Retina-attached slice recording reveals light-triggered tonic GABA signaling in suprachiasmatic nucleus

Abstract

Light is a powerful external cue modulating the biological rhythm of internal clock neurons in the suprachiasmatic nucleus (SCN). GABA signaling in SCN is critically involved in this process. Both phasic and tonic modes of GABA signaling exist in SCN. Of the two modes, the tonic mode of GABA signaling has been implicated in light-mediated synchrony of SCN neurons. However, modulatory effects of external light on tonic GABA signalling are yet to be explored. Here, we systematically characterized electrophysiological properties of the clock neurons and determined the spatio-temporal profiles of tonic GABA current. Based on the whole-cell patch-clamp recordings from 76 SCN neurons, the cells with large tonic GABA current (>15 pA) were more frequently found in dorsal SCN. Moreover, tonic GABA current in SCN was highly correlated with the frequency of spontaneous inhibitory postsynaptic current (sIPSC), raising a possibility that tonic GABA current is due to spill-over from synaptic release. Interestingly, tonic GABA current was inversely correlated with slice-to-patch time interval, suggesting a critical role of retinal light exposure in intact brain for an induction of tonic GABA current in SCN. To test this possibility, we obtained meticulously prepared retina-attached SCN slices and successfully recorded tonic and phasic GABA signaling in SCN neurons. For the first time, we observed an early-onset, long-lasting tonic GABA current, followed by a slow-onset, short-lasting increase in the phasic GABA frequency, upon direct light-illumination of the attached retina. This result provides the first evidence that external light cue can directly trigger both tonic and phasic GABA signaling in SCN cell. In conclusion, we propose tonic GABA as the key mediator of external light in SCN.

Keywords: Light triggered tonic GABA; Retina-attached SCN; Spatiotemporal analysis; Whole-cell patch.

Fig. 1

Spatio-temporal profiling of tonic GABA in SCN. a Differential interference contrast (DIC) image of freshly isolated mouse brain with glass pipette whole-cell patch clamped to a neuron. 3V; third ventricle, SCN; Suprachiasmatic nucleus, OX; Optic chiasm. Scale bar: 100 $\mu$m b Representative trace of tonic GABA current recording in a SCN neuron and list of information we gathered c Summary bar graphs of tonic current analyzed by period categories. Bar graphs are shown as mean and error of 0.95 confidence intervals. d Summary bar graphs of dorsal and ventral tonic current (left), sIPSC Frequency (middle), sIPSC Amplitude (right). Bar graphs are shown as mean and error of 0.95 confidence intervals. e Pearson’s correlation analysis of tonic current with sIPSC amplitude, sIPSC frequency, mediolateral relative distance, ventrodorsal relative distance, sliced time, patched time, slice-to-patch time interval and membrane capacitance. f Tonic current scatter plot on patched time grouped by tonic current size: “Small” (blue), “Medium” (gray), “Large” (red). Dashed lines indicate decision boundaries for three groups. Dark shaded area, ZT 12–24. g Logistic regression analysis with “Large” and “Small” group with sIPSC frqeuncy and slice-to-patch time interval. A line divided by blue and red areas represent decision boundary with accuracy of 89.8%. h Tonic current scatter plot on 2D virtual SCN map, colors and size indicates group and size of tonic current respectively. i Histogram and gaussian kernel density estimation(KDE) on radius axis from SCN center. SCN center is calculated by 2d centroid of virtual SCN area. Core and shell was divided at the peak of KDE. j Probability of occurrence of each group in dorsal or ventral (left) and core or shell (right). k Summary bar graph of topologically divided groups of tonic current. l, m Scatter polar charts of tonic current groups. 0${}^{\circ }$, 90${}^{\circ }$, 180${}^{\circ }$ and 270${}^{\circ }$ respectively indicates lateral, dorsal, medial and ventral directions. Group “Small” ($0\le I<5$ pA, (l) n = 34); Group “Medium” ($5\le I<15$ pA, n = 22) (m), “Large” ($I>15$ pA, n = 15) (n) o, Probability of occurrence and cosine fitting in 8 different radial divisions: Group “Small”, regression coefficient r${}^2$=0.80, phase shift $\theta$ = 319.2${}^{\circ }$ (top); Group “Medium”, regression coefficient r${}^2$ = 0.58, phase shift $\theta$=166.7${}^{\circ }$ (middle); Group “Large”, regression coefficient r${}^2$ = 0.51, phase shift $\theta$ = 93.8${}^{\circ }$ (bottom).

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