Volume 23, Issue 8, Pages 2245-2253 (May 2018) Trpv4 Mediates Hypotonic Inhibition of Central Osmosensory Neurons via Taurine Gliotransmission  Sorana Ciura, Masha Prager-Khoutorsky, Zahra S. Thirouin, Joshua C. Wyrosdic, James E. Olson, Wolfgang Liedtke, Charles W. Bourque  Cell Reports  Volume 23, Issue 8, Pages 2245-2253 (May 2018) DOI: 10.1016/j.celrep.2018.04.090 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2018 23, 2245-2253DOI: (10.1016/j.celrep.2018.04.090) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 dn-Trpv1 Does Not Mediate Hypotonicity Detection In Situ (A) Steady-state current-voltage (I-V) relations in voltage clamped ONs (Vhold −60 mV) in control and hypotonic solution (−25 mOsm). Panels show effects in cells isolated from WT OVLT without (left) or with SB366791 (3 μM), or from a Trpv1−/− mouse. (B) Plots show changes in membrane conductance relative to control induced by hypotonicity in all cells (filled gray circles). Population average (±SEM) is shown by blue circles; ∗∗∗p < 0.001. (C) Ratemeters show effects of hypotonicity (blue bars) on firing rate (FR) recorded from ONs in explants from WT and Trpv1−/− mice. Traces below are excerpts (10 s) of AP firing before, during, and after the stimulus. (D) Scatterplots show changes in the firing rate (ΔFR) induced by various hypotonic stimuli. Each blue circle shows ΔFR in one cell. Black symbols and gray areas represent SEM of control firing rate values. Solid lines are linear regressions. Cell Reports 2018 23, 2245-2253DOI: (10.1016/j.celrep.2018.04.090) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 Hypotonicity Inhibits ONs via GlyRs (A) Immunoreactive taurine (left) is detected in the OVLT region, below the median preoptic nucleus (MnPO; schematic at right; see also Figure S1). (B) Micrograph shows immunolabeled GlyRs present in neuronal somata (NeuN-positive; red) and processes in the OVLT. (C) Extracellular recordings show the effect of taurine (1 mM) on an OVLT ON in a WT hypothalamic slice. Note that the inhibitory effect is antagonized by strychnine (1 μM). (D) Bar graphs show mean (±SEM) firing rate in ONs with and without taurine and in the presence or absence of strychnine. ∗p < 0.05; ns, not significant. (E) Plot shows mean (±SEM) taurine concentration detected in the solution perfusing microdissected OVLTs. Data show average values from 3 experiments, which each contained tissue from 5 mice. Hypotonic stimulation (−50 mOsm; blue area) significantly increased taurine release (∗∗p < 0.01). (F) Excerpts of AP firing in ONs before (Control) and during application of a hypotonic stimulus (−25 mOsm; blue area) in explants superfused with artificial cerebrospinal fluid (ACSF), ACSF containing 3 mM Kynurenate and 10 μM bicuculline (synaptic block), or synaptic block plus 1 μM strychnine. Graphs at right plot ΔFR in all cells tested (open circles) and group means ± SEM (blue circles). p shown above plots. Cell Reports 2018 23, 2245-2253DOI: (10.1016/j.celrep.2018.04.090) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 Hypotonic Inhibition of ONs Requires HAACs in Glial Cells (A) Ratemeters show the effects of hypotonicity (−25 mOsm; blue area) on firing rate in ONs recorded in explants superfused with ACSF, ACSF + DCPIB (20 μM), or explants superfused with ACSF that were pre-treated with 50 μM fluorocitrate (FC, 90 min; Figure S2). (B) Bar graphs plot mean (±SEM) firing rate before (Control) and during the hypotonic stimulus (blue) for conditions shown in (A). (C) Whole-cell voltage-clamp recordings (VH −60 mV; voltage upper; current lower) from a cultured glial cell (left) and an ON acutely isolated from the OVLT of a Trpv1−/− mouse. Cells were exposed to hypotonicity (−50 mOsm; blue shading) then 20 μM DCPIB was added to the bath (purple bar). (D and E) Bar graphs show mean (±SEM) values of membrane conductance (G) measured in glia (D) and ONs (E). ∗p < 0.05; ∗∗∗p < 0.005; ns, not significant. Cell Reports 2018 23, 2245-2253DOI: (10.1016/j.celrep.2018.04.090) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 Hypotonicity Inhibits ONs via Trpv4 (A) Micrographs show immunoreactive Trpv4 (green), GFAP (red), and merge, in a section of mouse OVLT (see also Figure S3). (B) Pseudo-color images represent the F340/380 ratio of FURA-2 loaded glia used to measure changes in [Ca2+]i levels caused by hypotonicity (−50 mOsm; lower) before (Control), during application of HC-067047 (middle), and after wash. (C) Superimposed plots show dynamic changes in F340/380 caused by hypotonicity in a subset of cells. (D) Plots show peak hypotonicity-induced changes in F340/380 (ΔF340/380) in all glial cells from WT (left) and Trpv4−/− mice. Blue bars show mean ± SEM (E) Plots show ΔF340/380 in responsive WT glial cells in the absence (Control) and presence of 1 μM HC-067047 (right). Blue bars show mean ± SEM ∗∗∗p < 0.001. (F) Voltage-clamp recordings show effects of hypotonicity (−50 mOsm, blue traces) on currents evoked by voltage steps to −60 and +40 mV in glia from WT or Trpv4−/− mice. WT cells were patched with electrodes containing normal internal solution (WT), internal solution supplemented with BAPTA (WT+BAPTA), or normal internal solution and external solution containing 1 μM HC-067047 (WT + HC). (G) Bar graphs show mean (±SEM) changes in membrane conductance (ΔG) induced by hypotonicity. (H) Ratemeters show effects of hypotonicity (−25 mOsm; blue shading) on the firing rate from ONs in explants from Trpv1−/− or Trpv1/4−/− mice. (I) Bar graphs show mean (±SEM) firing rate recorded from ONs under control (white) and hypotonic conditions (blue) in explants from Trpv1−/− and Trpv1/4−/− mice. ∗p < 0.05, ∗∗∗p < 0.005, ns not significant. Cell Reports 2018 23, 2245-2253DOI: (10.1016/j.celrep.2018.04.090) Copyright © 2018 The Author(s) Terms and Conditions