Possible Involvement of the Rat Hypothalamo‐Neurohypophysial/‐Spinal Oxytocinergic Pathways in Acute Nociceptive Responses

Oxytocin (OXT)‐containing neurosecretory cells in the parvocellular divisions of the paraventricular nucleus (PVN), which project to the medulla and spinal cord, are involved in various physiological functions, such as sensory modulation and autonomic processes. In the present study, we examined OXT expression in the hypothalamo‐spinal pathway, as well as the hypothalamo‐neurohypophysial system, which includes the magnocellular neurosecretory cells in the PVN and the supraoptic nucleus (SON), after s.c. injection of saline or formalin into the hindpaws of transgenic rats that express the OXT and monomeric red fluorescent protein 1 (mRFP1) fusion gene. (i) The numbers of OXT‐mRFP1 neurones that expressed Fos‐like immunoreactivity (‐IR) and OXT‐mRFP1 intensity were increased significantly in the magnocellular/parvocellular PVN and SON after s.c. injection of formalin. (ii) OXT‐mRFP1 neurones in the anterior parvocellular PVN, which may project to the dorsal horn of the spinal cord, were activated by s.c. injection of formalin, as indicated by a significant increases of Fos‐IR and mRFP1 intensity intensity. (iii) Formalin injection caused a significant transient increase in plasma OXT. (iv) OXT, mRFP1 and corticotrophin‐releasing hormone mRNAs in the PVN were significantly increased after s.c. injection of formalin. (v) An intrathecal injection of OXT‐saporin induced hypersensitivity in conscious rats. Taken together, these results suggest that the hypothalamo‐neurohypophysial/‐spinal OXTergic pathways may be involved in acute nociceptive responses in rats.     

Electrophysiological Effects of Kainic Acid on Vasopressin‐Enhanced Green Fluorescent Protein and Oxytocin‐Monomeric Red Fluorescent Protein 1 Neurones Isolated from the Supraoptic Nucleus in Transgenic Rats

The supraoptic nucleus (SON) contains two types of magnocellular neurosecretory cells: arginine vasopressin (AVP)‐producing and oxytocin (OXT)‐producing cells. We recently generated and characterised two transgenic rat lines: one expressing an AVP‐enhanced green fluorescent protein (eGFP) and the other expressing an OXT‐monomeric red fluorescent protein 1 (mRFP1). These transgenic rats enable the visualisation of AVP or OXT neurones in the SON. In the present study, we compared the electrophysiological responses of AVP‐eGFP and OXT‐mRFP1 neurones to glutamic acid in SON primary cultures. Glutamate mediates fast synaptic transmission through three classes of ionotrophic receptors: the NMDA, AMPA and kainate receptors. We investigated the contributions of the three classes of ionotrophic receptors in glutamate‐induced currents. Three different antagonists were used, each predominantly selective for one of the classes of ionotrophic receptor. Next, we focused on the kainate receptors (KARs). We examined the electrophysiological effects of kainic acid (KA) on AVP‐eGFP and OXT‐mRFP1 neurones. In current clamp mode, KA induced depolarisation and increased firing rates. These KA‐induced responses were inhibited by the non‐NMDA ionotrophic receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3(1H4H)‐dione in both AVP‐eGFP and OXT‐mRFP1 neurones. In voltage clamp mode, the application of KA evoked inward currents in a dose‐dependent manner. The KA‐induced currents were significantly larger in OXT‐mRFP1 neurones than in AVP‐eGFP neurones. This significant difference in KA‐induced currents was abolished by the GluK1‐containing KAR antagonist UBP302. At high concentrations (250–500 μm ), the specific GluK1‐containing KAR agonist (RS)‐2‐amino‐3‐(3‐hydroxy‐5‐tert‐butylisoxazol‐4‐yl) propanoic acid (ATPA) induced significantly larger currents in OXT‐mRFP1 neurones than in AVP‐eGFP neurones. Furthermore, the difference between the AVP‐eGFP and OXT‐mRFP1 neurones in the ATPA currents was approximately equal to the difference in the KA currents. These findings suggest that the GluK1‐containing KARs may be more highly expressed in OXT neurones than in AVP neurones. These results may provide new insight into the physiology and synaptic plasticity of SON neurones.     

Fluorescent Visualisation of the Hypothalamic Oxytocin Neurones Activated by Cholecystokinin‐8 in Rats Expressing c‐fos‐Enhanced Green Fluorescent Protein and Oxytocin‐Monomeric Red Fluorescent Protein 1 Fusion Transgenes

The up‐regulation of c‐fos gene expression is widely used as a marker of neuronal activation elicited by various stimuli. Anatomically precise observation of c‐fos gene products can be achieved at the RNA level by in situ hybridisation or at the protein level by immunocytochemistry. Both of these methods are time and labour intensive. We have developed a novel transgenic rat system that enables the trivial visualisation of c‐fos expression using an enhanced green fluorescent protein (eGFP) tag. These rats express a transgene consisting of c‐fos gene regulatory sequences that drive the expression of a c‐fos‐eGFP fusion protein. In c‐fos‐eGFP transgenic rats, robust nuclear eGFP fluorescence was observed in osmosensitive brain regions 90 min after i.p. administration of hypertonic saline. Nuclear eGFP fluorescence was also observed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) 90 min after i.p. administration of cholecystokinin (CCK)‐8, which selectively activates oxytocin (OXT)‐secreting neurones in the hypothalamus. In double transgenic rats that express c‐fos‐eGFP and an OXT‐monomeric red fluorescent protein 1 (mRFP1) fusion gene, almost all mRFP1‐positive neurones in the SON and PVN expressed nuclear eGFP fluorescence 90 min after i.p. administration of CCK‐8. It is possible that not only a plane image, but also three‐dimensional reconstruction image may identify cytoplasmic vesicles in an activated neurone at the same time.     

LPS-induced Fos expression in oxytocin and vasopressin neurons of the rat hypothalamus

The aim of this study was to examine the involvement of the hypothalamic oxytocin (OXT) and vasopressin (AVP) neurons in acute phase reaction using quantitative dual-labeled immunostaining with Fos and either OXT and AVP in several hypothalamic regions. Administration of low dose (5 μg/kg) and high dose (125 μg/kg) of LPS induced intense nuclear Fos immunoreactivity in many OXT and AVP neurons in all the observed hypothalamic regions. The percentage of Fos-positive nuclei in OXT magnocellular neurons was higher than that of AVP magnocellular neurons in the supraoptic nucleus (SON), the magnocellular neurons in the paraventricular nucleus (magPVN), rostral SON (rSON), and nucleus circularis (NC), whose axons terminate at the posterior pituitary for peripheral release. The percentage of Fos-positive nuclei in AVP parvocellular neurons in the paraventricular nucleus (parPVN) was higher than that of OXT parvocellular neurons, whose axons terminate within the brain for central release. Moreover, the percentage of Fos-positive nuclei in AVP magnocellular neurons of the SON and rSON was significantly higher than that of the magPVN and NC when animals were given LPS via intraperitoneal (i.p.)-injection. This regional heterogeneity was not observed in OXT magnocellular neurons of i.p.-injected rats or in either OXT or AVP magnocellular neurons of intravenous (i.v.)-injected rats. The present data suggest that LPS-induced peripheral release of AVP and OXT is due to the activation of the magnocellular neurons in the SON, magPVN, NC, and rSON, and the central release of those hormones is in part derived from the activation of parvocellular neurons in the PVN. It is also suggested that the activation of AVP magnocellular neurons is heterogeneous among the four hypothalamic regions, but that of OXT magnocellular neurons is homogenous among these brain regions in response to LPS administration.     

Response of Arginine Vasopressin-Enhanced Green Fluorescent Protein Fusion Gene in the Hypothalamus of Adjuvant-Induced Arthritic Rats

Arginine vasopressin (AVP) and corticotrophin-releasing hormone (CRH) in the parvocellular neurosecretory cells of the paraventricular nucleus (PVN) play a major role in activating the hypothalamic-pituitary-adrenal axis, which is the main neuroendocrine response against the many kinds of stress. We examined the effects of chronic inflammatory/nociceptive stress on the expression of the AVP-enhanced green fluorescent protein (eGFP) fusion gene in the hypothalamus, using the adjuvant arthritis (AA) model. To induce AA, the AVP-eGFP rats were intracutaneously injected heat-killed Mycobacterium butyricum (1 mg/rat) in paraffin liquid at the base of their tails. We measured AVP, oxytocin and corticosterone levels in plasma and changes in eGFP and CRH mRNA in the hypothalamus during the time course of AA development. Then, we examined eGFP fluorescence in the PVN, the supraoptic nucleus (SON), median eminence (ME) and posterior pituitary gland (PP) when AA was established. The plasma concentrations of AVP, oxytocin and corticosterone were significantly increased on days 15 and 22 in AA rats, without affecting the plasma osmolality and sodium. Although CRH mRNA levels in the PVN were significantly decreased, eGFP mRNA levels in the PVN and the SON were significantly increased on days 15 and 22 in AA rats. The eGFP fluorescence in the SON, the PVN, internal and external layers of the ME and PP was apparently increased in AA compared to control rats. These results suggest that the increases in the concentrations of ACTH and corticosterone in AA rats are induced by hypothalamic AVP, based on data from AVP-eGFP transgenic rats.     

Specific expression of optically active reporter gene in arginine vasopressin-secreting neurosecretory cells in the hypothalamic-neurohypophyseal system

The anti-diuretic hormone arginine vasopressin (AVP) is synthesised in the magnocellular neurosecretory cells (MNCs) in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of the hypothalamus. AVP-containing MNCs that project their axon terminals to the posterior pituitary can be identified using immunohistochemical techniques with specific antibodies recognising AVP and neurophysin II, and by virtue of their electrophysiological properties. Recently, we generated transgenic rats expressing an AVP-enhanced green fluorescent protein (eGFP) fusion gene in AVP-containing MNCs. In this transgenic rat, eGFP mRNA was observed in the PVN and the SON, and eGFP fluorescence was seen in the PVN and the SON, and also in the posterior pituitary, indicating transport of transgene protein down MNC axons to storage in nerve terminals. The expression of the AVP-eGFP transgene and eGFP fluorescence in the PVN and the SON was markedly increased after dehydration and chronic salt-loading. On the other hand, AVP-containing parvocellular neurosecretory cells in the PVN that are involved in the activation of the hypothalamic-pituitary adrenal axis exhibit robust AVP-eGFP fluorescence after bilateral adrenalectomy and intraperitoneal administration of lipopolysaccharide. In the median eminence, the internal and external layer showed strong fluorescence for eGFP after osmotic stimuli and stressful conditions, respectively, again indicating appropriate transport of transgene traslation products. Brain slices and acutely-dissociated MNCs and axon terminals also exhibited strong fluorescence, as observed under fluorescence microscopy. The AVP-eGFP transgenic animals are thus unique and provide a useful tool to study AVP-secreting cells in vivo for electrophysiology, imaging analysis such as intracellular Ca²⁺ imaging, organ culture and in vivo monitoring of dynamic change in AVP secretion.     

Modulation of oestrogen receptor-beta mRNA expression in rat paraventricular and supraoptic nucleus neurones following adrenal steroid manipulation and hyperosmotic stimulation

Magnocellular neurosecretory neurones in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei express oestrogen receptor beta (ERbeta) but not ERalpha. In the PVN, ERbeta is strongly expressed in the ventromedial parvocellular neurones projecting to the brainstem. We used quantitative in situ hybridization, with (35)S-labelled riboprobes, to study heterologous regulation by manipulating adrenal steroid hormones (72 h after adrenalectomy +/- corticosterone replacement; repeated stress: halothane inhalation, environmental cold, immobilization, each daily for 3 days) in male rats. Adrenalectomy increased ERbeta mRNA expression in the magnocellular PVN and SON, by 2.2 and 2.5-fold, respectively, with no effect in the ventromedial parvocellular PVN neurones. Corticosterone replacement partially prevented the increases in ERbeta mRNA expression in magnocellular PVN and SON neurones. Repeated stress over 72 h had no effect on ERbeta mRNA expression in the magnocellular PVN or SON, but increased expression 1.4-fold in the ventromedial parvocellular PVN neurones. Although consequences of hydromineral balance derangement after adrenalectomy may stimulate magnocellular neurones, strongly stimulating the neurones by giving intact male rats 2% saline to drink for 72 h decreased ERbeta mRNA expression in the magnocellular PVN and SON neurones by approximately 60%, and in the ventromedial parvocellular PVN neurones by 13%. Thus, ERbeta mRNA expression is negatively regulated by basal glucocorticoid secretion in magnocellular PVN and SON neurones, and positively regulated by stress in ventromedial parvocellular PVN neurones. However, ERbeta mRNA expression in magnocellular neurones is negatively linked to hyperosmotic stimulation of the neurones. The 6.25-fold variation in ERbeta mRNA expression in magnocellular neurones from salt-loading to adrenalectomy could alter their sensitivity to oestrogens. Consequently, regulation of oxytocin and vasopressin neurone activity via ERbeta is expected to vary according to their functional state and, in particular, on basal glucocorticoid actions.     

A c‐fos‐Monomeric Red Fluorescent Protein 1 Fusion Transgene is Differentially Expressed in Rat Forebrain and Brainstem after Chronic Dehydration and Rehydration

We have previously shown that an acute osmotic stimulation induces the expression of a c‐fos and monomeric red fluorescent protein 1 (mRFP1) fusion transgene in osmosensitive rat brain areas, including the supraoptic (SON) and paraventricular nuclei (PVN). However, the effects of chronic stimuli, such as dehydration, have not been investigated. In the present study, the expression patterns of the c‐fos‐mRFP1 fusion gene in the forebrain and the brainstem of male and female transgenic rats were studied in seven experimental groups: ad lib. water (euhydration), water deprivation for 12, 24 or 48 h (dehydration) and water deprivation for 46 h + ad lib. water for 2, 6 or 12 h (rehydration). The number of cells that express nuclear mRFP1 fluorescence was quantified in the hypothalamus, the circumventricular organs and the brainstem. Compared to the euhydrated state, the number of transgene expressing cells significantly increased in all forebrain areas and in the rostral ventrolateral medulla after dehydration and 2 h of rehydration. In the nucleus of the solitary tract and area postrema, the number of mRFP1 fluorescent cells was markedly increased after 2 h of rehydration. Although the number of mRFP1 fluorescent cells in the organum vasculosum laminae terminalis, median preoptic nucleus and subfornical organ remained significantly increased after 6 h of rehydration, reaching control levels after 12 h of rehydration, the number of mRFP1 fluorescent cells in the SON and the PVN reached control levels after 6 h of rehydration. There were no significant differences between male and female rats. These results show that the expression of the c‐fos‐mRFP1 fusion gene changes in the forebrain and the brainstem not only after acute osmotic stimulation, but also after chronic osmotic stimulation. Interestingly, these studies reveal the differential activation of different neuronal groups over the time course of dehydration and rehydration.     

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.     

Activity‐Dependent Notch Signalling in the Hypothalamic‐Neurohypophysial System of Adult Mouse Brains

Notch signalling has a key role in cell fate specification in developing brains; however, recent studies have shown that Notch signalling also participates in the regulation of synaptic plasticity in adult brains. In the present study, we examined the expression of Notch3 and Delta‐like ligand 4 (DLL4) in the hypothalamic‐neurohypophysial system (HNS) of the adult mouse. The expression of DLL4 was higher in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) compared to adjacent hypothalamic regions. Double‐labelling immunohistochemistry using vesicular GABA transporter and glutamate transporter revealed that DLL4 was localised at a subpopulation of excitatory and inhibitory axonal boutons against somatodendrites of arginine vasopressin (AVP)‐ and oxytocin (OXT)‐containing magnocellular neurones. In the neurohypophysis (NH), the expression of DLL4 was seen at OXT‐ but not AVP‐containing axonal terminals. The expression of Notch3 was seen at somatodendrites of AVP‐ and OXT‐containing magnocellular neurones in the SON and PVN and at pituicytes in the NH. Chronic physiological stimulation by salt loading, which remarkably enhances the release of AVP and OXT, decreased the number of DLL4‐immunoreactive axonal boutons in the SON and PVN. Moreover, chronic and acute osmotic stimulation promoted proteolytic cleavage of Notch3 to yield the intracellular fragments of Notch3 in the HNS. Thus, the present study demonstrates activity‐dependent reduction of DLL4 expression and proteolytic cleavage of Notch3 in the HNS, suggesting that Notch signalling possibly participates in synaptic interaction in the hypothalamic nuclei and neuroglial interaction in the NH.     

The use of heat-induced hydrolysis in immunohistochemistry on angiotensin II (AT1) receptors enhances the immunoreactivity in paraformaldehyde-fixed brain tissue of normotensive Sprague-Dawley rats

The research on components of the renin-angiotensin system delivered a broad image of angiotensin II-binding sites. Especially, immunohistochemistry (IHC) provided an exact anatomical localization of the AT(1) receptor in the rat brain. Yet, controversial results between in vitro receptor autoradiography and IHC as well as between immunohistochemical studies using various antisera started a vehement discussion concerning specificity and cross-reactivity of these antisera. In particular the magnocellular subdivision of the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) provided controversial results on the localization of AT(1) receptors. Both areas are known for angiotensin II-induced release of vasopressin (VP) and oxytocin (OXT). To evaluate the significance of the appropriate method of antigen retrieval and its relevance for the detection of AT(1) receptors we performed IHC on AT(1) receptors in paraformaldehyde-fixed and paraffin-embedded brain tissue of Sprague-Dawley rats using either the detergent Triton X-100 or microwave oven heating. This study demonstrates that heat-induced hydrolysis enhances the quality and quantity of immunoreactivity (IR) in IHC on AT(1) receptors. In the organum vasculosum lamina terminalis and in the parvocellular subdivisions of the PVN we report a distribution of AT(1)-like-IR similar to that observed with other methods. However, in addition, we provide evidence that distinct AT(1)-like-IR is also localized in few magnocellular neurons of the PVN and in few parvocellular neurons of the dorsal SON but not in magnocellular neurons of the SON. Moreover, parallel IHC indicates that few magnocellular OXT- or VP-releasing neurons of the PVN as well as parvocellular OXT-releasing neurons of the SON do also contain AT(1) receptors.     

Expression of corticotropin releasing factor (CRF), urocortin and CRF type 1 receptors in hypothalamic-hypophyseal systems under osmotic stimulation

The expression of corticotropin releasing factor (CRF) and urocortin in hypothalamic magnocellular neurones increases in response to osmotic challenge. To gain a better understanding of the physiological roles of CRF and urocortin in fluid homeostasis, CRF, urocortin and CRF type 1 receptor (CRFR-1) gene expression was examined in the hypothalamic-hypophyseal system usingin situ and double-label in situ hybridization following chronic salt loading. CRFR-1 expression was further examined by immunohistochemistry and receptor binding. Ingestion of hypertonic saline by Sprague-Dawley rats for 7 days induced CRF mRNA exclusively in the oxytocin neurones of the magnocellular paraventricular nucleus (PVN) and the supraoptic nucleus (SON), but induced CRFR-1 mRNA in both oxytocin and vasopressin-containing magnocellular neurones. Hypertonic saline treatment also increased urocortin mRNA expression in the PVN and the SON. In the SON, urocortin was localized to vasopressin and oxytocin neurones but was rarely seen in CRF-positive cells. Changes in CRFR-1 mRNA expression in magnocellular neurones by hypertonic saline treatment were accompanied by changes in CRFR-1 protein levels and receptor binding. Hypertonic saline treatment increased CRFR-1-like immunoreactivity in the magnocellular PVN and SON, and decreased it in the parvocellular PVN. CRF receptor binding in the PVN and SON was also increased in response to osmotic stimulation. Finally, hypertonic saline treatment increased CRFR-1 mRNA, CRFR-1-like immunoreactivity and CRF receptor binding in the intermediate pituitary. These results demonstrate that the increase in the expression of CRF and urocortin message in magnocellular neurones induced by salt loading is accompanied by an increase in CRF receptor levels and binding in the hypothalamus and intermediate pituitary. Thus, CRF and urocortin may exert modulatory effects locally within magnocellular neurones as well as at the pituitary gland in response to osmotic stimulation.     

A comparison of the hemodynamic effects produced by electrical stimulation of subnuclei of the paraventricular nucleus

The paraventricular nucleus of the hypothalamus (PVN) is composed of magnocellular and parvocellular subdivisions. Magnocellular neurosecretory neurons project to the neurohypophysis while parvocellular neurons send monosynaptic axonal projections to autonomic regulatory areas in the brainstem and spinal cord. In the present study, we investigated the hemodynamic effects produced by selective magnocellular or parvocellular stimulation. In urethane anesthetized rats with intact baroreflexes, magnocellular and parvocellular stimulation produced only slight differences in hemodynamic responses, however, following acute sinoaortic denervation a clear difference was observed. Parvocellular stimulation produced an increase in arterial pressure and vasoconstriction in gut, kidney and skeletal muscle. Magnocellular stimulation produced little effect on arterial pressure and marked vasodilation in the hindquarters. Blockade of peripheral vasopressin vascular receptors did not affect the vasoconstrictor response produced by stimulation of PVN. These data are consistent with the hypothesis that the long descending neural projections of the parvocellular PVN subserve a selective vasoconstrictor function.     

Oxytocin receptor gene loss influences expression of the oxytocin gene in C57BL/6J mice in a sex‐ and age‐dependent manner

Parental care and sensory stimulation are critical environmental factors that influence oxytocin (OXT) and its receptor (OXTR). Because developmental Oxt mRNA expression is enhanced by sensory‐rich early life experience and reduced by sensory deprivation, we predicted that compared to wild‐type (WT) littermates, mice with congenital loss of OXTR (OXTR KO), as a genetically induced deprivation, would show impaired Oxt mRNA expression in the offspring hypothalamus during development. Oxt mRNA levels of male and female OXTR KO mice were not different from WT littermates from postnatal day (P)0 to P6, although, by P8, OXTR KO showed significantly decreased Oxt mRNA expression in the hypothalamus compared to WT littermates. At P14, male and female OXTR KO mice had significantly decreased Oxt mRNA expression specifically in the paraventricular nucleus (PVN), but not the supraoptic nucleus (SON), compared to WT littermates. We investigated whether this effect persisted in adulthood (P90) and found a significant genotype by sex interaction where male OXTR KO mice displayed a reduction in Oxt expression specific to the PVN compared to male WT littermates. By contrast, male and female OXTR KO adults had increased Oxt mRNA levels in the SON. These findings suggest that OXTR plays a role in developmental Oxt mRNA expression with sex by genotype interactions apparent at adulthood. We then measured OXT and neural activation in the PVN and SON at P14. We observed more OXT‐immunoreactive cells in the PVN of OXTR KO mice but significantly fewer c‐Fos immunoreactive cells. There were no genotype differences in immunoreactivity for OXT and no c‐Fos activity in the SON at P14. Combined, these data suggest that OXTR WT P14 mice have more PVN activity and are more likely to release OXT than OXTR KO mice. Future experiments are warranted to understand which OXTR‐expressing neural circuits modulate the development of the PVN oxytocin system.     

Distribution of MT1 melatonin receptor immunoreactivity in the human hypothalamus and pituitary gland: colocalization of MT1 with vasopressin, oxytocin, and corticotropin-releasing hormone

Melatonin is implicated in numerous physiological processes, including circadian rhythms, stress, and reproduction, many of which are mediated by the hypothalamus and pituitary. The physiological actions of melatonin are mainly mediated by melatonin receptors. We here describe the distribution of the melatonin receptor MT1 in the human hypothalamus and pituitary by immunocytochemistry. MT1 immunoreactivity showed a widespread pattern in the hypothalamus. In addition to the area of the suprachiasmatic nucleus (SCN), a number of novel sites, including the paraventricular nucleus (PVN), periventricular nucleus, supraoptic nucleus (SON), sexually dimorphic nucleus, the diagonal band of Broca, the nucleus basalis of Meynert, infundibular nucleus, ventromedial and dorsomedial nucleus, tuberomamillary nucleus, mamillary body, and paraventricular thalamic nucleus were observed to have neuronal MT1 receptor expression. No staining was observed in the nucleus tuberalis lateralis and bed nucleus of the stria terminalis. The MT1 receptor was colocalized with some vasopressin (AVP) neurons in the SCN, colocalized with some parvocellular and magnocellular AVP and oxytocine (OXT) neurons in the PVN and SON, and colocalized with some parvocellular corticotropin-releasing hormone (CRH) neurons in the PVN. In the pituitary, strong MT1 expression was observed in the pars tuberalis, while a weak staining was found in the posterior and anterior pituitary. These findings provide a neurobiological basis for the participation of melatonin in the regulation of various hypothalamic and pituitary functions. The colocalization of MT1 and CRH suggests that melatonin might directly modulate the hypothalamus-pituitary-adrenal axis in the PVN, which may have implications for stress conditions such as depression.     

Dopamine–oxytocin interactions in penile erection

Dopamine and oxytocin have established roles in the central regulation of penile erection in rats; however, the neural circuitries involved in a specific erectile context and the interaction between dopamine and oxytocin mechanisms remain to be elucidated. The medial preoptic area (MPOA), supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus may serve as candidate sites because they contain oxytocin cells, receive dopaminergic inputs and have been implicated in mediating masculine sexual behavior. Double immunofluorescence revealed that substantial numbers of oxytocin cells in the MPOA, SON and PVN possess dopamine D₂, D₃ and D₄ receptors. In anaesthetized rats, using intracavernous pressure as a physiological indicator of erection, blockade of lumbosacral oxytocin receptors (UK, 427843) reduced erectile responses to a nonselective dopamine agonist (apomorphine), suggesting that dopamine recruits a paraventriculospinal oxytocin pathway. In conscious males in the absence of a female, penile erection elicited by a D₂/D₃ (Quinelorane) but not D₄ (PD168077) agonist was associated with activation of medial parvocellular PVN oxytocin cells. In another experiment where males were given full access to a receptive female, a D₄ (L‐745870) but not D₂ or D₃ antagonist (L‐741626; nafadotride) inhibited penile erection (intromission), and this was correlated with SON magnocellular oxytocin neuron activation. Together, the data suggest dopamine’s effects on hypothalamic oxytocin cells during penile erection are context‐specific. Dopamine may act via different parvocellular and magnocellular oxytocin subpopulations to elicit erectile responses, depending upon whether intromission is performed. This study demonstrates the potential existence of interaction between central dopamine and oxytocin pathways during penile erection, with the SON and PVN serving as integrative sites.     

Regulation by osmotic stimuli of galanin-R1 receptor expression in magnocellular neurones of the paraventricular and supraoptic nuclei of the rat

Neurones of the supraoptic nucleus (SON) and the magnocellular and parvocellular divisions of the paraventricular nucleus (PVN) express galanin and [125I]galanin binding sites. Although the precise role(s) of galanin in these different cell populations is still unknown, it has been shown to regulate the electrophysiological, neurochemical and secretory activity of magnocellular neurones.In light of the well-described effects of hyperosmotic stimuli, such as salt-loading on magnocellular neurone activity and galanin synthesis and release, and the recent identification of multiple galanin receptors in brain, this study assessed the possible regulation of galanin receptor subtype expression in the PVN/SON of salt-loaded, dehydrated and food-deprived rats. Gal-R1 mRNA was abundant in the SON (and magnocellular PVN) of control rats and levels were increased in these same cells after 4 days of salt-loading (2% NaCl solution as drinking water) or water deprivation. The density of specific [125I]galanin(1-29) binding and the intensity of Gal-R1-like immunostaining were also increased in the characteristically enlarged, magnocellular neurones of the PVN and SON after these treatments. Gal-R2 mRNA was detected in the parvocellular PVN, but levels were not altered by the hyperosmotic stimuli. In contrast, food deprivation (4 days), which has been shown to reduce levels of several neurochemical markers in magnocellular neurones, produced a significant reduction in Gal-R1 (and galanin) mRNA levels in the SON, but no consistent change in neurone size, [125I]galanin binding levels, or Gal-R1 immunostaining. Along with previous findings from this and other laboratories, these data suggest that the expression of galanin and Gal-R1 receptors is regulated in parallel with functional and morphological changes in hypothalamic magnocellular neurones. Furthermore, Gal-R1 immunoreactivity was primarily detected in somatodendritic areas and thus galanin may influence the activity of these cells, particularly vasopressin synthesis/release, via autocrine or paracrine activation of Gal-R1 receptors, especially during long-lasting stimulation.     

Corticotropin-releasing factor type-1 receptor mRNA is not induced in mouse hypothalamus by either stress or osmotic stimulation

In rats, acute stress substantially increases corticotropin-releasing factor (CRF) type 1 receptor (CRFR-1) mRNA expression in the paraventricular nucleus (PVN) and osmotic stimulation induces both CRF and CRFR-1 mRNA in magnocellular PVN and supraoptic nucleus (SON). However, these phenomena have not been analysed in other species. We compared CRF and CRFR-1 expression in rat and mouse hypothalamus. Male C57BL/6 mice and Wistar rats were exposed to acute restraint stress for 3 h, or to hypertonic saline ingestion for 7 days. Restraint stress increased CRF and c-fos mRNA expression in both rat and mouse PVN. CRFR-1 mRNA was barely detectable in controls, whereas restraint stress substantially increased CRFR-1 mRNA in rat PVN, but not in mouse. Hypertonic saline ingestion induced CRF mRNA in magnocellular PVN and SON of the rat, but did not alter CRF mRNA levels in mouse hypothalamus. CRFR-1 mRNA was also induced in magnocellular PVN and SON of the rat in response to osmotic stimulation, but not in mouse. Immunohistochemistry demonstrated that CRFR-1-like immunoreactivity (ir) was distributed within parvocellular and magnocellular PVN of mouse and rat. CRFR-1-ir in rat PVN was increased by acute stress and osmotic stimulation. By contrast, these treatments did not alter CRFR-1-ir in mouse PVN. Combined immunohistochemistry and in situ hybridization revealed that CRFR-1-ir was most frequently colocalized to CRF in mouse PVN, whereas only a small percentage of oxytocin and vasopressin-producing cells coexpressed CRFR-1-ir. These results indicate that (i) by contrast to rats, neither acute stress nor osmotic stimulation induces CRFR-1 mRNA expression in the mouse PVN; (ii) osmotic stimulation does not alter CRF mRNA expression in parvocellular and magnocellular neurones of mouse PVN; and (iii) acute stress increases c-fos and CRF mRNA to a similar degree in mouse and rat PVN. Thus, differences may exist between mouse and rat in the regulation of CRF and CRFR-1 gene expression in hypothalamus following stress and osmotic stimulation.     

Effect of oxytocin on the behavioral activity in the behavioral despair depression rat model

Oxytocin (OXT), a nonapeptide posterior hormone of the pituitary, is mainly synthesized and secreted in the hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON). The present study was to investigate in which level, brain or periphery, OXT effecting on the behavioral activity in the behavioral despair depression rat model. The results showed that (1) either the forced swimming or the tail suspension significantly increased OXT concentration in the brain (PVN, SON, frontal cortex, hippocampus, amygdala, lumbar spinal cord) and in the periphery (posterior pituitary and serum); (2) intraventricular injection (icv) of OXT decreased the animal immobility time, whereas OXT receptor antagonist-desGly-NH₂, d(CH2)₅[D-Tyr₂, Thr-sup-4]OV (icv) increased the animal immobility time in a dose-dependent manner in forced swimming test (FST) and in tail suspension test (TST); (3) neither OXT nor OXT receptor antagonist (intravenous injection) influenced the animal immobility time in FST and in TST. OXT levels were increased in several areas of the brain and in the periphery following the behavioral despair, one stressor, yet pre-treatment with OXT appeared to be beneficial in term of reducing immobility time. The data suggested that behavioral despair could enhance OXT synthesis and secretion not only in the brain but also in the periphery, and OXT in the brain rather than the periphery played a role in the behavioral despair depression.     

The neurohypophysial oxytocin and arginine vasopressin system is activated in a knee osteoarthritis rat model

Osteoarthritis (OA) causes chronic joint pain and significantly impacts daily activities. Hence, developing novel treatment options for OA has become an increasingly important area of research. Recently, studies have reported that exogenous, as well as endogenous, hypothalamic‐neurohypophysial hormones, oxytocin (OXT) and arginine‐vasopressin (AVP), significantly contribute to nociception modulation. Moreover, the parvocellular OXT neurone (parvOXT) extends its projection to the superficial spinal dorsal horn, where it controls the transmission of nociceptive signals. Meanwhile, AVP produced in the magnocellular AVP neurone (magnAVP) is released into the systemic circulation where it contributes to pain management at peripheral sites. The parvocellular AVP neurone (parvAVP), as well as corticotrophin‐releasing hormone (CRH), suppresses inflammation via activation of the hypothalamic‐pituitary adrenal (HPA) axis. Previously, we confirmed that the OXT/AVP system is activated in rat models of pain. However, the roles of endogenous hypothalamic‐neurohypophysial hormones in OA have not yet been characterised. In the present study, we investigated whether the OXT/AVP system is activated in a knee OA rat model. Our results show that putative parvOXT is activated and the amount of OXT‐monomeric red fluorescent protein 1 positive granules in the ipsilateral superficial spinal dorsal horn increases in the knee OA rat. Furthermore, both magnAVP and parvAVP are activated, concurrent with HPA axis activation, predominantly modulated by AVP, and not CRH. The OXT/AVP system in OA rats was similar to that in systemic inflammation models, including adjuvant arthritis; however, magnocellular OXT neurones (magnOXT) were not activated in OA. Hence, localised chronic pain conditions, such as knee OA, activate the OXT/AVP system without impacting magnOXT.