Oestrogen-dependent hypothalamic oxytocin expression with changes in feeding and body weight in female rats.

Oxytocin (OXT) is produced in the hypothalamic nuclei and secreted into systemic circulation from the posterior pituitary gland. In the central nervous system, OXT regulates behaviours including maternal and feeding behaviours. Our aim is to evaluate whether oestrogen regulates hypothalamic OXT dynamics. Herein, we provide the first evidence that OXT dynamics in the hypothalamus vary with sex and that oestrogen may modulate dynamic changes in OXT levels, using OXT-mRFP1 transgenic rats. The fluorescence intensity of OXT-mRFP1 and expression of the OXT and mRFP1 genes in the hypothalamic nuclei is highest during the oestrus stage in female rats and decreased significantly in ovariectomised rats. Oestrogen replacement caused significant increases in fluorescence intensity and gene expression in a dose-related manner. This is also demonstrated in the rats' feeding behaviour and hypothalamic Fos neurons using cholecystokinin-8 and immunohistochemistry. Hypothalamic OXT expression is oestrogen-dependent and can be enhanced centrally by the administration of oestrogen.

Oxytocin-monomeric red fluorescent protein 1 synthesis in the hypothalamus under osmotic challenge and acute hypovolemia in a transgenic rat line.

We generated a transgenic rat line that expresses oxytocin (OXT)-monomeric red fluorescent protein 1 (mRFP1) fusion gene to visualize the dynamics of OXT. In this transgenic rat line, hypothalamic OXT can be assessed under diverse physiological and pathophysiological conditions by semiquantitative fluorometry of mRFP1 fluorescence intensity as a surrogate marker for endogenous OXT. Using this transgenic rat line, we identified the changes in hypothalamic OXT synthesis under various physiological conditions. However, few reports have directly examined hypothalamic OXT synthesis under hyperosmolality or hypovolemia. In this study, hypothalamic OXT synthesis was investigated using the transgenic rat line after acute osmotic challenge and acute hypovolemia induced by intraperitoneal (i.p.) administration of 3% hypertonic saline (HTN) and polyethylene glycol (PEG), respectively. The mRFP1 fluorescence intensity in the paraventricular (PVN) and supraoptic nuclei (SON) was significantly increased after i.p. administration of HTN and PEG, along with robust Fos-like immunoreactivity (co-expression). Fos expression showed neuronal activation in the brain regions that are associated with the hypothalamus and/or are involved in maintaining water and electrolyte homeostasis in HTN- and PEG-treated rats. OXT and mRFP1 gene expressions were dramatically increased after HTN and PEG administration. The plasma OXT level was extremely increased after HTN and PEG administration. Acute osmotic challenge and acute hypovolemia induced upregulation of hypothalamic OXT in the PVN and SON. These results suggest that not only endogenous arginine vasopressin (AVP) but also endogenous OXT has a key role in maintaining body fluid homeostasis to cope with hyperosmolality and hypovolemia.

Expression of hypothalamic feeding-related peptide genes and neuroendocrine responses in an experimental allergic encephalomyelitis rat model.

Experimental allergic encephalomyelitis (EAE) is considered to be a useful animal model of human multiple sclerosis (MS). However, among the various symptoms of MS, the mechanisms contributing to inflammatory anorexia remain unclear. In the present study, we used an EAE rat model to examine changes in expression levels of hypothalamic feeding-related peptide genes and neuroendocrine responses such as the hypothalamo-neurohypophysial system and the hypothalamo-pituitary-adrenal (HPA) axis. The weight gain and cumulative food intake in EAE rats in the early days after immunization was significantly lower than that of the control group. The expression of orexigenic peptide genes Npy and Agrp were significantly increased, whereas the levels of anorectic peptide genes (Pomc and Cart) were significantly decreased in the hypothalamus of EAE rats. There was also a significant increase in the mRNA and plasma oxytocin (OXT) but not of arginine vasopressin (AVP) in the supraoptic and paraventricular nuclei (PVN) of EAE rats at days 12 and 18 after immunization. The expression of corticotropin-releasing hormone (Crh) and Avp was downregulated and upregulated, respectively, in the parvocellular division of the PVN at day 12 after immunization. The expression level of Pomc in the anterior pituitary significantly increased, accompanied by increased plasma corticosterone levels, at days 6, 12, and 18 after immunization. These results suggest that inflammatory anorexia in rat EAE may be caused by activation of the OXT-ergic pathway and HPA axis via changes in the expression of hypothalamic feeding-related peptides, including Avp but not Crh.

Acute Mono-Arthritis Activates the Neurohypophysial System and Hypothalamo-Pituitary Adrenal Axis in Rats.

Various types of acute/chronic nociceptive stimuli cause neuroendocrine responses such as activation of the hypothalamo-neurohypophysial [oxytocin (OXT) and arginine vasopressin (AVP)] system and hypothalamo-pituitary adrenal (HPA) axis. Chronic multiple-arthritis activates the OXT/AVP system, but the effects of acute mono-arthritis on the OXT/AVP system in the same animals has not been simultaneously evaluated. Further, AVP, not corticotropin-releasing hormone (CRH), predominantly activates the HPA axis in chronic multiple-arthritis, but the participation of AVP in HPA axis activation in acute mono-arthritis remains unknown. Therefore, we aimed to simultaneously evaluate the effects of acute mono-arthritis on the activity of the OXT/AVP system and the HPA axis. In the present study, we used an acute mono-arthritic model induced by intra-articular injection of carrageenan in a single knee joint of adult male Wistar rats. Acute mono-arthritis was confirmed by a significant increase in knee diameter in the carrageenan-injected knee and a significant decrease in the mechanical nociceptive threshold in the ipsilateral hind paw. Immunohistochemical analysis revealed that the number of Fos-immunoreactive (ir) cells in the ipsilateral lamina I-II of the dorsal horn was significantly increased, and the percentage of OXT-ir and AVP-ir neurons expressing Fos-ir in both sides of the supraoptic (SON) and paraventricular nuclei (PVN) was increased in acute mono-arthritic rats. in situ hybridization histochemistry revealed that levels of OXT mRNA and AVP hnRNA in the SON and PVN, CRH mRNA in the PVN, and proopiomelanocortin mRNA in the anterior pituitary were also significantly increased in acute mono-arthritic rats. Further, plasma OXT, AVP, and corticosterone levels were significantly increased in acute mono-arthritic rats. These results suggest that acute mono-arthritis activates ipsilateral nociceptive afferent pathways at the spinal level and causes simultaneous and integrative activation of the OXT/AVP system. In addition, the HPA axis is activated by both AVP and CRH in acute mono-arthritis with a distinct pattern compared to that in chronic multiple-arthritis.

Changes in gene expressions of hypothalamic neuropeptides controlling feeding behaviors in bilateral nephrectomized rats.

Anorexia is one of the most widespread eating disorders that appears to contribute to malnutrition in patients with advanced kidney dysfunction. The changes of neuropeptides controlling feeding behaviors synthesized in the hypothalamus under several physiological condition could induce anorexia. While several mechanisms underlying uremic anorexia have been proposed, the changes of hypothalamic neuropeptides controlling feeding behaviors of uremic patients are poorly understood. The gene expressions of hypothalamic neuropeptides controlling feeding behaviors were evaluated after bilateral nephrectomy, which is a model of acute kidney dysfunction, by in situ hybridization histochemistry. Food consumption decreased markedly in bilateral nephrectomized rats. The mRNA levels of corticotrophin-releasing hormone, proopiomelanocortin, cocaine- and amphetamine-regulated transcript, which suppress feeding behavior, were significantly higher in bilateral nephrectomized rats than in sham-operated rats. On the other hand, the mRNA levels of Agouti-related peptide, neuropeptide Y, melanin-concentrating hormone, and orexin, which promote feeding behavior, were significantly lower in bilateral nephrectomized rats than in sham-operated rats. In addition, the plasma level of leptin, which has an anorexic effect, increased after bilateral nephrectomy. The results suggest that hypothalamic neuropeptides controlling feeding behaviors may be involved in the development of anorexia in bilateral nephrectomized rats. This report is the first step to elucidating the physiological mechanisms of anorexia in patients with kidney dysfunction.

Effects of acute kidney dysfunction on hypothalamic arginine vasopressin synthesis in transgenic rats.

Acute loss of kidney function is a critical internal stressor. Arginine vasopressin (AVP) present in the parvocellular division of the paraventricular nucleus (PVN) plays a key role in the regulation of stress responses. However, hypothalamic AVP dynamics during acute kidney dysfunction remain unclear. In this study, we investigated the effects of bilateral nephrectomy on AVP, using a transgenic rat line that expressed the AVP-enhanced green fluorescent protein (eGFP). The eGFP fluorescent intensities in the PVN were dramatically increased after bilateral nephrectomy. The mRNA levels of eGFP, AVP, and corticotrophin-releasing hormone in the PVN were dramatically increased after bilateral nephrectomy. Bilateral nephrectomy also increased the levels of Fos-like immunoreactive cells in brainstem neurons. These results indicate that bilateral nephrectomy upregulates the AVP-eGFP synthesis. Further studies are needed to identify the neural and/or humoral factors that activate AVP synthesis and regulate neuronal circuits during acute kidney dysfunction.

Centrally administered kisspeptin suppresses feeding via nesfatin-1 and oxytocin in male rats.

Kisspeptin (KP), known as a hypothalamic neuropeptide, plays a critical role in the regulation of not only reproduction but also food intake. The anorectic neuropeptides, nesfatin-1 and oxytocin (OXT), are expressed in central nervous system, particulaly in various hypothalamic nuclei, and peripheral tissue. We examined the effects of the intracerebroventricular (icv) administration of KP-10 on feeding and nesfatin-1-immunoreactive (ir) or OXT-ir neurons in the rat hypothalamus, using Fos double immunohistochemistry in male rats. Cumulative food intake was remarkably decreased 0.5-3 h after icv administration of KP-10 (6.0 µg) compared to the vehicle treated and the KP-10 (3.8 µg) treated group. The icv administration of KP-10 significantly increased the number of nesfatin-1-ir neurons expressing Fos in the supraoptic nucleus (SON), paraventricular nucleus (PVN), arcuate nucleus (ARC), dorsal raphe nucleus, locus coeruleus, and nucleus tractus solitarius. The decreased food intake induced by KP-10 was significantly attenuated by pretreatment with the icv administration of antisense RNA against nucleobindin-2. After icv administration of KP-10, the percentages of OXT-ir neurons expressing FOS were remarkably higher in the SON and PVN than for vehicle treatment. The KP-10-induced anorexia was partially abolished by pretreatment with OXT receptor antagonist (OXTR-A). The percentage of nesfatin-1-ir neurons expressing Fos-ir in the ARC was also decreased by OXTR-A pretreatment. These results indicate that central administration of KP-10 activates nesfatin-1- and OXT neurons, and may play an important role in the suppression of feeding in male rats.

Effects of hypergravity on the gene expression of the hypothalamic feeding-related neuropeptides in mice via vestibular inputs.

The effects of hypergravity on the gene expression of the hypothalamic feeding-related neuropeptides in sham-operated (Sham) and vestibular-lesioned (VL) mice were examined by in situ hybridization histochemistry. Corticotrophin-releasing hormone (CRH) in the paraventricular nucleus was increased significantly in Sham but not in VL mice after 3 days of exposure to a 2 g environment compared with a 1 g environment. Significant decreases in pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript and significant increases in neuropeptide Y, agouti-related protein in the arcuate nucleus and orexin in the lateral hypothalamic area were observed in both Sham and VL mice. After 2 weeks of exposure, CRH and POMC were increased significantly in Sham but not in VL mice. After 8 weeks of exposure, the hypothalamic feeding-related neuropeptides were comparable between Sham and VL mice. These results suggest that the hypothalamic feeding-related neuropeptides may be affected during the exposed duration of hypergravity via vestibular inputs.

Involvement of central nesfatin-1 neurons on oxytocin-induced feeding suppression in rats.

Peripheral anorectic hormones, such as peptide YY (PYY) and oxytocin (OXT), suppress food intake. A newly identified anorectic neuropeptide, nesfatin-1, is synthesized in both peripheral tissue and the central nervous system, particularly by various nuclei in the hypothalamus and brainstem. Here, we examined the effects of intraperitoneal (ip) administration of PYY3-36, OXT, and OXT analog, on nesfatin-1-immunoreactive (ir) neurons in the rat hypothalamus and brainstem, using Fos double fluorescence-immunohistochemistry. The ip administration of OXT and OXT analog significantly increased the number of nesfatin-1-ir neurons expressing Fos-ir in the paraventricular nucleus, the arcuate nucleus, and the nucleus tractus solitarius, but not in the supraoptic nucleus, the lateral hypothalamic area, and the area postrema. No differences in the percentage of nesfatin-1-ir neurons expressing Fos in the nuclei of the hypothalamus and brainstem were observed, between rats treated with vehicle or those treated with PYY3-36. The decreased food intake, induced by OXT and OXT analog, was attenuated significantly by pretreatment with intracerebroventricular administration of antisense nesfatin-1. These results suggested that nesfatin-1-expressing neurons in the hypothalamus and brainstem may play a role in sensing the peripheral level of OXT and its suppression of feeding in rats.

Comparison of the induction of c-fos-eGFP and Fos protein in the rat spinal cord and hypothalamus resulting from subcutaneous capsaicin or formalin injection.

We evaluated whether a c-fos-enhanced green fluorescent protein (eGFP) transgenic rat line, which expresses the c-fos and eGFP fusion gene, can be useful for the study of nociceptive pathways and processing. Capsaicin solution (15%) or formalin (5%) was subcutaneously injected bilaterally into the hind paws (100µL per each paw) of adult male c-fos-eGFP transgenic or wild-type rats. Control rats were injected with ethanol or physiological saline respectively. Transgenic and wild-type rats were perfused at 1.5, 3 and 6h post injection, with some transgenic rats being perfused 24h post injection. A comparison of eGFP in transgenic rats and Fos-like immunoreactivity (LI) in wild-type rats was made in the dorsal spinal cord, paraventricular nucleus (PVN) and supraoptic nucleus (SON). Oxytocin-LI (OXT-LI) was carried out to examine the activation of OXT neurons in the PVN and SON. Following capsaicin or formalin treatment, eGFP was maximally expressed at 6h in the spinal cord and 3h in the PVN and SON, whereas, Fos-LI was maximally expressed at 1.5h in all the regions we analyzed. Induction of eGFP in the OXT neurons was observed after capsaicin or formalin treatment, while Fos-LI in the OXT neurons was observed only after formalin treatment. These results demonstrate that the peak induction of c-fos-eGFP following exposure to acute nociceptive stimuli was delayed by around 1.5-4.5h, but more sensitive than endogenous Fos, suggesting that the c-fos-eGFP rat line can be useful for the study of nociceptive pathways and processing.

Effects of peripherally administered cholecystokinin-8 and secretin on feeding/drinking and oxytocin-mRFP1 fluorescence in transgenic rats.

Peripheral administration of cholecystokinin (CCK)-8 or secretin activates oxytocin (OXT)-secreting neurons in the hypothalamus. Although OXT is involved in the regulation of feeding behavior, detailed mechanism remains unclear. In the present study, we examined the central OXTergic pathways after intraperitoneally (i.p.) administration of CCK-8 and secretin using male OXT-monomeric red fluorescent protein 1 (mRFP1) transgenic rats and male Wistar rats. I.p. administration of CCK-8 (50µg/kg) and secretin (100µg/kg) decreased food intake in these rats. While i.p. administration of CCK-8 decreased water intake, i.p. administration of secretin increased water intake. Immunohistochemical study revealed that Fos-Like-Immunoreactive cells were observed abundantly in the brainstem and in the OXT neurons in the dorsal division of the parvocellular paraventricular nucleus (dpPVN). We could observe marked increase of mRFP1 fluorescence, as an indicator for OXT, in the dpPVN and mRFP1-positive granules in axon terminals of the dpPVN OXT neurons in the nucleus tractus solitarius (NTS) after i.p. administration of CCK-8 and secretin. These results provide us the evidence that, at least in part, i.p. administration of CCK-8 or secretin might be involved in the regulation of feeding/drinking via a OXTergic pathway from the dpPVN to the NTS.