I-V relationships of light evoked synaptic inputs to J-RGCs.

(A) Representative average synaptic currents recorded from a J-RGC under different holding potentials. Traces are the average of 6 repeats, with different colors indicating different holding potentials. Arrow, onset of a dark spot stimulus. (B) I-V curve from A at the time of peak conductance (magenta dashed line in A). This is the I-V relationship for a mixture of all synaptic inputs. (C and D) The same as A and B but with the bath application of PTX + STR to block all the inhibitory inputs. The reversal potential for remaining currents was calculated to be 9.7 ± 3.3 mV, n = 3 cells. Data for this figure are in S2 Data. (PDF)</p

A working model for the generation of J-RGCs’ direction selectivity.

Inhibition: only SACs on the dendrite side of J-RGCs make synaptic connections. PDs of different SAC sectors are indicated by the red arrow heads. Excitation: slow bipolar cells connect to the proximal dendrites of J-RGCs, while fast bipolar cells connect to the distal dendrites, resulting in more temporal overlap of excitatory inputs for PD motion, and less overlap thus less summation during ND motion. S1/S2, sub-lamina 1/2 of the IPL. BC, bipolar cell. Ge, excitatory conductance. Gi, inhibitory conductance. IPL, inner plexiform layer; J-RGC, J-type retinal ganglion cell; ND, null direction; PD, preferred direction; SAC, starburst amacrine cell.</p

Simulation of the Vm change responses during motion using actual or modified synaptic conductance as inputs.

(A) Simulated J-RGCs’ responses with actual (left) and modified (middle and right) synaptic inputs. Similar to Fig 3B except Vstart = Einh. Shaded area around the traces, mean ± SEM. Ge, excitatory conductance. Gi, inhibitory conductance. (B and C) Peak depolarization of simulated responses to PD and ND motion under the DSe (B) and DSi (C) conditions across different Vstart. Responses under the control condition are included for comparison. (D) DSI values of simulated responses under control, DSe and DSi conditions across different Vstart. Error bars, SEM. n = 100 trials. Data for this figure are in S2 Data. (PDF)</p

Direction selectivity of J-RGCs at different speeds.

(A and D) Representative EPSCs (A) and IPSCs (D) recorded from a J-RGC during PD and ND motion at 800 μm/s (left) and 2,000 μm/s (right). Traces are aligned to the estimated time when the leading edge of the moving spot entered the RF center (dotted line). Shaded area around the traces, mean ± SEM, n = 10 trials. (B, C, E, and F) Summary of the peak amplitudes and DSI values of motion evoked OFF EPSCs (B and C) and IPSCs (E and F). In B and C, n = 13 cells; in E and F, n = 9 cells. (G) The spiking responses of a J-RGC to PD and ND motion at 800 μm/s (left) and 2,000 μm/s (right). (H) Comparison of DSI values at 800 μm/s and 2,000 μm/s. n = 12 cells. Dotted line: DSI = 0.3. (I) Polar plots for the average spiking responses of a J-RGC to motion in 8 directions at 800 μm/s and 2,000 μm/s. n = 11 trials. (J) Comparison of direction selectivity measured by vector sum magnitudes of J-RGCs’ responses to 8 directions of motion at 800 μm/s and 2,000 μm/s. Dotted line: vector sum magnitude = 0.12. n = 6 cells. Error bars, SEM. In B, C, E, F, H, and J, paired t test; *, p p p S2 Data. (PDF)</p

Representative responses to individual OFF bar flashes at different positions along the PD-ND axis.

Representative responses to individual OFF bar flashes at different positions along the PD-ND axis.</p

J-RGCs receive DS synaptic inputs during motion stimulus.

(A) The responses of a J-RGC to a small square black dot moving across its RF center in 8 different directions. The average responses are summarized in the center polar plot. A diagram of the J-RGC’s dendrites is also shown to indicate the correlation between the morphological asymmetry and the preferred motion direction. Right, raster plots and PSTHs of the same J-RGC’s responses to 10 repeats of the PD and ND motion. (B) DSI values of J-RGCs’ responses to the moving spot stimulus under different luminance levels (see Methods). Dotted line: DSI = 0.3, threshold for DSGCs. n = 6/10/4 cells for optical density = 4.0/3.0/2.0 group. (C and E) Representative IPSCs (C) and EPSCs (E) recorded from a J-RGC during PD and ND motion. Traces are aligned to the estimated time when the leading edge of the moving spot entered the RF center (dotted line). Shaded area around the traces, mean ± SEM, n = 10 trials. (D and F) Comparison of IPSCs (D) and EPSCs (F) between PD and ND motion. Left, peak amplitudes; right, total charges. n = 20 cells. (G) Representative IPSCs (top) and EPSCs (middle) evoked by a spot contrast step stimulus (bottom). Shaded area around the traces, mean ± SEM, n = 7 trials. (H and I) IPSC (H) and EPSC (I) amplitudes from the ON and OFF pathways. n = 11 cells. (J) Representative IPSCs evoked by a black moving bar stimulus. Traces are aligned to the estimated time when the leading edge of the moving bar entered the RF center (dotted line). Shaded area around the traces, mean ± SEM, n = 9 trials. (K and L) Comparison of IPSC amplitudes from the responses to the leading (K) and trailing edge (L) between PD and ND motion. n = 6 cells. Error bars, SEM. In D, F, K, and L, paired t test; *, p p S1 Data. DS, direction-selective; DSGC, direction-selective ganglion cell; DSI, direction selectivity index; EPSC, excitatory postsynaptic current; IPSC, inhibitory postsynaptic current; J-RGC, J-type retinal ganglion cell; ND, null direction; PD, preferred direction; RF, receptive field.</p

J-RGCs’ direction selectivity under different drug conditions and luminance levels using the vector sum method.

(A) Polar plots of a J-RGC’s average spiking responses to motion in 8 directions under control condition and after bath application of PTX and PTX + STR. n = 10 trials. (B) Polar plots of a J-RGC’s average spiking responses to motion in 8 directions before and after bath application of DCG-IV. N = 10 trials. (C–E) Comparison of J-RGCs’ direction selectivity measured by the vector sum magnitudes of the motion responses before and after bath application of PTX (C, n = 6 cells), PTX+STR (D, n = 8 cells), and DCG-IV (E, n = 7 cells). Paired t test; **, p p (F) Vector sum magnitudes of J-RGCs’ responses to the moving spot stimulus under different luminance levels. Data from 23 randomly chosen OFF RGCs and 23 genetically defined ON-OFF DSGCs [57] are included for reference. n = 6/11/3 J-RGCs for optical density = 4.0/3.0/2.0 group. Dotted lines in C–F: vector sum magnitude = 0.12, mean + 2 × SD of the OFF RGC group. Error bars, SEM. Data for this figure are in S2 Data. (PDF)</p

Underlying data for S1–S3 and S5–S9 and S11 Figs.

Motion is an important aspect of visual information. The directions of visual motion are encoded in the retina by direction-selective ganglion cells (DSGCs). ON-OFF DSGCs and ON DSGCs co-stratify with starburst amacrine cells (SACs) in the inner plexiform layer and depend on SACs for their direction selectivity. J-type retinal ganglion cells (J-RGCs), a type of OFF DSGCs in the mouse retina, on the other hand, do not co-stratify with SACs, and how direction selectivity in J-RGCs emerges has not been understood. Here, we report that both the excitatory and inhibitory synaptic inputs to J-RGCs are direction-selective (DS), with the inhibitory inputs playing a more important role for direction selectivity. The DS inhibitory inputs come from SACs, and the functional connections between J-RGCs and SACs are spatially asymmetric. Thus, J-RGCs and SACs form functionally important synaptic contacts even though their dendritic arbors show little overlap. These findings underscore the need to look beyond the neurons’ stratification patterns in retinal circuit studies. Our results also highlight the critical role of SACs for retinal direction selectivity.</div

Underlying data for Figs 1–6.

Motion is an important aspect of visual information. The directions of visual motion are encoded in the retina by direction-selective ganglion cells (DSGCs). ON-OFF DSGCs and ON DSGCs co-stratify with starburst amacrine cells (SACs) in the inner plexiform layer and depend on SACs for their direction selectivity. J-type retinal ganglion cells (J-RGCs), a type of OFF DSGCs in the mouse retina, on the other hand, do not co-stratify with SACs, and how direction selectivity in J-RGCs emerges has not been understood. Here, we report that both the excitatory and inhibitory synaptic inputs to J-RGCs are direction-selective (DS), with the inhibitory inputs playing a more important role for direction selectivity. The DS inhibitory inputs come from SACs, and the functional connections between J-RGCs and SACs are spatially asymmetric. Thus, J-RGCs and SACs form functionally important synaptic contacts even though their dendritic arbors show little overlap. These findings underscore the need to look beyond the neurons’ stratification patterns in retinal circuit studies. Our results also highlight the critical role of SACs for retinal direction selectivity.</div

Asymmetric connections between J-RGCs and SACs.

(A) Optogenetic activation of ChR2+ SACs (left) and the representative photocurrents recorded from a SAC (right, holding potential −67 mV). (B) Optogenetic activation of ChR2+ J-RGCs (left) and the representative photocurrents (middle, holding potential −67 mV) and IPSCs (right, holding potential 0 mV) recorded from a J-RGC under the control condition (purple) or the drug cocktail condition (red). The drug cocktail contained APV, CNQX, STR, and HEX to block non-GABAergic synaptic transmissions in the retina. The same cocktail was used in all the following optogenetic experiments in C–H. (C) Representative IPSCs recorded from J-RGCs, evoked by blue LED pulse flashes with ChR2- (left) and ChR2+ (right) SACs in the retina. (D) Comparison of the peak amplitudes of IPSCs recorded from J-RGCs with ChR2− and ChR2+ SACs. Unpaired t test; **, p n = 6 cells for each group. (E) Representative optogenetically evoked IPSCs recorded in a J-RGC before and after bath application of DCG-IV. (F) Summary of optogenetically evoked IPSC peak amplitudes before and after bath application of DCG-IV. Paired t test; *, p n = 5 cells. (G) Experimental diagram for mapping the inputs from SACs (top) and representative results (bottom). The experiment maps the spatial distribution of SACs that provide inhibitory inputs to a J-RGC: Small spots of LED light pulse were positioned along the J-RGC’s PD-ND axis at different distances from its soma and evoked IPSCs recorded in the J-RGC. The spots are represented by their center locations and the distribution of light intensity within each spot (approximated by the cyan gaussian curves, the solid curve indicating the spot that evoked the strongest IPSCs). The full size of the J-RGC’s dendritic field along the PD-ND axis is shown on top for comparison. Example IPSC traces evoked by the LED spots at several locations are shown to highlight the spatial asymmetry around the soma. (H) Summary of the input strength from SACs along J-RGCs’ PD-ND axis. The normalized peak amplitudes of IPSCs are plotted against the locations of the spot centers. Distance > 0: on the dendrite side; distance t test; **, p p n = 5/5/4/5/6/5/5/6/5 cells at distances −200/−100/−50/−25/0/25/50/100/200 μm. (I) The measured input strength from SACs along J-RGCs’ PD-ND axis (magenta, same as in H) and the spatial distribution of relative intensity for the LED spot used for the measurement (cyan). (J) Hotspots (black arrows) of SAC inputs revealed by deconvolution using the inputs in I. (K) Schematic diagram to illustrate the results in J. The 2 black arrows indicate the locations of J-SAC synapses and the upstream SAC soma. LED light at these locations can elicit IPSCs in J-RGCs much more efficiently than that at other locations. S1/S2, sub-lamina 1/2 of the IPL. Error bars, SEM. In A–C, E, and G, shaded area around the traces, mean ± SEM, n = 10 trials. Data for this figure are in S1 Data. GABA, gamma-aminobutyric acid; HEX, hexamethonium; IPSC, inhibitory postsynaptic current; J-RGC, J-type retinal ganglion cell; SAC, starburst amacrine cell; STR, strychnine.</p