Fig. 3.

GABA_A inhibition disturbs the phase and period organization caused by long or short day length. (A) Time-lapse images of normalized Bmal1-ELuc oscillations in the LD-entrained SCNs are shown for every 3 h, color-coded to indicate the peak expression with dark red and lower-than-50% expression with dark blue (color bar on the bottom right). The clear D-V cluster formation and the short oscillation period can be seen in the control LD-entrained SCN (Upper). Application of the GABA_A blocker gabazine (GBZ) lengthens the free-running period in culture while making the cluster separation less clear (Middle). Similarly, physical separation by a surgical cut (Inset) lengthens the free-running period in the LD SCN (Lower). (Scale bar, 100 µm.) (B and C) The effect on the free-running period is observed consistently in a larger number of samples through whole-field luminometry using a photomultiplier tube. GBZ has no effect on the SD SCN culture (B) but in the LD SCN culture (C) GBZ increases the period (*P < 0.05, Student’s t test; n indicated in the bar graph). (D) The asymmetric repulsive coupling model can explain the phase and period organization under LD entrainment. With increased repulsive coupling in the D-SCN, the phase gap between D- and V- SCNs increases and the free-running period becomes shorter, as seen in the LD SCN. The light or dark shade indicates the region of vehicle or drug application. Simulation parameters are described in Materials and Methods and SI Appendix, Table S2.