Ferret VAChT-SAP treatment disrupts receptive field size of On- and Off-center dLGN neurons. (A) On and Off responsive units are center-surround organized and of a single center-type, either On-center or Off-center in both saline and Ferret VAChT-SAP-treated groups (scale inset equals 2°² of visual angle). (A,B) Treatment with Ferret VAChT-SAP leads to enlarged receptive fields for both On-center and Off-center neurons recorded in the dLGN (means ± SEMs). Statistics reflect two-tailed P-values calculated from independent two sample t-tests.

Ferret VAChT-SAP treatment disrupts spontaneous retinal activity during P15 to P25. (A) Raster plots from MEA recordings depict abnormal spontaneous retinal activity in Ferret VAChT-SAP-treated retinae compared to saline controls. (B) Correlation index analysis of RGC output shows that correlations are reduced at all inter-pair intervals in Ferret VAChT-SAP versus saline groups. (C,D) Ferret VAChT-SAP reduces overall activity levels (C) and overall RGC burst frequency (D) compared to saline controls. (E,F) Treatment with Ferret VAChT-SAP increases RGC burst duration (E) and decreases the number of spikes/burst (F). Panels (B-F) reflect means ± SEMs for grouped data from recordings at P15 (n = 2), P20 (n = 2), and P25 (n = 2). Statistics reflect two-tailed P-values resulting from independent two-sample t-tests.

Ferret VAChT-SAP treatment does not disrupt physiological On and Off organization in the dLGN. On and Off responsive units are located along runs of same center-type response in both saline and Ferret VAChT-SAP recovery animals. (A) Distributions of same center-type run lengths are not significantly different between the groups. (B) Nearest neighbor analysis of On and Off organization reveals that, for both saline and Ferret VAChT-SAP groups, On and Off units exhibit significantly greater clustering with neighbors of the same center-type than would be predicted by a randomized distribution of the cell populations. (C) The proportions of On/Off/Mixed responses recorded in the dLGNs of saline and Ferret VAChT-SAP cohorts are not different for any of the response types. Statistics in (C) reflect two-tailed P-values resulting from independent two-sample t-tests. N.S., not significant.

Effects of Ferret VAChT-SAP treatment on other retinal neuron types. (A,B) Treatment with Ferret VAChT-SAP leads to ablation of calbindin(+) amacrine cells and horizontal cells. (C,D) Calretinin(+) RGCs and HCs are also ablated by immunotoxin treatment. (E,F) Recoverin(+) photoreceptors appear qualitatively similar in both groups, but the retinal sheet is thinner in immunotoxin-treated retinae compared to controls. Data reflect means ± SEMs. Statistics reflect two-tailed P-values calculated from independent two sample t-tests. 'Merge' panels are counterstained with DAPI to reveal cytoarchitecture. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PRL, photoreceptor layer. Scale bars: 100 μm.

Effects of Ferret VAChT-SAP treatment on starburst amacrine and retinal ganglion cell ablation. (A,B) Treatment with Ferret VAChT-SAP leads to ablation of ChAT(+) starburst amacrine cells (SACs) and loss of vesicular acetylcholine transporter (VAChT) expression. (C-F) Brn3a(+) and NeuN(+) retinal ganglion cells (RGCs) are also ablated, although to a lesser degree than SACs, by Ferret VAChT-SAP treatment ((C,E) photomicrographs; (D,F) quantification). Data reflect means ± standard errors of the mean. Statistics reflect two-tailed P-values calculated from independent two sample t-tests. 'Merge' panels are counterstained with DAPI to reveal cytoarchitecture. GCL, ganglion cell layer; IPL, inner plexiform layer, INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PRL, photoreceptor layer. Scale bars: 100 μm.

Ferret VAChT-SAP disrupts development of On and Off sublamination. (A) On and Off retinogeniculate afferents in both dLGNs (left and right panels) at P25 are separated by intersublaminar spaces into distinct sublaminae in saline controls (arrows in (A), upper panels), but sublaminae are absent in Ferret VAChT-SAP-treated ferrets (A, lower panels). (A,C) The size of contralateral and ipsilateral layers is reduced by Ferret VAChT-SAP treatment, but eye-specific segregation is normal and the interlaminar space is unaffected. (B-D) Treatment with Ferret VAChT-SAP prevents the normal expression of ABAKAN (indicated by arrowheads in upper panels of (B)) in the intersublaminar space between On and Off sublaminae, but not in the interlaminar space between eye-specific layers. A, major contralateral layer; A1, major ipsilateral layer; On, On-center sublamina; Off, Off-center sublamina; C, minor contralateral layer; C1, minor ipsilateral layer; C2, minor contralateral layer; C3, retinal-afferent-free layer; W, geniculate wing; MIN, medial intralaminar nucleus. Panels (C,D) reflect means ± SEMs; statistics reflect two-tailed P-values resulting from independent two-sample t-tests. Coordinate insets in (A,B) reflect anterior-posterior and medial-lateral orientation within the dorsal thalamus. Scale bars: 200 μm. N.S., not significant.