Volume 93, Issue 4, Pages 767-776.e6 (February 2017) Different Modes of Visual Integration in the Lateral Geniculate Nucleus Revealed by Single-Cell-Initiated Transsynaptic Tracing  Santiago B. Rompani, Fiona E. Müllner, Adrian Wanner, Chi Zhang, Chiara N. Roth, Keisuke Yonehara, Botond Roska  Neuron  Volume 93, Issue 4, Pages 767-776.e6 (February 2017) DOI: 10.1016/j.neuron.2017.01.028 Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Single-Cell-Initiated Transsynaptic Tracing from LGN Principal Cells (A) Schematic of the experimental design. AAV expressing Cre-GFP was injected into V1 (left). A GFP-positive LGN cell was electroporated with a combination of three plasmids and Alexa-594 dye (middle). EnvA-SADΔG-mCherry rabies virus was injected into the LGN (right). (B) Two-photon image of a GFP-expressing LGN cell after electroporation (left, GFP only; middle, Alexa-594 only; right, combined; arrow, pipette). (C) TdTomato-expressing neuron in LGN, 3 days post-electroporation (left, whole brain; right, zoom-in to LGN; arrow, axon). (D) A cluster of ganglion cells in the retina 12 days after electroporation. Retina, with four indentations to flatten it, is outlined by dotted lines. (E) Ganglion cells of the cluster in (D) traced and color-coded based on morphological type. See Figure S1. Neuron 2017 93, 767-776.e6DOI: (10.1016/j.neuron.2017.01.028) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 LGN-Projecting Ganglion Cell Types Each row refers to a different type of ganglion cell observed in single-cell tracing. (A) Dendritic arbors of three example ganglion cells (only one for type 13). (B) Side projection of a representative ganglion cell (gray, antibody against ChAT; red dotted lines, ChAT strata). The boxes on the right represent the ten strata of the inner plexiform layer (black squares, dendrites of respective ganglion cell type; gray squares, ChAT strata). See Figure S2. Neuron 2017 93, 767-776.e6DOI: (10.1016/j.neuron.2017.01.028) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Monocular Clusters of Presynaptic Ganglion Cells (A) Distribution of ganglion cell types in single-cell (gray) or bulk tracing (white). (B) Distribution of number of ganglion cell types per monocular cluster (gray) or per pair of binocular clusters (white, ipsi- and contralateral combined). (C) The deviation of measured number of ganglion cell types from what is expected by a random draw (ipsilateral and contralateral binocular clusters shown separately). p values from Monte-Carlo simulations, ∗∗p < 0.01, ∗∗∗p < 0.001; n.s., not significant. (D) A representative relay-mode cluster. (E) Reconstruction of the ganglion cells in (D). (F) Dendritic stratification of the ganglion cells in (D). Each column refers to one presynaptic ganglion cell. Number on black boxes refers to the corresponding ganglion cell type. (G) The distribution of ganglion cells in relay-mode clusters based on dendritic stratification. Strata 1 and 2, OFF-sustained; 3 and 4, OFF-transient; 6 and 7, ON-transient; 8 and 9, ON-sustained responses. (H) A representative combination-mode cluster. (I) Reconstruction of the ganglion cells in (G). (J) Dendritic stratification of the ganglion cells in (H). (K) The distribution of ganglion cells in combination-mode clusters based on dendritic stratification. Cells were counted in all layers in which they stratify (G and K). See Figures S3–S6. Neuron 2017 93, 767-776.e6DOI: (10.1016/j.neuron.2017.01.028) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Binocular Clusters of Presynaptic Ganglion Cells (A) Representative binocular clusters of an LGN cell. (B) Dendritic stratification patterns of the ganglion cells in (A). (C) The distribution of ganglion cells in binocular clusters based on dendritic stratification; gray, ipsilateral; white, contralateral clusters. Cells were counted in all layers in which they stratify. (D) Pairwise comparison of ganglion cell type dominance between pairs of ipsilateral and contralateral clusters. (E) Comparison between the expected (black bars) and observed (red line) distribution of the mean Z score of absolute differences in ganglion cell count (|ipsilateral − contralateral|). Expected distribution based on binomial model. (F and G) Pairwise comparison of (F) number of ganglion cell types, and (G) Z scores of the number of ganglion cell types between pairs of ipsilateral and contralateral clusters. Red, c > i, more cells in the contralateral eye; black, i > c, more cells in ipsilateral eye. Blue lines in (G), mean Z scores. Lines connect corresponding cluster pairs (D, F, and G). (H) Pairwise comparison of the measured and expected number of ganglion cell types shared between an ipsilateral and corresponding contralateral cluster. Expected values are based on the random draw hypothesis with conditional probabilities to correct for reduced numbers of ganglion cell types ipsilaterally (Figure S8D). p values from Wilcoxon signed-rank test in (D) and (F) and Monte-Carlo simulations in (E) and (G) (two-sided) and (H) (left-tailed), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n.s., not significant. See Figures S7 and S8. Neuron 2017 93, 767-776.e6DOI: (10.1016/j.neuron.2017.01.028) Copyright © 2017 The Author(s) Terms and Conditions