Conditional Knock-Out of Vesicular GABA Transporter Gene from Starburst Amacrine Cells Reveals the Contributions of Multiple Synaptic Mechanisms Underlying Direction Selectivity in the Retina

Asymmetric inhibition between SACs and pDSGCs is impaired in Vgat^flox/floxCTD mice. A, Voltage-clamp traces from null-side SAC-DSGC pairs in CTD (Control) and Vgat^flox/floxCTD (KO) mice showing cholinergic EPSCs (red) and GABAergic IPSCs (black) evoked in DSGCs by depolarizing SACs. Dashed lines indicate the onset of voltage step in SACs. Top schematic diagram shows the relative soma locations in null-side pairs. Black arrow indicates the DSGC's preferred direction. Thicker red arrow indicates stronger GABAergic inputs from null-side SAC. B, Examples of null-side SAC-evoked IPSCs of pDSGCs in Control and KO groups exhibiting residual inhibition. C, Examples of preferred-side SAC-evoked IPSCs in Control and KO groups exhibiting residual inhibition. Top schematic diagram shows the relative soma locations in preferred-side pairs. D, Summary plot of IPSC peak amplitudes in pDSGCs evoked by preferred and null side SACs. Individual data points and statistical summary (mean ± SEM) are shown. Control–Pref: 49.5 ± 13.7 pA, n = 7 cells, 5 mice; Control–Null: 299.7 ± 35.0 pA, n = 16 cells, 8 mice; KO–Pref: 14.8 ± 5.1 pA, n = 13 cells, 8 mice; KO–Null: 44.3 ± 11.3 pA, n = 20 cells, 11 mice. E, As in D, summary plot of cholinergic EPSC peak amplitudes. Control–Pref: 167.3 ± 22.4 pA, n = 7 cells, 5 mice; Control–Null: 204.0 ± 23.4 pA, n = 16 cells, 8 mice; KO–Pref: 183.3 ± 25.0 pA, n = 13 cells, 8 mice; KO–Null: 187.2 ± 27.4 pA, n = 20 cells, 11 mice. F, Examples of IPSCs in pDSGCs evoked by uncaging of Rubi-GABA. Synaptic transmission was blocked by 300 μm CdCl₂. Vertical lines indicate the start of illumination by a 470 nm LED of a 400 μm spot centered on the DSGC. G, Summary plot of IPSC peak amplitudes (left) and charge transfer (right) in pDSGCs during uncaging of Rubi-GABA. Individual data points and statistical summary (mean ± SEM) are shown. Control: peak amplitude 17.8 ± 2.2 pA, charge transfer 1892.7 ± 245.6 pA/ms, n = 12 cells, 4 mice; KO: peak amplitude 19.6 ± 1.9 pA, charge transfer 2253.5 ± 254.8 pA/ms, n = 9 cells, 3 mice.

Inhibitory inputs onto pDSGCs in Vgat^flox/floxCTD mice lose direction selectivity. A, Voltage-clamp recordings of a pDSGC in Vgat^flox/floxTD (Control, Cell 1) and of 3 cells in Vgat^flox/floxCTD mice (KO, Cell 2–4) showing inhibitory currents evoked by the leading (On) and trailing (Off) edges of a bright bar moving in the preferred and null directions. Dashed lines separate the leading and trailing edge responses. B, For the On (left) and Off (right) components indicated in (A), summary plots of IPSC peak amplitudes and total charge transfer evoked by null-direction motion against those evoked by preferred-direction motion. Control mice include Vgat^flox/floxTD, Vgat^flox/floxD, and CTD for this figure and subsequent figures. Diagonal lines are unity lines. C, Summary plots showing IPSC amplitudes and total charge transfer in the preferred and null directions for control (n = 17 cells, 10 mice) and KO (n = 17 cells, 10 mice) groups. On: control preferred 300.3 ± 31.1 pA, control null 624.1 ± 52.8 pA, KO preferred 336.2 ± 42.0 pA, KO null 344.5 ± 36.9 pA; Off: control preferred 196.3 ± 30.2 pA, control null 472.7 ± 45.0 pA, KO preferred 250.4 ± 36.0 pA, KO null 274.3 ± 33.2 pA; total charge transfer: control preferred 108.3 ± 18.0 pA/s, control null 333.5 ± 34.9 pA/s, KO preferred 161.7 ± 23.3 pA/s, KO null 154.6 ± 23.3 pA/s.

Spiking activity remains direction selective in a subpopulation of pDSGCs in Vgat^flox/floxCTD mice. A, Cumulative frequencies of spike DSI from control and Vgat^flox/floxCTD (KO) mice. Dashed line indicates cutoff between non-DS and DS groups of pDSGCs in KO mice (see Results and Materials and Methods). Numbers 1–5 indicate individual cells belonging to control, non-DS, and DS groups. B, Polar plots and DSI values of spiking activity for cells labeled 1–5 in A illustrate a non-DS cell from a KO mouse (#3), weakly tuned cells in control (#1) and KO (#4) mice, and strongly tuned cells in control (#2) and KO (#5) mice. Colored lines represent individual repetitions and black lines represent the mean number of spikes evoked by bars moving in 12 directions. Red lines in the center represent the vector sum of spiking activity. The maximum number of spikes is shown in the upper right of each plot. C, Summary plot showing DSI and vector sum values in control (n = 33 cells, 20 mice) and KO-DS (n = 18 cells, 15 mice) groups. DSI control: 0.59 ± 0.03; KO-DS: 0.37 ± 0.04; vector sum control: 0.35 ± 0.03; KO-DS: 0.20 ± 0.02.

Different patterns of excitatory and inhibitory inputs underlie direction-selective spiking of pDSGCs. A, Example cell from a control mouse showing its spiking activity and excitatory and inhibitory currents evoked by preferred (red) and null (black) direction motion. The preferred direction is determined from spiking activity. Top four rows: traces from electrophysiological recordings of spikes and excitatory (inward) and inhibitory (outward) currents. Lighter traces are three individual repetitions and dark traces are the mean traces of the three repetitions. Lower three rows: polar plots of spiking responses and peak amplitudes of EPSCs and IPSCs. The On responses evoked by the leading edge of the moving bar are shown in this and subsequent examples in this figure. B, Example cell from the non-DS group in a Vgat^flox/floxCTD mouse. Excitatory inputs onto this cell are not direction selective. The relative timing between excitation and inhibition is similar for the preferred and null direction motion. C, Example cell from the DS group that received direction-selective excitatory inputs and slightly delayed inhibitory inputs in the preferred direction motion.

The excitatory but not inhibitory inputs onto the pDSGCs from the DS group in Vgat^flox/floxCTD mice are directionally tuned to the posterior direction. A, Summary plot showing the log ratio of IPSC amplitudes in the null and preferred directions (log(N/P)) for On and Off components for control (n = 17 cells, 11 mice), non-DS (On: n = 6 cells, Off: n = 11 cells), and DS (On: n = 14 cells, Off: n = 11 cells, On and Off data from 18 mice) groups. Data are represented as mean ± SEM. On: Control 0.34 ± 0.04, non-DS 0.07 ± 0.05, DS −0.04 ± 0.04; Off: Control 0.45 ± 0.04, non-DS 0.05 ± 0.04, DS −0.08 ± 0.04. B, Scatter plots of IPSC log(N/P) ratio versus spike DSI for On (left) and Off (right) components in the DS group. DSI values for On and Off spiking activity are calculated separately. Dashed lines indicate linear regression fit for this and subsequent plots. C, Summary plot showing the log ratio of EPSC amplitudes in the preferred and null directions [log(P/N)] for On and Off components for control (n = 17 cells, 11 mice), non-DS (On: n = 6 cells, Off: n = 11 cells), and DS (On: n = 14 cells, Off: n = 11 cells, On and Off data from 18 mice) groups. On: Control 0.13 ± 0.06, non-DS −0.08 ± 0.04, DS 0.12 ± 0.03; Off: Control 0.17 ± 0.04, non-DS −0.01 ± 0.02, DS 0.24 ± 0.03. D, Scatter plots of EPSC P/N ratio versus spike DSI for On (left) and Off (right) components in the DS group. E, Summary plot showing maximum EPSC amplitudes in pDSGCs from control and KO groups. Filled diamonds represent mean ± SEM. On: Control 320.5 ± 25.0 pA, KO 332.1 ± 34.4 pA; Off: Control 315.8 ± 22.7 pA, KO 357.0 ± 46.9 pA. F, Histograms of preferred directions calculated from spiking activity in control mice (top) and spiking activity (middle) and EPSC amplitude (bottom) in KO mice. The preferred directions of EPSCs are determined from the vector sums of peak amplitudes from 12 directions. The visual coordinates are labeled on the top of the histograms. G, Summary plot showing the log of EPSC P/N ratio before (−) and after (+) gabazine application in control and the DS group in Vgat^flox/floxCTD mice (KO-DS). Control mice: before gabazine, 0.23 ± 0.04; after gabazine, 0.05 ± 0.02, n = 20 cells, 8 mice; KO-DS: before gabazine, 0.14 ± 0.02; after gabazine, −0.02 ± 0.02, n = 8 cells, 5 mice. On and Off components are pooled together.

Relation between spiking activity and the time course of excitation and inhibition onto pDSGCs in Vgat^flox/floxCTD mice. A, Summary plots showing the temporal offset of t_peak or t_cm between EPSCs and IPSCs in pDSGCs evoked by the leading edge of a bright moving bar from the control, non-DS, and DS groups (Δt_peak: control, 149.0 ± 14.35 ms, n = 20 cells; non-DS, −10.0 ± 11.2 ms, n = 6 cells; DS, 86.7 ± 16.7 ms, n = 14 cells; Δt_cm, control: 136.7 ± 22.7 ms; non-DS, 3.5 ± 9.6 ms; DS, 56.8 ± 23.4 ms. B, Temporally aligned spike-density histograms (top), current-density histograms (middle) for EPSCs (red, inward) and IPSCs (blue, outward), and the relative difference between excitatory and inhibitory conductances (Δg, bottom) of a pDSGC in preferred and null directions of a moving bar stimulus. For B and C, 100 ms bins are used. Time windows of On and Off spikes are indicated by dashed lines. C, Cross-correlograms for the neuron in B. The spiking activity, EPSC, and Δg covary synchronously, as indicated by the correlation coefficient peaking at the t = 0 bin. The two smaller peaks at both sides result from the cross-correlation between On and Off responses during the moving bar stimulus. D, Summary plots showing the cross-correlation coefficient at t = 0 between spiking and EPSC and between spiking and Δg in the preferred and null directions (preferred direction: spike vs EPSC, 0.87 ± 0.04; spike vs Δg, 0.72 ± 0.09; null direction: spike vs EPSC, 0.80 ± 0.04; spike vs Δg, 0.56 ± 0.09, n = 8 cells).

Cholinergic component of the EPSCs in the DS group from the Vgat^flox/floxCTD mice is direction selective. A, Mean EPSC traces (average over three trials) of a pDSGC in the DS group evoked by a moving bar in the preferred and null directions. EPSCs were recorded before (top) and after (middle) 0.08 μm DHβE was applied through perfusion. The subtracted trace (bottom) represents the DHβE-sensitive cholinergic component of the EPSCs. B, Polar plot of the peak amplitude of the On responses for the total (black), DHβE-insensitive glutamatergic (blue), and DHβE-sensitive cholinergic (red) components from the cell in A. P, Posterior; A, anterior; S, superior; I, inferior. C, For On responses, scatter plots of EPSC peak amplitudes evoked by null-direction motion against those evoked by preferred-direction motion for the total (left), DHβE-insensitive (middle), and DHβE-sensitive (right) components. Diagonal lines are unity lines. D, Summary plot showing the log ratio of EPSC amplitudes in the preferred and null directions [log(P/N)] for the On response for the total (left), DHβE-insensitive (middle), and DHβE-sensitive (right) components. Total: 0.17 ± 0.03; DHβE-insensitive: 0.09 ± 0.02; DHβE-sensitive: 0.23 ± 0.05; n = 11 cells, 8 mice. E, As in C, scatter plots for Off responses. F, As in D, summary plot showing the log ratio of EPSC amplitudes in the preferred and null directions [log(P/N)] for the Off response. Total: 0.20 ± 0.03; DHβE-insensitive: 0.08 ± 0.05; DHβE-sensitive: 0.21 ± 0.03.