DREADD-induced activation of subfornical organ neurons stimulates thirst and salt appetite

Abstract

The subfornical organ (SFO) plays a pivotal role in body fluid homeostasis through its ability to integrate neurohumoral signals and subsequently alter behavior, neuroendocrine function, and autonomic outflow. The purpose of the present study was to evaluate whether selective activation of SFO neurons using virally mediated expression of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) stimulated thirst and salt appetite. Male C57BL/6 mice (12-15 wk) received an injection of rAAV2-CaMKII-HA-hM3D(Gq)-IRES-mCitrine targeted at the SFO. Two weeks later, acute injection of clozapine N-oxide (CNO) produced dose-dependent increases in water intake of mice with DREADD expression in the SFO. CNO also stimulated the ingestion of 0.3 M NaCl. Acute injection of CNO significantly increased the number of Fos-positive nuclei in the SFO of mice with robust DREADD expression. Furthermore, in vivo single-unit recordings demonstrate that CNO significantly increases the discharge frequency of both ANG II- and NaCl-responsive neurons. In vitro current-clamp recordings confirm that bath application of CNO produces a significant membrane depolarization and increase in action potential frequency. In a final set of experiments, chronic administration of CNO approximately doubled 24-h water intake without an effect on salt appetite. These findings demonstrate that DREADD-induced activation of SFO neurons stimulates thirst and that DREADDs are a useful tool to acutely or chronically manipulate neuronal circuits influencing body fluid homeostasis.

Keywords: Fos; angiotensin II; electrophysiology; hypothalamus; sodium.

Fig. 1.

A: low- and high-power digital images of HA immunofluorescence in SFO-x (i and ii) and SFO (iii and iv) mice. Scale bars, 100 μm. B: cumulative water intake of SFO mice (n = 13) after injection of CNO (3.0 mg/kg sc) or vehicle (0.25 ml sc). C: 60-min cumulative water intake of SFO (n = 13) or SFO-x (n = 12) mice plotted as a function of CNO dose. D: 120-min water and 0.3 M NaCl intakes of SFO mice after injection of CNO (3.0 mg/kg sc) or vehicle (0.25 ml sc). All values are means ± SE. *P < 0.01 vs. vehicle, ^#P < 0.05 vs. 0 mg/kg CNO or SFO-x, †P < 0.05, water vs. 0.3 M NaCl.

Fig. 2.

A, top: Fos immunoreactivity in the SFO after injection of CNO in SFO-x (i) or SFO (ii) mice. The Fos-immunoreactive nuclei overlapped with HA-positive cells in SFO mice (iii and iv). Bottom: mean ± SE number of Fos-positive nuclei in the SFO of mice injected with CNO or vehicle. B, top: digital images of Fos immunoreactivity in median preoptic nucleus (MnPO), organum vasculosum of the lamina terminalis (OVLT), supraoptic nucleus (SON), and hypothalamic paraventricular nucleus (PVH) of SFO and SFO-x mice after injection of CNO. Bottom: mean ± SE number of Fos-positive nuclei in the SFO of mice injected with CNO or vehicle. *P < 0.01 vs. vehicle, n = 4 for all groups. Scale bars, 100 μm for all images except Aiv (25 μm).

Fig. 3.

A: 2 examples of single-unit recordings in SFO mice. SFO neurons were responsive to either intracerebroventricular ANG II (left) or 0.5 M NaCl (right). Injection of CNO (0.3 mg/kg iv) significantly increased SFO neuronal discharge. B: whole cell patch-clamp recording of SFO neuron demonstrates that bath application of CNO depolarizes V_m and increases cell discharge. Note that hyperpolarizing potentials are not readily visible in this example because of higher baseline noise. C: confocal image of Neurobiotin-filled SFO neuron and HA-Tag immunofluorescence. *P < 0.05 SFO vs. SFO-x.

Fig. 4.

Chronic administration of CNO through drinking tubes significantly increased 24-h water intake but not 0.3 M NaCl intake of SFO mice. CNO did not affect water or 0.3 M NaCl intake of SFO-x mice. Values are means ± SE. ΔWater or 0.3 M NaCl intakes were calculated by the difference between the 24-h intake and the average baseline intake (day −3 to 0). *P < 0.05 vs. baseline or SFO-x mice.