PyrNs respond to gradual changes in display luminance. A Overview of expression pattern in a 6 dpf id2b:gal4,uas:egfp,HuC:lynTagRFP-t larva. id2b-positive neurons are labeled in cyan, while axon tracts and neuropil are labeled in red. Upper right: reconstructions of the three neuron types labeled in the id2b:gal4 transgenic. Lower right: region of analysis that includes the deep tectal neuropil and shallow SPV. B Maximum projection whole-brain image of a 7dpf id2b:gal4,uas:gcamp6s larva with a single mTomato-labeled PyrN (red arrowhead). C Magnified view of boxed region in B containing a mTomato-labeled PyrN. D 40° rotated view around X-axis of boxed region in B. Note three distinct neurite stratifications in SM, SFGS, and SGC layers. E Standard deviation (StDev) projection image of a multiphoton timeseries acquired from region indicated in B during presentation of 10s ramp stimulus. Pixels with higher values correspond to regions that underwent large variations in fluorescence intensity during recording interval. Red arrowhead indicates mTomato labeled PyrN in B?D. F Ramp-evoked responses in neurons labeled 1?4 in E during presentation of 10s and 30s ramp stimuli. Red trace corresponds to the mTomato labeled PyrN in B?E. Note that all four neurons exhibit strong cycle locked responses that peak near the minimum stimulus light intensity. G Output of linear SVM classifier run on data acquired from 127 PyrNs in 6 larvae during presentation of 10s ramp stimulus. Note ON, OFF, and DUAL responses and a subset of neurons that did not respond to visual stimulation. H Confusion matrix demonstrating SVM performance compared to investigator-determined ?true? classification. I Average responses to 10s ramp stimulus recorded from 100 active PyrNs in G classified by the linear SVM classifier as ON, OFF, or DUAL. Fourth panel depicts merged overlay of three response types. Note sequential activation of DUAL, ON, and OFF PyrNs during each cycle of the luminance ramp. Scale bar, 50?m in A, 75?m in B, 30?m in C?D, and 25?m in E. TL Torus longitudinalis, SPV stratum periventriculare

A subset of RGC inputs to tectum encode gradual changes in display luminance. A Retinal input layers of the tectal neuropil visualized in the left tectum of a 7dpf atoh7:gcamp6s larva. For clarity non-retinal layers are not labeled. B Threshold image of GCaMP6s signal in A. C Image in A with colored overlay denoting active ROIs detected during presentation of 30s ramp stimulus. D Layer distribution of ramp-responsive RGC ROIs. Data from 367 ROIs detected in 7 larvae. One-way ANOVA with Tukey?s multiple comparisons test was performed, p value <0.0001 is for each pairwise comparison between SFGS5/6 and every other group. E Average responses of ON, OFF, and DUAL response classes detected by linear SVM trained on PyrN 30s ramp data. Data from 545 ROIs detected in 6 larvae. F Comparison of response class distribution between PyrNs and RGC inputs. Note greater proportion of ON responsive units among RGC inputs compared to PyrNs. N=30 neurons from 5 larvae, two-way ANOVA, interaction effect p=0.002976, with posthoc unpaired t test. G Comparison of response onset (LUX at half-max) in PyrNs and RGC ROIs during 10s ramp stimulus presentation. Two-way ANOVA, interaction effect p=0.1657, with posthoc unpaired t test. H Comparison of response duration (width at half-max) in PyrNs and RGC ROIs during 10s ramp stimulus presentations. Two-way ANOVA, interaction effect p<0.0001, with posthoc unpaired t test. Note: for t test comparisons in F?H only p values that reached significance are shown. Scale bar: 50?m in A?C

PyrNs exhibit binocular responses. A Schematic overview of left and right stimulus presentation experiment. B StDev projection image of a timeseries acquired from a id2b:gcamp6s larva during ramp stimulus presentation to the left eye. Note the presence of active PyrNs in both the contralateral OT (black arrowheads) and ipsilateral OT (red arrowheads). C StDev projection image of same tectum as B during 10s ramp stimulus presentation to the right eye. Note the presence of active PyrNs in both the contralateral OT (black arrowheads) and ipsilateral OT (red arrowheads). D Schematic overview of stimulus presentation to the right eye of enucleated larvae. Lower panel is a transmitted light image of an 8dpf larva in which the left eye was surgically removed at 3dpf. E StDev projection image of a timeseries acquired from a left eye-enucleated larva during 10s ramp stimulus presentation to the right eye. F Ramp-evoked responses in contralateral neurons (c1-3) and ipsilateral neurons (i1-3) of tectum shown in E. Note that all six neurons exhibit consistent responses to each ramp stimulus cycle. G Response class distribution in left tectum (normal retinal input) and right tectum (no retinal input) of left eye-enucleated larvae. N = 26 and 19 neurons from 9 larvae for each condition, two-way ANOVA, interaction effect p=0.985, with posthoc unpaired t test. H, I Comparison of response onset (LUX at half max) and response duration (LUX at half max) in the left and right tectum of left eye-enucleated larvae presented with 10s ramp stimuli. Two-way ANOVA, interaction effects p=0.607 and 0.946, with posthoc unpaired t test. Note: for t test comparisons in G?I only p values that reached significance are shown. Scale bar: 50?m in B, C, and E

TL is the site of binocular integration. A Maximum projection image acquired from a 7dpf Tg(hspGGFF23C,uas:gcamp6s,uas:mcherry) triple transgenic larva. Note labeled cell bodies in TL and axonal plexus in OT. B Coronal view of green bracketed region in A. Note that axons from TL target the SM layer, directly beneath the skin overlying the tectum. C Magnified view of white bracketed region in B (mcherry channel only). Arrowheads denote SMTL neurons with axons extending into ipsilateral OT. D ON, OFF, and DUAL response classes identified in hspGGFF23C+ SMTL neurons. E Comparison of response class distribution in id2b+ PyrNs and hspGGFF23C+ SMTL neurons. Note greater proportion of ON responsive units among hspGGFF23C+ TL neurons. Two-way ANOVA, interaction effect p<0.0001, with posthoc unpaired t tests. F Comparison of response onset (LUX at half-max) in PyrNs and SMTLs in response to 10s ramp stimulus. Two-way ANOVA, interaction effect p=0.9317, with posthoc unpaired t tests. G Comparison of response duration (width at half-max) in PyrNs and SMTLs in response to 10s ramp stimulus. Two-way ANOVA, interaction effect p=0.989, with posthoc unpaired t tests. Note: for t test comparisons in E?G only p values that reached significance are shown. H Magnified view of anterior TL in a left eye-enucleated 7dpf Tg(hspGGFF23C,uas:gcamp6s) larva. Upon stimulus presentation to the right (intact) eye, three SMTLs in right TL exhibited visual responses (1?3). I Responses in SMTL neurons 1?3 in H during presentation of 10s ramp stimulus to the right (intact) eye. Note that the three neurons exhibited consistent responses to each ramp cycle. J Quantification of percent of SMTLs in left and right TL of enucleated larvae that responded during 10s ramp stimulus presentation to the right (intact) eye. N = 12 larvae, paired t test. Scale bar: 60?m in B, C, 30?m in D, and 20?m in K

TL is required for PyrN binocularity. A Maximum projection image of a 6 dpf Tg(vglut2a:dsred, id2b:gcamp6s) double transgenic larva prior to laser ablation of TL. B Larva in A at 7dpf, 1 day following targeted laser ablation of TL at 6 dpf. Note normal brain structure and similar density of GCaMP6s+ PyrNs in OT. C Sideview images of green-boxed regions in A and B. Note reduction in vglut2a:dsred+ cells in TL ablated larva. Graph at right depicts quantification of TL ablation using area measurements obtained from sideview projections. N = 6 larvae, paired t test. D Single confocal images at Z-planes through dorsal TL indicated by blue lines in C. Note strong reduction in vglut2a:dsred+ cells in TL ablated larva. E StDev projection image of timeseries acquired from a left eye enucleated larva with intact TL. Black arrowheads denote active neurons in contralateral OT with intact retinal input, red arrowheads denote active neurons in ipsilateral OT devoid of retinal input. Note similar proportion of active PyrNs in contra and ipsi OT. F StDev projection image of timeseries acquired from a left eye enucleated and TL-ablated larva. Black arrowheads denote active neurons in contralateral OT with intact retinal input, red arrowheads denote active neurons in ipsilateral OT devoid of retinal input. Note reduced number of active neurons in ipsilateral OT compared to contra OT. G Percentage of active neurons located in right/ipsilateral tectum during stimulation of right eye in enucleated larvae and larvae that underwent both enucleation and TL ablation. n=21 and 20 larvae, p=0.0033, unpaired t test. H Quantification of active PyrNs (expressed as a percentage of total id2b+ neurons) in ipsilateral vs. contralateral tectum in left eye-enucleated larvae during 10s ramp presentation to right eye. N=10 larvae, paired t test. I Quantification of active PyrNs (expressed as a percentage of total id2b+ neurons) in ipsilateral vs. contralateral tectum in larvae that underwent both enucleation and TL ablation during 10s ramp presentation to right eye. Note strong reduction in active PyrNs in all but one larva. N=8 larvae, paired t test. Scale bar: 75?m in A?B, 50?m in C?D, and 60?m in E?F

Visual receptive fields of PyrNs, SMTLs, and RGCs. A Overview of experimental design to monitor ramp-evoked responses in elements of the tectum-TL circuit. Left and right displays are used to present single 15° squares within a 6x4 checkerboard in a pseudorandom pattern. GCaMP6s signal intensity was used to quantify response strength at each position. Position response strength was subsequently used to generate RF maps. B Maximum projection image of an image timeseries acquired in tectum of a id2b:gcamp6s larva presented with RF mapping stimuli. C Example RF maps for the four PyrNs indicated in B. Note large, complex RFs and asymmetries in RF size between left and right visual fields. D Quantification of RF size in contralateral and ipsilateral RFs of individual PyrNs. Note high degree of variability in RF size and contra/ipsi ratio. E Distribution of contra/ipsi RF size ratio for 55 PyrNs recorded in 7 larvae. F Comparison of RF size in PyrNs, SMTLs, and RGCs. One-way ANOVA with Tukey?s multiple comparisons test, p<0.0001 for comparisons between each indicated group and the contra-PyrN group. G Maximum projection image of an image timeseries acquired in TL of a gff23c:gcamp6s larva presented with RF mapping stimuli. Arrow denotes SMTL with visible axon extending towards the right tectal lobe. H Example RF maps for the four SMTLs indicated in G. Note large, but discrete RFs and asymmetries in RF size between left and right visual fields. I Maximum projection image of an image timeseries acquired in the left tectum of a atoh7:gcamp6s larva presented with RF mapping stimulus to the right eye. Overlaid on this image are active RGC ROIs detected in response to presentation of a 30s luminance ramp stimulus to the right eye. Arrows denote four RGC ROIs at distinct positions along the A?P axis. J Example RF maps for the four RGC ROIs indicated in I. Note compact, discrete RFs and nasal-temporal distribution of RF positions match anteroposterior position of ROIs in the tectum. Scale bar: 75?m in B, 25?m in G, and 40?m in I

Model for spatial summation in the TL-PyrN circuit. A Dorsal view image of a Tg(HuC:lynTagRFP-t) larval brain. In this transgenic, all axon tracts and neuropil areas are fluorescently labeled. Image grayscale was inverted for clarity. Overlayed on the midbrain are reconstructions of a single SMTL (orange), a single RGC axon (cyan), and a single PyrN (red, blue, and green). B Magnified view of SMTL, RGC, and PyrN morphologies. Note large, sparsely branched SMTL axon that forms excitatory inputs (+) onto the SM PyrN dendrite (red) and the compact RGC axon that forms excitatory input (+) onto the SFGS PyrN dendrite (blue). C Model of how spatial summation in the TL-tectal circuit could be mediated by neural convergence at the SMTL-PyrN synapse. Direct RGC input to PyrNs specifies response class. In parallel, the same RGCs provide input to TLPNs that drive visual responses in TL. Three SMTLs with intermediate sized RFs converge onto the same dendrite. Spatial summation of inputs with partially overlapping RFs results in a large, compound RFs in PyrNs