Fig. 1
AAV-Cre-induced loss of paranodal junction in the internal capsule. a Schematic diagrams showing the design of the AAV-EGFP-2A-Cre constructs. The 2A peptide is cleaved just after translation, and EGFP and Cre recombinase are expressed independently. b, c Time course and schematic drawing of our experiments. To examine whether site-directed loss of paranodal junctions causes a delay in cortically evoked electromyograms (EMGs), a pair of bipolar stimulating electrodes was chronically implanted into the motor cortex (MCx), and EMG recording electrodes were placed in the triceps brachii muscles contralateral to the cortical stimulation electrodes in 6-week-old NfasccKO and Nfasc+/+ mice. AAV5-EGFP-2A-Cre was injected into the internal capsule (IC) of the mice at week 0. Evoked EMGs by cortical stimulation (Stim.) were recorded every week before (week 0) and after (from week 1 to week 13) AAV injection. d Representative micrograph of AAV-mediated EGFP expression (green) in the internal capsule 12 weeks after AAV injection. White dotted lines indicate the boundaries of the internal capsule (IC). Scale bar 250 μm. e Doubly staining by in situ hybridization for PLP mRNA (blue) and immunostaining with an anti-GFP antibody (brown) in the IC 12 weeks after AAV injection. Arrowheads indicate double positive cells for both PLP mRNA and GFP. The inset is a magnified view of the cell indicated by the arrow. Scale bar 50 μm (25 μm in inset). f Immunofluorescence staining with an anti-Caspr (green) and anti-Na+ channel (red) antibodies in sections containing the IC of Nfasc+/+ (left) or NfasccKO (right) mice 12 weeks after AAV injection. Each inset shows a magnified view of the immunolabeling indicated by the arrow. Scale bar 20 μm (5 μm in inset). Quantification of the number (g) and length (h) of Caspr-positive paranodes in Nfasc+/+ and NfasccKO mice injected with AAV5-EGFP-2A-Cre vector (Student’s t test, number [t = 4.623, df = 4, **p < 0.01], length [t = 5.319, df = 4, **p < 0.01]; n = 3 mice each). i Quantification of the number of Na+ channel-positive nodes in Nfasc+/+ and NfasccKO mice injected with AAV5-EGFP-2A-Cre vector (Student’s t test, t = 1.504, df = 4, p = 0.2069; n = 3 mice each). All data are shown as mean ± SEM