Arginine vasopressin (AVP) depletion in neurons of the suprachiasmatic nuclei affects the AVP content of the paraventricular neurons and stimulates adrenocorticotrophic hormone release

Arginine vasopressin (AVP) produced in the hypothalamic suprachiasmatic nuclei (SCN) plays a role in establishing neuroendocrine rhythms and, in particular, in regulating the corticotrope axis rhythm. It has recently been shown that AVP from SCN inhibits corticosteroid release. In order to investigate the influence of suprachiasmatic AVP on the different peptidergic systems through the hypothalamus, SCN neurons containing AVP were functionally lesioned by using toxins associated with a cytotoxic monoclonal antibody (MAb) raised against AVP. Six days later, the AVP contents and AVP mRNA were measured in different hypothalamic and extrahypothalamic sites. Adrenocorticotrophic hormone (ACTH) concentration was also measured in plasma. Microinjection of the AVP-MAb/toxin mixture into SCN brought about a significant decrease in the AVP expression in SCN. This is demonstrated by the decrease in the AVP immunoreactive content (24%, P < 0.01) and the decrease of AVP hybridized mRNA (33%, P < 0.01). This points to the efficiency of the microinjection in decreasing the production of AVP in the injection area. Modifications of the AVP contents in the two subdivisions of the hypothalamic paraventricular nucleus (PVN) were also observed. AVP contents decreased in the parvocellular subdivision (pPVN); this is coherent with the AVP depletion in SCN since pPVN is the major site of the SCN hypothalamic efferences. AVP content and AVP mRNA increased in the magnocellular subdivision (mPVN); this also confirms the difference in AVP synthesis regulation according to the PVN subdivisions. The microinjection did not modify AVP expression in supraoptic nuclei or oxytocin (OT) immunoreactive content in the main hypothalamic OT containing sites. Plasma ACTH values were double (P < 0.02) the values measured under non-specific IgG treatment 10 hr after lights on. This probably resulted from the stimulation of the hypothalamo-pituitary-adrenal system since corticotrophin-releasing hormone (CRH) mRNA increased simultaneously by 24% (P < 0.05) in the PVN and the immunoreactive CRH content of the median eminence significantly decreased (26%, P < 0.05). Overall, our data confirm that AVP produced in the SCN inhibits the CRH-adrenocorticotrope axis in normal conditions, probably because of SCN projections of AVP neurons on the PVN. J. Neurosci. Res. 50:565–574, 1997. © 1997 Wiley-Liss, Inc.     

Exaggerated response of arginine vasopressin-enhanced green fluorescent protein fusion gene to salt loading without disturbance of body fluid homeostasis in rats

We examined the effects of chronic salt loading on the hypothalamic expressions of the enhanced green fluorescent protein (eGFP), arginine vasopressin (AVP) and oxytocin (OXT) genes in AVP-eGFP transgenic rats that expressed eGFP in the hypothalamic AVP-containing neurones. In these rats, salt loading for 5 days caused a marked increase of the eGFP fluorescence in the magnocellular divisions of the paraventricular nucleus (PVN), the supraoptic nucleus (SON) and the internal layer of the median eminence. Expression of the eGFP gene was increased seven- to eight-fold in the PVN and SON of salt-loaded rats in comparison with euhydrated rats. By contrast, none of these changes were observed in the suprachiasmatic nucleus. The expression of the AVP and OXT genes was increased 1.5- to two-fold in the PVN and SON of salt-loaded nontransgenic (control) and transgenic rats. There were no differences in the expression levels of the AVP and OXT genes in the PVN and SON between nontransgenic (control) and transgenic animals under normal conditions and after salt loading. In the posterior pituitary gland, the intensity of the eGFP fluorescence did not change after salt loading for 5 days, but increased after 10 days of salt loading. Upon salt loading, significant increases in the plasma AVP concentrations, plasma osmolality and plasma Na+ were observed. Furthermore, there were no significant differences in changes of water intake, food intake, urine volume, urine osmolality, urine Na+ concentrations, and the body weights in both models under normal or salt-loaded conditions. Our results show that the response of the AVP-eGFP fusion gene to chronic salt loading is exaggerated, and humoral responses such as AVP and OXT and the body fluid homeostasis are maintained in AVP-eGFP transgenic rats. The AVP-eGFP transgenic rat gives us a new opportunity to study the dynamics of the AVP system in vivo.     

Unchanged Amounts of Vasopressin mRNA in the Supraoptic and Paraventricular Nucleus during Aging and in Alzheimer's Disease

The paraventricular (PVN) and supraoptic nucleus (SON) demonstrate a striking stability with respect to cell numbers during aging and Alzheimer’s disease (AD). Vasopressin (AVP) neurons even become activated during aging as judged from several parameters for neuronal activity, such as increased AVP plasma levels, enlarged nucleolar as well as cell size and an increased size of the Golgi apparatus in AVP-neurons. The activation possibly occurs as compensation for an age-related loss of AVP-receptors in the kidney. As a specific marker for AVP synthesis, we used quantitative in situ hybridization and estimated total amounts of AVP-mRNA in the entire SON and PVN of 14 control subjects and 14 AD patients that were matched for age, fixation time, postmortem delay and storage time of the tissue in paraffin. Following quantification, no differences were observed in total amounts of AVP-mRNA in the SON or PVN between young and old controls or between young and old AD patients, nor between the entire group of controls and AD patients. A significant negative correlation was found between the volume of the AVP-mRNA signal in the AD SON and age while the total amount of mRNA remained the same. This suggests a redistribution of cells or cell compartments in aging. A significant positive relation in both SON and PVN of AD patients was found between storage time of the paraffin-embedded tissue and the total amount of AVP-mRNA. A significant positive relation was present in the PVN, but not SON between pH of the cerebrospinal fluid, which is a marker for agonal state and the total amount of AVP mRNA. The present unchanged AVP-mRNA levels in SON and PVN confirm earlier observations on the stability of cell numbers in these nuclei in aging and AD. Although on the basis of other parameters, AVP-mRNA upregulation was expected, gradual, chronic stimulation over prolonged periods of time may, possibly, induce alternative mechanisms of regulation such as changes in translatability or in mRNA stability.     

Differential regulation of oxytocin and vasopressin messenger ribonucleic acid levels by gonadal steroids in postpartum rats

We previously reported that sequential estradiol and progesterone exposure followed by progesterone withdrawal increases oxytocin (OT), but not arginine vasopressin (AVP), messenger ribonucleic acid (mRNA) in the hypothalamic paraventricular nucleus (PVN) of the rat. Substitution of testosterone for progesterone and subsequent testosterone withdrawal in the estrogen-primed rat increases PVN AVP mRNA levels. At the end of pregnancy (day 21), rats are exposed to high estrogen and declining progesterone and testosterone concentrations. Coincident with these changes in circulating gonadal steroid hormones are increases in OT and AVP mRNAs. If progesterone levels are sustained at term, OT levels are attenuated and if testosterone is sustained, AVP mRNA levels are attenuated. Immediately postpartum, however, OT and AVP mRNA levels decline compared to term levels. To further determine the role of estrogen in the regulation of OT and AVP mRNAs, we performed two experiments. In the first experiment, we administered estrogen during the peripartum period to determine if estrogen supplementation prevents the relative attenuation of OT and AVP mRNAs that is seen after parturition. Day 18 pregnant rats were given estradiol-filled or empty capsules and sacrificed on day 2 of lactation. By Northern analysis, significant differences in PVN AVP, but not OT, mRNA were found between the estrogen- and sham-treated lactational animals, P < 0.02. In the second experiment, we determined if sustaining estrogen after progesterone is removed in steroid-treated ovariectomized rats is essential for the increase in OT mRNA. Ovariectomized rats were given either empty capsules or sequential estradiol- and progesterone-filled capsules and both were sustained for 12 days. When progesterone-filled capsules were removed, estradiol-filled capsules were either removed or left in place, and the animals were sacrificed 48 h later. PVN OT mRNA was analyzed by Northern blot hybridization. OT mRNA was increased in both of the steroid-treated groups to the same degree, compared to sham-treated animals, P = 0.04. In summary, estrogen supplementation during early lactation prevents the attenuation of PVN AVP, but not OT, mRNA after parturition. In the estrogen-primed ovariectomized rat, it is not necessary to sustain estrogen to see the effects of progesterone withdrawal upon PVN OT mRNA.     

Hypothalamic Galanin‐Like Peptide Rescues the Onset of Puberty in Food‐Restricted Weanling Rats

Galanin‐like peptide (GALP) is a known mediator of metabolism and reproduction; however, the role that GALP plays in the onset of puberty is unknown. First, we tested the hypothesis that central GALP administration could rescue puberty in food‐restricted weanling rats. GALP treatment in food‐restricted rats of both sexes rescued the timing of the onset of puberty to that seen in ad lib. fed controls. Second, we tested whether GALP translation knocked‐down in ad lib. fed, prepubertal rats would alter the timing of puberty. Knock‐down females, but not males, showed a significant (P < 0.01) delay in the onset of puberty compared to controls. Third, we sought evidence that the role of GALP in pubertal onset is mediated by the kisspeptin system. In situ hybridisation analyses showed a significant (P < 0.01) reduction in Kiss1 mRNA within the hypothalamic arcuate nucleus in food‐restricted rats compared to ad lib. fed controls and this reduction was prevented with i.c.v. GALP administration. Furthermore, analyses of Fos‐immunoreactivity (‐IR) after i.c.v. GALP treatment did not elicit Fos‐IR within any kisspeptin neurones, nor are GALP and kisspeptin peptides or mRNA colocalised. These data demonstrate that hypothalamic GALP infusion maintained the onset of puberty in food‐restricted weanling rats, although probably not via direct innervation of kisspeptin neurones.     

Cold and novel environment stress affects AVP mRNA in the paraventricular nucleus, but not the supraoptic nucleus: An in Situ hybridization study

The distribution and area of label for arginine vasopressin (AVP) mRNA or peptides were studied in rats exposed to cold or novel environments. In situ hybridization histochemistry was employed to detect AVP mRNA in hypothalamic frozen sections with a 45-mer photobiotinylated oligonucleotide probe. The storage of the peptide in both the hypothalamus and the pituitary was determined by immunohistochemistry. Label for mRNA or peptide was then quantified by the Cue-3 color image analysis system. Exposure to 4°C for 30 min caused a 3.5-fold increase in the label for AVP mRNA in the paraventricular nucleus (PVN) compared with that of control rats. This was correlated with a 2-fold elevation in serum ACTH. In addition, rats exposed to 30 min of a novel, thermoneutral (24°C) environment showed a 1.2- to -2.3-fold enhancement of the label for AVP mRNA in the PVN. In contrast, no changes were seen in the supraoptic nucleus (SON) following exposure to either cold or novel environments. Furthermore, neither stress caused significant changes in the storage of AVP peptide in the PVN, SON, median eminence, and posterior lobe of pituitary. This in vivo study demonstrates that PVN and SON neurons respond differentially to cold and novel environment exposures. The elevation of serum ACTH is correlated with the increased level of label for AVP mRNA in the rat hypothalamus, which suggests that AVP may play a role in the regulation of pituitary—adrenal responses to cold and novel environment stresses.     

Oxytocin in the Central Amygdaloid Nucleus Modulates the Neuroendocrine Responses Induced by Hypertonic Volume Expansion in the Rat

The present study investigated the involvement of the oxytocinergic neurones that project into the central amygdala (CeA) in the control of electrolyte excretion and hormone secretion in unanaesthetised rats subjected to acute hypertonic blood volume expansion (BVE; 0.3 M NaCl, 2 ml/100 g of body weight over 1 min). Oxytocin and vasopressin mRNA expression in the paraventricular (Pa) and supraoptic nucleus (SON) of the hypothalamus were also determined using the real time‐polymerase chain reaction and in situ hybridisation. Male Wistar rats with unilaterally implanted stainless steel cannulas in the CeA were used. Oxytocin (1 μg/0.2 μl), vasotocin, an oxytocin antagonist (1 μg/0.2 μl) or vehicle was injected into the CeA 20 min before the BVE. In rats treated with vehicle in the CeA, hypertonic BVE increased urinary volume, sodium excretion, plasma oxytocin (OT), vasopressin (AVP) and atrial natriuretic peptide (ANP) levels and also increased the expression of OT and AVP mRNA in the Pa and SON. In rats pre‐treated with OT in the CeA, previously to the hypertonic BVE, there were further significant increases in plasma AVP, OT and ANP levels, urinary sodium and urine output, as well as in gene expression (AVP and OT mRNA) in the Pa and SON compared to BVE alone. Vasotocin reduced sodium, urine output and ANP levels, although no changes were observed in plasma AVP and OT levels or in the expression of the AVP and OT genes in both hypothalamic nuclei. The results of the present study suggest that oxytocin in the CeA exerts a facilitatory role in the maintenance of hydroelectrolyte balance in response to changes in extracellular volume and osmolality.     

Subtle sex differences in vasopressin mRNA expression in the embryonic mouse brain

Arginine vasopressin (AVP) is a neuropeptide which acts centrally to modulate numerous social behaviors. One receptor subtype through which these effects occur is the AVP 1a receptor (AVPR1A). The modulatory effects of Avp via the AVPR1A varies by species as well as sex, since both AVP and the AVPR1A tend to be expressed more prominently in males. Beyond these neuromodulatory effects there are also indications that the AVP system may play a role in early development to, in part, organize sex‐specific neural circuitry that is important to sexually dimorphic social behaviors in adulthood. However, to date, AVP's role in early development is poorly understood, particularly with respect to its differential effect on males and females. In order to determine the timing and distribution of the AVP system in early brain development, we examined the brains of male and female C57BL/6J mice between embryonic day (E) 12.5 and postnatal day (P) 2 and quantified Avp and Avpr1a mRNA using qPCR and AVPR1A protein using receptor autoradiography. The mRNA for Avp was measurable in males and females starting at E14.5, with males producing more than females, while Avpr1a mRNA was found as early as E12.5, with no difference in expression between sexes. AVPR1A binding was observed in both sexes starting at E16.5, and while there were no observed sex differences, binding density and the number of neuroanatomical areas did increase over time. These data are significant as they provide the first whole‐brain characterization of the vasopressin system in the embryonic mouse. Further, these findings are consistent with data from other species, that have documented a sex difference in the vasopressin system during early brain formation.     

Sexually dimorphic expression of calcitonin gene-related peptide (CGRP) immunoreactivity by rat mediobasal hypothalamic neurons

Although the hypothalamic arcuate nucleus is a sexually dimorphic region of the rat brain, there are no reports of sex differences in the number of neurons containing specific neuropeptides within this structure. As cells synthesizing calcitonin gene-related peptide (CGRP) have been shown to exhibit sex differences in other steroid-receptive regions of the rat brain, we examined whether the CGRP-immunoreactive cells located in the mediobasal hypothalamus may also be sexually dimorphic. Immunostaining of sections from male and female colchicine-treated rats revealed a small population of CGRP-immunoreactive cells distributed throughout the arcuate nucleus. Immunoreactive cells were also detected in the lateral hypothalamic perifornical region, dorsomedial, posterior periventricular and ventral tuberomammillary nuclei, and zona incerta. Cell count analysis revealed approximately twice as many CGRP-immunoreactive cell profiles in the rostral (P < 0.01), middle (P < 0.001), and caudal (P < 0.01) thirds of the arcuate nucleus of male rats compared with females. A significant sex difference in immunoreactive cell numbers (male > female) was also detected within the caudal dorsomedial nucleus (P < 0.05) but not in the posterior periventricular nucleus, perifornical region and zona incerta. Although fibers immunoreactive for CGRP were identified in low density throughout the mediobasal hypothalamus, only female rats displayed prominent fiber staining in the periventricular region. Double-labelling immunofluorescence experiments revealed that the CGRP-immunoreactive cells within the zona incerta, but not the hypothalamus, were also immunoreactive for tyrosine hydroxylase; at least 60% of the A13 dopaminergic neurons co-express CGRP. These results provide evidence that sex differences exist in the number of specific neuropeptide-synthesizing cells within the hypothalamic arcuate nucleus and provide further examples of cell populations expressing CGRP immunoreactivity in a sexually dimorphic manner. © 1996 Wiley-Liss, Inc.     

Amphetamine treatment affects the extra‐hypothalamic vasopressinergic system in a sex‐ and nucleus‐dependent manner

The lateral septum (LS), a brain structure implicated in addictive behaviours, regulates the activation of dopaminergic neurones in the ventral tegmental area. Vasopressinergic projections from the extended amygdala to the LS, which are sexually dimorphic, could be responsible for the vulnerability to addiction in a sex‐dependent manner. The present study aimed to investigate the modulatory effects of amphetamine (AMPH) on the expression of vasopressin (AVP) in the vasopressinergic extra‐hypothalamic system in sensitised male and female rats. Adult male and female Sprague‐Dawley rats underwent an AMPH‐locomotor sensitisation protocol. Acute AMPH increased AVP mRNA expression in the medial amygdala (MeA), whereas AMPH‐induced sensitisation increased AVP mRNA expression in the bed nucleus of the stria terminalis (BNST) only in females. Interestingly, the increase in AVP expression in BNST was higher in oestrus females compared to dioestrus females and acute AMPH resulted in a decrease in AVP levels in the LS, only in males. Thus, there are complex and region‐specific interactions between AMPH and the extra‐hypothalamic vasopressinergic system in the brain, underlying possible alterations in different behaviours caused by acute and chronic AMPH exposure.     

Sex-Steroid Control of Galanin in the Rat Hypothalamic-Pituitary Axis

To further investigate how sex steroids regulate galanin (GAL) in the rat pituitary and hypothalamus, we examined the effects of prepubertal gonadectomy (Gx) and long-term (9 weeks) replacement with estradiol (E₂) or testosterone (T) on pituitary and hypothalamic GAL concentrations in Wistar rats (5–6/group). Sham-operated animals served as controls (CTR). Pituitary GAL concentration was markedly higher in random-cycling CTR-females than in CTR-males (1391 ± 247 vs 39 ± 5 pg/mg protein, P<0.01) and decreased after Gx only in females (20 ± 3 pg/mg protein, P<0.01). E₂ strongly increased pituitary GAL in Gx-females and Gx-males (4470 ± 365 and 3853 ± 347 pg/mg protein, P<0.01), whereas T had no effect. Inversely, hypothalamic GAL was higher in CTR males than in CTR females (5.4 ± 0.3 vs 4.0 ± 0.5 ng/mg protein, P<0.05), and decreased significantly after gonadectomy in males (3.7 ± 0.2 ng/mg protein, P<0.01). The only steroid treatment that significantly modified hypothalamic GAL in Gx animals was administration of E₂ to females (5.7 ± 0.4 ng/mg protein, P<0.01 vs non-treated Gx). We also studied in hypophysectomized (Hx) rats (8/group) the effects of sex steroids on hypothalamic GAL concentration and distribution. The low hypothalamic GAL concentration observed in male and female Hx rats (1.0 ± 0.1 ng/mg protein) was significantly increased by T in males and in females (respectively, by 40% and by 50%, P<0.02) and by E₂ in males (by 60%, P<0.02). Immunohistochemistry showed that both E₂ and T increased GAL labeling in fibers restricted to the lateral regions of the median eminence of male and female Hx rats, a distribution corresponding to that of GnRH-immunoreactive fibers. In conclusion, in addition to the marked stimulatory effect of estradiol on pituitary GAL, endogenous and exogenous sex steroids also modulate hypothalamic GAL in male and female rats. Both E₂ and T may exert a stimulatory influence on hypothalamic GAL concentration and histochemical analysis shows that sex steroids enhance GAL immunoreactivity in a subset of neurons that closely overlaps the distribution of GnRH neurons.     

Vasopressin and the regulation of evaporative water loss and body temperature in the cat

The role of vasopressin as a possible mediator of the inhibition of evaporative water loss (EWL) in dehydrated, heat-stressed cats has been examined by intravenous (i.v.) and intracerebroventricular (i.c.v.) injections of arginine vasopressin (AVP). In normally hydrated cats exposed to an ambient temperature (Ta) of 38°C, neither EWL nor body temperature (Tb), measured in the hypothalamus, was significantly altered by i.v. AVP infusion. Measurements of plasma osmolality (pOsm), pAVP and cerebrospinal fluid AVP (csfAVP) were made normally hydrated cats at Tas of 25 and 38°C and after dehydration for 1–4 days at these temperatures. The relationship between pOsm and pAVP can be described equally well by either a linear model or a log-linear model (r = 0.81 for both models). The pOsm-csfAVP relationship is best described by alog-linear model (r = 0.80). A possible role for intracranially released AVP in ody temperature regulation and control of EWL was examined by injecting various doses of AVP into the lateral ventricles of normally hydrated cats. No effect of AVP injection on Tb was observed at either a Ta of 23°C or 38°C. EWL was also unaffected by i.c.v. AVP administration at a Ta of 38°C. To confirm futher that intracranial AVP is not responsible for elevation of Tb and reduction of EWL during dehydration and heat-stress, specific antiserum to AVP was injected into the ventricle of dehydrated animals at a Ta of 38°C. No sinificant effect on either Tb or EWL was measured subsequent to antiserum infusion. These negative findings indicate that AVP does not suppress EWL by either a peripheral or a central action and is therefore not responsible for lowered EWL and elevated Tb seen in dehydrated heat-stressed cats.     

Repetitive noxious neonatal stimuli increases dentate gyrus cell proliferation and hippocampal brain‐derived neurotrophic factor levels

Neonatal noxious stimulation has been proposed to model pain triggered by diagnostic/therapeutic invasive procedures in premature infants. Previous studies have shown that hippocampal neurogenesis rate and the behavioral repertoire of adult rats may be altered by neonatal noxious stimuli. The purpose of this study was to evaluate whether noxious stimulation during neonatal period alters the nociceptive response and dentate gyrus neurogenesis when compared to rats subjected to a single noxious stimulus in late infancy. Plasma corticosterone and hippocampal brain‐derived neurotrophic factor (BDNF) levels were measured. Neurogenesis in the dentate gyrus was evaluated in adolescent rats (postnatal day 40; P40) exposed twice to intra‐plantar injections of Complete Freund's adjuvant (CFA) on P1 and P21 (group P1P21) or P8 and P21 (P8P21) or exposed once on P21 (pubertal). On P21, one subset of animals received 5‐bromo‐2′‐deoxyuridine (BrdU) and was euthanized on P40 for identification of proliferating cells in the dentate gyrus. Another subset was sampled for thermal response or plasma corticosterone measurement and hippocampal BDNF levels. Proliferative cell rate in dentate gyrus was the highest in all re‐exposed groups (P < 0.001), except for P8 females (P8P21F), revealing also a sex difference, where P8P21 males showed higher rate than females (P < 0.001). Stimulated groups took longer than CTL animals to lick the paws (P < 0.001), regardless of the age when the noxious stimulus was applied. Re‐exposed groups had lower corticosterone plasma level (P1P21 M and F, P8P21M) than controls. On the contrary, hippocampal BDNF was increased in males from both re‐exposed groups. These results show that infant noxious stimulation in neonatally previously stimulated rats is related to high proliferation in the DG and this association seems to be modified by the animal's sex. The new generated dentate granule cells in the hippocampus may have a role in the long‐term behavioral responses to neonatal nociceptive stimulation. Noxious stimulation in the neonatal period results in sex‐dependent neurogenic response. © 2013 Wiley Periodicals, Inc.     

Androgen and Estrogen Effects on Vasopressin Messenger RNA Expression in the Medial Amygdaloid Nucleus in Male and Female Rats

Vasopressin messenger RNA (AVP mRNA) expression in the medial amygdala and bed nucleus of the stria terminalis (BST) is almost completely dependent on gonadal steroids. In the BST, the effects of gonadal steroids on AVP mRNA expression are sexually dimorphic. Males have more cells that express AVP mRNA and more AVP mRNA per cell than females. Here we test whether this is also true for the MA. In gonadectomized rats that were treated with testosterone, males had more cells that were labeled for AVP mRNA than females. However, the labeling per cell did not differ between males and females. To assess contribution of testosterone metabolites to these differences, male and female rats were gonadectomized and implanted with empty tubing, or tubing filled with dihydrotestosterone (DHT), estradiol (E), or E plus DHT (E + DHT). The pattern of steroid effects on AVP mRNA expression in the MA was similar in both sexes. Hardly any labeled cells were found in rats with empty implants or rats treated with DHT. Significantly more labeled cells were found in rats treated with E, and even more cells in rats treated with E + DHT. The number of AVP mRNA-labeled cells was higher in males than in females for E as well as E + DHT treatment, but the labeling per cell did not differ between sexes. These data suggest that the number of MA cells that can express AVP mRNA is higher in males than in females, but the estrogen and androgen responsiveness of individual AVP mRNA-expressing cells in the MA does not differ between sexes.     

Influence of Testosterone on LHRH Release,LHRH mRNA and Proopiomelanocortin mRNA in Male Sheep1

The mechanism whereby testosterone (T) reduces pulsatile LHRH and LH release is unknown. We tested the hypothesis that hypothalamic levels of LHRH mRNA decrease and proopiomelanocortin (POMC) mRNA increase coincident with reduced LHRH release induced by either long-term or short-term T treatment in male sheep. Experiment 1 examined the effect of long-term T exposure on LHRH and LH release and LHRH and POMC mRNA levels. Yearling Suffolk rams were castrated and assigned to one of four treatments: 1) castrated (n = 4); 2) castrated, portal cannula (n = 5); 3) castrated +T (n = 4) and 4) castrated+T, portal cannula (n = 4). T-treated males received ten 10-cm silastic T-implants immediately after castration. Surgical placement of devices for collecting hypophyseal-portal blood occurred 2 to 3 months after castration. Seven to 10 days after surgery, blood samples were collected at 10-min intervals for 8h from portal cannulated males or for 5 h from non-cannulated males to assess pulsatile LHRH and/or LH release. Immediately after blood sample collection, hypothalamic tissue was collected for in situ measurement of LHRH or POMC mRNA. T-treatment decreased (P < 0.01) mean LHRH and LH and decreased (P < 0.01) LHRH and LH pulse frequency. T did not significantly affect (P > 0.10) silver grain area per LHRH neuron, but decreased (P < 0.01) silver grain area per POMC neuron. Portal cannulation tended to decrease (P= 0.057) silver grain area per LHRH neuron without significantly affecting (P > 0.10) LHRH cell numbers while reducing (P < 0.01) silver grain area per POMC neuron and POMC cell numbers. A second experiment examined the effect of 72 h of T-infusion on LHRH and POMC mRNA levels. Castrated yearling males were assigned to receive either vehicle (n = 4) or T (768 ug/kg/day;n=4). Blood samples were collected at 10 min intervals for 4h prior to and during the final 4 h of infusion. Infusion of T decreased (P < 0.01) mean LH and LH pulse frequency. T did not significantly affect (P > 0.10) silver grain area per LHRH neuron or LHRH cell numbers. T reduced (P < 0.01) silver grain area per POMC neuron without affecting (P > 0.10) POMC cell number. We reject our hypothesis and conclude that reduced LHRH or heightened POMC gene expression are not mechanisms whereby T reduces pulsatile LHRH release in male sheep.     

Sex differences in brain MRI abnormalities and neurodevelopmental outcomes in a rat model of neonatal hypoxia-ischemia

Purpose/aim of the study: Hypoxic-ischemic brain injury (HIBI) is associated with high mortality and neurodevelopmental deficits. We explored gender influence in a HIBI rat model. Materials and methods: Sprague–Dawley rats underwent HIBI on postnatal day (P) 7. Nervous reflexes, footprints, Morris water maze performances and magnetic resonance imaging (MRI) were analyzed. Results: Mortality rate was higher in HIBI males (20%) than in females (12.5%). Growth rate was slower in the HIBI group (p < 0.05), but was similar between HIBI males and females. HIBI rats showed impaired performances in the cliff aversion reflex, negative geotaxis reflex and gait tests at P14 (p < 0.05), but not at P9 or P11. There were no significant differences for the cliff aversion reflex and gait tests between genders. Negative geotaxis reflex impairment at P14 was more severe in HIBI males (p < 0.05). Step length and toe distance contralateral (but not ipsilateral) to the cerebral damage were shorter in HIBI rats, and were shorter in HIBI males than females (p < 0.05). Morris water maze latency time and swimming distance were longer in the HI group than in controls, and prolonged in HIBI males compared with females (p < 0.05). In the HIBI group, MRI showed more severe injury at P10 and P67 in males than in females (p < 0.05). Conclusions: Male rats are more vulnerable to the detrimental consequences of HIBI, with more severe nervous reflex deficits, brain injury, memory impairment and hemiplegic paralysis than female rats. Serial neurobehavioral follow-up is still executed on the HIBI infants who is absent of detectable abnormalities in early children.     

Ontogeny of expression and glucocorticoid regulation of the arginine vasopressin gene in the rat hypothalamic paraventricular nucleus

Corticotropin-releasing factor and arginine vasopressin (AVP) are the two major hypothalamic factors that regulate anterior pituitary adrenocorticotropin secretion during stress. We have previously reported that the expression of the corticotröpin-releasing factor gene in the hypothalamus and its regulation by glucocorticoids were not mature during the first week of life in the rat, i.e. during the stress non-responsive period. In this report, we studied the ontogeny of expression of the AVP gene in the hypothalamic paraventricular nucleus in rats using in situ hybridization. AVP mRNA was detected as early as day 20 of gestation (E20) both in the parvocellular and the magnocellular portion of the paraventricular nucleus. The levels of expression of the AVP gene increased steadily from E20 to the third day after birth (P3) and remained stable from P3 to P14. Bilateral surgical adrenalectomy induced an increase in AVP mRNA levels in the parvocellular portion of the paraventricular nucleus, but not in the magnocellular portion, in both 7-day-old and 14-day-old rats, suggesting that the glucocorticoid regulation of the AVP synthesizing neurons of the paraventricular nucleus is mature in the developing postnatal rat.     

Vasopressin in the cerebrospinal fluid of febrile children with or without seizures

Immaturity in water and electrolyte balance in the brain has been considered to increase the susceptibility of young animals and children to febrile convulsions (FCs). Arginine-vasopressin (AVP) is involved in the regulation of several centrally mediated events such as modulation of fever and the ease with which water permeates into and out of the brain. To evaluate the possible role of AVP in the control of water balance and susceptibility to convulsions during fever we measured the AVP concentration in the cerebrospinal fluid (CSF) and plasma of febrile children with or without convulsions. The febrile population consisted of 47 children, of whom 29 experienced seizures during fever. Seven children with epileptic symptoms and 18 children without seizures were included as nonfebrile controls. The CSF AVP concentration in febrile children without seizures and in nonfebrile convulsive children was significantly lower (0.60 ± 0.07 pmol / 1, mean ± SEM,P < 0.01 and 0.65 ± 0.19 pmol/l,P < 0.05, respectively) than in nonfebrile children without convulsions (0.83 ± 0.06 pmol/1). However, the levels of CSF AVP were not significantly different in children with FCs (0.71 ± 0.06 pmol/1) compared with other groups. CSF AVP correlated with the CSF osmolality (r = 0.33, P = 0.02). No statistical differences in plasma AVP levels between the groups could be found. The present data provide support for the hypothesis of synchronous regulation of osmolality and AVP concentration in CSF. During fever the concentration of CSF AVP was lower in nonconvulsive children compared with nonfebrile nonconvulsive children. CSF AVP levels were not affected in febrile children by convulsions.     

Differences in hypothalamic serotonin between estrous phases and gender: an in vivo microdialysis study

The aim of the present study was to assess whether there are gender differences in (1) levels of extracellular serotonin (5-HT) in the forebrain, and (2) the effect on 5-HT of a reuptake inhibitor, paroxetine, or a releasing drug, fenfluramine. In vivo microdialysis was used to measure 5-HT in the hypothalamus of male and regularly cycling female rats. Hypothalamic 5-HT was significantly lower in estrous females (0.83±0.05 pg/sample, n=33) than in male rats (1.04±0.06 pg, n=38). Levels in diestrous females (0.98±0.09 pg, n=38) were not significantly different from males. Paroxetine (1 mg/kg) increased hypothalamic 5-HT in males, and diestrous and estrous females to 2 pg/sample. However, the increase in hypothalamic 5-HT produced by a maximally effective dose of paroxetine (10 mg/kg) was significantly greater in male rats and during diestrous than during estrous.

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-Fenfluramine (10 mg/kg) evoked an increase in extracellular 5-HT to 15 pg/sample in all groups. A higher dose of

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-fenfluramine (20 mg/kg) produced a significantly greater increase in hypothalamic 5-HT in males than in females during estrous or diestrous. These results are consistent with other evidence that during estrous, when rats are responding to peak levels of estrogen and progesterone, 5-HT release is decreased.