DREADD-induced inhibition of the MnPO affects drinking behavior and neuroendocrine function in adult male rats

Purpose: Angiotensin II (Ang II) is a peptide hormone that contributes to body fluid balance and hypertension. 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. Methods: We used Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to test the role of the MnPO in thirst and neuroendocrine responses to Ang II in adult male Sprague-Dawley rats (250-300g). Rats were anesthetized with isoflurane and stereotaxically injected with an 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 for 4 weeks. Rats were anesthetized with inactin (10 mg/kg ip) and transcardially perfused 90 minutes after CNO and Ang II treatments. Brains were processed for cFos and mCherry immunohistochemistry. Results: DREADD-injected rats treated with CNO during Ang II exposure had a significantly attenuated drinking response compared to vehicle treatments or to control virus injected rats treated with CNO and Ang II (pIn vitro loose-cell voltage clamp recordings from DREADD-transfected MnPO slices indicated focal CNO (10 uM) application significantly reduces firing rates of these neurons. In situ hybridization experiments of DREADD-transfected MnPO neurons and vesicular glutamate transporter 2 indicated neurons transfected with the DREADD virus containing the CaMKIIa promotor are largely glutamatergic (89.17+1.32%). Conclusion: The results indicate CNO-induced inhibition of excitatory, CaMKIIa-expressing MnPO neurons influences drinking behavior and neuroendocrine function.