Simultaneous extracellular recordings of RGC axons and SC neurons combined with optotagging identifies GABAergic neurons in VGAT-ChR2 mice.

A, Recording configuration for tangential electrode insertion and optotagging in the visual layers of mouse SC. The optogenetic fiber is inserted perpendicularly to the Neuropixels probe to activate GABAergic cells in VGAT-ChR2 mice. RGC afferents (orange) project onto GABAergic (blue) and non-GABAergic (black) neurons in SC. B, Multi-unit response (MUA) to optogenetic stimulation along the 384 recording sites during the presentation of a black screen. The large spatial extend of the optogenetic activation is evident. Gray vertical bar = light artifacts induced by the LED stimulation. C, Visually evoked MUA during the presentation of a sparse noise stimulus along the recording sites. Receptive field contours of recording sites with high signal-to-noise ratio. The color code reflects the location within the SC. Scale bar represents 10 degree. D, Spatiotemporal waveform profiles. Single channel waveforms identified at the peak channel (top) and multi-channel waveforms (bottom) for RGC axon (left, orange) and inhibitory (middle, blue) and excitatory (right, gray) SC neurons. Receptive fields (RFs) indicate visually responsive neurons. E, Identification of SC cell-types via optotagging. Raster plots and peri-stimulus time histograms (PSTHs) for single-neuron responses to blue light pulses (100ms) presented under baseline conditions. Excitatory SC neurons (EXNs, bottom) and RGCs (top) do not respond to the LED pulse, while GABAergic SC neurons respond to the light pulse with an increase in spiking response (middle). Optogenetic stimulation period is highlighted in blue. The colored scale bars on the right represent 20 Hz firing rate. F, PSTHs and raster plots for different cell types shown in E in response to a natural movie stimulus (10s, 30 trials). Note the high firing rate in RGCs. G, Top: Proportion of identified GABAergic (INs, blue), non-GABAergic (EXNs, gray) SC neurons and retinal axons (RGCs, orange) populations. Note that around one third of the captured SC neurons are GABAergic (n = 326 RGCs, n = 468 EXNs, n = 212 INs). Bottom: Mean firing rates in response to a natural movie stimulus presented for 10s, 30 trials. Two-sided Wilcoxon rank-sum test.

Retinal innervation is similarly strong to excitatory and inhibitory SC neurons.

A, Monosynaptically connected RGC-SC EXN (gray) and RGC-SC IN (blue) pairs are identified via cross-correlation analysis (CCG). B, CCGs of connected RGC-SC EXN and RGC-SC IN pairs sorted by their peak latency (n = 214 RGC-SC EXN, n = 91 RGC-SC IN, n= 11 recordings). C, Connectivity matrix from a single recording. Gray marks indicate connections onto excitatory SC neurons, blue marks indication connections onto inhibitory SC neurons. RGC axons and SC neurons are sorted by their peak channel along the electrode. D, Distribution of peak channel distances between RGC axons and connected SC neurons (p = 0.0328, two-sided Wilcoxon rank-sum test). Inset shows pie chart of identified RGC-SC IN and RGC-SC EXN pairs. E, Elicited SC spiking in response to firing of a presynaptically connected retinal ganglion cell (RGC). Raster plot shows SC firing to 1000 randomly selected RGC spikes. Both SC cell types show robust activation upon RGC spiking (top) but also weaker connections can be found (bottom). F, Synaptic efficacy as a measure for connection strength for RGC-SC EXN and RGC-SC IN connected pairs (p = 0.053, n = 214 RGC-SC EXN, n = 91 RGC-SC IN). G, Peak latency responses for RGC-SC EXN and RGC-SC IN pairs triggered on retinal spiking (p = 0.321). Two-sided Wilcoxon rank-sum test.

Characterization of functional similarity between retinocollicular connected pairs.

A, Spatiotemporal receptive fields (STRF) evoked by a dark sparse noise stimulus for RGC-SC EXN and RGC-SC IN connected pairs. The functional similarity of the RGC axon and the postsynaptic SC neuron is characterized by the correlation coefficient rSD. B, Visually evoked activity in response to a natural movie stimulus for the connected pairs shown in A together with their correlation coefficient values rNM. C, The overall functional similarity between the presynaptic RGC and the postsynaptic SC neurons is reflected in the similarity index calculated from the averaged correlation coefficients (rSD + rSL + rNM)/3 (p = 0.245, two-sided Wilcoxon rank-sum test, n = 85 RGC-SC EXN pairs, n = 29 RGC-SC IN pairs). D, Relationship between similarity index and connection efficacy (RGC-SC EXN r = 0.29, p = 0.007; RGC-SC IN r = 0.471, p = 0.01; Pearson correlation coefficient test; n = 85 RGC-SC EXN pairs, n = 29 RGC-SC IN pairs).

Paired-spike dynamics: Second retinal spikes are more efficient in driving SC response.

A, Schematic illustrating the temporal dynamics between two successive RGC spikes. Pairs of RGC spikes with a minimum inter-spike interval (ISI) of 5 ms and maximum ISI of 30 ms were included if there were no preceding spikes before the 1st RGC for a dead time of at least 30ms. B, Cross-correlograms (CCG) of example RGC-SC EXN and RGC-SC IN pairs calculated from spike trains selected for 1st and 2nd RGCs and the corresponding raster plots to 1000 trials triggered on RGC spikes. C, Scatter plot of efficacies for 1st versus 2nd retinal spikes. The majority of connected pairs (185/214 RGC-SC EXN pairs; 73/91 RGC-SC IN pairs) showed paired-spike enhancement in response to 2nd RGC spikes. D, Paired-spike ratio (PSR) for RGC-SC EXN and RGC-SC IN connected pairs. The paired pulse enhancement is stronger in SC-EXNs (n = 214 RGC-SC EXN; n = 91 RGC-SC IN, p = 2.581ξ10-5, two-sided Wilcoxon rank-sum test). E, Correlation of 1st RGC efficacy with the PSR for connected pairs (r = 0.3314 for n = 214 RGC-SC EXN pairs and r = 0.4767 for n = 91 RGC-SC IN pairs; Pearson correlation coefficient test).

Connection contribution is higher in connected RGC-SC EXN pairs.

A, Example CCGs of monosynaptically connected RGC-SC EXN (top) and RGC-SC IN (bottom) pairs. Connected pairs were identified by their peaks in the CCGs (left). Raster plots of RGC spiking activity triggered on 1000 SC spikes (right). B, Population data on contribution measures for RGC-SC EXN and RGC-SC IN pairs (n = 114 RGC-SC EXN pairs, n=91 RGC-SC IN pairs, p = 0.0003, two-sided Wilcoxon rank-sum test). C, Relationship between functional similarity index and connection contribution (Pearson correlation coefficient test; n = 85 RGC-SC EXN pairs, n = 29 RGC-SC IN pairs). D, Correlation of mean firing rate (to natural movie stimulus) and contribution for RGC-SC EXN and RGC-SC IN pairs (Pearson correlation coefficient, two-sided Wilcoxon rank-sum test, n = 214 RGC-EXN and n = 91 RGC-IN connected pairs; n = 11 penetrations from 9 mice).

Spike waveform features analysis for GABAergic and non-GABAergic neuron populations in the SC.

A, Top: Illustration of features extracted from single-channel waveforms (magenta circles indicate the trough and peak). Middle and bottom: Single-channel mean waveforms for non-GABAergic (gray; n = 468) and GABAergic (blue, n = 212, n = 9 mice) SC populations (mean±SEM). B-D, Distribution of waveform features extracted from single-channel waveforms for inhibitory and excitatory SC populations. Peak-to-trough duration represents the time between trough and peak (p = 4.05ξ10-10). Peak-to-trough ratio represents the ratio between amplitudes of peak and trough (p = 4.45ξ10-9). Amplitude is the absolute difference between trough and peak (p = 1.38ξ10-9). E, Distribution of waveform spread along the probe extracted from multichannel waveforms for GABAergic and non-GABAergic SC populations (p = 1.58ξ10-4, n = 468 EXNs, n= 212 INs, Two-sided Wilcoxon rank-sum test).