An offset ON–OFF receptive field is created by gap junctions between distinct types of retinal ganglion cells

Abstract

In the vertebrate retina, the location of a neuron’s receptive field in visual space closely corresponds to the physical location of synaptic input onto its dendrites, a relationship called the retinotopic map. We report the discovery of a systematic spatial offset between the ON and OFF receptive subfields in F-mini-ON retinal ganglion cells (RGCs). Surprisingly, this property does not come from spatially offset ON and OFF layer dendrites, but instead arises from a network of electrical synapses via gap junctions to RGCs of a different type, the F-mini-OFF. We show that the asymmetric morphology and connectivity of these RGCs can explain their receptive field offset, and we use a multicell model to explore the effects of receptive field offset on the precision of edge-location representation in a population. This RGC network forms a new electrical channel combining the ON and OFF feedforward pathways within the output layer of the retina.

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Fig. 1: F-mini-ON and F-mini-OFF RGCs have both ON and OFF light responses.
Fig. 2: RF ON and OFF subfields measured by flashed spots are spatially offset.
Fig. 3: Alignment between ON and OFF strata of bistratified RGCs.
Fig. 4: Heterotypic gap junctions among F-mini RGCs are confirmed by immunohistochemistry.
Fig. 5: F-mini-ON and F-mini-OFF RGCs are electrically coupled to each other by gap junctions.
Fig. 6: F-mini-ON RGCs receive ON input from chemical synapses and OFF input from electrical synapses.
Fig. 7: F-mini-ON RGCs RF offset is captured by a morphological model.
Fig. 8: Multicell model of object localization shows an advantage of offset ON–OFF RFs.

Data availability

Data for ganglion cell typology in the mouse is available at http://RGCTypes.org/. A subset of the datasets that support the findings of this study are available at https://github.com/SchwartzNU/ProjectData_Fmini. The remainder of the datasets are available from the corresponding author upon reasonable request.

Code availability

Software code for the analyses supporting the findings of this work is available at https://github.com/SchwartzNU/ProjectData_Fmini. Visual and electrical stimulus code is available at https://github.com/Schwartz-AlaLaurila-Labs/sa-labs-extension and https://symphony-das.github.io.

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Acknowledgements

We thank the entire Schwartz Laboratory group for discussions, advice and support. We thank B. Novich for generously providing the FOXP1 antibody. Imaging work was performed at the Northwestern University Center for Advanced Microscopy, generously supported by a National Cancer Institute cancer center support grant (P30 CA060553) awarded to the Robert H. Lurie Comprehensive Cancer Center. Multiphoton microscopy was performed on a Nikon A1R multiphoton microscope, acquired through the support of the National Institutes of Health (NIH; 1S10OD010398-01). This work was supported by grants from the NIH National Eye Institute (F31 EY029593 and T32 EY025202) and an NIH Director’s New Innovator (DP2) award (EY026770).

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Authors

Contributions

S.C. and G.W.S performed the experiments. S.C. analyzed data and constructed models. S.C. and G.W.S. designed research and wrote the paper.

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Correspondence to Gregory W. Schwartz.

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The authors declare no competing interests.

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Peer review information Nature Neuroscience thanks Bart Borghuis, Stuart Trenholm, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Coupled cells are immunoreactive for F-mini RGC markers.

Images of the ganglion cell layer in a patch of retina in which a single F-mini-ON RGC was filled with Neurobiotin (magenta arrowhead). Left panel shows the Neurobiotin channel, with three brightly labelled coupled cells (white arrowheads) and three dimly labelled cells that likely represent second-order connections (magenta asterisks). Middle panel shows the same region with immunoreactivity for FOXP1, which labels F-mini-OFF RGCs, but does not label F-mini-ON RGCs15. Right panel shows immunoreactivity for FOXP2, which labels both F-mini RGC types. This experiment was performed on five F-mini RGC networks in four retinas: four F-mini-ON RGCs and one F-mini-OFF RGC injected. Three networks were stained for FOXP2 and FOXP1; two networks for FoOXP2 only. Neurobiotin labeled 9.0 ± 6.4 somas per retina, and was found in varying amounts in neurons; indicating first and second order connectivity. FOXP2 was present in 43 of 45 RGCs that were labeled with Neurobiotin. Coupled cells from these networks that could be morphologically identified by using the visible primary dendrites, and all showed the expected patterns of FoxP1 expression. 8/8 F-mini-ON RGCs were FOXP1 negative and 14/14 F-mini-OFF RGCs were FOXP1 positive.

Extended Data Fig. 2 Example RF maps from F-mini-ON and F-mini-OFF RGCs.

Receptive field maps of peak response to 40 μm flashed spots over the RF area, averaged over 2 or 3 repeats. a, A GJ coupled F-mini-ON and F-mini-OFF recorded simultaneously. b, Another such RGC pair. c, Two unconnected F-mini-ON RGCs. d, Two unconnected F-mini-OFF RGCs. On all plots, the cross markers are at the center of mass of responses over the 80th percentile (ON, white; OFF, black). Color scale is in mV change from baseline. All scale bars are 100 µm.

Extended Data Fig. 3 Alignment between ON and OFF strata of bistratified RGCs.

Offset values in µm from each bistratified RGC in Eyewire by type, followed by Eyewire anatomical type name in parentheses. Offsets are measured as a vector from proximal/inner COM to distal/outer COM, which in most RGCs is ON to OFF dendrites. Mean and SD of offsets are shown by red crosses. All figure data is from the Eyewire dataset8, exported via the Eyewire Museum mesh tool. Meshes were flattened and offset computationally with parameters fit by eye to maximize flatness.

Extended Data Fig. 4 Immunohistochemistry for three types of Connexin at RGC contact points shows negative results.

Three connexins were evaluated for presence at the regions of contact between an F-mini-ON and multiple F-mini-OFF RGCs, n = 1 of each experiment. a,b, Full depth maximum intensity projection images of a Neurobiotin-filled F-mini-ON RGC (magenta),the connected F-mini-OFF RGCs (cyan), and a cell of unclassified type due to insufficiently filled dendrites (yellow). Tracing, segmentation, and masking were performed manually. Image brightness was scaled separately by cell type for illustration here but not for analysis. c,d Thin projection images of regions in orange squares in a,b showing an example RGC crossing point with yellow square for spatial reference. Stack depth is 3.5 µm. e-g, The same region and depth as in c,d, showing the IHC channels for the three connexin proteins. h, Quantification of overlap between connexin images and RGC contact region masks. Values are similar before and after a 90 degree rotation of the connexin image. Points mark the overlap of the single F-mini-ON RGC with each F-mini-OFF RGC in the image.

Extended Data Fig. 5 Noise correlations between F-mini-ON and F-mini-OFF RGCs.

a, Traces from a simultaneously recorded pair of F-mini-ON (magenta) and F-mini-OFF (cyan) RGCs in current clamp in darkness (no stimulus). b-e. Example cross correlation of the simultaneous voltage from the cells in a. Brown trace is for shuffled trials. Shaded regions are SEM across trials. Time shift is F-mini-ON - F-mini-OFF (positive values are F-mini-ON earlier). b, Results in darkness. c, Results in darkness in the presence of MFA. d, Results under randomly moving object light stimulation. e, Results under the same light stimulation in the presence of MFA. f, Population data showing peak cross-correlation in control and in MFA. Values in MFA are significantly lower than corresponding values in control (n = 4 cell pairs, p = 0.0068, paired-sample one-tailed t-test). g, Full width at half max and h, time shift (right) of cross correlation peak in control conditions. Error bars in f-h are SEM across cell pairs and points are each cell pair. i, Relationship between cross-correlation peak and coupling coefficient in darkness measured from current injections as in Fig. 2e-h. Box plots in f,g,h show maximum, 75th percentile, median, 25th percentile, and minimum.

Extended Data Fig. 6 MFA does not selectively eliminate OFF responses in non-F-mini RGCs.

a, Example of an ON-OFF direction selective RGC responding to the onset and offset of a dark spot from a mean luminance of 2000 R*/rod/s in control conditions (black) and in MFA (green). b, Population data of spike counts and c, subthreshold potential responses to an OFF light step as in a for 3 ON-OFF DS RGCS. Baseline voltage level shift mean in control RGCs was −59.9 to −61.8 mV (n = 3 cells). Box plots in b,c show maximum, 75th percentile, median, 25th percentile, and minimum.

Extended Data Fig. 7 A single cell model generates responses similar to those observed in F-mini-ON RGCs.

a, Diagram of single cell receptive field offset model showing the parameters for each of four RGC input component pathways. b, Responses of the model to flashed spots of varying sizes showing a qualitative match of surround properties to F-mini-ON RGCs as seen in Extended Data Fig. 2a. c, Measured direction selectivity mean in F-mini-ON and ON-OFF DS RGCs, varying over speed (error bars are SD). Individual F-mini-ON RGCs are shown in gray (n = 103 F-mini-ON and n = 279 ON-OFF DS). d, Model response DSI over object speed showing similar DSI magnitude and low-speed preference properties to measured responses. e, (upper) Orientation selectivity of the population of F-mini-ON RGCs. Dashed lines are published means for OS and control RGCs28,29. (lower) Distribution of OS preference angle. g, Moving bar DS preference angle distribution across retina space of F-mini-ON RGCs. Blue = left eye, green = right eye. D,V,N,T denote dorsal, ventral, nasal, and temporal, respectively.

Extended Data Fig. 8 Multi-cell model results are robust over several parameters.

a, Illustration of the difference of gaussians RF map used in the single cell model, with an ellipse at the central 2σ contour. b, Diagram of RF offset and scaling properties in the model: the diameter (D) and the offset ratio (F) between ON (magenta) and OFF (cyan) sub-fields. c, Heatmap of vertical position error (for horizontally oriented stimuli) across models with a range of RF size (D) and RF offset ratio (F). Black and magenta points are the parameters used in the following panels and those in Fig. 5d–f. d, Absolute error, e, vertical error change ratio, and f, horizontal error change ratio for the three RF models across a range of cell density. g, Absolute error, h, vertical error change, and i, horizontal error change ratio for the three RF models across a range of noise values.

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Cooler, S., Schwartz, G.W. An offset ON–OFF receptive field is created by gap junctions between distinct types of retinal ganglion cells. Nat Neurosci 24, 105–115 (2021). https://doi.org/10.1038/s41593-020-00747-8

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