DREADD (Designer Receptors Exclusively Activated by Designer Drugs)‐induced activation of subfornical organ neurons stimulates thirst and salt appetite

Circumventricular organs such as the subfornical organ play a pivotal role in body fluid homeostasis through the ability of these neurons to sense neurohumoral signals and subsequently regulate thirst, salt appetite, neuroendocrine function, and sympathetic outflow. Previous studies have largely relied on lesions or infusion of drug antagonists into a structure that is adjacent to the 3 rd ventricle. Recently, a new approach known as DREADD ( D esigner R eceptors E xclusively A ctivated by D esigner D rugs) has been developed to manipulate neuronal activity through virally‐mediated expression of a G‐protein coupled receptor that becomes activated after administration of the exogenous ligand Clozapine N‐Oxide (CNO). The purpose of this study was to investigate whether DREADD expression in SFO neurons could be a useful tool to manipulate body fluid homeostasis. We hypothesized that excitation of SFO neurons using DREADD‐Gq would stimulate thirst and salt appetite. At least 2 week before experiments, male C57/Bl6 mice (12–15 weeks) received an SFO injection of rAAV2‐CaMKII‐HA‐hM3D(Gq)‐IRES‐mCitrine (1×10 12 molecular/mL, 50nL). Subsequent injection of CNO (0.25mL, sc) into mice (n=13) with robust expression of the DREADD in SFO (verified immunocytochemically) produced a dose‐dependent increase in water intake: 0.0 mg/kg (0.02±0.01 mL) vs 0.06 mg/kg (0.08±0.03mL, P<0.05) vs 0.1 mg/kg (0.21±0.05mL, P<0.05) vs 1mg/kg (0.32±0.09mL, P<0.05) vs 3 mg/kg (0.32±0.07mL, P<0.05). To test whether DREADD‐induced activation of SFO neurons induces salt appetite, mice (n=13) were given access to distilled water, 0.3M NaCl solution, and 0.01% Na chow for 1 week. Subsequent injection of CNO (3mg/kg, sc) versus vehicle significantly increased the ingestion of water (0.36±0.08 vs 0.05±0.01 mL, respectively; P<0.01) and 0.3M NaCl (0.14±0.04 vs 0.03±0.01mL, respectively; P<0.05). A final set of experiments verified that injection of CNO activated SFO neurons through expression of the early‐intermediate gene Fos. Injection of CNO (3mg/kg, sc) versus vehicle (0.25mL, sc) significantly increased the number of Fos‐positive nuclei in the SFO (159±49 vs 2±1 cells, respectively; n=4/group; P<0.01). Mice with DREADD expression outside of the SFO (n=12) did not increase water intake, salt appetite, or Fos expression after injection of CNO. Collectively, these findings demonstrate that DREADD can be used experimentally to manipulate neuronal activity of SFO neurons and ultimately reveals that direct excitation of SFO neurons stimulates thirst and salt appetite.