Circadian Behavioral Responses to Light and Optic Chiasm-Evoked Glutamatergic EPSCs in the Suprachiasmatic Nucleus of ipRGC Conditional vGlut2 Knock-Out Mice

Wheel-running behavior of Hopkins-Cre mice. Wheel-running activity records (double-plotted) of representative Control mice (Opn4^+/+; Vglut2^loxP/loxP; A–D) generated from the Hopkin-Cre line maintained under L12:D12 (100 lux:0 lux) for 84 d followed by DD for 114 d and then L12:D12 (1000 lux:0 lux) for 73 d.

Wheel-running behavior of Hopkins-Cre mice. Wheel-running activity records (double-plotted) of cKO mice (Opn4^Cre/+; Vglut2^loxP/loxP; A–F) generated from the Hopkins-Cre line maintained under L12:D12 (100 lux:0 lux) for 84 d followed by DD for 114 d and then L12:D12 (1000 lux:0 lux) for 73 d.

Wheel-running behavior of Salk-Cre and Hopkins-Cre mice. Wheel-running activity records (double-plotted) of Control (Opn4^+/+; Vglut2^loxP/loxP; A), cKO (Opn4^Cre/+; Vglut2^loxP/loxP; B–H) and dKO (Opn4^Cre/Cre; Vglut2^loxP/loxP; I, J) mice maintained under L12:D12 conditions. Animals shown in A, C–H were maintained initially under 100 lux:0 lux for 37 d followed by 1000 lux:0 lux for 40 d. Animals shown in B, I, J were maintained initially under 1000 lux:0 lux for 35 d followed 100 lux:0 lux for 22 d.

Evoked and spontaneous EPSCs in SCN neurons. Voltage-clamp recordings of eEPSCs in SCN neurons of (A) WT; (B) control (Opn4^+/+; Vglut2^loxP/loxP); (C) cKO (Opn4^Cre/+; Vglut2^loxP/loxP); and (D) dKO (Opn4^Cre/Cre; Vglut2^loxP/loxP) mice. EPSCs (membrane potential clamped at -60 mV) were evoked by stimulation of the optic chiasm in the absence of CNQX (left recording in A–D) and in the presence of AMPA/kainate antagonist CNQX (20 µM, right recording in A–D). Each recording in A–D shows a test current followed by a stimulus artifact and the eEPSC. sEPSC are shown in the recordings on the right. The corresponding actograms for each of the mice (B, C) are shown in Figure 4B (B), G (C), and I (D). E, A histogram showing the distribution of the stimulation voltage required to evoke the threshold EPSC in WT (n = 50 neurons, black bars) and cKO mice (n = 52, gray bars); there was no significant difference between the two groups.

The relationship between eEPSC amplitude and stimulus strength in WT and cKO SCN neurons. Dependence of eEPSC amplitude (pA) on the strength of stimulus (V) applied to the optic chiasm in WT and Opn4^Cre/+ mice (A–F). A, C, E, WT (n = 35 SCN neurons). B, D, F, cKO (Opn4^Cre/+; Vglut2^loxP/loxP; n = 41 SCN neurons). A, B, Each line on the graph represents voltage-dependent changes of eEPSC for an individual neuron. C, D, Scatter histograms showing the distribution of EPSC amplitudes evoked by stimulation. E, F, The results of each fitted linear mixed-effect model (lines) were used to analyze the eEPSC amplitude (pA) model at the population level (averaged over all animal-specific random effects) for the WT and Opn4^Cre/+ mice (E, F). The plotting symbols show the geometric mean amplitude at each stimulation amplitude. Error bars are omitted as there is no unique or unambiguous way to define SE when multiple sources of variation are present.

Plots showing all the data points for each of the eEPCS parameters analyzed. The small black circles are the individual data points, the horizontal lines indicate the 25th quartile, the median, and the 75th quartile. The large black circles indicate data points lying outside 1.5 times the interquartile range. The whiskers indicate the upper and lower values.