Fig. 2: ELS modifies intrinsic firing rates of orexin neurons in a sex specific manner.

A Representative viral targeting of orexin neurons in the LH, scale bar = 1 mm & 25 µm. B Validation of AAV-2/8-miniHCRT-tdTomato construct, scale bar = 50 µm. C Representative neurons from acute slice preparation under brightfield and Cy3 filter cube fluorescence with representative spontaneous firing rates of 3-4 Hz. D, E Representative traces of spontaneous excitatory post-synaptic currents (sEPSC) and spontaneous inhibitory post-synaptic currents (IPSCs) from orexin neurons in voltage-clamp (−70mV) configuration from Naïve and ELS mice. Male mice orexin neuron. F sEPSC peak amplitude (unpaired t test: Naïve = −38.14pA, ±3.81 (N = 3, n = 8 cells), ELS = −36.35pA, ±1.39 (N = 2, n = 5 cells), p = 0.728). G Event frequency (unpaired t test: Naïve =1.022 Hz, ±1.44 (N = 3, n = 10 cells), ELS = 1.215 Hz, ±2.30 (N = 2, n = 5 cells), p = 0.616). H Inter-event interval (unpaired t test: Naïve=1665 ms, ±340 (N = 3, n = 10 cells), ELS = 1478 ms, ±537 (N = 2, n = 5 cells), p = 0.765). I Female mice orexin neuron sEPSC peak amplitude (unpaired t test: Naïve = −37.57pA, ±3.32 (N = 3, n = 7 cells), ELS = −36.31 pA, ±1.50 (N = 3, n = 10 cells), p = 0.705). J Event frequency (Mann–Whitney test: Naïve = 0.836 Hz, ±0.183 (N = 3, n = 7 cells), ELS = 0.853 Hz, ±0.485 (N = 3, n = 10 cells), p = 0.720). K Inter-event interval (unpaired t test: Naïve = 1306 ms, ±257 (N = 3, n = 7 cells), ELS = 2049 ms, ±550 (N = 3, n = 10 cells), p = 0.303). Male mice orexin neuron. L sIPSC peak amplitude (unpaired t test: Naïve =−106.8 pA, ±15.1 (N = 2, n = 7 cells), ELS = −103.8 pA, ±10.1 (N = 2, n = 7 cells), p = 0.871). M Event frequency (unpaired t test: Naïve =1.022 Hz, ±0.135 (N = 2, n = 7 cells), ELS = 1.215 Hz, ±0.246 (N = 2, n = 8 cells), p = 0.230). N Inter-event interval (unpaired t test: Naïve=980.1 ms, ±169 (N = 2, n = 7 cells), ELS = 1489 ms, ±555 (N = 2, n = 7 cells), p = 0.398). Female mice orexin neuron. O sIPSC peak amplitude (unpaired t test: Naïve = − 116.5 pA, ±8.77 (N = 2, n = 9 cells), ELS = −76.9 pA, ±8.34 (N = 2, n = 9 cells), p = 0.0064). P Event frequency (unpaired t test: Naïve = 1.012 Hz, ±0.203 (N = 2, n = 7 cells), ELS = 1.230 Hz, ±0.203 (N = 2, n = 9 cells), p = 0.468). Q Inter-event interval (Mann–Whitney test: Naïve = 1431 ms, ±373 (N = 2, n = 7 cells), ELS = 824.5 ms, ±194 (N = 2, n = 9 cells), p = 0.252). R Representative traces of spontaneous firing rates of orexin neurons from Naïve and ELS male mice. S Representative traces of spontaneous firing rates of orexin neurons from Naïve and ELS female mice. T Resting membrane potentials (RMP) of orexin neurons from Naïve and ELS male mice (unpaired t-test: Naïve =−56.65 mV, ±1.04 (N = 3, n = 12 cells), ELS = −48.36 mV, ±1.78 (N = 4, n = 11 cells), p = 0.0005). U Spontaneous firing rates of orexin neurons from Naïve and ELS male mice (unpaired t test: Naïve = 3.85 Hz, ±0.459 (N = 4, n = 12 cells), ELS = 6.789 Hz, ±1.13 (N = 4, n = 12 cells), p = 0.025. V Instantaneous AP frequency of orexin neurons from Naïve (N = 5, n = 14) and ELS (N = 5, n = 11) male mice in response to 5 pA incremental current injections (two-way repeated measures ANOVA; F(1,23) = 40.60, p = < 0.001). Naive N = 4, n = 14 cells, ELS N = 4, n=11cells. W Resting membrane potentials (RMP) of orexin neurons from Naïve and ELS female mice (unpaired t test: Naïve =−54.19 mV, ±1.53 (N = 4, n = 16 cells), ELS = −49.66 mV, ±0.930 (N = 4, n = 16 cells), p = 0.0169). X Spontaneous firing rates of orexin neurons from Naïve and ELS female mice (unpaired t test: Naïve = 6.91 Hz, ±0.807 (N = 4, n = 15 cells), ELS = 3.648 Hz, ±0.618 (N = 4, n = 14 cells), p = 0.0037). Y Instantaneous AP frequency of orexin neurons from Naïve (N = 3, n = 9) and ELS (N = 3, n = 10) female mice in response to 5 pA incremental current injections (two-way repeated measures ANOVA; F(1,17) = 15.67, p = < 0.01). N = mice, n = cells. Bar charts = mean ± S.E.M. ns = not significant, *P < 0.05, **P < 0.01.