Distinct temporal spiking response properties of ON-transient versus ON-sustained RGCs. (A) Maximum intensity projection z-stack fluorescence images of ON-T (left, cyan) and ON-S (right, magenta) RGCs dialyzed with Alexa Fluor 594 during whole-cell patch-clamp recording. Scale bar 50 µm. (B) Characteristic spiking responses to light step increment of example ON-T and ON-S RGCs. (C) Example spiking responses to repeated trials of spatially uniform (300 µm spot diameter) Gaussian noise stimulus (top black trace) presented with a mean luminance of ∼2400 P*/Scone/s (∼3000 R*/rod/s). ON-T (cyan) and ON-S (magenta) RGCs were recorded from sequentially in the same piece of retina. PSTHs (bottom) calculated from 20 repetitions of noise stimulus. (D) ON-T and ON-S STAs derived from responses to non-repeated noise stimuli. Colored lines and shaded areas show mean ± SEM STA waveforms from ON-T (n= 15) and ON-S (n= 13) RGCs. Note time axis is reversed to facilitate comparison with linear filters in Figures 2 and 4. (E) Quantification of STA kinetics in different RGCs. Open circles show measurements from individual cells and filled circles with error bars show mean ± SEM values. Significance values from Wilcoxan rank sum tests.

Distinct excitatory synaptic input to ON-T and ON-S RGCs. (A) Excitatory synaptic currents measured in example ON-T (cyan) and ON-S (pink) RGCs in response to same Gaussian noise stimulus as in Fig. 1. Traces show average currents from ten repetitions of the same stimulus and are baseline subtracted to facilitate comparison. (B) Linear filters computed from responses to non-repeated noise stimulus presentation. Lines and shaded areas show mean ± SEM of responses from ON-T (n= 13) and ON-S (n= 15) RGCs. (C) Quantification of filter kinetics in different RGCs. (D) Excitatory currents measured from example ON-T and ON-S RGCs in response to a 0.5 second step increase in light intensity (50% contrast; mean response to five repetitions of stimulus). (E) Quantification of step response kinetics (n= 19 ON-T RGCs; n= 13 ON-S RGCs). In (C) and (E) open circles show measurements from individual cells and filled circles with error bars show mean ± SEM values. Significance values from Wilcoxan rank sum tests.

Identification of bipolar cell subtypes presynaptic to ON-T RGCs. (A) Maximum-projections z-stacks of two dye-filled ON-T RGCs (green) in wholemount retina from Gjd2-EGFP (red) transgenic mice. (B) Traced skeletons of dendritic arbors of the two ON-T RGCs from EM volume. (C) An EM micrograph showing a ribbon synapse (arrowhead) on a BC axonal terminal onto the dendrite of a ON-T RGC. (D) An example skeleton for each presynaptic BC type (types 5i, 5t, 5o, X, 6 and 7) of ON-T RGCs. Top: whole-mount view, Bottom: side view. (E) Bar plot showing the proportion of input synapses to ON-T RGCs from each BC type. Mean ± SEM (n=3 RGCs, 2 from this study and one from Ding et al. (2016)).

Bipolar cell subtypes presynaptic to ON-T and ON-S RGCs have indistinguishable temporal response properties. (A) Maximum-projection z-stacks of fluorescent dye-filled bipolar cells targeted for electrophysiological recording in retinal slices from Gjd2-EGFP (left), Grm6-tdTomato (middle), and Gus8.4-EGFP (right) transgenic mice. (B) Responses to spatially uniform Gaussian noise stimulation (same as in Figs. 1-2) in example type 5i (cyan), type 6 (magenta) and type 7 (green) bipolar cells. Voltage traces are average responses to ten repetitions of the noise stimulus. (C) Linear filters computed from responses to non-repeated noise stimulus presentation. Lines and shaded areas show mean ± SEM of responses (type 5i, n= 11; type 6, n= 7; type 7, n= 6). (D) Quantification of response kinetics from linear filters. No significant differences across types (p = 0.19 zero-cross times; p= 0.89 biphasic index; Kruskal-Wallis test). (E) Responses to step light increment. Lines and shaded areas show peak-normalized mean ± SEM responses across type 5i bipolar cells (n= 8) and type 6 bipolar cells (n= 5). (F) Quantification of step response kinetics. Time to peak and steady-state response amplitude were not different between type 5i and type 6 bipolar cells (p= 0.72 and p= 0.35, respectively; Wilcoxan rank sum test).

Presynaptic inhibition does not establish kinetic differences between bipolar cell inputs to ON-T versus ON-S RGCs. (A) Example EM micrograph showing a ribbon dyad (red arrowhead) between a BC and an ON-T RGC and an amacrine cell, with an adjacent feedback inhibitory synapse (yellow asterisk). (B) Bar plots showing the percentage of RGC/AC ribbon dyads with feedback inhibition. Mean ± SEM (n=2 ON-S RGCs, 2 ON-T RGCs) (C) Excitatory currents measured in response to light step increment in control conditions (cyan) and in the presence of GABA receptor antagonists (10 µM SR95531 and 50 µM TPMPA; dashed gray) in an example ON-T RGC. Traces are mean response from six repeats of stimulus presentation. (D) Summary of steady-state amplitudes measured from step responses in control or with GABA receptor antagonists. No significant difference between conditions (p= 0.063; Wilcoxan signed rank test) (E) Control excitatory currents (cyan) or with addition of strychnine (1 µM; dashed gray) to bath in an example ON-T RGC. (F) Summary of strychnine experiments. No significant difference between conditions (p= 0.438; Wilcoxan signed rank test). In (D) and (F), open circles with lines show measurements from individual ON-T RGCs. Filled circles with error bars show mean ± SEM.

Distinct glutamate input kinetics at ON-S vs ON-T RGC dendrites. (A) ON-S RGC dendrites expressing iGluSnFR in a KCNG-Cre mouse were filled with sulforhodamine 101 (left) after measuring their spiking response to a 200 µm spot extracellularly (Right). Yellow bands indicate stimulus duration. (B) iGluSnFR expressing ON-S and ON-T dendrites at different depths of the inner-plexiform layer (Left) were imaged using two-photon microscopy. Example iGluSnFR signals extracted from small regions of interest (ROIs 1-8) from ON-S and ON-T RGC dendrites illustrate the kinetic differences in their respective inputs. Black, mean responses; gray, individual trials. (C) Distribution of steady-state response amplitudes of the individual (gray) and average (black) ROIs from different recordings done from ON-S and ON-T dendritic field of views evoked by a 200 µm static spot (n = 7 FOVs, 6 retinas for ON-S and 5 FOVs, 5 retinas for ON-T RGC dendrites, *p<0.001; t-test).

Stronger flash-evoked suppression of excitatory currents in ON-T versus ON-S RGCs. (A) Paired flash-evoked excitatory currents measured at different inter-flash intervals (50, 100, 200, 400 ms) in an example ON-T (top; cyan) and an ON-S (middle, magenta) RGC. Bottom black traces show voltage responses to same stimulus measured in an example type 5i bipolar cell. Arrows at top indicate timing of first flash (black) and second flashes (gray). Flashes were 400% contrast and 10 ms duration. Traces show mean responses to five repeats of each paired flash interval. (B) Filled circles with error bars show mean ± SEM paired-flash ratios at different inter-flash intervals across all ON-T RGCs (cyan; n= 6), ON-S RGCs (magenta; n= 5) and type 5i bipolar cells (black; n= 6).

Bipolar cell subtype-specific differences in ribbon size. (A) 3D reconstructions showing segmented axon stalk and axonal terminal (gray) and all the synaptic ribbons within the axonal terminal a type 5i (cyan) and a type 6 (magenta) BC. (B) Violin plot showing distribution of ribbon volumes (each data point is a ribbon) for the type 5i and type 6 BC in (A). The boxes indicate the interquartile range (thick lines, 25%-75%), median value (white dot), and 1.5 times interquartile range (thin line). Shaded gray areas represent rotated kernel density plots. (C) 3D reconstructions showing segmented dendritic arbor (gray) and all the synaptic ribbons presynaptic to these segments of ON-T (cyan) or ON-S (magenta) RGC dendrites. (D) Violin plot with scattered dots showing ribbon volumes for each ribbon presynaptic to a stretch of reconstructed ON-T or ON-S RGC dendrite. Each ribbon (dot) is colored differently for each BC type. (E) An EM micrograph (left) and 3D reconstruction (right) of a ribbon and its adjacent vesicles. The ribbon is indicated by the red arrow in the EM image. In the 3D reconstruction, yellow dots indicate the adjacent vesicles surrounding the presynaptic ribbon (red) in the BC terminals (gray). Postsynaptic ON-T RGC is in blue. (F) Scatter plot showing ribbon size in volume in relationship with the number of adjacent vesicles. Gray line represents the linear regression model fitted to the data. Linear regression: β = 6.98e−06, p < 0.001, r2 = 0.66.

Noise-evoked responses are biased towards large amplitude events in ON-T compared to ON-S RGCs. (A) Spike event peak spike rate cumulative distributions for ON-T (magenta; n= 6 cells; 131 spike events) and ON-S RGC (cyan; n= 7 cells; 300 spike events). Firing rates from average responses to a repeated noise stimulus (see Fig. 1B-C) were normalized to the maximal firing rate in each cell before combining events across cells. (B) EPSC amplitude cumulative distributions for ON-T (n= 13 cells; 275 EPSCs) and ON-S (n= 10 cells; 365 EPSCs) RGCs. Same stimulus and normalization procedure as in (A).

Linear-nonlinear models of excitatory synaptic input to ON-T and ON-S RGCs. (A) Linear filters computed from responses to non-repeated noise stimulus presentation. Lines show filters from individual ON-T (cyan; n= 13) and ON-S (magenta; n= 15) RGCs. Filters were normalized to the peak amplitude of the primary inward component to facilitate visual comparison of temporal responses. Same cells as shown in average filters in Fig. 2B. (B) Nonlinearities derived by plotting the predicted response (convolution of linear filter and stimulus) against the measured excitatory currents for each RGC shown in (A). Linear predictions were calculated using non-normalized filters.

Characterization of Gjd2-EGFP+ bipolar cells. (A) Vertical section of the Gjd2-EGFP mouse line showing EGFP positive bipolar cells axons (green) stratifying in the ON sublamina of the IPL, co-labeled with cholinergic amacrine cell (magenta, ChAT antibody). Transmitted light image of the retina in blue. (B) Vertical section of the Gjd2-EGFP mouse line showing EGFP positive bipolar cells (green) colocalized with the T5 marker CaBP5 (magenta). (C) En-face image of a whole mount retina from the GJD2 mouse line showing EGFP positive bipolar cells bodies (green) co-labeled with the T5 bipolar cell marker CaBP5 (magenta). (D) Same field as in (C) with cell bodies of EGFP positive bipolar cells outlined (green) by application of a digital edge outlining filter in ImageJ. (E) Position of cell body centroids for EGFP positive bipolar cells in a larger field of view (20X objective) outlined by green circles. (F) Density recovery profile analysis showing the density profile as a function of distance from the reference neuron for EGFP positive bipolar cells (green histograms) and for an equal amount of neurons randomly positioned in the same space (grey histograms) showing the presence of a characteristic exclusion distance for EGFP positive bipolar cells, hallmark of a retinal mosaic.

ON-T and ON-S RGC dendrites ramify in distinct IPL sublaminae. (A) Side projections from 2P z-stack images of dye-filled RGCs (ON-T: top, cyan; ON-S: bottom, magenta) and EGFP signal (yellow) in retinas from Gjd2-EGFP mice. RGCs are same cells as shown in en face view in Fig. 1A (B) Quantification of fluorescence signals from dye filled RGCs in Gjd2-EGFP mouse retina as in (A). Colored lines and shaded areas show mean ± SEM fluorescence intensity with respect to distance from peak EGFP (yellow) fluorescence intensity (n= 6 ON-T, cyan and n= 4 ON-S, magenta, RGCs).

Gjd2-EGFP+ type 5 bipolar cells provide ∼40% of total synapses onto ON-T RGCs. (A) An example ON-T RGC biolistically labeled by Cerulean Fluorescent Protein (blue) and PSD95-mCherry (green) in retina from a Gjd2-EGFP (red) transgenic mouse. Top: en face view, Bottom: side views. (B) Top: Dot map of PSD95-mCherry apposed (magenta) or not apposed (green) to GFP+ BC axon terminals. Bottom: Magnified view of the raw images and the dot map for a segment of dendritic arbor in the while box. (C) Quantification of total excitatory synapse density across the dendritic arbor of ON-T RGCs. (D) Quantification of excitatory synapses in ON-T dendrites that are adjacent to GFP+ axon terminals.

Classification of three subtypes of type 5 bipolar cells. (A) Wholemount views of the axon terminals of type 5i, type 5t and type 5o bipolar cells in the SBFSEM volume in Fig. 3. The tiling of the space validates the categorization of the three subtypes of type 5 bipolar cells. (B) Identification of Gjd2-EGFP+ bipolar cells as type 5i bipolar cells, supported by correlated light (left) and electron (right) microscopy images. The reconstructed skeleton of the type 5i bipolar cell in (B) is delineated by dashed lines in (A).

Linear-nonlinear models of bipolar cell voltage responses. (A) Individual linear filters for all type 5i (cyan; n= 11), type 6 (magenta; n= 7), and type 7 (green; n=6) bipolar cells computed from responses to non-repeated noise stimulus presentation. Filters were normalized to the peak amplitude of the primary inward component to facilitate visual comparison of temporal responses. Same cells as shown in average filters in Fig. 4B. (B) Nonlinearities derived by plotting the predicted response (convolution of linear filter and stimulus) against the measured voltage response for each bipolar cell shown in (A). Linear predictions were calculated using non-normalized filters.

Presynaptic inhibition does not affect response kinetics but does affect spatial integration for excitatory inputs to ON-T RGCs (A) Step-evoked (100% contrast, 300 µm spot diameter, 500 ms duration) excitatory synaptic currents in example ON-T (cyan) and ON-S RGCs measured in ACSF. Traces show mean responses to ten trials and are baseline subtracted to facilitate comparison. (B) Summary of light step-evoked steady-state response amplitude for different stimulus sizes measured in ACSF. Note that excitatory inputs in ON-T are more transient than those in ON-S RGCs across all stimulus sizes, but responses in both RGC types are more sustained than when measurements were performed using Ames’ solution (compare to Fig. 2D-E). (C) Summary of total response, measured as the integrated excitatory current response during the light step, versus stimulus spot size. Responses were normalized to the maximal response in each cell prior to averaging. Excitation in ON-T RGCs is tuned to smaller spot sizes than in ON-S RGCs and this tuning relies in part on GABAergic inhibition. Symbols with error bars in (B) and (C) show mean ± SEM (ON-T control n = 12; ON-S control n = 6; ON-T GABA-R block n = 5; ON-T Gly-R block n = 5).