DREADD‐Induced Inhibition of the MnPO Affects Drinking Behavior and Neuroendocrine Function in Adult Male Rats

Abstract

Angiotensin II (Ang II) is a peptide hormone that becomes elevated in patients with obstructive sleep apnea and during chronic intermittent hypoxia. Forebrain circumventricular organs (CVOs) are sensitive to circulating Ang II and project to the median preoptic nucleus (MnPO). The MnPO projects to the paraventricular nucleus (PVN) and contributes to elevated sympathetic tone and thirst. In the present study, we use Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to test the role of the MnPO in the drinking and neuroendocrine responses to Ang II in adult male rats. Prior to the drinking tests, adult male Sprague‐Dawley rats (250–300g) were anesthetized with isoflurane and stereotaxically injected with a Cre‐independent inhibitory (Gi) DREADD (rAAV5‐CaMKIIa‐hM4D(Gi)‐mCherry) or control (rAAV5‐CaMKIIa‐mCherry) virus in the MnPO. After 2 weeks of recovery, each rat was administered 10 mg/kg of exogenous clozapine‐N‐oxide (CNO) ip to inhibit DREADD expressing cells or vehicle ip followed by 2 mg/kg Ang II sc twice per week (at least 72 h apart) for 4 weeks. Rats transfected with the Gi DREADD in the MnPO and subsequently treated with CNO during Ang II exposure had a significantly attenuated drinking response compared to vehicle treatments or to animals transfected with the control virus injected with CNO and Ang II (p<0.050). Interestingly, Gi DREADD injected rats also did not have as robust of a drinking response to Ang II during subsequent treatments with vehicle after the first CNO treatment, indicating a possible protective effect. After the testing period, rats were anesthetized with inactin (100 mg/kg ip) and transcardially perfused 90 minutes following CNO and Ang II treatments. Brains were processed for cFos and mCherry immunohistochemistry. Overall, MnPO transfection with the Gi DREADD and subsequent CNO treatment decreased cFos staining in the MnPO (p=0.002), magno‐ and parvo‐cellular subregions in the PVN (p<0.050), supraoptic nucleus (p=0.017), and rostral ventrolateral medulla (p=0.004) compared to controls. Plasma samples were also taken to measure plasma Arginine Vasopressin (AVP) on the day rats were perfused. Ang II injections significantly increased plasma AVP and this increase was significantly attenuated by CNO in rats injected in the MnPO with the Gi DREADD (p=0.009). In vitro loose‐cell voltage clamp recordings were obtained from MnPO neurons transfected with Gi DREADD. Focal CNO (10 uM) application significantly reduced the firing rates of MnPO neurons transfected with Gi DREADD but not unlabeled neurons in the same slice or neurons transfected with the control virus. In situ hybridization experiments analyzing the colocalization of DREADD transfected MnPO neurons and vesicular glutamate transporter 2, indicated that neurons transfected with the DREADD virus containing the CaMKIIa promotor are largely glutamatergic (89.17±1.32%). The results indicate that CNO‐induced inhibition of excitatory, CaMKIIa‐expressing MnPO neurons influences activity in downstream regions controlling drinking behavior, neuroendocrine function, and autonomic regulation.Support or Funding InformationSupported by NIH P01 HL088052 (JTC) and T32 AG020494 (ABM)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.