Somatostatin Actions on Rat Supraoptic Nucleus Oxytocin and Vasopressin Neurones

Magnocellular neurones in the supraoptic nucleus (SON) receive major afferent inputs from the brainstem that have been implicated in the regulation of oxytocin and vasopressin secretion from the posterior pituitary. Notably, at parturition, some neurones that project from the nucleus tractus solitarii (NTS) in the brainstem directly to the SON are activated. Many of these are noradrenergic and regulate oxytocin secretion during parturition, whereas others contain somatostatin and their role is unclear. In the present study, we report that, at parturition, somatostatin mRNA expression in the NTS is significantly increased compared to pregnancy, suggesting an active role for these neurones at that time. Intracerebroventricular somatostatin infusion significantly increased plasma oxytocin secretion in both virgin female and pregnant rats. Intracerebroventricular somatostatin increased SON oxytocin and vasopressin neurone firing‐rates, and increased Fos expression in the SON and paraventricular nucleus and in the subfornical organ. Retrodialysis of somatostatin onto the ventrally exposed SON also increased vasopressin neurone firing rate but, unexpectedly, decreased oxytocin neurone firing rate. The experiments indicate that somatostatin neurones in the NTS are activated during parturition but, because the direct effects of somatostatin on oxytocin neurones are inhibitory, this direct pathway does not appear to contribute to enhanced oxytocin release at this time, although indirect somatostatin effects may do so.     

Oxytocin and vasopressin immunoreactivity in rabbit hypothalamus during estrus, late pregnancy, and postpartum

Mother rabbits construct an elaborate maternal nest before parturition and display a single, brief, daily nursing bout throughout lactation. These features present a unique model for investigating the relevance of changes in neuroendocrine secretion associated with pregnancy and parturition for the regulation of maternal behavior. In the present study we analyzed changes in the location, somal size, and number of oxytocin (OT)and arginine vasopressin (AVP)-immunoreactive (IR) neurons in the hypothalamus of rabbits in estrus, late pregnancy (day 29), and postpartum day 1. From estrus to late pregnancy, the number of OT-IR neurons increased in the scattered cell groups located in the lateral hypothalamic area (LHA), but not in the magnocellular nuclei, i.e., paraventricular nucleus (PVN) and supraoptic nucleus (SON). On postpartum day 1 the increase in the number of OT-IR neurons was sustained in the LHA and became apparent also in the main body of the PVN, in which the number of OT-IR neurons doubled. Increases in the somal size of OT-IR cells were seen in all three nuclei only on postpartum day 1. No OT-IR cells were found in the suprachiasmatic nucleus (SCN). From late pregnancy and into postpartum day 1 increases in the somal size of AVP-IR neurons were detected in the PVN, SON, and LHA but not in the SCN. The number of AVP-IR neurons increased between late pregnancy and postpartum day 1 in the SON only. The changes observed in OT and AVP expression in specific hypothalamic nuclei may be related to specific somatic and behavioral events occurring around the time of parturition, e.g., nest-building, maintenance of homeothermy, elevation of blood volume, and nursing in mother rabbits.     

Progesterone regulation of GABAA receptor plasticity in adult rat supraoptic nucleus

Marked plasticity in GABAA receptor signalling occurs in adult oxytocin neurons of the supraoptic nucleus (SON) through the modulation of GABAA receptor alpha subunits during pregnancy. The present studies were undertaken to examine the potential mechanisms underlying this plasticity. In vivo microdialysis experiments in conscious rats revealed that no significant changes in extracellular GABA concentrations occurred within the SON over the last two days of pregnancy and the time of parturition itself. In situ hybridization studies examined the effects of gonadal steroid manipulation upon the GABAA receptor subunits expressed by SON neurons (alpha1, alpha2, beta2 and gamma2 subunits) and demonstrated that cellular levels of the alpha1 subunit were increased following 8 days oestrogen and progesterone treatment. Estrogen alone or allopregnanolone, the progesterone derivative, had no effect on alpha1 subunit mRNA expression in the SON. Immunocytochemical experiments demonstrated progesterone receptors in many neural populations but not within the SON of late pregnant rats. These studies indicate that alterations in endogenous GABA release within the SON are unlikely to be responsible for the GABAA receptor plasticity exhibited by oxytocin neurons in late pregnancy. Rather, data demonstrate that the fluctuating concentrations of progesterone during pregnancy act indirectly on SON neurons to modulate alpha1 subunit mRNA expression. Together, these experiments provide evidence for the ligand-independent induction of GABAA receptor plasticity in the adult brain by progesterone.     

Projection target‐specific action of nicotine in the caudal nucleus of the solitary tract

The brainstem nucleus of the solitary tract (NTS) is the key integrating relay in the central processing of sensory information from the thoracic and from most subdiaphragmatic viscera. Modulation of neuronal excitability and synaptic activity in the NTS by nicotinic agents can have potent effects on vital physiological functions, such as feeding, digestion, respiration, and blood circulation. Caudal NTS neurons demonstrate considerable heterogeneity in projection targets, synaptic properties, and expression of nicotinic acetylcholine receptors (nAChRs). However, despite its heterogeneity, the caudal NTS may contain discrete subsets of neurons with unique projection target‐specific properties. To test this hypothesis, we used in vivo fluorescent tracing and ex vivo patch‐clamp electrophysiology to evaluate responsiveness to nicotine of anatomically identified caudal NTS neurons that project to the hypothalamic paraventricular nucleus (PVN) and the brainstem caudal ventrolateral medulla (CVLM). The results of this study demonstrate that responsiveness to nicotine correlates with where the neurons project. Specifically, PVN‐projecting caudal NTS neurons respond to nicotine only presynaptically (i.e., via activation of presynaptic nAChRs and potentiation of synaptic release of glutamate), suggesting indirect, glutamate‐dependent effects of nicotine on the PVN‐projecting NTS circuitry. By contrast, CVLM‐projecting caudal NTS neurons exhibit only limited presynaptic, but dominant somatodendritic, responsiveness to nicotine, suggesting that the effects of nicotine on the CVLM‐projecting NTS circuitry are direct and largely glutamate independent. Understanding the relationships among function‐specific brainstem/hypothalamic neuronal networks, nuclei, and individual neurons could help develop therapies targeting identifiable neuronal circuits to offset impaired autonomic homeostasis. © 2014 Wiley Periodicals, Inc.     

The magnocellular oxytocin system,the fount of maternity: adaptations in pregnancy

Oxytocin secretion from the posterior pituitary gland is increased during parturition, stimulated by the uterine contractions that forcefully expel the fetuses. Since oxytocin stimulates further contractions of the uterus, which is exquisitely sensitive to oxytocin at the end of pregnancy, a positive feedback loop is activated. The neural pathway that drives oxytocin neurons via a brainstem relay has been partially characterised, and involves A2 noradrenergic cells in the brainstem. Until close to term the responsiveness of oxytocin neurons is restrained by neuroactive steroid metabolites of progesterone that potentiate GABA inhibitory mechanisms. As parturition approaches, and this inhibition fades as progesterone secretion collapses, a central opioid inhibitory mechanism is activated that restrains the excitation of oxytocin cells by brainstem inputs. This opioid restraint is the predominant damper of oxytocin cells before and during parturition, limiting stimulation by extraneous stimuli, and perhaps facilitating optimal spacing of births and economical use of the store of oxytocin accumulated during pregnancy. During parturition, oxytocin cells increase their basal activity, and hence oxytocin secretion increases. In addition, the oxytocin cells discharge a burst of action potentials as each fetus passes through the birth canal. Each burst causes the secretion of a pulse of oxytocin, which sharply increases uterine tone; these bursts depend upon auto-stimulation by oxytocin released from the dendrites of the magnocellular neurons in the supraoptic and paraventricular nuclei. With the exception of the opioid mechanism that emerges to restrain oxytocin cell responsiveness, the behavior of oxytocin cells and their inputs in pregnancy and parturition is explicable from the effects of hormones of pregnancy (relaxin, estrogen, progesterone) on pre-existing mechanisms, leading through relative quiescence at term inter alia to net increase in oxytocin storage, and reduced auto-inhibition by nitric oxide generation. Cyto-architectonic changes in parturition, involving evident retraction of glial processes between oxytocin cells so they get closer together, are probably a response to oxytocin neuron activation rather than being essential for their patterns of firing in parturition.     

Regional suppression by water intake of c-fos expression induced by intraventricular infusions of angiotensin II

Intracerebroventricular (i.c.v.) infusions of angiotensin II (AII) reliably induced c-fos expression in the supraoptic (SON) and paraventricular (PVN) nuclei, as well as other areas of the basal forebrain including the OVLT, subfornical organ (SFO), and bed nucleus (BNST). Double-labelling showed that AII-induced c-fos was observed in both vasopressin (AVP-) and oxytocin (OXY)-containing neurons of the SON and PVN in male rats. Allowing rats to drink water after AII infusions suppressed c-fos expression both AVP- and OXY-stained magnocellular neurons. Intragastric infusions of water were also effective, showing that oro-pharyngeal stimuli were not critical. Maximal suppression occurred in rats in whom water had been infused intragastrically about 5 min before i.c.v. AII infusions, suggesting that changes in osmolarity were responsible. i.c.v. AII also induced c-fos expression in a number of brainstem structures, including the solitary nucleus (NTS), lateral parabrachial nucleus (LPBN), locus coeruleus (LC), and the area postrema (AP). These results indicate that AVP and OXY-containing neurons in the magnocellular parts of the SON and PVN alter their immediate-early gene response to AII after water intake, and that this does not depend upon oro-pharyngeal factors. Furthermore, AII can induce c-fos expression in a number of brainstem nuclei associated with autonomic function, and these do not respond to water intake.     

Allopregnanolone and induction of endogenous opioid inhibition of oxytocin responses to immune stress in pregnant rats

In virgin rats, systemic administration of interleukin (IL)-1β (i.e. to mimic infection), increases oxytocin secretion and the firing rate of oxytocin neurones in the supraoptic nucleus (SON). However, in late pregnancy, stimulated oxytocin secretion is inhibited by an endogenous opioid mechanism, preserving the expanded neurohypophysial oxytocin stores for parturition and minimising the risk of preterm labour. Central levels of the neuroactive metabolite of progesterone, allopregnanolone, increase during pregnancy and allopregnanolone acting on GABA(A) receptors on oxytocin neurones enhances inhibitory transmission. In the present study, we tested whether allopregnanolone induces opioid inhibition of the oxytocin system in response to IL-1β in late pregnancy. Inhibition of 5α-reductase (an allopregnanolone-synthesising enzyme) with finasteride potentiated IL-1β-evoked oxytocin secretion in late pregnant rats, whereas allopregnanolone reduced the oxytocin response in virgin rats. IL-1β increased the number of magnocellular neurones in the SON and paraventricular nucleus (PVN) expressing Fos (an indicator of neuronal activation) in virgin but not pregnant rats. In immunoreactive oxytocin neurones in the SON and PVN, finasteride increased IL-1β-induced Fos expression in pregnant rats. Conversely, allopregnanolone reduced the number of magnocellular oxytocin neurones activated by IL-1β in virgin rats. Treatment with naloxone (an opioid antagonist) greatly enhanced the oxytocin response to IL-1β in pregnancy, and finasteride did not enhance this effect, indicating that allopregnanolone and the endogenous opioid mechanisms do not act independently. Indeed, allopregnanolone induced opioid inhibition over oxytocin responses to IL-1β in virgin rats. Thus, in late pregnancy, allopregnanolone induces opioid inhibition over magnocellular oxytocin neurones and hence on oxytocin secretion in response to immune challenge. This mechanism will minimise the risk of preterm labour and prevent the depletion of neurohypophysial oxytocin stores, which are required for parturition.     

Changes in Pro-Opiomelanocortin and Pre-proenkephalin mRNA Levels in the Ovine Brain during Pregnancy, Parturition and Lactation and in Response to Oestrogen and Progesterone

In the female sheep opioids act centrally to influence both oxytocin release and maternal behaviour. We have used in situ hybridization histochemistry to investigate the changes in mRNA expression of the two opioid precursor genes, proopiomelanocortin (POMC) and pre-proenkephalin (PPE), in discrete hypothalamic nuclei as a function of pregnancy, parturition and lactation and following treatment with oestrogen and progesterone. Quantitative in situ hybridization histochemistry demonstrated that POMC mRNA expression in the arcuate nucleus (ARC) decreased at parturition and increased during lactation compared to late pregnant and ovariectomized animals. Oestradiol and progesterone treatments increased POMC mRNA expression compared to ovariectomized controls. Pre-proenkephalin mRNA expression was quantified in three discrete hypothalamic nuclei, the ventromedial nucleus (VMN), the paraventricular nucleus (PVN) and the suprachiasmatic nucleus (SCN). In the VMN, PPE mRNA expression increased during lactation compared to late pregnancy and parturition. Expression levels during late pregnancy and parturition were decreased compared to ovariectomized animals. Oestradiol increased, and progesterone decreased, PPE mRNA levels compared to ovariectomized controls. Combined progesterone followed by oestrogen treatment produced significant increases in PPE mRNA expression. In the PVN, PPE expression increased at parturition compared to late pregnant, lactating and ovariectomized animals. Expression levels in late pregnant animals were decreased compared to lactating or ovariectomized ones. However, sex steroid treatment produced no changes in PPE expression in the PVN. No changes were observed in PPE mRNA expression in the SCN in response to any of the experimental conditions. This data shows that both POMC and PPE mRNA levels are altered in the sheep brain during pregnancy, parturition and lactation and in response to sex steroids, although the direction of the changes is not always the same and in the case of PPE only the VMN and PVN are affected. Levels of gene expression found following exogenous steroid treatment do not precisely mirror those found during pregnancy, parturition and lactation and this suggests that factors other than changing sex steroids levels are involved. In the context of maternal behaviour it is interesting to note that PPE mRNA expression increases at parturition in the PVN when oxytocin mRNA expression levels are also increased.     

Nitric oxide synthase increases in hypothalamic magnocellular neurons after salt loading in the rat. An immunohistochemical and in situ hybridization study

Magnocellular hypothalamic neurons of the paraventricular (PVN) and supraoptic (SON) nuclei have been shown to contain a wide variety of messenger molecules in addition to vasopressin and oxytocin, including the nitric oxide (NO)-synthesizing enzyme (NOS). In this paper we have investigated the effects of salt loading on the expression of NOS by means of immunohistochemistry and in situ hybridization. The results show an increase in the number of NOS-immunoreactive (IR) neurons both in the PVN and the SON after 5 and 14 days of salt loading. Several of these neurons were double labelled with vasopressin antiserum. In situ hybridization showed a marked increase in the number of neurons expressing NOS mRNA and a stronger signal in individual neurons. The present results suggest a role for NO in the magnocellular hypothalamic system after salt loading.     

Effects of cholecystokinin (CCK)-8 on hypothalamic oxytocin-secreting neurons in rats lacking CCK-A receptor

Peripheral administration of cholecystokinin (CCK)-8 selectively activates oxytocin (OXT)-secreting neurons in the supraoptic (SON) and the paraventricular nuclei (PVN) with the elevation of plasma OXT level in rats. We examined the effects of intravenous (iv) administration of CCK-8 on the neuronal activity of hypothalamic OXT-secreting neurons and plasma OXT level in Otsuka Long-Evans Tokushima Fatty (OLETF) rats that have a congenital defect in the expression of the CCK-A receptor gene. In situ hybridization histochemistry (ISH) for c-fos mRNA revealed that the expression of the c-fos gene was not induced in the SON, the PVN, the nucleus of the tractus solitarius (NTS) and the area postrema (AP) 30 min after iv administration of CCK-8 (20 and 40 microg/kg) in OLETF rats. In Long-Evans Tokushima Otsuka (LETO) rats (controls), c-fos mRNA was detected abundantly in those nuclei 30 min after iv administration of CCK-8 (20 microg/kg). Immunohistochemistry for c-fos protein (Fos) showed that the distributions of Fos-like immunoreactivity (LI) were identical to the results obtained from ISH. Dual immunostaining for OXT and Fos revealed that Fos-LI was mainly observed in OXT-secreting neurons in the SON and the PVN of LETO rats 90 min after iv administration of CCK-8 (20 microg/kg). Radioimmunoassay for OXT and arginine vasopressin (AVP) showed that iv administration of CCK-8 did not cause significant change in the plasma OXT and AVP levels in OLETF rats, while iv administration of CCK-8 caused a significant elevation of plasma OXT level without changing the plasma AVP level in LETO rats. These results suggest that peripheral administration of CCK-8 may selectively activate the hypothalamic OXT-secreting neurons and brainstem neurons through CCK-A receptor in rats.     

Contribution of the vagus nerve and lamina terminalis to brain activation induced by refeeding

Following refeeding, c-fos expression is induced in a particular set of brain regions that include the nucleus of the solitary tract (NTS), parabrachial nucleus (PB), central amygdala (CeA), paraventricular hypothalamic nucleus (PVH), supraoptic nucleus (SON) and the circumventricular organs. Within the PVH, the expression is particularly intense in the magnocellular division of the nucleus and it is as yet not clear how this activation occurs. The respective contribution of the vagus afferents and lamina terminalis, which conveys signals entering the brain through the forebrain circumventricular organs, has been investigated in rats subjected to a unilateral cervical vagotomy (UCV) or a unilateral lesion of the fibres running within the lamina terminalis (ULT) and projecting to the neuroendocrine hypothalamus. UCV significantly decreased postprandial c-fos expression in the NTS, PB, CeA and parvocellular division of the PVH. In contrast, ULT impaired postprandial activation of the magnocellular neurons in the PVH and SON. The present study also characterized the types of neurons activated in the PVH and SON during refeeding. In the magnocellular regions, arginine-vasopressin (AVP) neurons were activated upon refeeding whereas there was no apparent induction of Fos expression in oxytocin cells. In the parvocellular PVH, postprandial Fos was induced only in 30% of the corticotrophin-releasing factor (CRF) and AVP neurons. The results of the present study suggest that the postprandial activation of the brain requires the integrity of both the vagal- and lamina terminalis-associated pathways.     

Changes in Oxytocin Immunoreactivity and mRNA Expression in the Sheep Brain during Pregnancy, Parturition and Lactation and in Response to Oestrogen and Progesterone

The effects of pregnancy, parturition and lactation and exogenous treatments with oestradiol and progesterone on oxytocin (OXY) immunoreactivity and gene expression in the sheep brain were investigated. Immunocytochemistry was used to demonstrate that increased OXY-immunoreactivity occurred in cells of the paraventricular (PVN) and supraoptic nuclei (SON), the bed nucleus of the stria terminalis (BNST), the anterior commissural nuclei (ACN) and the periventricular part of the medial preoptic area (PvMP). Oxytocin immunoreactive terminals were also seen in the accessory olfactory nucleus, the glomerular and peri-glomerular layers of the olfactory bulb, the lateral septum, the zona incerta and the pars compacta of the substantia nigra. Compared to ovariectomized and late pregnant animals, the intensity of immunoreactivity was increased in all of these oxytocinergic elements at parturition, during lactation and following exogenous treatment with oestradiol. The OXY-immunoreactivity was also more intense in late pregnant animals compared to ovariectomized ones. Quantitative in situ hybridization histochemistry showed that cells in the PVN, SON, BNST and PvMP all showed significantly increased expression of OXY mRNA in animals at parturition and during lactation compared to late pregnant or ovariectomized animals. Expression levels in late pregnant animals were also significantly higher than in ovariectomized ones. Progesterone treatment significantly increased OXY mRNA in the PVN, SON, BNST and PvMP whereas oestradiol treatment was only effective in the PVN, BNST and PvMP. Combined treatment with these steroids did not significantly increase OXY mRNA levels in comparison with their administration alone. These results show that OXY-immunoreactivity and mRNA expression are at their highest in the sheep brain when maternal behaviour is induced. The increased synthesis/storage of the peptide at parturition may be due to changes in circulating concentrations of both progesterone and oestradiol during late pregnancy.     

Previous maternal experience potentiates the effect of parturition on oxytocin receptor mRNA expression in the paraventricular nucleus

In sheep, central oxytocin release at parturition induces maternal behaviour which is thought to be mediated by changes in the expression of central oxytocin receptors. The distribution, effects of parturition, previous maternal experience and hormonal status on the distribution of an oxytocin receptor was investigated using immunocytochemistry and in situ hybridization. In ewes with no previous maternal experience, parturition induced significant increases in oxytocin receptor mRNA expression in the anterior olfactory nucleus, medial preoptic area, ventromedial hypothalamus, lateral septum, medial amygdala, bed nucleus of the stria terminalis and diagonal band of Broca. In maternally experienced ewes, parturition induced additional increases in two areas, the paraventricular nucleus and the Islands of Calleja. The changes in progesterone and oestrogen that occur during late pregnancy and parturition appear to contribute to increases in expression in the anterior olfactory nucleus, Islands of Calleja, medial preoptic area, ventromedial hypothalamus, bed nucleus of the stria terminalis and diagonal band of Broca, but not in the paraventricular nucleus, lateral septum and medial amygdala. These results demonstrate that progesterone and oestrogen priming enhance oxytocin receptor mRNA expression in a number of regions in the olfactory system, hypothalamus and limbic brain. These effects appear to be independent of maternal experience. Parturition increases oxytocin receptor mRNA expression in all the areas influenced by hormonal priming and the lateral septum, medial amygdala and paraventricular nucleus. Maternal experience also enhances expression of oxytocin receptor mRNA in the paraventricular nucleus and the Islands of Calleja. Because the paraventricular nucleus is the main source of oxytocin release in the brain, this upgrading of autoreceptors as a result of maternal experience may serve to enhance release of this peptide in projection sites regulating maternal behaviour.     

Sequential Exposure to Estrogen and Testosterone (T) and Subsequent Withdrawal of T Increases the Level of Arginine Vasopressin Messenger Ribonucleic Acid in the Hypothalamic Paraventricular Nucleus of the Female Rat

The hypothalamic peptides arginine vasopressin (AVP) and oxytocin (OT) have been implicated as mediators of socio-sexual behaviors in addition to their roles in osmolar homeostasis (AVP), milk ejection and uterine contractility (OT). Within 24  h of parturition, OT and AVP messenger ribonucleic acid (mRNA) levels increase in the hypothalamic paraventricular, and to a lesser degree, the supraoptic nucleus (PVN and SON) of the rat. We previously reported that the prepartum increase in OT mRNA is related to the spontaneous decline in progesterone levels prior to parturition. We also reported that increases in PVN and SON OT mRNA can be induced by exposing the ovariectomized rat to a steroid regimen that mimics the steroid milieu of pregnancy, namely sequential estrogen and progesterone and subsequent progesterone withdrawal. Levels of PVN and SON AVP mRNAs were not affected by progesterone withdrawal in late pregnant rats or the steroid regimen that increased OT mRNA in ovariectomized rats. These observations suggest that other factors, perhaps hormonal, may influence AVP mRNA levels. A decline in testosterone coincident with waning progesterone levels also occurs prepartum. Since peak levels of AVP mRNA prepartum coincide with the prepartum decline in testosterone, we questioned whether declining testosterone levels are important for the increase in AVP mRNA levels.     

A combined electron microscopic HRP and immunocytochemical study of the limbic projections to rat hypothalamic nuclei containing vasopressin and oxytocin neurons

Light microscopic studies in our laboratory have indicated that the lateral septum, amygdala, and ventral subiculum project in a perinuclear fashion to the paraventricular (PVN), supraoptic (SON), and suprachiasmatic (SCN) nuclei (Oldfield et al., '82; Silverman and Oldfield, '84). In the present paper a combined anterograde HRP and immunocytochemical procedure has been used to determine the connectivity between these limbic efferents and peptide-containing processes emanating from the above mentioned hypothalamic nuclei. Synaptic associations were found to exist between efferents from (1) the septum and both vasopressin (VP)- and oxytocin (OX)-positive dendrites derived from cells in the PVN and SON, (2) the septum and VP dendrites dorsal to the SCN, (3) the ventral subiculum and both VP and OX dendrites arising from the PVN and SON, and (iv) the amygdala and VP dendrites from the PVN. These observations help clarify an apparent discrepancy between electrophysiological data, in which limbic efferents have been shown to influence the activity of VP and OX neurons in the PVN and SON, and anatomical evidence which indicates only a perinuclear innervation from these sites not encroaching on the hypothalamic nuclei themselves. In each case the synaptic connections are made on dendrites external to the nucleus: those lateral and ventrolateral to the PVN, dorsal to the SON, and dorsal or dorsolateral to the SCN.     

Adenovirus-mediated gene delivery to cells of the magnocellular hypothalamo-neurohypophyseal system

The objective of the present study was to define the optimum conditions for using replication-defective adenovirus (Ad) to transfer the gene for the green fluorescent protein (GFP) to the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei and cells of the neurohypophysis (NH). As indicated by characterizing cell survival over 15 days in culture and in electrophysiological whole cell patch-clamp studies, viral concentrations up to 2 x 10(7) pfu/coverslip did not affect viability of transfected PVN and NH cultured cells from preweanling rats. At 2 x 10(7) pfu, GFP gene expression was higher (40% of GFP-positive cells) and more sustained (up to 15 days). Using a stereotaxic approach in adult rats, we were able to directly transduce the PVN, SON, and NH and visualize gene expression in coronal brain slices and in the pituitary 4 days after injection of Ad. In animals receiving NH injections of Ad, the virus was retrogradely transported to PVN and SON neurons as indicated by the appearance of GFP-positive neurons in cultures of dissociated cells from those brain nuclei and by polymerase chain reaction and Western blot analyses of PVN and SON tissues. Adenoviral concentrations of up to 8 x 10(6) pfu injected into the NH did not affect cell viability and did not cause inflammatory responses. Adenoviral injection into the pituitary enabled the selective delivery of genes to the soma of magnocellular neurons. The experimental approaches described here provide potentially useful strategies for the treatment of disordered expression of the hormones vasopressin or oxytocin.     

The effect of air puff stress on c‐Fos expression in rat hypothalamus and brainstem: central circuitry mediating sympathoexcitation and baroreflex resetting

Psychological stress evokes increases in sympathetic activity and blood pressure, which are due at least in part to an upward resetting of the baroreceptor‐sympathetic reflex. In this study we determined whether sympathetic premotor neurons in the rostral ventrolateral medulla (RVLM), which have a critical role in the reflex control of sympathetic activity, are activated during air puff stress, a moderate psychological stressor. Secondly, we identified neurons that are activated by air puff stress and that also project to the nucleus tractus solitarius (NTS), a key site for modulation of the baroreceptor reflex. Air puff stress resulted in increased c‐Fos expression in several hypothalamic and brainstem nuclei, including the paraventricular nucleus (PVN), dorsomedial hypothalamus, perifornical area (PeF), periaqueductal gray (PAG), NTS and rostral ventromedial medulla, but not in the RVLM region that contains sympathetic premotor neurons. In contrast, neurons in this RVLM region, including catecholamine‐synthesizing neurons, did express c‐Fos following induced hypotension, which reflexly activates RVLM sympathetic premotor neurons. The highest proportion of NTS‐projecting neurons that were double‐labelled with c‐Fos after air puff stress was in the ventrolateral PAG (29.3 ± 5.5%), with smaller but still significant proportions of double‐labelled NTS‐projecting neurons in the PVN and PeF (6.5 ± 1.8 and 6.4 ± 1.7%, respectively). The results suggest that the increased sympathetic activity during psychological stress is not driven primarily by RVLM sympathetic premotor neurons, and that neurons in the PVN, PeF and ventrolateral PAG may contribute to the resetting of the baroreceptor‐sympathetic reflex that is associated with psychological stress.     

Acute myocardial infarction activates magnocellular vasopressin and oxytocin neurones

Myocardial infarction (MI) is a leading cause of death worldwide. For those who survive the acute insult, the progressive dilation of the ventricle associated with chronic heart failure is driven by an adverse increase in circulating levels of the antidiuretic hormone, vasopressin, which is secreted from hypothalamic supraoptic (SON) and paraventricular nuclei (PVN) nerve terminals. Although increased vasopressin neuronal activity has been demonstrated in the latter stages of chronic heart failure, we hypothesised that vasopressin neurones become activated immediately following an acute MI. Male Sprague‐Dawley rats were anaesthetised and an acute MI was induced by ligation of the left anterior descending coronary artery. After 90 minutes of myocardial ischaemia, brains were collected. Dual‐label immunohistochemistry was used to quantify the expression of Fos protein, a marker of neuronal activation, within vasopressin‐ or oxytocin‐labelled neurones of the hypothalamic PVN and SON. Fos protein and tyrosine hydroxylase within the brainstem were also quantified. The results obtained show that the expression of Fos in both vasopressin and oxytocin neurones of the PVN and SON was significantly elevated as soon as 90 minutes post‐MI compared to sham rats. Moreover, Fos protein was also elevated in tyrosine hydroxylase neurones in the nucleus tractus solitarius and rostral ventrolateral medulla of MI rats than sham rats. We conclude that magnocellular vasopressin and oxytocin neuronal activation occurs immediately following acute MI, rather than in the later stages of chronic heart failure. Therefore, prompt vasopressin antagonist therapy as an adjunct treatment for acute MI may impede the progression of ventricular dilatation, which remains a key adverse hallmark of chronic heart failure.     

Gonadal regulation of PrRP mRNA expression in the nucleus tractus solitarius and ventral and lateral reticular nuclei of the rat

We investigated the prolactin-releasing peptide (PrRP) gene expression quantitatively in the rat brain and the involvement of estrogen and progesterone using in situ hybridization. The strongest signals were observed in the nucleus tractus solitarius (NTS), which showed approximately 70% of total PrRP mRNA in the brain. Moderate expression was observed in the ventral and lateral reticular nuclei (VLRN) of the medulla oblongata. PrRP mRNA signals in the hypothalamic ventromedial- and dorsomedial nuclei showed only 5% of total signals. The PrRP mRNA expression among female rats showing normal gonadal cycle and male rats showed that the highest levels were in female rats in proestrus. Administration of estrogen or progesterone after ovariectomy induced an increase in PrRP mRNA expression in the NTS. PrRP mRNA content in the NTS increased with the progress of the pregnancy and reached a peak on the 14th day, the mid-period of pregnancy, when plasma progesterone increases. We also observed the colocalization of PrRP and estrogen receptor alpha in the neurons distributed in the NTS by double labeling immunocytochemistry. These findings indicate that PrRP gene expression is regulated by gonadal steroid hormones in the medulla oblongata, and parts of PrRP synthesizing neurons are considered to be directly influenced by estrogen in the NTS.