Overview of the experimental setup and recording procedure.

(a) Two-photon imaging of ganglion cell layer (GCL) somata in the whole-mounted ex vivo mouse retina. (b) Schematic ex vivo whole-mounted retina (dot marks optic disc; d, dorsal; t, temporal; v, ventral; n, nasal). (c) Two representative recording fields from (b) showing GCL somata loaded with Ca2+ indicator OGB-1 (Methods). (d) Representative Ca2+ activity from cells in the GCL (white circles in (c)) in response to chirp (left) and moving bar stimulus (right) (black, mean; gray, s.d.). (e) Timeline of experimental procedure; for details, see text. IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OS+IS, outer and inner segments; DETA/NO, nitric oxide (NO) donor.

Functional classification of mouse RGC types.

(a) Illustration of the random forest classifier (RFC) to predict cell type labels for Ctrl 1 of both datasets. For each cell, Ca2+ responses to chirp and moving bar, soma size, and p-value of permutation test for direction selectivity (left) constitute the input to the RFC (center) to predict a cell type label, i.e., a type GX (right). For details, see Methods and (80). (b) Pooled heat map of unsorted cell responses to chirp and moving bar stimulus from both Ctrl 1 datasets after quality filtering (QIMB>0.6 or QIchirp>0.45, and classifier confidence score ≥ 0.25). The color bar indicates normalized response activity. (c) Heat map from (b), but sorted according to their assigned type. (d) Distribution of RGC types predicted by the RFC classifier for both Ctrl 1 of the control(Ctrl 1 (Ctrl); solid black), of DETA/NO-dataset (Ctrl 1 (NO); dotted black), and for the dataset from Baden et al. (5) (red). (e) Representative RGC type response averages to chirp and moving bar (Ctrl, black; training dataset, red). (f) Correlation matrix of type mean responses per RGC type between Ctrl and Baden et al. (5) dataset for chirp (left) and moving bar (right). Dashed boxes indicate functional groups (Off, On-Off, Fast On, Slow On, and Uncertain RGCs; see (5)). The color bar indicates the Pearson correlation coefficient.

Certain RGC types are affected by adaptational and/or NO-induced effects, while others are unaffected.

(a) Left: Two representative mean Ca2+ responses of sequentially recorded RGC types showing no differences between Ctrl 1 (black) and Ctrl 2 (orange) (top: G1; bottom: G21). Right: Corresponding histograms displaying the differences between the average traces of the sequentially recorded cell of the respective cell types. Zero indicates no difference between the response of the same cell across both recordings, whereas negative values indicate a decreased and positive values an increased activity. n.s.: not significant; ***: p<0.001; One-sample T-test. (b) Two representative RGC types that show decreased (top: G23) and increased (bottom: G31) response activity during Ctrl 2. n.s.: not significant; ***: p<0.001; One-sample T-test. (c) As in (a), but between sequentially recorded Ctrl 1 (black) and DETA/NO (green) (top: G2; bottom: G17). n.s.: not significant; ***: p<0.001; One-sample T-test. (d) As (c), but showing two cell types that display a decreased (top: G28) and increased (bottom: G32) activity when perfused with DETA/NO. n.s.: not significant; ***: p<0.001; One-sample T-test. (e) Box plots of trace differences of all sequentially recorded cells of all RGC types from control(ΔCtrl: ΔRCtrl2-Ctrl1; orange) and NO-dataset (ΔDETA/NO: ΔRNO-Ctrl1; green). Bold numbers indicate RGC types with >10 sequentially recorded cells per dataset and condition. Dashed line shows zero baselines, i.e., no difference between traces. Diamond symbols represent outliers. Gray and white background blocks summarize the larger functional groups for better visualization (Off, On-Off, Fast On, Slow On, Uncertain RGCs). ***: p<0.001; Mann-Whitney U-Test.

Disentangling NO-induced effects from adaptational response changes reveals type-specific NO modulation.

(a) Left: Difference between sequentially recorded Ctrl 2 and Ctrl 1 RGC traces per type subdivided into 8 features (ΔCtrl: ΔRCtrl2-Ctrl1). Color code indicates response increase (red), no change (white), and decrease (blue) for Ctrl 2. Middle: Difference between DETA/NO and Ctrl (ΔDETA/NO: ΔRNO-Ctrl1). Right: Difference between the two heatmaps (ΔRNO-Ctrl1 ΔRCtrl2-Ctrl1). Asterisks indicate significant differences of the trace differences of all cells per feature and per cell type between ΔCtrl and ΔDETA/NO using independent two-sided T-test and Bonferroni correction for multiple tests; *: p<0.0003. (b) Example chirp traces are categorized into unaffected (top two types: G1, G12), adaptational (two middle types: G22, G24), and NO-affected (bottom: G32). Left traces show exemplary responses per type from the control-dataset (black: Ctrl; orange: Ctrl 2) and NO-dataset (black: Ctrl; green: DETA/NO). (c) Subdividing the chirp (left) and moving bar (right) stimuli into 8 features for detailed feature analysis. The chirp is subdivided into 6 features ((1) on, (2) off, (3) low frequency, (4) high frequency, (5) low contrast, and (6) high contrast); the moving bar into 2 ((7) on and (8) off).

Functional clustering of the G32 reveals three distinct types that are differently affected by NO.

(a) Visual responses of G32 cells recorded from several experiments in response to the full-field chirp (left) and moving bar (right) stimuli. (b) Visual features extracted from chirp (top) and moving bar (bottom) stimuli using sparse PCA on the responses. Color indicates the weight of each feature. (c) Top: Bayesian Information Criterion (BIC) as function of number of clusters. Arrow indicates the lowest BIC and the number of clusters to choose. Bottom: Number of cells per predicted cluster. (d) Cells sorted according to their assigned cluster. Cells at the bottom were discarded. (e) Mean responses of the 3 corresponding clusters for the chirp (left) and moving bar (right). (f) Left: Sequentially recorded mean responses of the 3 clusters to Ctrl 1 (black) and Ctrl 2 (orange). Right: Cluster mean responses to Ctrl 1 (black) and DETA/NO (green) (g) Left: Trace difference between Ctrl 2 & Ctrl 1 (orange) and DETA/NO & Ctrl (green) for the 3 clusters (clusters 1-3 from top to bottom). Right: Correlation coefficient between Ctrl 2 & Ctrl 1 (orange) and DETA/NO & Ctrl 1 (green) for the 3 clusters. n.s.: not significant; **: p<0.01, ***: p<0.001; independent T-test & Mann-Whitney U-Test. (h) Left: Suppression index (SI) computed for Ctrl 1 (gray), Ctrl 2 (orange), and DETA/NO (green) for the 3 clusters. n.s.: not significant; **: p<0.01, ***: p<0.001; Kruskal-Wallis test & Dunnett’s test. Right: Difference of SI between Ctrl 2 & Ctrl 1 (orange) and DETA/NO & Ctrl 1 (green). n.s.: not significant; **: p<0.01, ***: p<0.001; independent T-test & Mann-Whitney U-Test.

Spatial receptive fields are not affected by NO across various RGC types.

(a) Representative estimated spatial receptive fields (sRF) of six RGC types. Top: Estimated sRFs to Ctrl 1. Cross indicates RF center; solid line indicates outline of the Gaussian fit of the RF center; dashed outline indicates corresponding Gaussian fit of the same cell to DETA/NO. Bottom: Same as top, but of DETA/NO condition. (b) RF diameter difference in percentage between DETA/NO and Ctrl 1. Only types with more than 5 sequentially recorded cells were included. One-sample T-test. (c) Surround index difference between DETA/NO and Ctrl 1. Only types with more than 5 sequentially recorded cells were included. One-sample T-test.

Multi-electrode recordings and pseudo-calcium transformation.

(a) Representative RGC type, G6 ‘(On-)Off ‘JAM-B’ mix, recorded from one retina forming a mosaic. Three exemplary response traces of the same type. Cells are indicated in the mosaic as blue. Left: tSTAs and sSTAs computed from their responses to a checkerboard stimulus. Right: PSTHs to the chirp stimulus. (b) As (a)), but displaying another type, G10 ‘On-Off local edge’. (c) Illustration of the transformation of PSTHs to pseudo-calcium traces using an OGB-1 filter described in Baden et al. (5) to match and identify RGC types. (d) Overlay of representative mean responses of three RGC types of the Baden et al. (5) dataset (red) and the assigned Ca2+-transformed (black) traces to the chirp stimulus. (e) Inter-cluster correlation matrix of PSTHs of each assigned RGC type within a group with its group mean. Only types with n>5 cells per type were included. Dashed boxes indicate functional groups (Off, On-Off, Slow On, and Uncertain RGCs; see (5)). Color bar indicates Pearson correlation coefficient. (f) Comparison of the distribution of predicted RGC types between the MEA(blue) and Ca2+-datasets (black).

NO only affects temporal features of three distinct clusters of G32.

(a) Mean Ca2+-transform responses to the chirp of the RGC type G32. Top: Sequentially recorded RGC responses to the Ctrl (black) and DETA/NO (green) conditions. Bottom: Sequentially recorded RGC responses to the Ctrl (black) and Wash-out (orange) conditions after DETA/NO application. (b) Left: Unclustered PSTHs of cells assigned to type G32. Right: Cell’s PSTHs were clustered and sorted into three distinct clusters. (c) Sequentially recorded mean responses of 3 clusters to Ctrl (black), DETA/NO (green), and Wash-out (orange). (d) Ellipse size of the fitted Gaussian of the sRF of the three conditions (Ctrl: black; DETA/NO: green; Wash-out: orange) for the 3 clusters (clusters i -iii; top to bottom). All tested conditions were not significant; Two-sided T-test. (e) Left: tRF kernels of the three conditions (Ctrl: black; DETA/NO: green; Wash-out: orange) for 3 clusters (clusters i -iii; top to bottom). Right: Full width at half minimum (FWHM) of the temporal RF kernels of the three conditions (Ctrl: black; DETA/NO: green; Wash-out: orange) for the 3 clusters. *: p<0.05; **: p<0.01, repeated measures ANOVA & Dunnett’s test.