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Sidekick 2 directs formation of a retinal circuit that detects differential motion

Abstract

In the mammalian retina, processes of approximately 70 types of interneurons form specific synapses on roughly 30 types of retinal ganglion cells (RGCs) in a neuropil called the inner plexiform layer. Each RGC type extracts salient features from visual input, which are sent deeper into the brain for further processing1,2,3,4. The specificity and stereotypy of synapses formed in the inner plexiform layer account for the feature-detecting ability of RGCs. Here we analyse the development and function of synapses on one mouse RGC type, called the W3B-RGC5,6. These cells have the remarkable property of responding when the timing of the movement of a small object differs from that of the background, but not when they coincide6. Such cells, known as local edge detectors or object motion sensors, can distinguish moving objects from a visual scene that is also moving6,7,8,9,10,11,12. We show that W3B-RGCs receive strong and selective input from an unusual excitatory amacrine cell type known as VG3-AC (vesicular glutamate transporter 3). Both W3B-RGCs and VG3-ACs express the immunoglobulin superfamily recognition molecule sidekick 2 (Sdk2)13,14, and both loss- and gain-of-function studies indicate that Sdk2-dependent homophilic interactions are necessary for the selectivity of the connection. The Sdk2-specified synapse is essential for visual responses of W3B-RGCs: whereas bipolar cells relay visual input directly to most RGCs, the W3B-RGCs receive much of their input indirectly, via the VG3-ACs. This non-canonical circuit introduces a delay into the pathway from photoreceptors in the centre of the receptive field to W3B-RGCs, which could improve their ability to judge the synchrony of local and global motion.

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Figure 1: Sidekick proteins are expressed by subsets of retinal neurons.
Figure 2: Selective connectivity of VG3-ACs and W3B-RGCs.
Figure 3: Decreased synaptic connectivity in Sdk2 mutants.
Figure 4: Excitatory input reaches W3B-RGCs via VG3-ACs.

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Acknowledgements

We thank E. Soucy and J. Greenwood for assistance with constructing the two-photon microscope, the Genome Modification Facility at Harvard for generating mouse lines, and E. Feinberg for insight into the ion selectivity of ChR2. This work was supported by grants from the NIH (NS029169 and EY022073) to J.R.S., NSERC (Canada) and Banting Postdoctoral Fellowships to A.K., a HHMI-Life Sciences Research Foundation Postdoctoral Fellowship to X.D., and an NIH fellowship (F31 NS055488) to Y.K.H.

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Authors and Affiliations

Authors

Contributions

A.K., M.Y. and J.R.S. planned experiments, analysed data and wrote the paper. A.K. performed electrophysiological and histological experiments, M.Y. performed genetic and histological experiments, X.D. developed methods and generated reagents and Y.K.H. generated reagents and performed in situ hybridization. The authors declare no competing interest.

Corresponding author

Correspondence to Joshua R. Sanes.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Expression of Sdk1 and Sdk2 in developing retina.

a, Double-label in situ hybridization for Sdk1 and Sdk2 at P10. Arrow indicates a retinal ganglion cell that expresses both Sdk genes. Label in INL includes amacrine and bipolar cells, as summarized in Fig. 1k. Other images (not shown) reveal that Sdk2 is also expressed by horizontal cells. b, In situ hybridization for Sdk1 and Sdk2 RNA at indicated postnatal ages. Scale bars, 10 μm.

Extended Data Figure 2 Generation and characterization of Sdk1 and Sdk2 knock-in mice

a, b, Targeting vectors used to generate Sdk1ce/ce (a) and Sdk2ce/ce (b) mice. c, HEK293 cells transfected with expression vectors encoding Sdk1 or Sdk2, followed by staining with mouse antibodies to Sdk1 and Sdk2. d, HEK293 cells transfected with expression vectors encoding Sdk1 or Sdk2, followed by immunoblotting with rabbit polyclonal antibodies to Sdk1 and a mouse monoclonal antibody to Sdk2 (CS22). e, Retinal sections from P30 Sdk1ce/ce and Sdk2ce/ce mice stained with mouse antibodies to Sdk1 and Sdk2. Signal on blood vessels is nonspecific. f, Reverse transcription PCR (RT–PCR) from Sdk1ce/+, Sdk1ce/ce , Sdk2ce/+ Sdk2ce/ce and wild-type (WT) mice. Total RNA was prepared from brain. G3PDH, glyceraldehyde-3-phosphate dehydrogenase. g, VG3-ACs and W3B-RGCs are Sdk1-negative. CreER expressed in P30 Sdk1ce/+ mouse was stained with antibodies to the oestrogen receptor and vesicular glutamate transporter 3 (VG3). Bottom row shows CreER and YFP double staining in a Sdk1ce/+; TYW3 mouse. h, Sdk2-expressing W3B cells express the RGC marker Brn3a, but not the amacrine cell marker AP2. Calretinin is expressed in all the SACs, a subpopulation of type II catecholaminergic cells (CAII) amacrine cells and some RGCs. i, SACs (ChAT-positive), type I catecholaminergic (tyrosine hydroxylase-positive), SEG (glycinergic) and nGnG (Satb2-positive35), amacrine cells do not express Sdk2. Scale bars, 10 μm.

Extended Data Figure 3 Optimization of optogenetic methods.

a, Sample current recorded from a ChR2–YFP-expressing HEK cell in response to a stationary PSF-sized spot of 920-nm laser stimulation for 7 ms. b, Plot of average peak current measured on ChR2–YFP-expressing HEK cells in response to a stationary point spread function (PSF)-sized spot of laser stimulation at 840 nm, 860 nm and 920 nm for a range of different powers. The 920 nm light produced the largest currents for the least power. c, Plot of the rise time (10–90%) of currents to a stationary, PSF-sized spot of laser stimulation at 840 nm, 860 nm and 920 nm for a range of different powers. The 920 nm light produced the shortest rise time for the least power. d, Cartoon of a HEK cell with four adjacent PSF-sized spots that start on the edge of the cell and extend off (left). Sample currents evoked by stimulation of these regions. Currents decrease in size as the PSF moves away from the cell. e, Quantification of the experiment illustrated in d for 5 cells. The curve was obtained in only one direction and reflected about the y axis to give a measure of the x–y spread of ChR2 excitation. f, Cartoon of a HEK cell stimulated by a stationary, PSF-sized spot at 10 adjacent 1-μm planes that extend from the cell surface to −10 μm. Sample currents evoked by this procedure at the indicated z-positions. Average peak current measured on four cells for this procedure (right) show the z-extent of ChR2 excitation. g, Sample current evoked in a ChR2-expressing RGC in response to stimulating an ROI (10 × 15) with a dwell time of 0.03 ms, taking a total time of 5 ms. h, Sample current evoked in a ChR2-expressing SAC in response to stimulating an ROI (10 × 15) with a dwell time of 0.03 ms, taking a total time of 5 ms. i, Average peak current evoked in RGCs and SACs in response to the stimuli shown in g and h (n = 6 cells). j, Average rise time (10–90%) of currents evoked on RGCs and SACs in response to the stimuli shown in g and h (n = 6 cells). k, Plot of average peak current measured in a W3B-RGC in response to stimulation of VG3-ACs with two-photon stimulation (920 nm) for a range of sample plane powers. Average current size shows a sigmoid relationship. Responses plateau at 25 mW sample plane power (n = 6 W3B-VG3 pairs in two animals). Ipeak versus power relationship from b has been re-plotted for comparison. l, Ipeak versus sample plane power measured on W3B-RGCs to VG3-AC replotted from k. Sample currents beside the curve were evoked by the powers indicated. Responses require a threshold amount of excitation in VG3-ACs; probably owing to calcium-dependent vesicle release mechanisms (n = 6 W3B-VG3 pairs in two animals).

Extended Data Figure 4 VG3-ACs form direct excitatory synapses on W3B-RGCs.

a, Average current (I)–voltage (V) plot of VG3-AC evoked currents on W3B-RGCs. Currents were normalized to the maximum inward current per cell (n = 10 VG3-W3B pairs). b, Currents are abolished by inhibitors of AMPA-type glutamate receptors (DNQX, 20 μM), but are unaffected by inhibitors of GABA (picrotoxin (PTX), 100 μM), glycine (strychnine (STR), 3 μM) receptors and gap junctions (18β-glycyrrhetinic acid (18β-GA), 25 μM) (n = 8–10 per condition). c, Latency of VG3-AC-evoked currents on W3B (n = 623 VG3s and 14 RGCs). d, Inward currents measured in HEK cells transiently transfected with constructs containing ChR2–GFP. The first, twenty-fifth and fiftieth responses to a train (1 Hz) of 60 stimuli are shown for HEK cells recorded in control and CdCl2-containing solution. e, Peak currents from the experiment shown in a, plotted as a percentage of the initial peak size. Currents in CdCl2 appear to decrease slightly over time. f, Quantification of peak currents measured in Cd2+-containing solution expressed as a percentage of those found in control solution. ChR2 activity is largely unaffected by the presence of CdCl2 (n = 6 cells, in control solution, 10 cells in 500 μM and 8 cells in 1 mM CdCl2 solution) g, Responses of a ChR2-positive RGC to stimulus train that alternated between two-photon excitation of ChR2 on the RGC soma and one-photon activation of photoreceptors above the RGC. The first, fifteenth and thirtieth responses in the train are shown. ChR2 responses are insensitive to the calcium channel blocker but light responses are not. h, Peak current measured on the final pulse of the train expressed as a percent of the first for the experiments performed like that in e. IChR2 is largely immune to the calcium channel blocker CdCl2 (n = 4 ChR2-positive RGCs). i, Heat map of VG3-AC responses measured in a W3B-RGC in control solution (left) and the same heat map measured in the presence of 500 μM CdCl2. VG3-AC responses initiated by soma stimulation require functional voltage gated calcium channels in the nerve terminals. j, Heat map of VG3-AC responses measured in the same W3B-RGC in CdCl2 solution shown in f in response to a 10 × 10 stimulus grid (grid square = 5 × 5 pixels) in the inner plexiform layer. Activating ChR2 on VG3-AC nerve terminals produces responses in W3B-RGCs in spite of silencing voltage-activated calcium channels globally. Scale bars, 65 ms and 34 μm. k, Currents recorded from W3B-RGCs (−60 mV) in response to stimulation of VG3-ACs at their somas (INL) or terminals (IPL) in control and CdCl2 (200μM) containing solution (left). Average peak current evoked by either soma (INL) or nerve terminal (IPL) stimulation in CdCl2 solution expressed as a percentage of that found in control (right, n = 276 VG3-ACs and 5 W3B-RGCs). Currents evoked by IPL stimulation in CdCl2-containing solution result from Ca2+ influx via ChR2 on VG3-AC terminals. VG3-ACs synapse directly with W3B-RGCs. l, Sample currents evoked in W3B-RGCs, W3D-RGCs and ooDSGCs by blue light (one-photon) stimulation of ChR2 positive VG3-ACs. Experiments were done in the presence of a cocktail of blockers: 10 μM ACET, to block the OFF pathway; 20 μM L-AP4, to block the ON pathway, and 300 μM hexamethonium, to block cholinergic nicotinic receptors19,51. m, Average peak currents from experiments like those in l (n = 15 W3B-RGCs, n = 7 W3D-RGCs and n = 13 ooDSGCs). These results from one-photon stimulation of a population of VG3-ACs, confirm the conclusion from two-photon stimulation of single VG3-ACs (Fig. 2): these amacrine cells innervate W3B-RGCs far more strongly than W3D-RGCs or ooDSGCs. By one-photon stimulation, the currents evoked in ooDSGCs are stronger than those in W3D-RGCs, whereas they are similar in the two-photon data. This difference probably arises from their larger dendritic size. On the basis of the dendritic diameter of VG3-ACs (50 μm; Figs 2c and 3r), W3B-RGCs (115 μm; Figs 2c and 3l), W3D-RGCs (125 μm; data not shown) and ooDSGCs (200 μm; ref. 37), we can estimate that dendrites of W3B-RGCs, W3D-RGCs and ooDSGCs overlap dendrites of 24, 29 and 100 VG3-ACs, respectively. Given the percentage connectivity in shown in Fig. 2, we estimate that each W3D-RGC is innervated by 10–15 VG3-ACs, whereas each ooDSGC is innervated by 35 VG3-ACs.

Extended Data Figure 5 Synaptic connectivity of VG3-ACs and W3B-RGCs.

a, b, Strength of connections as a function of distance from six interneuron types to W3B-RGCs (a) and VG3-ACs to four RGC types (b). Number of synaptic partners assayed shown above and sample currents shown below each graph. The W7 population contained six nearly-disconnected and ten connected pairs, presumably corresponding to the S1-laminating and S3-laminating W7 subsets. Normalized peak currents (Ipeak) from each pair were normalized to the average maximum response from VG3-AC–W3B-RGCs.

Extended Data Figure 6 Normal retinal architecture in Sdk2 mutants.

Sections of Sdk2ce/+ and Sdk2ce/ce retinas (P30) were stained with antibodies to cell-type-specific markers, synaptic components or fluorescent proteins. No differences between mutants and controls were detected in cells other than VG3-ACs and W3B-RGCs. a, Brn3a labels most RGCs. b, AP2 labels all amacrine cells. c, d, Synapsin I and SV2 are associated with synaptic vesicles. e,VGlut1 is concentrated in photoreceptor and bipolar terminals. f, PSD-95 family members, stained with anti-MAGUK, are associated with synaptic sites, usually excitatory postsynaptic densities. g, Synaptotagmin 2 is concentrated in bipolar cells types 2 and 6. h, Anti-HCN4 labels type 3a bipolar cells. i, Anti-disabled-1 (Dab1) labels AII amacrines. j, Anti-VAChT labels dendrites of SACs. k, Anti-calretinin labels subsets of RGCs and amacrine cells, including SACs. l, Anti-protein kinase C-α (PKCα) labels rod bipolar cells and a subset of amacrine cells. m, Anti-VGlut3 labels VG3-ACs. Sprouting is evident in the mutant. n, Single-cell reconstructions of VG3-ACs labelled sparsely with GFP in retinal cross-sections. o, Quantification of sprouting in mutant VG3-ACs. Scale bars, 10 μm.

Extended Data Figure 7 Morphological analysis of VG3-ACs and W3B-RGCs in Sdk2 mutants.

a, b, Dye-injected W3B-RGCs were imaged and skeletonized as described in Methods. Projections on rotated stacks counterstained with anti-VAChT antibodies are shown in h. c, d, Similar to a, b, except for VG3-ACs labelled sparsely with tdTomato and counterstained with anti-ChAT to label the somas of SACs. Scale bars, 25 μm. e, f, Mean intensity (±s.e.m.) of dye-labelled W3B-RGC dendrites (e) and VG3-AC dendrites (f) across the IPL from images such as those shown in ad. g, h, En face or laminar projections of skeletonized dendritic arbors from VG3-ACs labelled sparsely with tdTomato in Sdk2+/ce and Sdk2ce/ce retinae. i, j, En face or laminar projections of skeletonized dendritic arbors from dye-filled W3B-RGCs in Sdk2+/ce and Sdk2ce/ce retinae.

Extended Data Figure 8 Electrical and synaptic properties of Sdk2 mutant VG3-ACs and W3B-RGCs.

al, Strength of connections as a function of distance from three interneuron types (SACs, type 5 bipolar cells (BC5) and type II catecholaminergic cells (CAII)) to W3B-RGCs and from VG3-ACs to W3D-RGCs in Sdk2ce/+ (black) and Sdk2ce/ce (red) retinae. ad, Normalized peak current. eh, Percentage of connected pairs. il, Average current amplitudes in a 30-ms window after the stimulus pulse. mo, Latencies of currents detectable above noise. Number of pre- and postsynaptic partners assayed shown below each column. p, Responses evoked in a W3B-RGC (green dot) in a Sdk2 mutant after stimulation of 66 VG3-ACs (genotype: TYW3; Sdk2ce/ce ; Vglut3-cre; LOX-STOP-LOX-ChR2-tdTomato). q, Average latencies for currents detectable above noise in control. r, Sodium currents in W3B-RGCs Sdk2ce/+ and Sdk2ce/ce retinae. Sample currents evoked by a step from −60 mV to −5 mV (left), and average peak sodium current amplitude measured on W3B-RGCs in Sdk2ce/+ and Sdk2ce/ce retinae. s, Retinal cross-sections from wild-type mice and those that overexpress Sdk2 broadly using the Six3-cre driver and the Sdk2 swap transgene. Sdk2 is expressed strongly from the swap transgene, which is shown as a schematic below the micrographs. t, Strength of connections from VG3-ACs to W3B-RGCs in Sdk2ce/ce mutants in which Sdk2 expression was rescued in VG3-ACs. Data were derived from 121 VG3-ACs and 3 W3B-RGCs. Fit to the control data (Fig. 3a) re-plotted in blue.

Extended Data Figure 9 Effective deletion of VG3-ACs by diphtheria toxin.

a, Spike responses of W3B-RGCs in Sdk2ce/+ and Sdk2ce/ce retinae in response to a 100-μm flashing spot centred on the receptive field. OFF responses are strongly reduced in the absence of Sdk2. b, Inhibitory currents recorded (Vh = −5 mV) from W3B-RGCs in Sdk2+/ce and Sdk2ce/ce retinae in response to a full field flash for 1 s. c, Ipeak measured from excitatory currents in non-W3B-RGCs (n = 8 in 4 Sdk2ce/+ mice and 8 in 4 Sdk2ce/ce mice). d, e, Sample images of tdTomato-positive VG3-ACs in retinae from diphtheria-toxin-treated control (d) and Vglut3-cre; Rosa-CAG-LOX-STOP-LOX-DTR mice (e). Scale bar, 40 μm. f, Average firing rate recorded from W3B-RGCs in control (blue, n = 21) and diphtheria-toxin-treated Vglut3-cre;DTR/TYW3 (red, n = 12) retinae in response to small spots flashed for 1 s. Dark lines, average; shadowing denotes s.e.m. Bin width, 50 ms. g, Excitatory currents recorded (Vh = −65 mV) from W3B-RGCs in control and diphtheria-toxin-treated Vglut3-cre;DTR/TYW3 retinae in response to small spots flashed for 1 s. h, Inhibitory currents recorded (Vh = −5 mV) from W3B-RGCs in control and diphtheria-toxin-treated Vglut3-cre;DTR/TYW3 retinae in response to small spots flashed for 1 s.

Extended Data Figure 10 A delay line in the differential motion response.

a, Average latency to peak firing rate in W3B-RGCs and non-W3B-RGCs in response to a spot flashed over their receptive field centre (n = 21 non-W3B-RGCs and 18 W3B-RGCs). Spike responses on W3B-RGCs are delayed (**P <0.01, Student’s t-test). b, Possible mechanisms for the delay. In non-W3B-RGCs excitatory currents produced by bipolar cells drive the neuron to fire. In W3B-RGCs, spikes could be delayed because the interposition of VG3-ACs delays the onset of the excitatory postsynaptic current (EPSC) (W3B e delay) or because of a transient inhibitory postsynaptic current that arrives at the same time as the EPSC and delays the cell from reaching threshold (W3B i delay). c, d, Excitatory (c) and inhibitory (d) currents measured from non-W3B-RGCs and W3B-RGCs in response to a flashing spot centred on the receptive field or a full field flash. Dotted lines denote the stimulus onset, non-W3B-RGC EPSC onset and W3B-RGC EPSC onset (red). The onset of EPSCs in W3B-RGC lags behind those found on non-W3B-RGCs by 30 ms. No significant transient inhibition was observed in the receptive field centre (d). e, f, Histogram of latency to the onset of the ON (e) and OFF (f) excitatory current measured on W3B-RGCs (light blue, light red) and non-W3B-RGCs (blue, red). W3B-RGCs lag non-W3B-RGCs by 32–40 ms and also have higher variance about the mean (n = 29 non-W3B-RGCs and 27 W3B-RGCs). g, Average peak of inhibitory currents measured on W3B-RGCs in response to a 100-μm diameter flashing spot and a full field flash (n = 6 wild-type W3B-RGCs; *P <0.05, Student’s t-test). h, Slope of the light-evoked excitatory current between 10% and 90% of the peak. Slopes of currents in W3B-RGCs are significantly lower than those of non-W3B-RGCs (n = 29 non-W3B-RGCs and 27 W3B-RGCs; **P <0.001). The non-W3B-RGCs included W3D-RGCs, ooDSGCs, α-RGCs and some unidentified RGCs. i, Average maximal firing rates measured in W3B-RGCs to local, global and differential motion stimuli (n = 10 W3B-RGCs in 5 control mice, 6 W3B-RGCs in 4 Sdk2ce/ce mice and 9 W3B-RGCs in 3 diphtheria-toxin-treated Vglut3-cre;DTR/TYW3 mice; **P < 0.01, Student’s t-test). Firing rates in global and differential motion were normalized to that elicited in control W3B-RGCs with local motion stimuli for Fig. 4j.

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Krishnaswamy, A., Yamagata, M., Duan, X. et al. Sidekick 2 directs formation of a retinal circuit that detects differential motion. Nature 524, 466–470 (2015). https://doi.org/10.1038/nature14682

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