Figure 3.

DHPG-induced current in Pomc^Creneurons antagonized by the TRPC5 channel blockers. A-B.Ex vivo calcium imaging of POMC neurons in brain slices. A, left panel, spinning disk image of baseline GCaMP6 fluorescence in POMC neurons; right panel, bath perfusion of the mGluR1/5 agonist DHPG (5 μM) significantly increased intracellular Ca²⁺. This concentration is 1/10 of what is typically used in vitro, but the effects are still easily measured. Scale bar is 100 μm. B, the time course of the responses to increasing concentrations of DHPG represents averaged normalized fluorescence for all cells in a slice (n = 3 slices per concentration). C-D, representative traces of DHPG-induced inward current perfused with TTX (1 μM) in the absence (C) or presence (D) of TRPC4/5 channel blocker HC070 (100 nM). E-F, representative traces of DHPG-induced inward current perfused with TTX (1 μM) in the presence of TRPC1/4/5 channel blocker Pico 145 (1 μM) (E) or the store-operated Ca²⁺ channel inhibitor GSK7975A (10 μM) (F). 0.1% v/v DMSO vehicle control had no effect (data not shown). DHPG-induced inward currents were recorded in arcuate YFP labeled neurons (V_hold = −60 mV) from Pomc^Cre mice. G, the I–V relationship before and during the peak response from the same cell in F indicated that the reversal potential of the nonselective cation current was ∼ −30 mV. H, summary of the effects of GSK7975A, HC070 and Pico145 on DHPG-induced current (one-way ANOVA, effect of treatment, F_{(3, 32)} = 16.97, p < 0.0001; Bonferroni's multiple comparisons test a,b,c, p < 0.05; d, ns.). Scatter plots represent mean ± SEM. n = 11 control, n = 6 HC070, n = 5 pico145 and n = 14 GSK7975A treated neurons.