Phase Advancing Is a Common Property of Multiple Neuron Classes in the Mouse Retina

Optical recordings show phase-advanced responses in multiple ganglion cell populations. A, We made electrophysiological whole-cell recordings of excitatory current responses to flashed contrast spots to determine the flash duration that gave the maximum response amplitude without prolonging the response. We used this flash duration (5 monitor frames; 83 ms) throughout this study. B, Left, Two-photon fluorescence image of GCaMP6f-expressing cells in the ganglion cell layer. Right, Calcium responses of an example phase advancing OFF-type ganglion cell in the imaged population (gray circle in left panel; ttpPA = 56 ms, osPA = 119 ms). The vertical dashed black line indicates t₀, i.e., when the spot crossed the center of the imaging window. Arrowheads indicate the measured response onset time; the vertical cyan line shows the peak response time for the flashed spot. Shaded area represents ±SEM; scale bar = 10 μm. C, Example OFF-type ganglion cell with ttpPA = −104 ms considered not phase advanced, and osPA = 100 ms considered phase advanced. D, E, Population data histograms for all identified cell populations (OFF, ON, ON-OFF non-DS, and DS) showing response onset time (left) and response time-to-peak (right). Shaded columns (left) show cells classified as not phase advancing (<5 ms difference between response to moving vs flashed spot). Representative examples of phase advancing and nonphase advancing ON GC, ON-OFF GC, and DSGC populations are shown in Extended Data Figure 2-1. Scatter plots of response onset phase advancing versus response time-to-peak phase advancing for the recorded cell populations are shown in Extended Data Figure 2-2. A table in Extended Data Figure 2-3 summarizes the measurements of phase advancing for each group.

OFF-α-type and ON-α-type ganglion cells receive phase-advanced excitatory and inhibitory synaptic input. A, Two-photon fluorescence image of an OFF-α ganglion cell (top) and ON-α ganglion cell (bottom) filled with the red fluorophore Sulforhodamine 101 during targeted electrophysiological whole-cell recording (scale bar = 20 μm). Traces (right) show the cells’ respective excitatory current (V_hold = −60 mV), inhibitory current (V_hold = 15 mV), and membrane voltage response to a rightward (magenta line) and leftward (green line) moving spot, and a stationary flashed spot (black line). Spot velocity = 1340 μm s⁻¹; shaded area, ± SEM. B, Phase advancing of response onset time for the recorded OFF-α-type ganglion cell population. Measurements included spots moving at two speeds (670 vs 1340 μm s⁻¹). Box and whisker plot, median (red); 25th and 75th percentiles (top and bottom edges, blue); error bars, ±1 SD. Gray circles represent data from individual cells. Statistical comparison: Mann–Whitney U, all ****p < 0.0001 (left: U = 149, n = 68, 45; center: U = 102, n = 51, 30; right: U = 393, n = 52, 32). C, Same as B, for all recorded ON-α-type ganglion cells. All ****p < 0.0001 (left: U = 114, n = 56, 40; center: U = 140, n = 36, 22; right: U = 12, n = 16, 13). Response time-to-peak data for the recorded population is shown in Extended Data Figure 3-1.

LN receptive field model predicts timing of object motion-evoked responses in α GCs. Ai, Example linear (L) approximations of the spatial receptive field (RF; top, left) and temporal filter characteristic (bottom) of an OFF-α GC, measured using white noise checkerboard stimulation and reverse-correlation analysis. Model simulations used the temporal filter and a 2D difference-of-Gaussians fit to the measured spatial receptive field. Aii, Schematic of the LN model used to predict stimulus-evoked ganglion cell synaptic current and membrane voltage responses. The visual stimulus (left) was convolved with the measured spatiotemporal RF to obtain a linear response prediction. This linear response prediction was scaled nonlinearly (N) for each cell using the static nonlinear transfer function (green) obtained from the measured white noise response. The resulting LN model output (magenta) was compared with the measured response to the moving and flashed spot stimuli. B, LN model response (dark traces) and recorded response (light traces) for an example OFF-α ganglion cell. C, Scatterplot of the measured osPA values versus the modeled values based on excitatory current (left), inhibitory current (center), and membrane voltage response (right). Dashed line, unity; orange point represents the example cell shown in B. D, Same as C but for ttpPA values. E–G, Same as B–D, for ON-α ganglion cells.

Phase advancing in ON-α and OFF-α GCs does not require GABA_a-ergic inhibitory signaling. A, Excitatory (top) and inhibitory (bottom) currents obtained with electrophysiological whole-cell recordings from an example ON-α ganglion cell to moving and flashed spot stimuli under control conditions (black) and in the presence of gabazine (magenta). B, C, Response onset and time-to-peak for the recorded ON-α GC population under control conditions (black) and in the presence of gabazine (magenta). D–F, As A–C, for OFF-α GCs. Extended data figures show results obtained using the pharmacological blockers TPMPA (Extended Data Fig. 5-1) and strychnine (Extended Data Fig. 5-2).