorticoid hormones. Targets of glucocorticoid‐induced calmodulin may include the protein phosphatase calcineurin, calmodulin‐activated phosphodiesterase(s), and BK‐type potassium channels. The net result of calcium feedback inhibition is a reduction of Ca2+ available for the facilitation of secretory activity i.e. calcium‐induced desensitization. It is proposed that the intracellular calcium feedback loop outlined above also operates in the CNS components of negative corticosteroid feedback.
Identification and Characterization of AngiotensinIV Binding Sites in Rat Neurone and Astrocyte Cell Cultures
Authors: Greenland Karen , Wyse Bruce , Sernia Conrad
Published: October 2006

This study demonstrates the existence of the putative receptor for the hexapeptide (3–8) fragment of angiotensin II (AngIV) on rat astrocytes and neurons grown in cell culture. Binding of 125I‐AngIV was saturable and distinct from that of the AngII receptor subtypes. Equilibrium binding was attained in 15 min in astrocytes and 75 min in neurons at 22 °C. The bound peptide was confirmed by HPLC to be intact AngIV while the bound peptide was substantially degraded, even in the presence of peptidase inhibitors. Scatchard analysis of equilibrium binding was consistent with a two binding site model, revealing a high affinity and a low affinity binding site in both cell types. In neurons, the respective association constants (Ka) were 2.72±0.23 nM−1 and 727± 354 nM−1, with associated receptor densities of 109.30±58.87 and 1723±1167 fmol/mg protein. Similar analyses in astrocytes gave Kas of 5.71±2.85 nM−1 and 277±205 nM−1, and respective densities of 191.1±90.1 and 1425±1250 fmol/mg protein. However, the quantitative reliability of these binding isotherms may be influenced by the degration of unbound peptide. Competitive binding analysis was used to determine the specificity of the receptor site, with the relative order of affinities being AngIV>AngIII>AngII(4–8), and no displacement by AngII, losartan and PD123319 in either neurons or astrocytes. Autoradiography with 125I‐AngIV performed on neuronal cultures demonstrated that binding was confined to a subpopulation of the total cells. These data support the existence of a specific binding site for AngIV in both neurons and astrocytes, consistent with the properties of binding reported previously in the brain, and distinguish this site from the AngII receptor subtypes.
Sodium Pentobarbitone and the Suppression of Luteinizing Hormone Pulses in the Female Rat: the Role of Hypothermia
Authors: Strutton Paul H. , Coen Clive W.
Published: October 2006

The role of hypothermia in the suppression of pulsatile luteinizing hormone (LH) release by the barbiturate sodium pentobarbitone has been investigated in ovariectomized rats. Each animal was fitted with an intraperitoneal miniature radio transmitter to monitor core temperature and with an indwelling intravenous and intraperitoneal catheter. During the 6‐h sampling period the animal's core temperature was recorded automatically every 5 min and a 25 μl blood sample was obtained concurrently using an automated system. After the initial 3 h of sampling either the drug or the vehicle was administered via the intraperitoneal cannula from outside the cage, thus ensuring minimal disturbance to the animal. Administration of sodium pentobarbitone (40 mg/kg) at an ambient temperature of 21°C resulted in a significant hypothermia throughout the 3‐h post‐injection period. During this period there was a significant reduction in mean LH concentration, and in the frequency and amplitude of the LH pulses. When the drug was administered at an ambient temperature of 35°C there was no reduction in core temperature and no significant change in the LH pulse parameters. Vehicle treatment was without significant effect on core temperature or on the LH pulse parameters when administered at an ambient temperature of either 21°C or 35°C. These results indicate that the effects of this barbiturate on the pulsatile release of LH are secondary to the induced hypothermia and suggest that hypothermia per se may be able to disrupt LH pulses. It is therefore imperative to reassess the significance of previous studies that have implicated particular neurotransmitter systems in the control of LH pulses; unrecognized hypothermic effects of the treatments may have been the primary cause of the pulse suppression, rather than a direct involvement of the neurotransmitter in question in the regulation of LH releasing hormone. The neurophysiological process by which the hypothermic state may inhibit LH pulses remains to be determined.