Suppl. Fig. 1 A)Gross brain morphology ofWTand α2δ3 KOmice.Brains of adult WT and α2δ3 KO mice did not differ in size and shape. Scale in mm. B) Original Western Blot for α2δ3 (made in-house).
Suppl. Fig. 2A) Overview image of global DiI tracing in WT and α2δ3 KO brains with injection into the thalamus (th) revealed similar results between genotypes. DiI-positive fibers from the thalamus reached the primary sensory cortex (S1). Control staining for B) L1 and C) MAP2 and neurofilament (NF). ic (internal capsule). Scale bars A) 500 μm B, C) 100 μm.
Suppl. Fig. 3 Neurofilament (NF) and MAP2 positive processes represent discrete neurites. A) Double and control (without primary antibody) staining of NF (red) and MAP2 (green) do not overlap. B) Overlay of TAU1 (red) and NF (turquoise) and partially with myelin (green) and appropriate control staining (without primary antibody) suggests that labeled processes are axons. Scale bars50μm.
Suppl. Fig. 4 Neuron specific c-fos activity and antibody specificity.A) C-fos antibody specificity was shown exemplarily in a WT brain using the blocking peptide (heat stimulation (pain)). Blocking experiments showed a fluorescent signal for c-fos in the analyzed brain regions, while the signal disappeared when slices were incubated with the c-fos peptide. B) NeuN and c-fos double labeling showed the activation of cortical neurons after heat stimulation (pain) in the different analyzed brain regions of both, mutant and control mice. DG (dentate gyrus of the hippocampus); S1limb (primary somatosensory cortex; limb region); V1 (primary visual cortex). Scale bars A) 50 μm; B) 20 μm.
Suppl.Tab.1 Quantification of α1 expression. A) Measurements of cells/mm2 and mean fluorescence intensity and B) number of analyzed mice, slices, images, and cells.
Suppl. Tab.2Measurements of MRI volume data and quantification of areas inNISSL-stained sections, and quantification of MAP2 and neurofilamentstaining. A) Mean area in pixel of white matter structures using MRI images (first pair of brains) and B) number of analyzed images. C) Mean area in pixel of NISSL-stained structures and D) number of analyzed slices and E) mice. F) Mean gray value of MAP2 staining and mean thickness of cortical layers and G) number of analyzed mice, slices and images. H) Mean gray value of neurofilament (NF) staining and mean thickness of cortical layers and I) number of analyzed mice, slices and images.
Suppl. Tab.3Quantification of c-fos, GluT and GAD67 expression following stimulation in the dentate gyrus (DG), primary somatosensory cortex barrel field (S1BF), primary somatosensory cortex limb region (S1limb) and primary visual cortex (V1). A) Measurements of c-fos positive cells/mm2 in the control, heat pain and touch condition and B) number of analyzed mice, slices, regions and images. C) measurements of positive cells/mm2 for GluT+, GAD67+, c-fos+/GluT+, and c-fos+/GAD67+ after heat pain stimulation in the primary somatosensory cortex (S1) and primary visual cortex (V1) and D) number of analyzed mice, slices and images.
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