Fig. 2
Direct evidence of light-triggered tonic GABA signaling in SCN via retina-attached slice patch. a Sequence of optimized retina-attached brain extraction method. Numbers indicate sequential steps of prep. b Eye or retina attached brain. Scale bars: 5 mm (top left). Retina attached SCN slice. Scale bar: 1 mm (top right). Retina-attached SCN slice fixed with anchor and accessed with patch pipette. Red asterisk denotes the position of patched cell. Scale bar: 200 \(\mu\)m; SCN is indicated with white dotted line (lower) c Timeline of experiment. Blue box represent retina attached slicing and resting time and red box represent whole-cell patch clamp periods (top). Magnified whole-cell recording protocol with light illumination highlighted with yellow box (lower). d Raw trace of voltage clamping (Holding potential, 0 mV) during ambient light illumination from cell#1. Insets, high time resolution trace at 1 min, 8 min, 15 min. Inset scale: 200 ms, 10 pA (top). 1 min bin analysis of phasic GABA (blue, sIPSC frequency) and tonic GABA (red, holding current shift) from cell #1 (middle). 1 min bin analysis of sIPSC amplitude from cell #1 (bottom), mean and error of 0.95 confidence intervals. e Paired point plots of light-triggered GABA signaling from two cells in SCN: holding current (top left), sIPSC frequency (top middle), sIPSC amplitude (top right), relative holding current change compared to baseline (bottom left), relative sIPSC frequency change compared to baseline (bottom middle), relative sIPSC amplitude change compared to baseline (bottom right). “Base” is first bin of light illumination; “Peak” is bin of maximum value; “End” is last bin of light illumination. f Representative raw 1 min traces of cell#1, right before the light illumination start (left, black), 5 min after light termination (middle, red) and data point distribution for measurement of shifted holding current (right). Dashed lines indicate mode of histograms. g Comparison of before and after holding GABA current (red) and phasic sIPSC frequency (blue) of cell#1. h Graphical description of light-induced GABA signaling in SCN