Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Jan;24(1):105-115.
doi: 10.1038/s41593-020-00747-8. Epub 2020 Nov 23.

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

Sam Cooler  1   2 , Gregory W Schwartz  3   4
Affiliations

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

Sam Cooler et al. Nat Neurosci. 2021 Jan.

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.

PubMed Disclaimer

Conflict of interest statement

Competing interests

The authors declare no competing interests.

Figures

Extended Data Fig. 1
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 RGCs. 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
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
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 dataset, 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
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
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
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
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 Figure 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 RGCs,. (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
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.
Figure 1
Figure 1. F-mini-ON and F-mini-OFF RGCs have both ON and OFF light responses.
a, b, Images of F-mini-ON and F-mini-OFF RGCs from fixed tissue. Magenta and cyan color scheme for RGC types is consistent throughout. All cell images throughout are shown with the dorsal direction on the retina toward the top of the page. Targeted RGCs were traced and colored, overlaid on gray. Morphology was consistent in all images (n = 40, 20 cells) c, Stratification profiles of an F-mini-ON and an F-mini-OFF RGC from our cell fills (colored lines) and from the data in the Eyewire museum (black). Dashed lines indicate ON and OFF choline acetyltransferase (ChAT) bands. Shading shows the stratification regions of ON (yellow) and OFF (grey) bipolar cells (BCs) in the inner plexiform layer. d, F-mini-ON RGC in current clamp responding to the onset and offset of a positive contrast spot from darkness to 200 isomerizations R*/rod/s. e, F-mini-ON RGC responding to the leading and trailing edge of a positive contrast moving bar (140 μm x 500 μm, 1000 μm/s, 200 R*/rod/s). f, g, Same as d, e for an F-mini-OFF RGC. h, Spike counts in F-mini-ON RGCs responding to positive contrast spots of varying diameters. Mean onset responses in yellow; mean offset responses in black;shaded region is SEM (n = 172 cells). i, Same as h for F-mini-OFF RGCs (n = 85 cells). j, Spiking responses of F-mini-ON RGCs to a flashed spot at varying mean luminance, showing the variations in ON and OFF responses with light level (n = 3 cells, spot diameter = 130 μm). k, Spiking responses of F-mini-ON RGCs to onset of flashed spots of varying contrast from the background luminance, mean with shadedSD across cells (n = 8 cells).
Figure 2
Figure 2. RF ON and OFF subfields measured by flashed spots are spatially offset
a, Average responses of a F-mini-ON RGC to 30 μm spots of positive (orange) or negative (black) contrast at the indicated positions. b,c, Interpolated ON and OFF spatial RFs from the data in a. White and black crosses mark the ON and OFF centers-of-mass (COM), respectively. d, Population data showing the fractional overlap of the OFF RF relative to the ON RF for F-mini-ON RGCs (magenta) and other ON-OFF RGCs (grey) (n = 9, 14 cells, see Methods). e, Polar histograms showing the offset angle between the ON RF COM and the OFF RF COM for F-mini-ON RGCs (magenta) and other ON-OFF RGCs (grey). OFF ventral of ON is shown as a downward (ventral) angle. f, Image of the cell from a-c overlaid with its ON (orange) and OFF (black) RF contours. g, Average OFF RF for F-mini-ON RGCs aligned to the center-of-mass of each ON RF at the origin. Points are the center-of-mass for individual cells (n = 9). Scale bar is the same in a-c,f,g. Box plots in d show maximum, 75th percentile, median, 25th percentile, and minimum.
Figure 3
Figure 3. Alignment between ON and OFF strata of bistratified RGCs.
a, Example projection image of an F-mini-ON RGC from Eyewire, dendrites colored by stratification: green proximal/inner/ON, blue distal/outer/OFF, cyan between, red soma and axon. Centers of mass of inner and outer strata are marked by circled crosses. b, Dendritic offset distance by cell type, mean with SD, n = 16, 19, 23, 17, 7, 6, 3, 6 cells. RGC type names are followed by Eyewire anatomical types in parentheses. Magenta line is at 38 μm, the mean RF offset found in F-mini-ON RGCs. c, Offset values in μm from four small bistratified RGC types in Eyewire. Red crosses show mean and standard deviation of offsets.. Spatial data from the remaining four bistratified types is in Extended Data Fig. 3. Box plots in b show maximum, 75th percentile, median, 25th percentile, and minimum.
Figure 4
Figure 4. Heterotypic gap junctions among F-mini-RGCs are confirmed by immunohistochemistry
Image of the ganglion cell layer in a patch of retina in which a single F-mini-ON RGC was filled with Neurobiotin (arrow). RGC somas labeled by the dye are circled: cyan F-mini-OFF (first degree connections) and magenta F-mini-ON (filled RGC and second degree connections). The transcription factor FOXP2 is found in all F-mini RGCs (red); FOXP1 is found in F-mini-OFF RGCs (blue). Results were consistent over several images (n = 5 filled networks on 4 retinas).
Figure 5
Figure 5. F-mini-ON and F-mini-OFF RGCs are electrically coupled to each other by gap junctions.
a, An example illustrating heterotypic network connectivity. The F-mini-OFF RGC labeled ‘0’ was filled with Alexa Fluor 488 (cyan), revealing 7 coupled somas (white). b, Cell-attached recordings from each of the labeled somas shown in a. Cells 1 through 6 show clear signs of being neighboring F-mini-ON RGCs. The soma of Cell 7 is dimmer, and is likely a second-order connected F-mini-OFF RGC. c, Distribution of the number of dye coupled cells observed in Neurobiotin and Alexa Fluor 488 cell fills of F-mini-ON and F-mini-OFF RGCs, n = 13, 3 cells. d, Average voltage traces from an RGC pair in which one F-mini RGC was injected with current (top row) and the coupled F-mini RGC of the opposite type (bottom row) showed a response. Current injections were +50 pA (lighter traces) and −50 pA (darker traces). e, Voltage change relationship across the electrical synapse in both directions for the pair in d. f, Distribution of the coupling coefficient (slope of line in e) for all recorded pairs, in control conditions (top) and in the presence of MFA (bottom). g, Example of MFA abolishing voltage deflection, showing voltage in F-mini RGCs (for −50 pA injection in coupled cell) in control conditions and in the presence of MFA (green). Image in a is a composite of a maximum projection image of the F-mini-OFF dendrites in cyan with a maximum projection image of the ganglion cell layer in white. Cell ‘0’ in b was recorded in current clamp mode. Box plots in c show maximum, 75th percentile, median, 25th percentile, and minimum.
Figure 6
Figure 6. F-mini-ON RGCs receive ON input from chemical synapses and OFF input from electrical synapses.
a, Response of an F-mini-ON RGCs to an ON 120 μm spot before (black) and after MFA (green) application. Dashed line is −60 mV. b, Responses of the cell in a to a moving positive contrast bar before (black) and after MFA (green) application. c, (upper) Population data for F-mini-ON RGC spike responses to flashed spots as in a (n = 6 cells). Points connected by dotted lines are individual cells. (lower) OFF:ON ratio of in control and MFA conditions. d, Same as c for peaks of subthreshold voltage responses (n = 6 cells). e,f, Image of a recorded F-mini-ON RGC (magenta) before and after ablation of 3 of the coupled somas (cyan). g, Responses of an F-mini-ON RGC to an ON 120 μm spot at 2000 R*/rod/s before (black) and after ablation (red) of coupled somas. Dashed line is −60 mV. h, Responses of a different cell to a moving ON bar at 700 R*/rod/s before (black) and after ablation (red). i, (upper) Population data for F-mini-ON RGC spike responses to flashed spots as in c (n = 6 cells). (lower) OFF:ON ratio in control and ablation conditions. j, Same as i for subthreshold membrane voltage (n = 6 cells). Box plots in c,d,i,j show maximum, 75th percentile, median, 25th percentile, and minimum.
Figure 7
Figure 7. F-mini-ON RGCs RF offset is captured by a morphological model.
a, Traced microscope image from an experiment in which a single F-mini-ON RGC (magenta) was filled with Neurobiotin. Four coupled F-mini-OFF RGCs are in cyan. A spatial offset is apparent in the dendritic arbors of the two cell types relative to the soma of the F-mini-ON. b, Diagram of the imaging datasets used in the morphological model, described in d and e. c, RF model diagram (see Methods). Measurements of F-mini-ON coupled soma positions in d were randomly combined with convex polygon fits to the dendrites of F-mini-ON and F-mini-OFF RGCs in e, to create a purely anatomical prediction of the center-of-mass (COM) offset of ON and OFF RFs (red and blue crosses). d, Locations of labeled somas relative to the injected F-mini-ON RGCs (magenta circle) included both confirmed F-mini-OFF RGCs (squares) and RGC somas that were not further characterized (triangles) (n = 50 somas, 13 injected cells, colored by injected cell) Each point represents the position of a gap-junction labeled soma relative to the position of the filled F-mini-ON RGC. Results above suggest that all coupled cells were in fact F-mini-OFF RGCs, but only some of them (squares) were confirmed via electrophysiology or IHC. e, Area of dendrites relative to soma position for the measured population of F-mini-ON and F-mini-OFF RGCs (n = 38, 12 cells). Soma locations are marked by 50 μm crosses. f, Result from the model. Colored surface is mean OFF RF relative to the centered ON RF. True F-mini-ON RF offset data (black crosses) and format are from Fig. 2g.
Figure 8
Figure 8. Multi-cell model of object localization shows an advantage of offset ON-OFF RFs.
a, Schematics of the different RF structures and error metrics. b, RF activation map (left) for a single instantiation of the overlapped RF model. The stimulus is the black-white edge. Green circles show the positions of each RGC with the radius of the circle proportional to its response. Red cross indicates the center of mass of the RGC responses. Magnified portion (right) shows the ON (magenta) and OFF (cyan) subfields of each RF. Line thickness is proportional to activation of each subfield. c, RF activation map for the same positions and stimulus as in b, but for the offset RF model retaining total size. Orange lines in the magnified view (right) connect ON and OFF subfields from the same RGC. d, Absolute error in decoded edge position for each model. Points are means of 500 iterations of each model with different RGC and stimulus positions. Error bars for SEM are obscured by symbols; SD was similar for each point with a mean of 0.42 μm. e, Vertical component of the error in d shown as a change ratio relative to the overlapped RF result. f, Same as e for the horizontal component.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Schwartz GW et al. The spatial structure of a nonlinear receptive field. Nat. Neurosci. 15, 1572–1580 (2012). - PMC - PubMed
    1. Wassle H, Peichl L & Boycott BB Dendritic territories of cat retinal ganglion cells. Nature 292, 344–345 (1981). - PubMed
    1. Gauthier JL et al. Receptive Fields in Primate Retina Are Coordinated to Sample Visual Space More Uniformly. PLoS Biol. 7, e63–e63 (2009). - PMC - PubMed
    1. Roska B & Werblin F Vertical interactions across ten parallel, stacked representations in the mammalian retina. Nature 410, 583–587 (2001). - PubMed
    1. Masland RH The fundamental plan of the retina. Nat. Neurosci. 4, 877–886 (2001). - PubMed

References (Methods)

    1. Sato C, Iwai-Takekoshi L, Ichikawa Y & Kawasaki H Cell type-specific expression of FoxP2 in the ferret and mouse retina. Neurosci. Res. 117, 1–13 (2017). - PubMed
    1. Longair MH, Baker DA & Armstrong JD Simple Neurite Tracer: open source software for reconstruction, visualization and analysis of neuronal processes. Bioinformatics 27, 2453–2454 (2011). - PubMed
    1. Rousso DL, Gaber ZB, Wellik D, Morrisey EE & Novitch BG Coordinated actions of the forkhead protein Foxp1 and Hox proteins in the columnar organization of spinal motor neurons. Neuron 59, 226–240 (2008). - PMC - PubMed
    1. Lu MM, Li S, Yang H & Morrisey EE Foxp4: a novel member of the Foxp subfamily of winged-helix genes co-expressed with Foxp1 and Foxp2 in pulmonary and gut tissues. Mech. Dev. 119 Suppl 1, S197–202 (2002). - PubMed
    1. Bhattacharyya S et al. Using Gjd3-CreEGFP mice to examine atrioventricular node morphology and composition. Sci. Rep 9, 2106 (2019). - PMC - PubMed

Publication types