Fig. 5

Experimental set-up of an acute axonal degeneration model in vitro. A Experimental scheme for the in vitro live imaging procedure. On DIV 0, rat cortical neurons were transduced with the AAV.mScarlet-LC3, which were subsequently imaged on DIV 10. The medium was changed every two days. B Vector diagram of AAV.mScarlet-LC3. As an adeno-associated viral vector, it expresses mScarlet fluorophores fused to LC3. ITR: AAV-6 inverted terminal repeat. CB chicken beta-actin. CMV cytomegalovirus promoter. WPRE woodchuck hepatitis virus posttranscriptional regulatory element. bGH-pA bovine growth hormone-polyadenylation site. C Exemplary image of rat cortical neurons transduced with LC3 AAV vectors in the microfluidic chamber on DIV 10. D Representative clearly labeled autophagosomes on the axon transduced with the given LC3 AAV vector on DIV 10. Scale bar: 5 μm. E Schematic drawing of axotomy in microfluidic chambers on DIV 10. Each microfluidic chamber comprises 4 wells and 2 compartments connected by 450 μm long microgrooves. The soma compartment serves as the locus for seeding primary cortical neurons. Only axons have the ability to cross the microgrooves and reach the opposite side, named the axonal compartment. Axotomy is performed only in the axonal compartment to induce acute axonal degeneration