Fig. 2: 15q11.2 deletion decreases neurite complexity and length but not synaptic density.

A 3D reconstructions of 25X oil immersion confocal images of pAAV-hSyn-EGFP (SYNAPSYN-GFP) expressing representative neurons with corresponding Sholl intersections in control and 15q11.2 deletion neurons at DIC 80 and DIC 146. Panels on the left demonstrate reconstruction of GFP (green) with Sholl intersection overlay (multicolored spheres). Panels on the right demonstrate inverted images with Sholl intersection and neurite skeleton only. Scale bar 50 µm. B Quantification of Sholl analysis of N = 29 control cells from 4 lines and N = 26 deletion cells from 3 lines at DIC 80. Two-way ANOVA P < 0.0001. C Sholl analysis of N = 49 control cells from 4 lines and N = 45 deletion cells from 3 lines at DIC 146. Two-way ANOVA P < 0.0001. D Average Scholl intersections by line at 80 µm from data in B and C. Individual data points are means of 6–12 cells per donor line. Columns represent means ± SEM of n = three to four donor lines. Individual symbols are the mean of a single donor line. Two-way ANOVA P < 0.001. E Data from D presented by donor line. Columns represent mean per line and symbols are individual neurons. F Average total neurite length per cell is decreased in deletion compared to control donor lines. Symbols are average of cells from each donor line. Columns are means of N = 3 or 4 donor lines per condition and timepoint. G Data from F presented per donor line. Column is the mean per line and each symbol is a neuron. Two-way ANOVA P < 0.001. Tukey’s multiple comparisons * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001. H Representative 3D reconstructions of 63X confocal images of control (top) and 15q11.2 deletion (bottom) proximal neurites of SYNAPSIN-GFP (green) expressing neurons at DIC146 immunostained for either glutamatergic (left two columns) or GABAergic (right two columns) synapses. Glutamatergic synapses are identified based on immunostaining for vesicular glutamate transporter 1 (VGLUT1) (red) outside of the neurite and intracellular post synaptic density protein 95 (PSD95) (blue) (left panels). Panels on the right show the neurite mask generated by SYNAPSIN-GFP staining (green). The presynaptic puncta from glutamatergic synapses on to the masked neurons are generated from VGLUT1 staining outside of the mask (red spheres) that are within 1 µm of the PSD95 puncta within the neurite (blue spheres, masked by neurite). GABAergic synapses are identified by immunostaining for presynaptic glutamic acid decarboxylase 65 (GAD65) (red) and post synaptic scaffolding protein GEPHRYN (blue). Panels on the right show the mask generated by GFP staining (green). The presynaptic puncta from GABAergic synapses on to the masked neurons are generated from GAD65 staining outside of the mask and within 1 µm of the GEPHRYN puncta within the neurite. Post synaptic puncta are generated from GEPHRYN staining within the masked region. I Quantification of glutamatergic synapses, defined as pre and post synaptic puncta colocalizing within 1 µm of each other, normalized to the surface area of the masked region to determine density. J Quantification of glutamatergic synapses, defined as pre and post synaptic puncta colocalizing within 1 µm of each other, normalized to the surface area of the masked region to determine density. For I and J, each symbol type represents a different donor line with each symbol representing a neurite from a unique cell. Scale bar 3 µm. Not significant by two-tailed t test.