TMEM63B channel is the osmosensor required for thirst drive of interoceptive neurons

Fig. 1. TMEM63B is expressed in SFO and TMEM63B deletion impairs water appetite. a Left: schematic illustration showing SFO position in brain. D3V, dorsal 3rd ventricle. Right: immunochemical detection of HA expression in the SFO of WT and Tmem63bHA-fl/HA-fl mice. Scale bars, 100 μm. b Immunochemical detection of HA, nNOS and GAD in the SFO of Tmem63bHA-fl/HA-fl;Gad1-GFP mice. Scale bar, 50 μm. Arrowheads and arrows showing that cells containing HA signal overlapped with GAD (indicated by blue arrowheads) and nNOS (indicated by yellow arrow). c Percentages of HA-positive cells with nNOS or GAD, or percentages of nNOS- or GAD-positive cells with HA (n = 3 mice, 5 slices). d Experimental design to induce dehydrated thirst (water deprivation for 24 h) or osmotic thirst (intraperitoneal injection of 2 M Mannitol). e Time course of water licking (left) or 0.3 M NaCl licking (right) in the two-bottle test under water deprivation. f Quantification of water and NaCl licking for 60 min after water deprivation in Tmem63b+/+, Tmem63b+/− and Tmem63b−/− mice (n = 11−14 mice in each experiments). g Time course of licking after i.p. injection of 2 M mannitol. h Quantification of licking after mannitol…

Fig. 2. TMEM63B in SFO is essential for thirst appetite. a Schematic of virus injection into SFO of Tmem63bHA-fl/HA-fl mice to produce SFO-specific TMEM63B deletion. b Representative images validating knockout of TMEM63B by immunostaining HA. Scale bars, 50 μm. c Time course of water licking and 0.3 M NaCl licking under water deprivation in Tmem63bHA-fl/HA-fl mice injected with AAV-Cre or AAV-mCherry virus. d Quantification of licking in AAV-Cre- (n = 14 mice) and AAV-mCherry- (n = 12 mice) injected animals. e Schematic illustrating the strategy used to generate Tmem63bLacZ mouse line. f Schematic of virus injection into SFO of Tmem63bLacZ/LacZ mice to produce SFO-specific TMEM63B rescue. g Representative images validating re-expression of TMEM63B by immunostaining HA. Scale bars, 50 μm. h Time course of water licking or 0.3 M NaCl licking under water deprivation in Tmem63bLacZ/LacZ mice injected with AAV-Cre or AAV-EGFP. i Quantification of licking in AAV-Cre- (n = 8 mice) and AAV-EGFP- (n = 9 mice) injected animals. Data are means ± SEM, *P < 0.05, **P < 0.01; two-way repeated-measures ANOVA with Holm-Šídák post-hoc analysis for d and i.

Fig. 3. TMEM63B is required for osmotic activation of SFOTMEM63B neurons. a Schematic of viral labeling of SFOTMEM63B neurons. b Left: experimental design to excite SFOTMEM63B neurons. Right: FOS immunoreactivity showing NaCl (i.p.)-activated SFOTMEM63B neurons. Scale bars, 50 μm. Quantification: n = 8 mice for Tmem63bCre/+ mice and n = 7 for Tmem63bCre/Cre mice. c Representative traces of the effect of hypertonic solution on the AP firing of SFOTMEM63B neurons. Lower traces show firing rates. d Left: percentages of activated neurons after applying hypertonic solution (for Tmem63bCre/+: n = 37 neurons from 16 mice, for Tmem63bCre/Cre: n = 25 neurons from 9 mice). Right: firing rates of activated SFOTMEM63B neurons from Tmem63bCre/+ mice (n = 29 neurons, left) and Tmem63bCre/Cre mice (n = 6 neurons, right) shown in left, before (Iso) and after hypertonic stimulation (Hyp). e Mean firing rate for all tested neurons. f RMP in SFOTMEM63B neurons from Tmem63bCre/+ or Tmem63bCre/Cre mice. g Representative spike pattern of SFOTMEM63B neurons in response to depolarizing currents. Current steps in the trace were made in 10-pA increments from –10 pA to +40 pA. Vm, the membrane potential before…

Fig. 4. TMEM63B contributes to the osmosensory transduction of SFOTMEM63B neurons in response to hyperosmolarity. a Top: schema of the action potential (AP) firing pattern of SFOTMEM63B neurons from Tmem63bCre/+ (left) mice and Tmem63bCre/Cre mice (right) before and after hypertonic stimulation. Bottom: AP are clipped for clarity. Black dashed lines show membrane potential; membrane potential was obtained from voltage histograms (blue gaussian curve). Threshold (blue dashed lines), AP threshold, was determined at the point where the voltage trace reached 10 mV/ms. b Quantification of the threshold from Tmem63bCre/+ neurons (n = 37) and Tmem63bCre/Cre neurons (n = 25) before and after hypertonic stimulation. c Subthreshold depolarization (distance to threshold) before (Iso) and after hypertonic stimulation (Hyp) from Tmem63bCre/+ mice (n = 37) and Tmem63bCre/Cre mice (n = 25). d The changes of interspike interval of SFOTMEM63B neurons from Tmem63bCre/+ mice (n = 37) and Tmem63bCre/Cre mice (n = 25) before and after hypertonic stimulation. e Representative voltage depolarization of SFOTMEM63B neurons in response to hypertonic solution in the presence of TTX and synaptic blockers. f…

Fig. 5. TMEM63B deletion does not affect AngII activation of SFOTMEM63B neurons. a Representative traces of the effect of AngII on the AP firing of SFOTMEM63B neurons. Lower traces show firing rates. b Firing rates for SFOTMEM63B neurons from Tmem63bCre/+ mice (n = 14 neurons) and Tmem63bCre/Cre mice (n = 15 neurons), before (Ctr) and during AngII stimulation. c The activation ratio of firing rate after AngII stimulation shown in b. Data are means ± SEM, ****P < 0.0001; n.s., no significant; two-tailed paired t-test for b and two-tailed unpaired t-test for c.

Fig. 6. Hypoosmolarity reduces SFOTMEM63B neuronal activity. a Representative traces of the effect of hypotonic solution on the AP firing of SFOTMEM63B neurons. Lower traces show firing rates. b Quantification of firing rates of SFOTMEM63B neuron from Tmem63bCre/+ mice (n = 20 neurons, left) and Tmem63bCre/Cre mice (n = 18 neurons, right), before (Iso) and after hypotonic stimulation (Hypo). c The inhibition ratio of firing rate after hypotonic stimulation shown in b. Cells without inhibition are excluded. d Percentages of neurons displaying different responses after hypotonic stimulation shown in b. e Representative voltage fluctuation of SFOTMEM63B neurons in response to hypotonic solution in the presence of TTX and synaptic blockers. f Quantification of the hyperpolarized membrane potential from Tmem63bCre/+ neurons (n = 10) and Tmem63bCre/Cre neurons (n = 12) after hypotonic stimulation. Data are means ± SEM, *P < 0.05, **P < 0.01, n.s., no significant; two-tailed unpaired t-test for c, f; two-tailed paired t-test for b; χ2 test for d.