Chemogenetic Activation of Oxytocin Neurons Improves Pain in a Reserpine-induced Fibromyalgia Rat Model.

Fibromyalgia (FM) is a syndrome characterized by chronic pain with depression as a frequent comorbidity. However, efficient management of the pain and depressive symptoms of FM is lacking. Given that endogenous oxytocin (OXT) contributes to the regulation of pain and depressive disorders, herein, we investigated the role of OXT in an experimental reserpine-induced FM model. In FM model, OXT-monomeric red fluorescent protein 1 (OXT-mRFP1) transgenic rats exhibited increased depressive behavior and sensitivity in a mechanical nociceptive test, suggesting reduced pain tolerance. Additionally, the development of the FM-like phenotype in OXT-mRFP1 FM model rats was accompanied by a significant reduction in OXT mRNA expression in the magnocellular neurons of the paraventricular nucleus. OXT-mRFP1 FM model rats also had significantly fewer tryptophan hydroxylase (TPH)- and tyrosine hydroxylase (TH)-immunoreactive (ir) neurons as well as reduced serotonin and norepinephrine levels in the dorsal raphe and locus coeruleus. To investigate the effects of stimulating the endogenous OXT pathway, rats expressing OXT-human muscarinic acetylcholine receptor (hM3Dq)-mCherry designer receptors exclusively activated by designer drugs (DREADDs) were also assessed in the FM model. Treatment of these rats with clozapine-N-oxide (CNO), an hM3Dq-activating drug, significantly improved characteristic FM model-induced pathophysiological pain, but did not alter depressive-like behavior. The chemogenetically induced effects were reversed by pre-treatment with an OXT receptor antagonist, confirming the specificity of action via the OXT pathway. These results indicate that endogenous OXT may have analgesic effects in FM, and could be a potential target for effective pain management strategies for this disorder.

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.

AVP-eGFP was significantly upregulated by hypovolemia in the parvocellular division of the paraventricular nucleus in the transgenic rats.

Arginine vasopressin (AVP) is produced in the paraventricular (PVN) and supraoptic nuclei (SON). Peripheral AVP, which is secreted from the posterior pituitary, is produced in the magnocellular division of the PVN (mPVN) and SON. In addition, AVP is produced in the parvocellular division of the PVN (pPVN), where corticotrophin-releasing factor (CRF) is synthesized. These peptides synergistically modulate the hypothalamic-pituitary-adrenal (HPA) axis. Previous studies have revealed that the HPA axis was activated by hypovolemia. However, the detailed dynamics of AVP in the pPVN under hypovolemic state has not been elucidated. Here, we evaluated the effects of hypovolemia and hyperosmolality on the hypothalamus, using AVP-enhanced green fluorescent protein (eGFP) transgenic rats. Polyethylene glycol (PEG) or 3% hypertonic saline (HTN) was intraperitoneally administered to develop hypovolemia or hyperosmolality. AVP-eGFP intensity was robustly upregulated at 3 and 6 h after intraperitoneal administration of PEG or HTN in the mPVN. While in the pPVN, eGFP intensity was significantly increased at 6 h after intraperitoneal administration of PEG with significant induction of Fos-immunoreactive (-ir) neurons. Consistently, eGFP mRNA, AVP hnRNA, and CRF mRNA in the pPVN and plasma AVP and corticosterone were significantly increased at 6 h after intraperitoneal administration of PEG. The results suggest that AVP and CRF syntheses in the pPVN were activated by hypovolemia, resulting in the activation of the HPA axis.

Chemogenetic activation of endogenous arginine vasopressin exerts anorexigenic effects via central nesfatin-1/NucB2 pathway.

We examined whether the chemogenetic activation of endogenous arginine vasopressin (AVP) affects central nesfatin-1/NucB2 neurons, using a transgenic rat line that was previously generated. Saline (1 mL/kg) or clozapine-N-oxide (CNO, 1 mg/mL/kg), an agonist for hM3Dq, was subcutaneously administered in adult male AVP-hM3Dq-mCherry transgenic rats (300-370 g). Food and water intake were significantly suppressed after subcutaneous (s.c.) injection of CNO, with aberrant circadian rhythmicity. The percentages of Fos expression in nesfatin-1/NucB2-immunoreactive neurons were significantly increased in the hypothalamus and brainstem at 120 min after s.c. injection of CNO. Suppressed food intake that was induced by chemogenetic activation of endogenous AVP was ablated after intracerebroventricularly administered nesfatin-1/NucB2-neutralizing antibody in comparison with vehicle, without any alteration of water intake nor circadian rhythmicity. These results suggest that chemogenetic activation of endogenous AVP affects, at least in part, central nesfatin-1/NucB2 neurons and may exert anorexigenic effects in the transgenic rats.

Peripherally administered cisplatin activates a parvocellular neuronal subtype expressing arginine vasopressin and enhanced green fluorescent protein in the paraventricular nucleus of a transgenic rat.

Cisplatin is one of the most potent anti-cancer drugs, though several side effects can induce stress responses such as activation of the hypothalamic-pituitary adrenal (HPA) axis. Arginine vasopressin (AVP) and corticotrophin-releasing hormone (CRH) expressed in the parvocellular division of the paraventricular nucleus (pPVN) play an important role in the stress-induced activation of the HPA axis. We aimed to evaluate whether intraperitoneal (i.p.) administration of cisplatin could activate parvocellular neurons in the pPVN, using a transgenic rat model that expresses the fusion gene of AVP and enhanced green fluorescent protein (eGFP). Along with the induction of FosB, a marker of neuronal activation, i.p. administration of cisplatin significantly increased eGFP fluorescent intensities in the pPVN. In situ hybridization histochemistry revealed that AVP-eGFP and CRH mRNAs in the pPVN were increased significantly in cisplatin-treated rats. These results suggest that cisplatin administration increases neuronal activation and upregulates AVP and CRH expression in the pPVN.

Effect of oestrogen-dependent vasopressin on HPA axis in the median eminence of female rats.

The median eminence (ME) anatomically consists of external (eME) and internal (iME) layers. The hypothalamic neurosecretory cells terminate their axons in the eME and secrete their neurohormones regulating anterior pituitary hormone secretion involved in stress responses into the portal vein located in the eME. Magnocellular neurosecretory cells (MNCs) which produce arginine vasopressin (AVP) and oxytocin in the paraventricular (PVN) and supraoptic nuclei (SON) terminate their axons in the posterior pituitary gland (PP) through the iME. Here, we provide the first evidence that oestrogen modulates the dynamic changes in AVP levels in the eME axon terminals in female rats, using AVP-eGFP and AVP-DREADDs transgenic rats. Strong AVP-eGFP fluorescence in the eME was observed at all oestrus cycle stages in adult female rats but not in male transgenic rats. AVP-eGFP fluorescence in the eME was depleted after bilateral ovariectomy but re-appeared with high-dose 17ß-oestradiol. AVP-eGFP fluorescence in the MNCs and PP did not change significantly in most treatments. Peripheral clozapine-N-oxide administration induced AVP-DREADDs neurone activation, causing a significant increase in plasma corticosterone levels in the transgenic rats. These results suggest that stress-induced activation of the hypothalamic-pituitary-adrenal axis may be caused by oestrogen-dependent upregulation of AVP in the eME of female rats.