Figure 2. Diurnal changes in the spontaneous electrical activity of Rhabdomys pumilio SCN neurons.

(A) Whole-cell patch clamp recording setup showing bright-field image of a SCN coronal brain slice. The SCN (delineated by white dotted lines) can be observed above the optic chiasm (OC), on either side of the third ventricle (3V). Patch pipette targeting a SCN neuron is indicated by the red arrow and magnified in inset (a1). (B) Representative current-clamp traces of the different spontaneous excitability states recorded in R. pumilio SCN neurons: (i) highly depolarized cells, becoming silent (top trace) or displaying depolarized low-amplitude membrane oscillations (DLAMOs) (bottom trace); (ii) moderate resting membrane potential (RMP) with cells firing action potentials (APs) at high or low rate; and (iii) hyperpolarized-silent neurons. (C) Pie charts showing the percentages of SCN neurons in the different electrical states during the day and at night (χ²=21.498, ***p<0.001, Chi-square test). Mean RMP (D), spontaneous firing rate (SFR) (E) and input resistance (R_input) (F) of neurons recorded during the day (orange, n=67 for RMP and SFR, n=66 for R_input) and at night (blue, n=44 for RMP and SFR, n=43 for R_input). Data are expressed as mean ± SEM with each dot representing an individual neuron. ∗p < 0.05, ns: non-significant. RMP: F_{(1, 5.036)}=10.249, p=0.024; SFR: F_{(1, 7.027)}=5.998, p=0.044; R_input: F_{(1, 5.984)}=0.878, p=0.385, mixed-effects linear model. (G) Manual hyperpolarization of hyperexcited SCN neuron elicits a range of electrical states. Silent cell resting at highly depolarized state could be driven to display DLAMOs, fire APs, and become hyperpolarized-silent by injection of progressive steps of steady-state hyperpolarizing currents (from 0 to ~ −16 pA (red line); driving RMP from −32 mV to −60 mV).