Figure 12. Double CRISPR mutagenesis of Trpc5 attenuates slow excitatory postsynaptic potential (EPSP), increases rheobase, and shifts the F-I curve.

(A) High-frequency photostimulation (20 Hz) generated slow EPSP in Kiss1^ARH neuron from ovariectomized, control mouse. Red trace is slow EPSP after low-pass filtering. (B) Slow EPSP in Kiss1^ARH neuron from OVX, double sgTrpc5 -targeted (Trpc5 mut) mouse. (C) Summary of the effects of Trpc5 mutagenesis on slow EPSP amplitude in female mice. Unpaired t-test, t₍₂₇₎ = 5.916, ****p<0.0001. Data are expressed as mean ± SEM, with data points representing individual cells. (D) Double sgRNA mutagenesis of Trpc5 channels in Kiss1^ARH neurons significantly increased the RMP (control: –64.7 ± 1.4 mV versus Trpc5 mut: –71.1 ± 1.2 mV, unpaired t-test, t₍₇₃₎ = 3.524, ***p=0.0007). (E) Current ramp showing the increased rheobase in a Kiss1^ARH neuron from Trpc5 mut mice (control: 31.1 ± 1.2 pA, n = 31, versus Trpc5 mut, 35.3 ± 1.0 pA, n = 33, unpaired t-test, t₍₆₂₎ = 2.777, **p=0.0073). (F) Firing frequency vs. current (F-I) curves for control versus Trpc5 mut (two-way ANOVA: main effect of treatment [F_(1,52) = 13.04, p=0.0007], main effect of injected current [F_(8,416) = 291.3, p<0.0001] and interaction [F_(8,416) = 6.254, p<0.0001]; control, n = 26, Trpc5 mut, n = 28; post hoc Bonferroni test, *p<0.05; **p<0.01, and ***p<0.005, respectively). (G) Model simulations of the effects of a current ramp (50 pA/s) for OVX (left panel) and OVX female with reduced (muted) TRPC5 conductance (right panel), and (H) the associated firing frequency versus current curves. In the latter case, the TRCP5 conductance was halved, which is a conservative estimation of the CRISPR state in which the Trpc5 is much more mutated in Kiss1^ARH neurons (Figure 11D).