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. Author manuscript; available in PMC: 2022 Jun 7.
Published in final edited form as: Nat Neurosci. 2022 May 6;25(5):659–674. doi: 10.1038/s41593-022-01068-8

Extended Data Fig. 8. Characterizations of Nephronectin-positive-wide-field neurons (NPWFs) and their differential inputs from αRGCs versus ooDSGCs.

Extended Data Fig. 8

a, Sample DIC and fluorescent microscope images showing the targeted postsynaptic Npnt-FlpO-TdTomato neurons for connectivity tests. At least 30 times each experiment was repeated independently with similar results. Scale bar: 20 μm. b, The majority of the labeled Npnt-FlpO-TdTomato neurons are positive for Npnt antibody immunostaining (96.5±2.5% double-positive neurons among all TdTomato-positive neurons in b, n= 3 animals). The dotted line marks the pial surface. c, Npnt-FlpO-TdTomatao neurons have their somata positioning right above the top boundary of the SGI (dotted lines) marked by the vAChT. Scale bars: (b and c, 100 μm). d-g, Current-clamp recordings of NPWFs in response to (d) step-stimulation, 50pA per step for 1second, 10 steps per sweep, and (e) ramp-stimulation, 2.5pA/s for 20 seconds per trail, 3 trails per sweep. f, Voltage-clamp recordings of spontaneous EPSCs. g, A sample spontaneous ESPC trace from f. h, Single-cell morphologies of NPWFs (ESC1) were reconstructed after physiological recording and intracellular dye filling showing the dendritic complexity. Scale bar: 100 μm. i, j, SC-specific Npnt expression using immunohistochemistry (i) was absent in the SC-specific conditional knockout (j). Scale bar: 200μm. k-m, Sample immunohistochemistry images showing differential mWmC transfer from distinct RGC starter neurons onto retinorecipient neurons in the SC (mWmC, mCherry signal in red). k, Percentages of Npnt-positive wide-field neurons (NPWFs, ESC1) within connected neurons of the αRGCs and ooDSGCs. Data in this figure are presented as mean ± SEM. NPWFs were enriched in αRGC tracing datasets (77.0±8.2% double-positive neurons among all Npnt-positive cells in l, n=5 animals) but largely absent in ooDSGC tracing datasets (4.5±2.1% double-positive cells among all Npnt-positive neurons in m, n=5 animals). ****, p<0.0001, two-sided Student’s t-test. l, αRGC ouputomes labeled with Kcng4:mWmC. ESC1 neurons were immunostained for Npnt. m, ooDSGC tracing datasets in SC labeled with Cart:mWmC. ESC1 neurons stained with Npnt. Bottom panels are magnified view of inset from l and m labeled by solid square line.