bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and Localization of 23,000 and Glycosylated Rat Prolactin in Subcellular Fractions of Rat Anterior Pituitary and Purified Secretory Granules
Authors: Bollengier F. , Geerts A. , Matton A. , Mahler A. , Velkeniers B. , Hooghe‐Peters E. , Vanhaelst L.
Published: September 2006

Rat pituitary homogenates were submitted to differential and density gradient centrifugation. Subcellular fractions as well as the purified secretory granules were examined in electron microscopy, radioimmunological techniques, protease digestion, alkaline treatment and immunoblotting.

The global outcome of these experiments was that: 1) the glycosylated rPRL was foremost recorded in the crude secretory granular fraction, also in the microsomal fraction and the cytosol, but virtually not in the plasma membrane fraction; 2) in purified secretory granules glycosylated rPRL appeared as an array of near Mr, such as was formerly obtained by enzymatic deglycosylation; 3) protease digestion and ice‐cold alkaline treatment of the secretory granules showed that 23,000 rPRL appears in three different physicochemical states in these organelles: unsequestered within a closed system, membrane‐bounded and membrane‐bound, whereas glycosylated rPRL preferentially assumed the membrane‐bounded and bound state; 4) likewise treatment of microsomal vesicles showed that 23,000 and glycosylated rPRL are sequestered in these bodies, but apparently 23,000 rPRL appears as both integral membrane‐bound and released from the lumen, whereas glycosylated rPRL is chiefly retained as an integral membrane protein. 5) dopamine alters the pattern of glycosylation as well in Mr as in relative percentages of the molecular variants.

The systematical occurrence of the array of near Mr glycosylated rPRL variants in secretory granules and microsomal vesicles strongly indicates that glycosylated rPRL is biosynthesized as a pool of proteins with a different degree of glycosylation. On the basis of our data, we speculate that selection of definite molecular variants from this pool could play an important role in the biological function of 23,000 rPRL and that oligosaccharides could perhaps target the glycosylated forms of rPRL to specific sites of action.
The Orphan Receptor SF‐1 Binds to the COUP‐Like Element in the Promoter of the Actively Transcribed Oxytocin Gene
Authors: Wehrenberg Uwe , von Goedecke Stephanie , Ivell Richard , Walther Norbert
Published: September 2006

The factors regulating oxytocin expression have not yet been characterized in detail. Although direct control by ligand‐dependent binding of nuclear hormone receptors to the oxytocin promoter has been suggested, the presence of these receptors in the tissues expressing oxytocin has not been shown consistently. We have analyzed nuclear proteins from preovulatory bovine granulosa cells and corpus luteum, tissues actively expressing the oxytocin gene, and describe here the characterization of a tissuespecific factor binding to the conserved element in the oxytocin promoter that has been implicated in the control of this gene. This factor is the bovine homologue of SF‐1, an orphan receptor expressed specifically in steroidogenic tissues. It is suggested that SF‐1 binds to the oxytocin promoter in vivo and is involved in control of oxytocin gene expression possibly by interaction with other factors.
Increased Fos Expression in Oxytocin Neurons Following Masculine Sexual Behavior
Authors: Witt Diane M. , Insel Thomas R.
Published: September 2006

Induction of the c‐fos protein product (Fos) was used to immunocytochemically identify oxytocin (OT) neurons that may be activated during copulatory interactions. Fos induction was quantified in sexually‐experienced male rats after either (a) exposure to a testing arena recently vacated by an estrous female, (b) copulatory interactions such as mounting and intromission without ejaculation, or (c) mounting and intromissions culminating in ejaculation. In the parvocellular regions of the paraventricular nucleus of the hypothalamus (PVN), the number of neurons expressing Fos increased following either intromission (53%) or ejaculation (124%). Significant, but less striking, increases in the number of cells expressing Fos were noted in magnocellular regions of the PVN where intromission resulted in a 13% increase and ejaculation in a 49% increase in Fos. The number of perikarya immunoreactive for OT and AVP did not differ as a function of increasing sexual contacts. In control (novel arena) males, 33–73% of the Fos labeling occurred in OT cells. Sexual interactions did not enhance the number of double‐labeled cells in most parvocellular regions. However, in lateral parvocellular regions located in the most caudal aspects of the PVN, 31% of the Fos‐positive cells occurred in OT neurons in ejaculated males, while in control males none of the OT cells were double‐labeled. This PVN subdivision is known to consist of neurons that project to the brain stem and spinal cord at lumbar levels which contain motor neurons that regulate penile reflexes. The present data suggest a possible neurochemical circuit which incorporates oxytocinergic neurons in the mediation of masculine sexual responses.
Characterization and Localization of Mouse Hypothalamic Growth Hormone‐Releasing Factor and Effect of Gold Thioglucose‐Induced Hypothalamic Lesions
Authors: Miki Nobuhiro , Ono Masami , Asakawa‐Yasumoto Kumiko , Aoki Tomoko , Murata Yoji , Ishituka Yuichi , Demura Hirosi , Sasaki Fumihiko
Published: September 2006

Hypothalamic growth hormone‐releasing factor (GRF) in higher mammals, including human GRF, is a 44 amino acid residue peptide and is highly homologous in structure. By contrast, mouse GRF (mGRF) recently deduced by cDNA cloning consists of only 42 residues and shows relatively low homology to the GRFs of higher mammals and the same rodent species, rat. To characterize and localize the predicted mature mGRF peptide in the hypothalamus, we have generated its antiserum and developed a homologous radioimmunoassay. Immunoreactive mGRF in the acid hypothalamic extract was eluted as a single peak at a position identical to that of synthetic peptide on both gel filtration chromatography and reverse‐phase high‐performance liquid chromatography (HPLC). Secretion of immunoreactive mGRF from incubated hypothalami increased several fold in response to 50 mM K+, and this rise was abolished in the absence of medium Ca2+. Only a single peak of immunoreactive mGRF that coeluted with synthetic replicate was observed after the K+ ‐stimulated medium was extracted on Bond Elut C18 cartridges and applied on reverse‐phase HPLC. Immunohistochemistry identified many mGRF‐positive cell bodies in the arcuate nucleus and dense bundles of immunoreactive fibers in the median eminence. Treatment of mice with gold thioglucose (GTG), a chemical agent known to cause hypothalamic lesions, markedly depleted both content and in vitro secretion of immunoreactive mGRF. The decline in mGRF secretion was greater in GTG obese than in nonobese mice, whereas somatostatin secretion was not affected by GTG treatment. As compared to the previous findings obtained on rat GRF, mouse had approximately the same content of hypothalamic GRF, but retained only one third the capacity to release GRF in response to the same depolarizing stimulus.

These findings demonstrate that mouse hypothalamus contains and secretes a mGRF‐like molecule which has immunological and chromatographic characteristics identical to that predicted by molecular cloning. The depolarization‐evoked, Ca2+‐dependent release of immunoreactive mGRF and its neuronal localization in the arcuate nucleus‐median eminence region indicate that it functions as a hypophysiotropic hormone of physiological importance. GTG produces hypothalamic GRF deficiency, probably sparing hypophysiotropic somatostatin neurons, and could be used to generate a mouse model for human GRF‐deficient dwarfism.
Stress and Interleukin‐1 β‐Induced Activation of c‐fos, NGFI‐B ann CRF Gene Expression in the Hypothalamic PVN: Comparison Between Sprague‐Dawley, Fisher‐344 and Lewis Rats
Authors: Rivest Serge , Rivier Catherine
Published: September 2006

Various signals are known to activate the hypothalamic‐pituitary‐adrenal (HPA) axis, an event largely dependent on the release of corticotropin‐releasing factor (CRF) which originates mainly from the parvocellular paraventricular nucleus (PVN) of the hypothalamus. These signals include neurogenic stimuli such as exposure to mild electroshocks, and systemic stimuli like administration of cytokines. The HPA axis activity of Lewis rats has been reported to be hyporesponsive to such stimuli, but the exact mechanisms involved in this phenomenon are poorly understood. The present study investigated the effect of footshock exposure and central injection of interleukin (IL)‐1β, on CRF neuronal activity and gene expression in the PVN of adult male Sprague‐Dawley (SD), Fisher‐344 (F344) and Lewis (LEW) rats. The animals were deeply anesthetized and rapidly perfused transcardially with a solution of 4% paraformaldehyde 3 h after the beginning of the footshock session (1.5 mA, 2 s duration, 4/min over 1 h), or the i.c.v. injection of IL‐1β (100 ng in 10 μl). mRNA encoding the immediate ‘early’ genes (lEGs) c‐fos and NGFI‐B, as well as CRF, were assayed by in situ hybridization histochemistry, while the localization of Fos protein within CRF‐immunoreactive (ir) neurons in the PVN was determined using a dual immunostaining protocol. Both stress and IL‐1β induced robust Fos‐ir expression within the parvocellular division of the PVN in all 3 strains. The number of cells immunoreactive for both Fos and CRF proteins in the PVN was similar in SD, F344 and LEW rats following either challenge. While control animals did not display detectable levels of c‐fos or NGFI‐B mRNA in the PVN, both treatments induced significant expression of these transcripts in this hypothalamic nucleus and no significant differences were observed among SD, F344 and LEW rats. Relative levels of CRF mRNA in the PVN were also significantly and comparably increased following either stress or central IL‐1β treatment. In contrast, plasma ACTH and corticosterone levels were significantly higher in F344 and SD rats than in LEW animals during the stress session.

These results provide further evidence that physical stress and central IL‐1β can enhance expression of several lEGs, as well as CRF, within the parvocellular division of the PVN. These independent indices of functional activation within parvocellular CRF neurons appear to respond similarly to diverse challenges in adult male SD, F344 and LEW rats. Thus, these findings suggest that the hyporesponsiveness of LEW HPA axis to stress does not seem to be a consequence of a defect in afferent signals to the parvocellular PVN, at least in mature male rats.
Natriuretic Peptides Stimulate Cyclic GMP Production in an Immortalized LHRH Neuronal Cell Line
Authors: Olcese J. , Middendorff R. , Münker M. , Schmidt C. , McArdle C. A.
Published: September 2006

The role of cyclic 3′,5′‐guanosine monophosphate (cGMP) as a second messenger in LHRH neurons is not well understood. Recent studies involving nitric oxide, a direct activator of soluble guanylate cyclase (GC), have implicated cGMP in the regulation of LHRH secretion both in vivo and in vitro. Evidence for the membrane‐bound form of GC in LHRH neurons has thus far not been reported. In polymerase chain reaction screening of various cell lines for the natriuretic peptide receptors—which represent GCs—we identified both GC‐A and GC‐B cDNAs by southern blot hybridization in reverse transcribed and amplified extracts of the GT1‐7 cell line, an immortalized LHRH neuronal cell line. Subsequent experiments demonstrated that all of the natriuretic peptides elevated cGMP production with a rank order of potency: CNP > ANP > BNP. Time course studies revealed a rapid intracellular accumulation of cGMP following exposure to CNP with a peak at 2.5 min. CNP was some 200‐fold more potent than the NO donor, sodium nitroprusside, in stimulating cGMP accumulation in these cells. These data show for the first time the presence of functional mGCs on LHRH cells, and suggest that the natriuretic peptides may also participate in the regulation of LHRH activity.
Neuropeptide‐Y Stimulates Pituitary‐Adrenal Activity in Fetal and Adult Sheep
Authors: Brooks A. N. , Howe D. C. , Porter D. W. F. , Naylor A. M.
Published: September 2006

Corticotrophin‐releasing factor (CRF) and arginine vasopressin (AVP) are the primary neuropeptides regulating the secretion of ACTH from the anterior pituitary gland during fetal and adult life. However, a number of other neuropeptides including neuropeptide‐Y (NPY) appear to modulate the activity of this system. The potential role of NPY in the regulation of pituitary‐adrenal function was examined in fetal and adult sheep. Administration of NPY (6.5 μg) as a bolus injection into the third cerebral ventricle of adult ewes elicited a significant (P<0.05) increase in plasma concentrations of ACTH. In fetal sheep at day 125 gestation (term = 145 days) a five‐fold higher dose (30 μg) of NPY injected into the lateral cerebral ventricles also caused a significant increase in plasma concentrations of ACTH. The potential of NPY to influence ACTH secretion directly from the pituitary gland was investigated using primary cultures of fetal (day 130 gestation) and adult pituitary cells. CRF (10−10–10−7 M) caused a significant (P<0.01) dose‐related increase in ACTH secretion from both fetal and adult pituitary cells. Furthermore, the secretion of ACTH from adult cells was significantly greater (P<0.05) than that from fetal cells. NPY (10−10–10−7 M) had no effect on basal or CRF‐stimulated ACTH secretion from fetal or adult pituitary cells. Pre‐incubation of pituitary cells with cortisol (10−9 and 10−7 M) for three days significantly inhibited CRF‐stimulated ACTH secretion but had no effect on basal ACTH release. The simultaneous addition of NPY did not alter the ability of cortisol to inhibit CRF‐stimulated ACTH secretion.

These data show that exogenous administraiton of NPY stimulates pituitary‐adrenal activity in fetal and adult sheep. The lack of any direct pituitary effects of NPY suggests that this neuropeptide acts centrally within the brain to exert its effects on the pituitary‐adrenal axis.
Characterization of Membrane‐Associated Peptidase Activities Expressed by Endothelial Cells of the Ovine Median Eminence
Authors: Lew Rebecca A. , Tetaz Tim , Smith A. Ian
Published: September 2006

The capillary endothelial cells of the median eminence represent a potential site for the degradation/modification of both circulating and hypothalamic peptides passing through the hypophysial portal system toward the pituitary. This study examines endothelial cell peptidase expression in vitro by monitoring the metabolism of gonadotropin‐releasing hormone (GnRH) by cultured endothelial cells from sheep median eminence. Cleavage of GnRH by median eminence endothelial cell membranes generated GnRH1–5 as the primary stable product, which was then degraded to GnRH1–3 and free amino acids. Degradation of GnRH was completely inhibited by TPCK, ZnCI2 and N‐ethylmaleimide, and partially inhibited by EDTA and by a specific inhibitor of the metalloendopeptidase EC 3.4.24.15, CFP‐AAY‐pAB. Interestingly, an increase in GnRH1–9 production was seen with the latter inhibitors, suggesting a two‐step mechanism of GnRH degradation involving a primary cleavage at the Pro9‐Gly10‐NH2 bond, inhibitable by TPCK, ZnCI2, and NEM, followed by cleavage by EC 3.4.24.15 to generate GnRH1–5. Phosphoramidon and angiotensin converting enzyme inhibitors (as well as other non‐specific inhibitors) were without effect, indicating that endopeptidase EC 3.4.24.11 and angiotensin converting enzyme are not involved. Neither bovine aortic endothelial cell nor AtT‐20 cell membranes exhibited this pattern of peptidase activity. Degradation of GnRH by intact median eminence endothelial cells in culture was also observed, suggesting an extracellular orientation for these enzymes; the potential role of such peptidases in the fine regulation of both pituitary function and local blood flow is currently under investigation.
Opioids and Coupling of the Anterior Peri‐Third Ventricular Input to Oxytocin Neurones in Anaesthetized Pregnant Rats
Authors: Bull Philip M. , Douglas Alison J. , Russell John A.
Published: September 2006

In the pregnant rat the osmotic drive to oxytocin neurones is reduced and oxytocin secretion itself is inhibited by endogenous opioids. Coupling of the anterior peri‐third ventricular input pathway, involved in osmoregulation, to magnocellular oxytocin neurones was studied in urethane‐anaesthetized virgin and 21 day pregnant rats using electrical stimulation of the region anterior and ventral to the third cerebral ventricle (AV3V region) to drive the oxytocin neurones, and giving naloxone to prevent the action of any endogenous opioids on the system. Trains ofstimuli (0.5 mA, 1 ms pulses, 10 s on 10 s off, at either 10 Hz or 25 Hz for 10 or 2 min respectively) were given at 20 or 30 min intervals via an electrode stereotaxically‐implanted in the AV3V region, and femoral arterial blood plasma samples collected immediately before and after each stimulation were radioimmunoassayed for oxytocin concentration. The first (control) AV3V stimulation increased plasma oxytocin concentration reproducibly and similarly in virgin and 21‐day pregnant rats. Naloxone administered 10 min before the second stimulus increased basal plasma oxytocin concentration in virgin and pregnant rats and increased the oxytocin secretory response to 25 Hz AV3V stimulation in virgin but not pregnant rats, and the response was significantly greater in virgin rats. Naloxone reveals oxytocin secretion unrestrained by endogenous opioids, therefore it appears that there is an opioid‐independent reduction in the excitatory coupling of the AV3V input to oxytocin neurones which may explain the reduced osmoresponsiveness of oxytocin neurones at the end of pregnancy. In other experiments, morphine (μ‐opioid agonist; at 1 & 5 mg/kg 5 min before the second and third stimulation periods respectively) or U50,488 (κ‐opioid agonist; 0.5 and 2.5 mg/kg respectively) was given to test the opioid sensitivity of the oxytocin neurone response to stimulation of the AV3V input. Morphine did not significantly affect the oxytocin secretory response to 25 Hz AV3V stimulation in either virgin or pregnant rats but there was a significant dose related inhibition of the oxytocin secretory response to 10 Hz stimulation in both groups. U50,488 also inhibited the oxytocin secretory response to 25 Hz AV3V stimulation in a dose‐related manner in both virgin and pregnant rats. The inhibitory effects of morphine and U50,488 were not different between virgin and pregnant rats; this indicates that central opioid interactions with the AV3V input to oxytocin neurones are not changed in pregnancy. The initial similar oxytocin secretory response to AV3V stimulation in pregnant and virgin rats seems to be a product of a reduced effectiveness of the excitatory AV3V input to oxytocin neurones in late pregnancy and the previously established reduced posterior pituitary sensitivity to inhibitory endogenous opioids.
Steroidal and Photoperiodic Regulation of Opiate Binding in Male Golden Hamsters
Authors: Tubbiola Maureen L. , Bittman Eric L.
Published: September 2006

This study was designed to investigate interactions between daylength and testosterone (T) in the regulation of 3H‐naloxone binding which may contribute to seasonal changes in the negative feedback and behavioral effects of androgens in the golden hamster. Photoperiod influenced opiate binding in hamsters with intact gonads only in the medial amygdala. Castration elevated specific 3H‐naloxone binding in the medial amygdala, medial preoptic nucleus, bed nucleus of the stria terminalis, and in the basolateral amygdaloid nucleus of hamsters exposed to either long (14L:10D) or short (5L:19D) days. Exposure to SD renders hamsters less sensitive to T maintenance in reversing these effects. Delay of T replacement until 5 weeks after castration eliminated the ability of this androgen to reverse the influence of castration upon opiate receptors in the medial amygdala. Pinealectomy markedly increased 3H‐naloxone binding in short days in several brain areas.

The data demonstrate that androgens and photoperiod interact to regulate 3H‐naloxone binding, particularly in the medial amygdala. These effects may play a functionally relevant role in seasonal changes in the expression of sexual behavior and/or gonadotropin secretion.
Patch‐Clamp Analysis of Direct Steroidal Modulation of Glutamate Receptor‐Channels
Authors: Wong Michael , Moss Robert L.
Published: September 2006

Steroid hormones regulate the neuroendocrine and behavioral functions of the brain by using a number of diverse cellular mechanisms. Many steroids exert rapid electrophysiological effects on neurons, involving specific interactions with membrane components, such as neurotransmitter receptors. Previous studies suggest that the steroids, estrogen and pregnenolone sulfate (PS), might directly modulate glutamate receptors. The present experiments utilized patch‐clamp recording of glutamate receptor‐channels in excised membrane patches to test for direct modulation by these steroids. Characteristic single‐channel activity from N‐methyl‐D‐aspartate (NMDA) receptors could be elicited in both inside‐out and outside‐out patches excised from acutely dissociated hippocampal neurons. PS, but not 17ß‐estradiol, increased the open probability of NMDA channel activity in inside‐out and outside‐out patches. The PS‐induced increase in open probability could be attributed to an increase in both frequency of opening and mean open time of the NMDA receptor, though the effect on frequency of opening was more prominent. The non‐NMDA agonist, kainate, induced continuous shifts and increased noise in the baseline current of outside‐out patches, but rarely activated clearly resolvable single‐channel openings. 17ß‐estradiol and PS had no apparent effect on the kainate‐induced currents. These findings suggest that some steroids can directly modulate glutamate receptors, but other steroids may utilize indirect mechanisms for regulating glutamatergic synaptic transmission.
Electrical Stimulation of the Rat Periventricular Nucleus Influences the Activity of Hypothalamic Arcuate Neurones
Authors: Dickson Suzanne L , Leng Gareth , Robinson Iain C.A.F.
Published: September 2006

In rats, the release of growth hormone (GH) is inhibited during electrical stimulation of the periventricular nucleus but after the end of stimulation, there is a rebound ‘hypersecretion’ of GH. We examined the responses of arcuate neurones in pentobarbitone‐anaesthetized male rats, following electrical stimulation of the periventricular nucleus to test the hypothesis that the effects of periventricular nucleus stimulation on GH secretion are mediated via effects upon GH‐releasing hormone (GRF) neurones in the arcuate nucleus. The electrical activity of 2 groups of arcuate neurones were analysed before, during and after periventricular nucleus stimulation (10 Hz, 5 min, 0.5 mA biphasic, 0.5/1.0 ms): a) putative neurosecretory cells which were antidromically identified (AD) as projecting to the median eminence (n = 53) and b) non‐neurosecretory cells, identified by their spontaneous ‘bursting’ pattern of activity (n = 29). During stimulation predominantly inhibitory responses were observed in both AD and bursting cell groups. Of the 39 AD cells which were spontaneously active, 25 were inhibited during the periventricular nucleus stimulation, and 10 of these showed a rebound hyperactivation following the end of stimulation. Fifteen bursting cells were inhibited during stimulation and 4 of these displayed a rebound hyperactivation following the end of stimulation. Additional evidence was sought for the identity of these cells by testing their response to electrical stimulation of the basolateral amygdala (which has previously been shown to increase plasma GH concentration without influencing the release of other pituitary hormones). Six of the 10 AD cells which displayed the inhibition/rebound response to periventricular nucleus stimulation were also excited following electrical stimulation of the basolateral amygdala. We conclude that 1) electrical stimulation of the periventricular nucleus and the basolateral amygdala exert predominantly inhibitory and excitatory effects respectively upon the activity of arcuate neurones but for neither site were the effects of stimulation exclusively upon GRF neurones, and 2) the rebound hypersecretion of GH following PeN stimulation is likely to involve the rebound activation of arcuate neurones.
Systemic Osmotic Stimulation Increases Vasopressin and Oxytocin Release Within the Supraoptic Nucleus
Authors: Ludwig Mike , Callahan Michael F. , Neumann Inga , Landgraf Rainer , Morris Mariana
Published: September 2006

Vasopressin (VP) and oxytocin (OT) are released within the hypothalamic nuclear region in response to direct microdialysis with hypertonic solutions. Experiments were performed to determine whether systemic osmotic stimulation causes changes in intranuclear peptide release within the supraoptic nucleus (SON). A hypertonic sodium chloride solution was injected intraperitoneally (ip) or intravenously (iv) and microdialysis techniques were used to simultaneously monitor central and peripheral peptide release in urethane anesthetized rats. Systemic osmotic stimuli elicited increases in intranuclear peptide release which were delayed and long‐lasting, occurring over a 2.5 h period. In contrast, plasma peptide levels peaked at 30‐min after the stimulus. The results demonstrate that increased plasma sodium elicits an increase in VP and OT release into the extracellular space of the hypothalamic SON. The different patterns of peptide release in plasma and brain point toward the possibility of independently regulated release into the different compartments.
Hypothalamic‐Pituitary‐Adrenal Activation Following Endotoxin Administration in the Developing Rat: A CRH‐Mediated Effect
Authors: Shanks Nola , Meaney Michael J.
Published: September 2006

The present studies assessed hypothalamic‐pituitary‐adrenal (HPA) responses following immune activation with endotoxin (ip) in three‐day old Long Evans rats. Marked plasma corticosterone (B), adrenocorticotrophic hormone (ACTH) responses and biphasic fluctuations in plasma glucose were maximal at a dose of 0.05 mg/kg. HPA responses peaked between 3–5 h following immune challenge and plasma ACTH and B responses were greater in female than in male rat pups. Plasma levels of corticosterone binding globulin (CBG) were reduced in males and substantially increased in females during the peak HPA response. Changes in plasma glucose were biphasic with slight increases when ACTH and B levels were maximal, but hypoglycemia was evident once plasma B levels returned to resting values. Endotoxin challenge reduced median eminence corticotropin‐releasing hormone (CRH) levels at times corresponding with elevated HPA activity, and prior icv injection of the CRH antagonist, a‐helical CRH, significantly attenuated elevations in plasma ACTH and B. In addition, α‐helical CRH pretreatment completely blocked endotoxin‐induced changes in plasma CBG in both males and females. These findings support the view that endotoxin‐induced HPA activation in the neonate may occur via CRH.
Glucocorticoids, but not Dopamine, Negatively Regulate the Melanotrophic Activity of the Rabbit Pituitary Intermediate Lobe
Authors: Schimchowitsch Sarah , Plante Martin , Kienlen Pascal , Félix Jean‐Marc , Koch Bernard , Stoeckel Marie‐Elisabeth
Published: September 2006

The leporidae (rabbit and hare)pituitary intermediate lobe(IL)differs from that of other mammals by its neuroendocrine regulation. In particular, it is not submitted to the classic dopaminergic inhibitory control, which has been considered as a repressive factor for the expressionof glucocorticoid receptors (GR) in the mammalian IL. Hence, the present experiments aimed at examining the rabbit IL for the possible existence of GR. Using both immunocytochemistry and binding studies with (3H)‐dexamethasone, we localized GR in the nuclei of IL cells and showed the presence of saturable and high‐affinity type II receptor sites, with Kd∼3.9nM. Also, exposure of cultured IL cells to 10 nM dexamethasone (DEX) resulted in the blockade of melanocyte‐stimulating hormone(αMSH) secretion stimulated by oxytocin (OT). Importantly, the inhibitory effect was reversed by a 100‐fold excess of the glucocorticoid antagonist RU 38486. This is the first study which clearly demonstrates in a mammalian IL, namely the rabbit, the presence of functional GR, involved in the negative regulation of the melanotrophic activity of this gland.
The Secretion of α40‐MSH from Xenopus Melanotropes Involves Calcium Influx through ω‐Conotoxin‐Sensitive Voltage‐Operated Calcium Channels
Authors: Scheenen Wim J. J. M. , de Koning Harry P. , Jenks Bruce G. , Vaudry Hubert , Roubos Eric W.
Published: September 2006

The secretory activity of endocrine cells largely depends on the concentration of free cytosolic calcium. We have studied the mechanisms that are involved in supplying the calcium necessary for the secretion of α‐melanophore‐stimulating hormone (α‐MSH) from melanotrope cells in the pituitary intermediate lobe of the amphibian Xenopus laevis. Using whole‐cell voltage clamp, high‐voltage activated calcium currents were observed, with a peak current between 0 and +20 mV. Two types of Ca2 +‐currents appeared, depending on the experimental setup. An inactivating current, which was observed after a 10 msec depolarizing prepulse, resembled currents through N‐type channels as it was clearly inhibited by 1 μM ω‐conotoxin. The second type was a non‐inactivating current, which was blocked up to 50% by 1 μM nifedipine, indicating its L‐type nature. Only a small component of this inactivating current could be blocked by ω‐conotoxin. No evidence was found for the presence of transient, low‐voltage activated currents.

The spontaneous secretion of α‐MSH from superfused neurointermediate lobes was dependent on extracellular calcium, as low calcium conditions (10−4‐10−8 M) rapidly inhibited this process. Under these conditions, secretion was not affected by depolarizing concentrations of potassium chloride. The calcium ionophore A23187 increased secretion under low calcium conditions, but had no effect on spontaneous α‐MSH release. Treatment with CoCI2, a blocker of calcium channels, strongly inhibited the secretory process. These results suggest that spontaneous α‐MSH release depends on influx of calcium through voltage‐operated calcium channels. Nifedipine did not affect spontaneous secretion from lobes, nor did it affect potassium‐induced α‐MSH secretion from dispersed melanotropes. Also BAY‐K8644, a specific agonist of L‐type channels, did not influence α‐MSH release, neither under normal nor under low calcium conditions. On the other hand, ω‐conotoxin dose‐dependently inhibited α‐MSH release, to a maximum of 65% at a concentration of 5 μM, and inhibited potassiuminduced secretion by 40%. Thapsigargin, an agent that mobilizes calcium ions from intracellular stores, had no effect on spontaneous α‐MSH release under normal or low calcium conditions. From these results it is concluded that the spontaneous release of α‐MSH by melanotropes of X. laevis is effectuated by calcium influx through ω‐conotoxin‐sensitive, voltage‐operated N‐type calcium channels and that mobilization of calcium from intracellular stores does not play a major role in the regulation of this release.
Cell Specific Expression of a Vasopressin Transgene in Rats
Authors: Zeng Qi , Carter David A. , Murphy David
Published: September 2006

A transgene consisting of the rat vasopressin structural gene containing a reporter in exon III, flanked by 5 kb of upstream and 3 kb of downstream sequences, is expressed in vasopressinergic, but not oxytocinergic, magnocellular neurons of rats. Functionally appropriate physiological stimuli increase transgene expression in magnocellular neurons in an exaggerated fashion; the magnitude of the transgene response to osmotic challenge exceeds that of the endogenous gene by 10–15 fold. Magnocellular vasopressinergic neurons in the rat are now accessible to rational and precise genetic perturbation of function and regulation.
Increased Hypothalamic Somatostatin Expression in Mice Transgenic for Bovine or Human GH
Authors: Hurley David L. , Bartke Andrzej , Wagner Thomas E. , Wee Beth E. F. , Phelps Carol J.
Published: September 2006

Acute studies of GH removal by hypophysectomy or GH replacement in adult rats have shown that GH has a positive influence on its hypothalamic inhibitory hormone somatostatin (SRIH). The present study was undertaken to assess the effect of lifelong exposure to elevated GH on the development and differentiation of SRIH‐producing hypothalamic neurons, including comparison of differing GH levels and heterologous species of GH. Expression of somatostatin peptide and mRNA was evaluated using respective immunocytochemistry and in situ hybridization in brains of transgenic mice bearing constructs of either human (hGH) or bovine (bGH) linked to metallothionein (MT) promoter or bGH linked to phosphoenolpyruvate carboxykinase (PEPCK) promoter. Nontransgenic littermates served as controls. All transgenic constructs resulted in high levels of circulating heterologous GH and significantly elevated body weights. Both bGH levels and body weights were higher in PEPCK‐bGH than in MT‐bGH mice; mean weights were not different between MT‐bGH and MT‐hGH mice. Numbers of SRIH‐immunoreactive neurons in the hypophysiotropic periventricular nucleus (PeN) of transgenic mice showed a two‐fold increase (P<0.01) relative to control animals; the number of SRIH‐positive cells in the medial basal hypothalamus (MBH) was comparable for transgenic and control mice. Total SRIH mRNA in situ hybridization intensity also showed a two‐fold increase (P<0.05) in the PeN of all transgenic mice compared with controls, and was not elevated in the MBH. The higher levels of GH produced in PEPCK‐bGH transgenic mice led to greater weight gain, but not to greater SRIH expression than in other GH‐transgenic mice, suggesting that the increased SRIH cell number and mRNA in the PeN of MT‐GH‐transgenic mice may represent a plateau of maximal feedback stimulation. The results indicate that lifelong elevated heterologous GH in mice stimulates hypothalamic SRIH expression markedly. It is not known whether this mechanism is direct or indirect via a mediator of GH such as IGF, but the heterologous GH appears to be specific to these hypophysiotropic neurons.
Increased Cortisol Levels in Aging and Alzheimer's Disease in Postmortem Cerebrospinal Fluid
Authors: Swaab D. F. , Raadsheer F. C. , Endert E. , Hofman M. A. , Kamphorst W. , Ravid R.
Published: September 2006

The hypothalamo‐pituitary‐adrenal (HPA) axis is activated during aging and even more so in dementia. Increased levels of corticosteroids may be neurotoxic. Therefore we have investigated cortisol levels in cerebrospinal fluid (CSF) of Alzheimer patients and controls. Ventricular postmortem CSF was collected from clinically and neuropathologically well‐defined Alzheimer patients (n = 26) and control subjects (n = 21). In the group of Alzheimer patients the mean CSF total cortisol level was 83% higher than that in the controls. In presenile Alzheimer patients (< 65 years of age; n = 13) the CSF‐cortisol level was 5 times higher than that of presenile controls (n = 7). In contrast, senile Alzheimer patients (n = 13) and controls of over 65 years of age (n = 14) did not show a significant difference in CSF‐cortisol levels. The presence or absence of a difference in the cortisol‐CSF levels in, respectively, presenile or senile Alzheimer patients as compared to controls was due to the 3.5‐fold rise of CSF‐cortisol in control subjects over 65 years of age as compared with controls under 65 years of age. The CSF‐cortisol levels in presenile and senile Alzheimer patients were similar. No significant correlation was observed in the Alzheimer patients between age of onset of the dementia and CSF cortisol levels or duration of Alzheimer's disease and CSF cortisol levels.

The finding that in senile Alzheimer patients cortisol levels were similar to those of unaffected age‐matched controls does not seem to support the cortisol neurotoxicity hypothesis. On the other hand, it should be noted that postmortem ventricular CSF cortisol levels were found to be 13–16 times higher than lumbar puncture CSF cortisol levels of ambulatory patients. This means that the ventricular CSF levels probably reflect the reaction of the HPA‐axis to the process of dying rather than the basal levels of this system. The exact consequences of elevated HPA‐axis activity for the human brain should be studied in more detail.
Role of Neuropeptide Y in the Regulation of Tyrosine Hydroxylase Messenger Ribonucleic Acid Levels in the Male Rat Arcuate Nucleus as Evaluated by in situ Hybridization
Authors: Hong Min , Li Songyun , Pelletier Georges
Published: September 2006

Neuroanatomical data have clearly demonstrated the existence of synaptic contacts between neuropeptide Y (NPY) endings and tuberoinfundibular dopaminergic (TIDA) neurons in the rat arcuate nucleus. In order to determine the influence of NPY in the biosynthesis of dopamine, we have studied the effects of NPY and some NPY analogs on tyrosine hydroxylase (TH) gene expression in TlDA neurons in the male rat. The following peptides: NPY, PYY, [Leu31, Pro34]‐NPY (a Y, receptor agonist) and NPY13–36 (a Y2 receptor agonist) were injected into the left lateral ventricle of adult male rats. All the animals were perfused with 4% paraformaldehyde 4 h after injection. Cryostat sections through the arcuate nucleus were processed for quantitative in situ hybridization. The intracerebroventricular injection of NPY, PYY and [Leu31, Pro34]‐NPY induced an increased of 43, 33 and 42%, respectively, in the number of grains overlying TH neurons. On the other hand, the Y2 receptor agonist NPY13–36 did not influence mRNA levels. These data then strongly suggest that NPY positively regulates the genetic expression of TH in rat TlDA neurons via the Y, NPY receptor subtype.
Similarities and Discrepancies in the Signaling Pathway for Nerve Growth Factor in an Insulin Producing Cell Line and a Neural Crest‐Derived Cell Line
Authors: Tazi Abdelali , Czernichow Paul , Scharfmann Raphael
Published: September 2006

Like neuronal cells, insulin producing cells (beta cells) possess nerve growth factor (NGF) binding sites and express mRNA coding for the low‐ and high‐affinity NGF receptors, p75NGFR and Trk‐A respectively. Although the role of NGF on neuronal cells is well documented, its function on beta cells is still unknown. As a first step towards the elucidation of the role of NGF on beta cells, we have characterized both types of NGF receptors on INS‐1 cells, a beta cell line derived from a rat insulinorna and studied some early post‐receptor events by comparing the signaling pathway of NGF in those cells and in PC12 cells, a well characterized NGF‐responsive cell line. By polymerase chain reaction, immunocytochemistry, cross‐linking and Western blot analysis, we clearly demonstrated that Trk‐A and p75NGFR, the two NGF receptors expressed in INS‐1 cells and PC12 cells are similar. Moreover, upon NGF treatment, Trk‐A is phosphorylated on tyrosine residues in both cell types in the same dose‐ and time‐dependent manner. These data clearly demonstrate that the first step of NGF signal transduction is similar in PC12 and INS‐1 cells. Although early responsive genes like NGFI‐A and c‐fos are induced in both cell types upon NGF treatment, the induction of c‐jun expression is restricted to PC12 cells. Furthermore, the expression of late responsive genes, such as vgf and transin, which are induced by NGF in PC12 cells, are not induced in INS‐1 cells. In conclusion, although the initial steps of NGF signal transduction are similar in PC12 and INS‐1 cells, some of the later differ. These dissimilarities could suggest that NGF plays different roles in neuronal and pancreatic beta cells.
Hypothalamic‐Pituitary‐Adrenal Function in Chronic Intermittently Cold‐Stressed Neonatally Handled and Non Handled Rats
Authors: Bhatnagar Seema , Meaney Michael J.
Published: September 2006

Neonatally handled (H) animals, as adults, exhibit lower ACTH and corticosterone (B) responses to a number of acute stressors compared to their non‐handled (NH) counterparts. However, little is known about activity within the hypothalamic‐pituitary‐adrenal (HPA) axis of H and NH animals under conditions of chronic stress. We, therefore, examined HPA function in adult H and NH rats exposed to chronic intermittent cold stress (4 h of 4°C cold a day for 21 days; H CHR and NH CHR) and in control H and NH (H CTL and NH CTL) rats. H CTL and NH CTL animals displayed comparable ACTH and B responses to a single, acute exposure to cold. We found that H CHR animals exhibited lower levels of ACTH, but not B, during the 21st exposure to cold (the homotypic stressor) compared to the first exposure to cold in H CTL; however, ACTH and B levels in NH CHR were not different from those in NH CTL. In contrast, NH CHR animals hypersecreted ACTH and B in response to restraint (the novel, heterotypic stressor) compared to NH CTL and both H groups, whereas H CHR and H CTL animals did not differ in their responses to restraint. These endocrine responses were associated with increased basal median eminence levels of both CRH and AVP in H CHR and NH CHR relative to their control groups (with NH CHR exhibiting the highest absolute levels of each secretagogue), and with decreased glucocorticoid receptor densities in septum of both H CHR and NH CHR. In addition, the expected lower glucocorticoid receptor density in hippocampus and frontal cortex of NH rats compared to H rats was observed. We believe that the difference in glucocorticoid receptor density between H and NH animals in the hippocampus and frontal cortex aid the associated differences in secretagogue content in the median eminence are related to the hypersecretion of ACTH and B in the NH CHR relative to the other groups. Furthermore, we hypothesize that an active inhibitory process is involved in the adaptation of HPA responses of H CHR animals to the homotypic stressor, and present a working model of regulation of activity within the CRH/AVP neurons in the PVN.
Stimulation of CRH‐Mediated ACTH Secretion by Central Administration of Neurotensin: Evidence for the Participation of the Paraventricular Nucleus
Authors: Rowe Wayne , Viau Victor , Meaney Michael J. , Quirion Rémi
Published: September 2006

Central administration of neurotensin (NT) stimulates hypothalamic‐pituitary‐adrenal (HPA) activity in freely‐moving rats. Increases in adrenocorticotropin hormone (ACTH) and corticosterone (B) were observed 15 rnin following central NT administration and remained elevated for up to 4 h. Of the two NT fragments tested, NT1–8 and NT8–13 only NT8–13 was found to significantly elevate ACTH and B levels. Moreover, NT8–13 activated the HPA axis with a temporal profile similar to NT1–13, suggesting an interaction with the pharmacologically and molecularly characterized NT receptor.

Animals pre‐treated intravenously with the corticotropin‐releasing hormone (CRH) antagonist, α‐helical CRH, showed attenuated plasma ACTH and B responses to central NT administration. This indicates that CRH receptor activation is necessary for the stimulatory effects of NT on HPA function. Bilateral lesions of the paraventricular nucleus (PVN) of the hypothalamus significantly reduced NT‐induced stimulation of ACTH and B release suggesting that the PVN is essential for NT's stirnulatory action. Median eminence content studies indicated that acute central NT administration stimulates CRH, but not arginine vassopressin (AVP), release in animals examined 60 min following NT injection. Taken together, these findings suggest that the stimulatory effects of NT on HPA activity occur via specific NT receptors and that one site of action of NT is likely at the level of the PVN where NT elicits the release of CRH.
Regulation of Estrogen Receptor and Progesterone Receptor Messenger Ribonucleic Acid by Estrogen in the Brain of the Whiptail Lizard (Cnemidophorus uniparens)
Authors: Young Larry J. , Nag Pratip K. , Crews David
Published: September 2006

Receptive behavior in females vertebrates is controlled by hormones, principally estrogen, secreted by the ovary. Estrogen influences behavior by interacting with a specific estrogen binding protein, or receptor, located in target cells in certain hypothalamic nuclei. To better understand the molecular mechanisms involved in the control of receptive behavior in whiptail lizards, we investigated the effects of exogenous estrogen on t h e regulation of estrogen receptor and progesterone receptor expression in several regions of the brains of Cnernidophorus uniparens. First we determined a dosage of 17β‐estradiol 3‐benzoate (0.5 μg) which reliably induced receptive behavior in ovariectomized C. uniparens. Then using in situ hybridization, we examined the effects of that dosage on the expression of estrogen receptor and progesterone receptor mRNA in the brain 24 h after injection. Estrogen treatment resulted in a significant up‐regulation of estrogen receptor mRNA expression in the ventromedial nucleus of the hypothalamus and torus semicircularis, down‐regulation of estrogen receptor mRNA expression in the lateral septum, and no change in the periventricular nuclei of the hypothalamus, the periventricular nucleus of the preoptic area, and the dorsal hypothalamus. The same dosage resulted in increased progesterone receptor mRNA expression in the ventromedial nucleus of the hypothalamus and the periventricular nucleus of the preoptic area; no significant changes in progesterone receptor mRNA expression were observed in the periventricular nuclei of the hypothalamus or the torus semicircularis, although the differences in progesterone receptor expression in the torus semicircularis approached statistical significance. The up‐regulation of estrogen receptor gene expression by estrogen in the ventromedial nucleus of the hypothalamus of lizards is opposite to that reported in female rats in which estrogen down‐regulates estrogen receptor expression in the ventromedial nucleus of the hypothalamus. We propose that this may be related to differences in reproductive physiology between vertebrates which have extended follicular phases and prolonged periods of estrus and rats, which have an abbreviated follicular phase and brief periods of estrus.