bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of the Bed Nucleus of the Stria Terminalis in Tonic Regulation of Paraventricular Hypothalamic CRH and AVP mRNA Expression
Authors: Herman James P. , Cullinan William E. , Watson Stanley J.
Published: September 2006

The bed nucleus of the stria terminalis (BNST) occupies a central position in pathways regulating hypothalamo‐pituitary‐adrenocortical (HPA) stress regulation. The potential role of the BNST in tonic neural control of HPA function was assessed by examining effects of selective BNST lesions on expression of ACTH secretagogues in HPA‐integrative neurons of the medial parvocellular paraventricular nucleus. Anterior BNST lesions (ABN) involved major portions of the anteromedial, anterolateral, ventromedial, ventrolateral, dorsolateral and juxtacapsular subnuclei. These lesions resulted in significant (30%) decreases in corticotropin‐releasing hormone (CRH) mRNA expression across the rostrocaudal extent of the medial parvocellular PVN, with no accompanying changes in basal arginine vasopressin (AVP) mRNA levels. Posterior BNST (PBN) lesions involved large but subtotal damage to the posterior intermediate, posterior medial, posterior lateral and preoptic subnuclei; these lesions resulted in small but significant changes in CRH mRNA and slight increases in number of AVP mRNA‐producing parvocellular neurons. PBN effects on CRH mRNA expression were most pronounced at the caudal extent of the medial parvocellular zone, suggesting a topographic input from the posterior BNST to the PVN that is only partially compromised by PBN lesions. Analysis of individual cases revealed a correlation between damage of the anterolateral BNST and decreased CRH mRNA levels, and damage of the posterior intermediate and/or posterior medial BNST and increased CRH mRNA levels. The results suggest differential BNST input into HPA regulation, perhaps reflecting the diversity of limbic input into the BNST region.
Bicuculline Methiodide Influences the Central Nervous System to Produce Hyperglycemia in Rats
Authors: Nonogaki Katsunori , Iguchi Akihisa , Sakamoto Nobuo
Published: September 2006

The influence of bicuculline methiodide (BMI), a gamma‐aminobutyric acid (GABA) receptor antagonist, on central nervous system regulation of blood glucose homeostasis was studied in fed rats. Injection of BMI (1–10 nmol) into the third ventricle was found to produce hepatic venous hyperglycemia in a dose‐dependent manner. This change was associated with increased secretion of epinephrine and glucagon. The role of epinephrine in hyperglycemia was then studied in bilaterally adrenalectomized (ADX) rats injected with BMI. Plasma glucose concentration was found to increase in ADX rats although the level was approximately half that for intact rats and significantly higher than for controls. The increase in epinephrine and glucagon secretion seen in intact rats, but not in ADX rats, suggests BMI‐induced epinephrine release is responsible for the glucagon secretion. Three possible mechanisms are suggested to account for the rise in plasma glucose in the hepatic vein after injection of BMI: 1) that epinephrine is secreted by the adrenal medulla, 2) that epinephrine secretion stimulates glucagon secretion or 3) that there may be some direct innervation of the liver in rats.
Evidence for Multiple Forms of Melatonin Receptor‐G‐Protein Complexes by Solubilization and Gel Electrophoresis
Authors: Barrett P. , MacLean A. , Morgan P. J.
Published: September 2006

The daily production of melatonin from the pineal gland influences circadian and seasonal behaviour and physiology. To further understand how melatonin may function, it is important to characterize the receptor and signal transduction systems. Using the detergent digitonin, we were able to solubilize the receptor from the ovine pars tuberalis (PT) membrane. The receptor was isolated as a complex associated with its heterotrimeric G‐protein. In the solubilized state, pre‐bound 125l‐2‐iodomelatonin was stable at 4°C, but was displaceable by GTPγS. The receptor‐G‐protein complex could be separated by molecular mass using native polyacrylamide gel electrophoresis. We demonstrate that the receptor‐complex has a molecular mass of 525 kDa and differs from solubilized receptor‐complexes isolated from either the lizard brain, chicken brain or the ovine hippocampus. Furthermore the receptor complex isolated from the hippocampus had the lowest molecular mass of these tissues (365 kDa) and was found not to be sensitive to GTPγS. This may indicate the existence of a distinct non‐G‐protein coupled form of the receptor.
The Effects of Aging and Hormonal Manipulation on Amyloid Precursor Protein APP695 mRNA Expression in the Rat Hippocampus
Authors: Chao Helen M. , Spencer Robert L. , Frankfurt Maya , McEwen Bruce S.
Published: September 2006

In the rat hippocampus, neuronal morphology and survival are profoundly affected by adrenal steroids, and synaptic plasticity can be modulated by the ovarian sex steroids estrogen and progesterone, β‐amyloid peptides, which accumulate in neuritic plaques and are derived from the amyloid precursor protein (APR), have been shown to be both trophic and toxic for hippocampal neurons. Of the various APR isoforms, APP695 is the predominant form found in rat brain and the APP695 mRNA is abundantly expressed in the hippocampus. In order to investigate the hypothesis that APR may serve as a mediator of the steroid effects, we have monitored the hippocampal expression of APP695 mRNA by in situ hybridization, with aging and with steroid manipulation. In aged female rats we observed a decrease in the level of APP695 mRNA relative to young female rats, while no such age difference was evident in male rats. Physiological, surgical and pharmacological manipulation of glucocorticoids appeared to have no effect on APP695 mRNA levels in the hippocampus. Treatment of young, ovariectomized female rats with estrogen and progesterone, resulted in an increase in hippocampal APP695 expression compared to untreated, ovariectomized controls.
Estrogenic Induction of NADPH‐ Diaphorase Activity in the Preoptic Neurons Containing Estrogen Receptor Immunoreactivity in the Female Rat
Authors: Okamura Hiroaki , Yokosuka Makoto , Hayashi Shinji
Published: September 2006

Nitric oxide and estrogen have been shown to play a critical role in the control of female reproductive function. In order to determine an anatomical relationship between nitric oxide generating neurons and estrogen target neurons, NADPH‐diaphorase histochemistry was combined with estrogen receptor immunohistochemistry in the female medial preoptic area. While only a few weakly stained neurons for NADPH‐diaphorase were found in ovariectomized control rats, a drastic increase in NADPH‐diaphorase activity was observed in the medial preoptic nucleus of estradiol‐treated ovariectomized animals. The total number of NADPH‐diaphorase neurons in the estradiol‐treated group increased three‐fold relative to controls, and more than 80% of those neurons contained estrogen receptor‐immunoreactivity in their nuclei. Since neuronal NADPH‐diaphorase is nitric oxide synthase, the present result suggests that nitric oxide synthase activity can be positively regulated by estradiol in neurons containing estrogen receptor in the female medial preoptic nucleus.
Induction of Substance P‐immunoreactivity by Estrogen in Neurons Containing Estrogen Receptors in the Anterovental Periventricular Nucleus of Female but not Male Rats
Authors: Okamura Hiroaki , Yokosuka Makoto , Hayashi Shinji
Published: September 2006

Effects of gonadal steroids on numbers of neurons containing estrogen receptor (ER) and/or substance P (SP) were examined in the anteroventral periventricular nucleus (AVPV) of female and male rats by double‐labeling immunohistochemistry employing antibodies specific for ER and SP. Animals were gonadectomized and received subcutaneously either oil alone (Control group), sequential injections of estradiol benzoate and oil (EB + Oil group), or those of EB and progesterone (EB + P group). In the female control rat, a large population of ER‐immunoreactive (IR) cells were found clustered throughout the AVPV. They were counted more than 2,000 in total of 4 sections in this nucleus. On the contrary, SP‐IR neurons were scarcely observed in the same area of this group. Administration of estrogen to female animals decreased the total number of ER‐IR cells to 67% of the control group. In contrast to the supressive effect of estrogen to its own receptor, it induced SP‐IR neurons in the AVPV of the female. Approximately 50–80 SP‐IR neurons were counted in the 4 sections, and 59% of these neurons expressed ER‐IR material in their nuclei. In the female EB + P group, the number of ER‐IR neurons also decreased to 79% of the control group. Although the number of SP‐IR neurons in this group decreased to 32% of that in the EB + Oil group, a ratio of coexistence of ER‐IR material in these neurons increased to 75%. The male control group contained a smaller population of ER‐IR cells relative to the female control (1497 vs 2143). SP‐IR neurons were rarely observed as were in the female control. Administration of estrogen to the male also decreased the number of ER‐IR cells in a manner similar to that in the female. However, unlike the female, the steroid failed to induce the SP‐IR neurons in the male.

These results demonstrate sexual dimorphism in the AVPV not only in the number of ER‐IR neurons but also in the responsiveness of SP neurons to estrogen. They further provide anatomical evidence that a subset of SP neurons are regulated by estradiol in estrogen sensitive neurons in the female rat. The data also suggest that this peptide is involved in mechanisms of luteinizing hormone surge by mediating actions of gonadal steroids in the AVPV.
Glucocorticoid Regulation of c‐fos, c‐jun and Transcription Factor AP‐1 in the AtT‐20 Corticotrope Cell
Authors: Autelitano Dominic J.
Published: September 2006

Glucocorticoids (GC) are potent repressers of both basal and corticotropin releasing factor (CRF) stimulated transcription of the proopiomelanocortin (POMC) gene in corticotrope cells of the anterior pituitary. Despite the finding of a novel, high affinity glucocorticoid receptor (GR) binding site within the proximal region of the POMC promoter, the mechanism by which GC inhibit POMC transcription is still uncertain. Recent studies have described mechanisms whereby GC inhibit transcription of other genes via a direct interaction with components of the transcription factor AP‐1. Since it has been shown that CRF stimulates c‐fos in AtT‐20 corticotrope cells, and that c‐fos over‐expression elevates POMC transcription, the current study has investigated whether GC can repress c‐fos and c‐jun gene expression and AP‐1 DMA binding activity in AtT‐20 corticotrope cells. Acute treatment with doses of dexamethasone (DEX) that markedly inhibited nuclear POMC hnRNA had no effect on basal c‐fos mRNA expression, but resulted in a transient down regulation of c‐jun. In addition, acute DEX pretreatment significantly lowered CRF stimulation of POMC gene expression and attenuated the CRF stimulation of c‐fos mRNA by 25%. Although DEX treatment of AtT‐20 cells did not affect AP‐1 DNA binding capacity of nuclear extracts, DEX pretreatment blunted the stimulation of AP‐1 binding in response to CRF. In further studies, nuclear extracts from CRF‐treated cells were coincubated with nuclear extracts from control or DEX treated cells. High levels of DEX treated extracts led to a relative repression of CRF‐induced AP‐1 binding, suggesting that ligand‐activated GR may lower available AP‐1 levels by direct protein: protein interaction. Finally, the composition of AP‐1 in AtT‐20 nuclear extracts was found to be heterogeneous, with the variation dependent upon hormonal treatment. These data suggest that in the corticotrope cell relatively high levels of activated GR may influence CRF‐induced AP‐1 DNA binding via transient genomic actions on basal c‐jun and stimulated c‐fos and/or via direct protein: protein interactions.
Endogenous Glucocorticoids and the Induction and Spread of Monoarthritis in the Rat
Authors: Donaldson Lucy F. , McQueent Daniel S. , Seckl Jonathan R.
Published: September 2006

Using mono ‐ and bilateral tarsal arthritic models in the rat, we have previously shown increases in the expression of mRNAs encoding substance P and calcitonin gene‐related peptide (CGRP) in primary sensory neurons innervating inflamed joints. Dorsal root ganglion (DRG) neuropeptide content in rats is altered by glucocorticoids, and since glucocorticoids regulate the expression of preprotachykinin (PPT) gene, the substance P precursor in other tissues, these effects may be mediated at the level of transcription. Indeed adrenalectomy potentiates disease in polyarthritis although the relationship to joint disease itself is unclear. Secretion of corticosterone in both mono ‐and bilaterally inflamed rats showed a loss of the normal diurnal nadir with no elevation of evening values. However, there were no changes in glucocorticoid target organs (adrenal gland, thymus and spleen) suggesting the stress was intermittent. Adrenalectomy in mono ‐ and bilaterally inflamed rats did not significantly alter either the severity of inflammation or its spread. Bilaterally inflamed animals did, however, show reduced weight gain. Adrenalectomy had no effect on the induction of PPT and CGRP mRNA expression in innervating DRG neurons in monoarthritis (14 days after adjuvant injection), the unilateral increase in both PPT and CGRP mRNA expression in ADX animals being similar to SHAM arthritic rats. (PPT: ADX 140 ± 13 left; 99 ± 6 right % control; SHAM 160 ± 22 left, 100 ± 5 right % control. CGRP: ADX 177 ± 6 left, 97 ± 3 right % control; SHAM 147 ± 21 left, 100 ± 5 right % control). In bilaterally arthritic rats adrenalectomy potentiated the increase in CGRP mRNA expression (SHAM 168 ± 6% left, 271 ± 23% right; ADX 217 ± 38% left, 193 ± 4% right; control 100 ± 5%) but significantly attenuated the contralateral (right) increase in PPT mRNA expression seen in sham‐operated rats (SHAM 136 ± 21% left; 140 ± 11% right; ADX 145 ± 28% left, 86 ± 15% right; control 100 ± 5%). Inflammation‐associated changes in somatostatin mRNA expression were similar in sham‐operated and adrenalectomized rats. We conclude, therefore, that whilst adrenalectomy exerts subtle effects on the expression of PPT and CGRP mRNAs in DRG neurons in bilateral arthritis, the presence or absence of high circulating endogenous corticosteroids in these animals, with limited arthritis, do not modulate the arthritic process.
Detection of Neutral Endopeptidase (NEP, Enkephalinase, E.C.3.4.24.11) in Relation to Dopaminergic and Gonadoliberinergic Nerve Endings in the Median Eminence of the Male Rat: A Double Labeling Ultrastructural Study
Authors: Vandenbulcke Franck , Ciofi Philippe , Beauvillain Jean Claude
Published: September 2006

The existence of neutral endopeptidase (Enkephalinase, NEP, E.G.3.4.24.11) in membranes of nerve endings in the rat median eminence suggests that some neuropeptides have paracrine and/or autocrine actions in this region. In vitro, neutral endopeptidase is capable of hydrolysing a variety of regulatory peptides but in vivo, many works indicate that in the central nervous system this enzyme is highly implicated in the biological inactivation of enkephalins and tachykinins. In addition there is evidence that NEP is also involved in the inactivation of neurotensin in vivo.

The modulation of the release of gonadotrophin releasing hormone (GnRH) is one of the documented actions of enkephalins within the median eminence. However, it is at present unclear whether enkephalins act on dopamine endings, on GnRH endings or on both.

As the technical parameters and particularly the tissue fixation used to detect neutral endopeptidase are compatible with immunocytochemical detection of GnRH and tyrosine‐hydroxylase (the rate limiting enzyme in the synthesis of catecholamines), two double immunolabelings were realised at the ultrastructural level to determine if GnRH and dopamine nerve endings have the enzyme inserted within their plasma membrane.

Our study shows the presence of neutral endopeptidase on tyrosine‐hydroxylase‐immunoreactive nerve endings while presence of the enzyme on GnRH‐immunoreactive nerve endings is not demonstrated. Consequently, our results provide morphological arguments for possibilities of paracrine and/or autocrine actions by neuropeptides inactivated by neutral endopeptidase on tuberoinfundibular dopa‐minergic nerve endings. Conversely, action of the same peptides on GnRH boutons seems more unlikely.
Adenylyl Cyclase mRNA Expression does not Reflect the Predominant Ca2+/Calmodulin‐Stimulated Activity in the Hypothalamus
Authors: Mons Nicole , Cooper Dermot M. F.
Published: September 2006

Only three (Types I, II, V) of the six currently‐described subtypes of adenylyl cyclase are prominently expressed in the rat brain. These species are differently sensitive to Ca2+, βγ subunits of G‐proteins and protein kinase C. A knowledge of the susceptibility of the cAMP‐signalling system in particular brain regions to these diverse modes of regulation can shed light on the mechanism of action of the neurotransmitters that modify neuronal activity in such regions. Cyclic AMP is extensively involved in the physiological functions of the hypothalamus. We have used in situ hybridization histochemistry with synthetic oligonucleotides to examine the expression in the rat hypothalamus of the three major brain subtypes of adenylyl cyclase‐Ca2+/calmodulin‐stimulable (Type I), Ca2+‐insensitive (Type II) and Ca2+ ‐inhibitable (Type V). The hypothalamus expresses high levels only of Type II mRNA, particularly in the supraoptic and paraventricular nuclei. Curiously, the strong expression of the Ca2+‐insensitive Type II mRNA and the lack of expression of the major brain specific Type I mRNA does not correlate with the adenylyl cyclase activity, which is largely Ca2+/calmodulin stimulable in plasma membranes prepared from the hypothalamus.
Stress‐Specific Regulation of Corticotropin Releasing Hormone Receptor Expression in the Paraventricular and Supraoptic Nuclei of the Hypothalamus in the Rat
Authors: Luo Xun , Kiss Alexander , Makara Gabor , Lolait Stephen J , Aguilera Greti
Published: September 2006

Corticotropin releasing hormone (CRH), a major regulator of pituitary ACTH secretion, also acts as a neurotransmitter in the brain. To determine whether CRH is involved in the regulation of hypothalamic function during stress, CRH receptor binding and CRH receptor mRNA levels were studied in the hypothalamus of rats subjected to different stress paradigms: immobilization, a physical‐psychological model; water deprivation and 2% saline intake, osmotic models; and i.p. hypertonic saline injection, a combined physical‐psychological and osmotic model. In agreement with the distribution of CRH receptor binding in the brain, in situ hybridization studies using 35S‐labeled cRNA probes revealed low levels of CRH receptor mRNA in the anterior hypothalamic area, which were unaffected after acute or chronic exposure to any of the stress paradigms used. Under basal conditions, there was no CRH binding or CRH receptor mRNA in the supraoptic (SON) or paraventricular (PVN) nuclei. However, 2 h after the initiation of acute immobilization, CRH receptor mRNA hybridization became evident in the parvicellular division of the PVN, with levels substantially increasing from 2 to 4 h, decreasing at 8 h and disappearing by 24 h. Identical hybridization patterns of CRH receptor mRNA were found in the parvicellular PVN after repeated immobilization; levels were similar to those after 2 h single stress following immobilization at 8‐hourly intervals for 24 h (3 times), and very low, but clearly detectable 24 h after 8 or 14 days daily immobilization for 2 h. On the other hand, water deprivation for 24 or 60 h and intake of 2% NaCI for 12 days induced expression of CRH receptor mRNA in the SON and magnocellular PVN, but not in the parvicellular pars of the PVN. Both parvicellular and magnocellular hypothalamic areas showed CRH receptor mRNA following i.p. hypertonic saline injection, single (4 h after) or repeated at 8‐hourly intervals for 24 h (3 injections), or one injection daily for 8 or 14 days. Consistent with the expression of CRH receptor mRNA, autoradiographic studies showed binding of 125I‐Tyr‐oCRH in the parvicellular division of the PVN after immobilization; in the magnocellular division of the PVN after osmotic stimulation, and in the PVN and SON after i.p. hypertonic saline injection. The data show that stress‐specific activation of the parvicellular and magnocellular systems is associated with CRH receptor expression, and suggest a role for CRH in the autoregulation of hypothalamic function.
Limbic Regions Mediating Central Actions of Oxytocin on the Milk‐Ejection Reflex in the Rat
Authors: Ingrarn C. D. , Adams T. S. T. , Jiang Q. B. , Terenzi M. G. , Lambert R. C. , Wakerley J. B. , MOOS F.
Published: September 2006

Central oxytocin administration has a profound facilitatory effect on the patterning of the milk‐ejection reflex in the lactating rat. Lesion and microinjection studies indicate that this action is, in part, mediated via a population of limbic neurones in the bed nuclei of the stria terminalis and ventrolateral septum, which have been shown to possess oxytocin receptors and to be activated by selective oxytocin‐receptor agonists in vitro. In vivo electro‐physiological recordings reveal that some of these neurones display cyclical activity which is highly correlated to each milk ejection, and are rapidly activated following i.c.v. administration of oxytocin. coincident with the facilitation of milk ejection activity. A hypothetical model is proposed in which this population of limbic neurones serves to gate the activity of a pacemaker which, in turn, coordinates the bursting of hypothalamic magnocellular neurones. The oxytocin innervation of these neurones and their expression of oxytocin receptors increases in the post‐partum period, and the resultant enhanced sensitivity leads to a greater facilitatory response during lactation. Inhibitory opioid and noradrenergic inputs which converge on these oxytocin‐sensitive neurones may function to switch off the facilitatory circuit during periods of stress. Thus, this population of limbic neurones participates in the regulation of neuroendocrine activity during lactation by providing an appropriate degree of feedback to alter the patterning of the milk‐ejection reflex.
Naloxone Disinhibits Magnocellular Responses to Osmotic and Volemic Stimuli in Chronically Hypoosmolar Rats
Authors: Dohanics Janos , Verbalis Joseph G.
Published: September 2006

Normonatremic and chronically hyponatremic rats were pretreated with naloxone (5 mg/kg) or isotonic (1 50 mM) NaCI, then were given i.v. injections of 2 M NaCl (2 ml) or were hemorrhaged (20 ml/kg). Baseline and post‐stimulus blood samples were withdrawn through indwelling jugular venous catheters. Baseline levels of plasma vasopressin (AVP) and oxytocin (OT) were similar in both normonatremic and hyponatremic rats and did not change after naloxone pretreatment. Increases in plasma AVP and OT levels in response to both hypertonic saline and hemorrhage were markedly blunted in the hyponatremic rats compared to the normonatremic rats. Naloxone pretreatment caused augmented AVP and OT secretion in response to hypertonic saline stimulation and hemorrhage in both the normonatremic and hyponatremic rats; the magnitude of the naloxone augmentations in the hyponatremic rats were sufficient to normalize the OT response to hypertonic saline and both the OT and AVP responses to hemorrhage. Our results therefore suggest that endogenous opioids are likely involved in the inhibition of stimulus‐induced AVP and OT release that accompanies chronic hypoosmolality.
Hypothalamic Site‐Dependent Effects of Neuropeptide Y on Gonadotropin‐Releasing Hormone Secretion in Rhesus Macaques
Authors: Francis Pau K.‐Y. , Berria Matthew , Hess David L. , Spies Harold G.
Published: September 2006

In rodents and rabbits, neuropeptide Y (NPY) has a bimodal effect on gonadotropin‐releasing hormone (GnRH) secretion. lntracerebroventricular (icv) administration or direct infusion of NPY into the median eminence (ime) suppresses GnRH release in ovariectomized (OVX) animals, but stimulates GnRH release in intact or OVX animals treated with ovarian steroids. Specific ovarian steroiddependent NPY effects are, however, not obvious in non‐human primates. In OVX rhesus monkeys, icv administration of NPY has been shown to suppress luteinizing hormone (LH) secretion whereas ime infusion of NPY stimulates GnRH pulses. In such animals, estrogen replacement does not reverse the inhibitory NPY effect on LH release, although estrogen enhances the stimulatory NPY effect on GnRH secretion. These observations led us to speculate that the bimodal NPY effects in non‐human primates may depend on either the site of NPY action or the nature of the steroid milieu. This study utilized the push‐pull perfusion (PPP) technique to examine the effects of either ime or icv infusion of NPY on GnRH release in OVX monkeys treated with or without both ovarian steroids. Without exception, irne infusion of NPY increased GnRH concentrations in push‐pull perfusates regardless of the steroid status of the animals. In contrast, GnRH levels were reduced during icv infusion of NPY in both untreated and estrogen/progesterone‐treated, OVX monkeys. These results indicate that, unlike other mammalian species, in the rhesus monkey the stimulatory and inhibitory effects of NPY on GnRH release depend on the site of NPY infusion within the brain rather than the ovarian steroidal environment.
The Effect of Long‐Term Castration on the Neuronal and Physiological Responses to Acute or Repeated Restraint Stress: Interactions with Opioids and Prostaglandins
Authors: Chen Xihua , Herbert J.
Published: September 2006

Sixty min supine restraint stress induced a marked, but transient, hypothermic response in intact male rats, which tended to recover towards pre‐stress levels or slightly overshoot during the later stages of restraint. Castration reduced the initial hypothermia but increased overshoots. Baseline (pre‐stress) core temperature was also higher in castrated than intact rats, but the reduction in stress‐induced hypothermia was still present even when this difference had been taken into account. The hypothermic response was not altered during the course of 10 sessions of daily repeated restraint in either intact or castrated rats. Castration did not alter cardiac responses to restraint. Both intact and castrated rats showed marked tachycardia during the initial 12 min of restraint, followed by a gradual fall towards baseline values. Repeated restraint accentuated the second phase of the cardiac response, without modifying the initial tachycardia, in both intact and castrated animals. The response of blood corticosterone levels to the first period of restraint was unaltered by castration but the attenuation observed after 10 sessions of stress was more complete in castrated rats.

The neuronal c‐fos response 60 min after the last of the series of repeated restraints was less in the hypothalamic paraventricular nucleus, medial amygdala, and locus coeruleus compared with that following the first session, but not in the lateral septum or the bed nucleus of the stria terminalis. Castration did not change the c‐fos profile following the same number of restraint sessions. Castration depleted completely the vasopressinergic innervation in the lateral septum, diagonal band of Broca and medial amygdala.

Five mg/kg naloxone i.p. prior to the onset of stress resulted in persistent hypothermic responses throughout the stress period in intact males. This was reduced in castrated rats. Blockade of prostaglandins synthesis by 12 mg/kg indornethacin i.p. 3 h prior to stress had little effect on hypothermic stress responses.

The results of these experiments suggest that an androgen‐dependent process, possibly AVP‐containing systems in the forebrain, may regulate both baseline and stress‐induced levels of core temperature and have selective effects on components of the response to stress, including restraint‐induced hypothermia and the adaptation of corticoid responses to repeated restraint. Steroid‐dependent opioid systems may also be implicated in the hypotherrnic response to restraint stress.
Bilateral Lesions of the Suprachiasmatic Nuclei Alter the Nocturnal Melatonin Secretion in Sheep
Authors: Tessonneaud A. , Locatelli A. , Caldani M. , Viguier‐Martinez M. C.
Published: September 2006

The hypothalamic suprachiasmatic nuclei (SCN) constitute both the biological clock of many circadian rhythms, and the first relay in the transmission of light cues from the retina to the pineal gland, which releases, via nocturnal melatonin secretion, an endocrine expression of the daylength. The aim of the present work was to investigate the precise role of the SCN in the entrainment of the nocturnal rhythm of melatonin (MEL) in sheep. Bilateral lesions of the SCN were performed via a transsinusal surgical approach in 10 adult rams submitted to a constant photoperiod (16L:8D). Lesioned rams were compared to 4 sham and 2 control animals. Blood samples were collected 8 days before, 8 days after, and one month after surgery. Plasma MEL levels were estimated using direct radioimmunoassay. At the end of the experiment, histology and immunohistochemistry of the suprachiasmatic area were performed, and the extent of lesions was evaluated using a computerized image analysis system. Six rams exhibited a complete lesion of the SCN, and in the four remaining animals, the lesions were restricted to the anterior part of the SCN. For all animals, the nocturnal melatonin secretion was altered, but depending on the extent of the lesion, two types of results were observed:

Eight days after surgery, in 3 of the 4 rams bearing anterior lesions of the SCN (SCNJ, a nocturnal increase in melatonin secretion still occurred at dusk, but the duration of this secretion extended beyond the end of the night. One month after surgery, melatonin profiles were once again normal, as compared to sham animals. The fourth SCN, ram exhibited a cyclic MEL secretion not synchronized with the light cycle 8 days after surgery, and synchronized with dusk but not dawn one month after surgery.

Eight days after surgery, all the rams bearing complete lesions of the SCN (SCNZ) showed strongly altered MEL secretion. This secretion was continuous during the period of sampling, without any characteristic nocturnal peak. Nevertheless, one month after surgery, a nocturnal increase in melatonin secretion synchronized with the dusk was again observed, but the duration of the melatonin secretion was longer than the duration of the night.

These results confirm that the SCN mediates the nocturnal pineal gland activity, demonstrate that the anterior part of the sheep SCN is the main drive for the inhibition of the melatonin secretion at dawn and suggest that a positive input to the pineal gland may stimulate MEL secretion at dusk. The partial recovery of the cyclic secretion of melatonin after complete lesioning of the SCN suggests the existence of other structure(s) involved in this rhythmic function.
Increased Expression of Type 1 Angiotensin II Receptors in the Hypothalamic Paraventricular Nucleus following Stress and Glucocorticoid Administration
Authors: Aguilera Greti , Kiss Alexander , Luo Xun
Published: September 2006

Double staining in situ hybridization studies have shown that angiotensin II (All) type 1 receptors (AT1) in the hypothalamic paraventricular nucleus (PVN) are located primarily in corticotropin releasing hormone (CRH) neurons of the parvicellular subdivision. The purpose of these studies was to investigate the role of All regulating the hypothalamic‐pituitary adrenal (HPA) axis, by correlating AT1 receptor expression levels in the PVN with the known changes in activity of the HPA axis under different stress paradigms, and manipulation of circulating glucocorticoids. AT1 receptor mRNA was measured by in situ hybridization using 35S‐labelled cRNA probes and All binding by autoradiography using 125I[Sar1,lle8]All in slide mounted hypothalamic sections. AT1 receptor mRNA levels and All binding in the PVN were reduced by about 20% 18 h after adrenalectomy remaining at these levels up to 6 days after. This effect was prevented by corticosterone administration in the drinking water, or dexamethasone injection (100 mg, s.c., daily). Conversely, dexamethasone injection in intact rats caused a 20% increase in AT1 receptor mRNA in the PVN. AT1 receptor mRNA and binding in the PVN increased 4 h after exposure to stress paradigms associated with activation of the HPA axis (immobilization for 1 h, or i.p. injection of 1.5 M NaCl), and remained elevated after repeated daily stress for 14 days. Unexpectedly, two osmotic stress models associated with inhibition of the HPA axis (60 h water deprivation or 12 days of 2% saline intake) also resulted in increased AT1 receptor mRNA levels and All binding in the parvicellular PVN. In intact rats, the stimulatory effect of acute stress on AT1 receptor mRNA in the PVN was significantly enhanced by dexamethasone administration (100 μg, s.c., 14 h and 1 h prior to stress), while in adrenalectomized rats, with or without glucocorticoid replacement, stress reduced rather than increased, AT1 receptor mRNA. Dexamethasone, 100 μg, injected sc within 1 min the beginning of immobilization in adrenalectomized rats, increased AT1 receptor mRNA in the PVN to levels significantly higher than those after dexamethasone alone, indicating that the stress induced glucocorticoid surge is required for the stimulatory effect of stress on AT1 receptor mRNA. The data suggest that AT1 receptor expression in the PVN is under dual control during stress: stress‐activated inhibitory pathways and the stimulatory effect of glucocorticoids. The lack of specificity of the changes in AT1 receptor expression in the PVN following stressors with opposite effects on ACTH secretion (osmotic and physical‐psychological stress) does not support a role for All as a major determinant of the response of the HPA axis during stress.
Bacterial Lipopolysaccharide‐Induced Changes in FOS Protein Expression in the Rat Brain: Correlation with Thermoregulatory Changes and Plasma Corticosterone
Authors: Hare A. S. , Clarke G. , Tolchard S.
Published: September 2006

In the present study the regions of the brain showing an increase in the number of FOS protein stained cells 180 min following intravenous saline or bacterial lipopolysaccharide (LPS) treatment were investigated and correlated with changes in body temperature and plasma corticosterone levels. Particular attention was given to the possible involvement of the circumventricular organs and regions of the brainstem containing central noradrenergic neurones. LPS at doses of 0.35, 3.5 and 50 μg caused highly significant increases in FOS protein expression in the organum vasculosum of lamina terminalis, the area postrema and the subfornical organ compared with saline controls. Marked increases in bacterial lipopolysaccharide‐induced FOS protein expression were observed in the ventrolateral medulla, the nucleus of the solitary tract and the locus coeruleus which contain the A1, A2 and A6 noradrenergic neurones respectively. The changes in body temperature induced by LPS were found to be dependent upon the dose of LPS administered; the lowest dose employed (0.35 μg) induced an immediate and sustained fever, 3.5 μg LPS caused a biphasic response consisting of a hypothermic response followed by a febrile response, whereas 50 μg LPS induced a hypothermic response which then normalised by 160 min post‐injection. Intravenous saline injection had no significant effect on body temperature. The occurance of LPS‐induced hypothermia was coincident with increased FOS expression in the bed nucleus of stria terminalis, which houses vasopressinergic neurones involved in antipyresis, whereas in animals showing an LPS‐induced febrile response there was no significant difference in the number of FOS stained cells in the bed nucleus of stria terminalis compared with saline treated animals. LPS also caused marked increases in FOS protein expression in the parvocellular regions of the paraventricular nucleus (pPVN) of the hypothalamus, the central nucleus of the amygdala and the ventral septa1 area. Plasma corticosterone was unaffected by the lowest dose of LPS (0.35 μg), however the higher doses employed (3.5 and 50 μg) caused significant increases in plasma corticosterone which correlated with the increases in the number of FOS stained cells in the pPVN. The results of the present study suggest that, in addition to the organum vasculosum of lamina terminalis, the area postrema and subfornical organ may be important in the responses to antigenic challenge that are mediated by the central nervous system. They also add support to the possible involvement of the bed nucleus of stria terminalis in LPS‐induced hypothermia and of the involvement of the of the major noradrenergic cell groups (A1, A2 & A6) and a number of hypothalamic and extrahypothalamic forebrain regions in the interaction of immune and central nervous systems.
Effects of Dopamine D1 and D2 Receptor Agonists and Antagonists on Basal and Ethanol‐Modulated β‐Endorphin Secretion from Hypothalamic Neurons in Primary Cultures
Authors: Boyadjieva Nadka. I. , Sarkar Dipak. K.
Published: September 2006

In the present study, we determined the effects of dopamine receptor agonists and antagonists on basal and ethanol‐modulated β‐endorphin (β‐EP) secretion from hypothalamic neurons in primary cultures. Treatment with various concentrations of dopamine D1 agonist SKF 38393 and D1 antagonist SCH 23390 did not affect basal IR‐β‐EP release. However, dopamine D2 receptor agonist LY 141865 reduced basal immunoreactive (IR)‐β‐EP release in a concentration dependent manner. D2 receptor antagonist, sulpiride, on the other hand, stimulated basal IR‐β‐EP release and blocked LY 141865‐induced inhibition of IR‐β‐EP release in a concentration dependent manner. When the actions of these DA receptor agents on ethanol‐modulated IR‐β‐EP release were studied, both D1 and D2 receptor agents failed to affect ethanol‐modulated IR‐β‐EP release. These data suggest that the endogenous secretion of β‐EP from hypothalamic neurons is under the influence of an inhibitory dopaminergic system involving the D2 receptor. Furthermore, ethanol's effects on β‐EP secretion are not mediated by dopamine.
Cyclosporin A and FK506 are Potent Activators of Proopiomelanocortin‐Derived Peptide Secretion Without Affecting Corticotrope Glucocorticoid Receptor Function
Authors: Sheppard Karen E.
Published: September 2006

Unliganded glucocorticoid receptors (GR) are localized in the cytoplasm and are associated with heat shock protein (hsp)90, hsp70, and a member of the immunophilin family, FK506 binding protein 59 (FKBP59). Several members of the cyclophilin and FKBP families have now been shown to associate with unactivated steroid receptors, however the physiological role these immunophilins play in steroid receptor function is questionable. In the present study we have measured GR binding and nuclear translocation of activated receptor in corticotrope cells following treatment with the immunophilin ligands FK506 and cyclosporin A (CsA). Extensive GR binding studies in AtT20 cells, a mouse corticotrope tumor cell line failed to demonstrate an effect of FK506 or CsA on either the ability of GR to bind ligand, or on nuclear translocation of the liganded receptor at either a saturating or subsaturating dose of dexamethasone (DEX). Consistent with the binding data, functionally, neither CsA nor FK506 altered the glucocorticoid induced decrease in either proopiomelanocortin (POMC) derived peptide secretion or POMC heteronuclear (hn) RNA expression. Despite the fact these drugs did not modulate the actions of glucocorticoids on corticotrope cells, both FK506 and CsA were potent stimulators of basal β‐endorphin secretion (4–6 fold) from rat anterior pituitary cultures and AtT20 cells. In addition, FK506 and CsA potentiated β‐endorphin secretion induced by corticotropin releasing factor (CRF) and phorbol ester, but had no apparent acute (60 min) effect on POMC hnRNA levels. Unlike the acute actions of these immunosuppressant drugs, chronic (24 h) treatment lead to a decrease in cytoplasmic POMC mRNA with no apparent change in the amount of secreted β‐endorphin. Taken together these data suggest that FK506 and CsA do not alter GR activation or function in corticotrope cells, however, they are potent but short lived stimulators of POMC‐derived peptide secretion. The observation that CsA and FK506 stimulate POMC‐derived peptide secretion, and potentiate both phorbol ester and CRF induced secretion, suggests that these immunosuppressant drugs are acting upon a common point within these intracellular pathways.
Effect of Perinatal Hypothyroidism on Expression of Cytochrome C Oxidase Subunit I Gene, which is Cloned by Differential Plaque Screening from the Cerebellum of Newborn Rat
Authors: Koibuchi Noriyuki , Matsuzaki Shigeru , Ichimura Kaoru , Ohtake Hideki , Yamaoka Sadao
Published: September 2006

Early development of the central nervous system is influenced by several hormones including thyroid hormone. This study was designed to clone the gene whose expression is changed in association with perinatal hypothyroidism in the rat cerebellum. Rats were sacrificed at 15 day‐old postnatal age (P15) and their cerebella were removed. Poly (A)+ RNA was extracted to construct a cDNA library using λgt 10 cloning vector. Differential plaque screening was then performed using 32P‐labeled antisense cDNA synthesized from poly (A)+ RNA of the methimazole‐treated (hypothyroid) P15 rat cerebellum (hypothyroid probe), and of the euthyroid P15 rat cerebellum (euthyroid probe). The clones, which hybridized strongly to the euthyroid probe and weakly or not at all to the hypothyroid probe, were isolated. Sequence analysis of these clones revealed that all isolated clones encode cytochrome c oxidase subunit I (COX I), which is located in the mitochondria1 DNA. The decrease in COX I gene expression was not seen in the animals, which received methimazole treatment and daily replacement of thyroid hormone. In situ hybridization detection showed not only overall decrease in COX I gene expression but also change in distribution of hybridization signal in the cerebellar cortex of hypothyroid rat. Such change was not observed in the T4‐replaced animals. Based on the evidence that thyroid hormone greatly influences brain development, the results of the present study indicate that the terminal enzyme of mitochondria1 respiratory chain, COX I is one of the important target molecules regulated by thyroid hormone in the newborn rat cerebellum.
Inhibition of Hypothalamic Nitric Oxide Synthase Gene Expression in the Rat Paraventricular Nucleus by Food Deprivation is Independent of Serotonin Depletion
Authors: Ueta Yoichi , Levy Andrew , Chowdrey Hardial S. , Lightman Stafford L.
Published: September 2006

We have investigated the effects of food deprivation on nitric oxide synthase (NOS) transcript levels in the rat paraventricular (PVN) and supraoptic nuclei (SON), using in situ hybridization histochemistry. Food deprivation for 48 h significantly and consistently reduced NOS transcript prevalence by approximately 50% in both sites. Since there is considerable evidence for an important role of 5‐HT in feeding behaviour, we then examined the effect of food deprivation on NOS gene expression in the PVN following para‐chlorophenylalanine (PCPA)‐induced hypothalamic 5‐HT depletion. As starvation causes central down‐regulation of the thyroid axis, changes in thyrotropinreleasing hormone (TRH) and pituitary thyrotrophin (TSH) transcript prevalence were used as internal controls. PCPA pretreatment (200 mg/kg body weight as a single daily dose ip for 2 days) had no significant effect on basal levels of NOS, TRH or TSH transcripts, or on the effect of a subsequent 48 h fast, which significantly reduced all three. These results show for the first time, that food deprivation for 48 h significantly reduces NOS gene expression in the rat PVN and SON. Secondly, that basal levels and the fasting‐induced reductions in the prevalence of NOS, TRH and TSH transcripts were not affected by PCPA‐induced hypothalamic 5‐HT depletion. Therefore, at least under the experimental conditions used here, 5‐HT does not appear to be involved in setting baseline levels—or in the starvation‐induced inhibition of NOS or thyroid axis gene expression in the PVN.
Anorexia Induced by the Parasitic Nematode, Nippostrongylus brasiliensis: Effects on NPY and CRF Gene Expression in the Rat Hypothalamus
Authors: Horbury S. R. , Mercer J. G. , Chappell L. H.
Published: September 2006

Infections of the gastrointestinal nematode, Nippostrongylus brasiliensis, in the laboratory rat result in a characteristic biphasic anorexia which is followed by hyperphagia once the worm burden has been cleared. Despite the importance of parasite‐induced anorexia, relatively little is known of the underlying mechanisms. We have investigated the involvement of the central appetite drive in this anorexia by studying the gene expression of two neuropeptides with opposing actions on energy balance, NPY and CRF. Gene expression was assessed by in situ hybridization at 2, 8 and 16 days post‐infection (p.i.) in infected rats, in uninfected controls, and in a group with food intake restricted to match that taken voluntarily by the parasitized animals. The sampling intervals corresponded to each of the two phases of maximum anorexia and the period of compensatory hyperphagia. Surprisingly, we found that increases in NPY gene expression in the hypothalamic arcuate nucleus (ARC) accompany anorexia in rats infected with N. brasiliensis; there was a significant relationship between degree of anorexia and induction of NPY mRNA after 8 days of infection. Furthermore, ARC NPY mRNA levels in parasitized animals were similar to those in pair‐fed individuals with food intake restricted to match the infected rats. The number of larvae used to establish the infection affected both the degree of anorexia and the level of NPY mRNA at 8 days p.i. in a dose‐dependent manner. NPY gene expression remained elevated in infected rats during at least the initial stages of compensatory hyperphagia. This suggests that animals detect a state of energy deficit during the early stages of the infection, yet do not feed, but become hyperphagic coincident with worm loss. The failure of anorectic parasitized animals to feed in response to activation of the NPYergic system makes this a novel system in which to study the regulation of hypothalamic NPY by physiological challenge. There were no significant differences in CRF gene expression between the groups at any of the sampling intervals.