Fig. 2

High Shank2 expression identifies a distinct neuronal subpopulation in the spinal cord. Identification of shank2 expression in the spinal cord. a and b Shank2 IF staining revealed a synaptic pattern around cells in the dorsal and ventral horn, with a subset of Shank2 high expression neurons in laminae III, IV and V (mean intensity; 785 ± 99, 788 ± 100, 892 ± 241, 1097 ± 256, 941 ± 235, Laminae I–V respectively); (N = 3; n = Laminae I–III: 180; Laminae IV–V: 181 neurons), scale bar: 100 µm. c and d These Shank2^high expressing neurons were further divided into location from midline in laminae III, IV and V (median; 425 ± 138 vs. 375 ± 122 Shank2^high vs. Neu-N; (N = 3; n = Shank2^high: 78; Neu-N + : 155 neurons), scale bar: 50 µm. e and f High magnification confocal pictures reveal a continuous band of Shank2 around cells, however STED imaging shows the distinct punctuate patterns of Shank2 colocalised with Bassoon; (N = 3), scale bar E: 1 µm; scale bar F: 500 nm. g Sparse labeling of neurons in the dorsal laminae was performed by AAV9-CMV-GFP intraspinal injection, combined with Shank2 IF staining. Shank2 is highly distributed on cell-body and dendrites; (N = 3), scale bar: 1 µm. h Shank2 Western blot on cortex (Cx), hippocampus (Hp), striatum, (Str), cerebellum (Cb), spinal cord (Sc), dorsal root ganglia (Drg), positive control ( +) and negative control ( −), reveals 4 isoforms in the spinal cord located at 220–240, 160, 140 and 100 kD. The isoforms 160 and 100 were also detectable in other CNS regions like cortex, hippocampus, cerebellum, and Dorsal root ganglia. The cerebellum also showed the 220–240 isoform which was not found in cortex, hippocampus or DRG; (N = 3). i, j IF staining and western blot of WT and Shank2^−/− mice show an almost complete loss of Shank2^high expressing neurons in laminae III, IV and V, this loss corresponds to the loss of MW isoforms at 240 and 160 kD, the highest and lowest MW were still expressed; (N = 3), scale bar J: 100 µm