Figure 1.
Figure 1. snph KO cortical neurons display enhanced axon regrowth capacity. (A and B) A microfluidic chamber allows physical and fluidic separation of axons from cell bodies and dendrites. Cortical neurons were seeded in the soma chamber where cell bodies and dendrites are restricted to the soma chamber, whereas axons grow into the axon terminal chamber through the microgroove channels (MC; 450 µm in length). The device (a) allows quick and definitive detection of axons, (b) determines axon directionality, (c) reduces background signals from glial and neuronal cell bodies and dendrites, (d) permits axotomy along the edge of microgrooves by vacuum aspiration, and (e) measures axon regrowth. Neurons were stained with βIII-tubulin (A) or infected with lenti-YFP (B); axon removal in the terminal chamber by vacuum aspiration was confirmed by imaging live neurons expressing YFP before and after axotomy (B). (C–E) Images (C and D) and quantitative analysis (E) showing enhanced axon regrowth after axotomy in snph KO neurons. WT (C) and snph KO (D) cortical neurons were axotomized at DIV4 and allowed to regrow for 3 d. Axons were immunostained with βIII-tubulin at DIV7. Note that the normalized area of regenerating axons was significantly increased in snph KO neurons relative to controls (P < 0.001). (F and G) Images (F) and quantitative analysis (G) of WT and snph KO cortical neurons showing early phases of axon regrowth at 7 and 28 h after axotomy. (H and I) Representative images (H) and quantitative analysis (I) showing the formation of new growth cones after axotomy. Axonal terminals were costained with βIII-tubulin and Alexa Fluor 543 phalloidin 14 h after axotomy. Note that deleting snph facilitates the formation of growth cones (69.11 ± 2.90%, P = 0.0002) compared with WT (44.46 ± 1.92%) from injured axon tips. Imaging data were pooled from a total number of microgroove channels indicated within bars (E and G) taken from three pairs of littermates or a total number of axonal terminal chambers indicated within bars (I) and expressed as mean ± SE (Student’s t test).

snph KO cortical neurons display enhanced axon regrowth capacity. (A and B) A microfluidic chamber allows physical and fluidic separation of axons from cell bodies and dendrites. Cortical neurons were seeded in the soma chamber where cell bodies and dendrites are restricted to the soma chamber, whereas axons grow into the axon terminal chamber through the microgroove channels (MC; 450 µm in length). The device (a) allows quick and definitive detection of axons, (b) determines axon directionality, (c) reduces background signals from glial and neuronal cell bodies and dendrites, (d) permits axotomy along the edge of microgrooves by vacuum aspiration, and (e) measures axon regrowth. Neurons were stained with βIII-tubulin (A) or infected with lenti-YFP (B); axon removal in the terminal chamber by vacuum aspiration was confirmed by imaging live neurons expressing YFP before and after axotomy (B). (C–E) Images (C and D) and quantitative analysis (E) showing enhanced axon regrowth after axotomy in snph KO neurons. WT (C) and snph KO (D) cortical neurons were axotomized at DIV4 and allowed to regrow for 3 d. Axons were immunostained with βIII-tubulin at DIV7. Note that the normalized area of regenerating axons was significantly increased in snph KO neurons relative to controls (P < 0.001). (F and G) Images (F) and quantitative analysis (G) of WT and snph KO cortical neurons showing early phases of axon regrowth at 7 and 28 h after axotomy. (H and I) Representative images (H) and quantitative analysis (I) showing the formation of new growth cones after axotomy. Axonal terminals were costained with βIII-tubulin and Alexa Fluor 543 phalloidin 14 h after axotomy. Note that deleting snph facilitates the formation of growth cones (69.11 ± 2.90%, P = 0.0002) compared with WT (44.46 ± 1.92%) from injured axon tips. Imaging data were pooled from a total number of microgroove channels indicated within bars (E and G) taken from three pairs of littermates or a total number of axonal terminal chambers indicated within bars (I) and expressed as mean ± SE (Student’s t test).

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