LacZ Adeno-associated virus(AAV Serotype 8)

Corticospinal (CS) neurons in layer V of the sensorimotor cortex are essential for voluntary movement control. Here, researchers show that the semaphorin 3A (Sema3A)-neuropilinin-1 (Npn-1) signaling pathway is an essential negative regulator of CS axon collateral formation in the mouse spinal cord in both sexes. Sema3A is expressed in the ventral spinal cord, whereas CS neurons express Npn-1, suggesting that Sema3A may prevent CS axons from entering the ventral spinal cord. Indeed, ectopic expression of Sema3A in the spinal cord in vivo inhibits CS axon collateral formation, and Sema3A or Npn-1 mutant mice have ectopic CS axon collateral formation within the ventral spinal cord compared with littermate controls. Finally, Npn-1 mutant mice exhibit impaired skilled movements, likely due to abnormal CS connection formation in the ventral spinal cord. These genetic findings suggest that Sema3A–Npn-1 signaling-mediated inhibition of CS axon collateral formation is essential for proper CS circuit formation and the ability to perform skilled motor behaviors.

To visualize CS axon collaterals, Emx1-Cre mice were crossed with a conditional GFP reporter mouse strain (Emx1-Cre; ccGFP). GFP+ CS axonal collaterals were observed in the lumbar spinal cord of P14 Emx1-Cre;ccGFP mice. Most of these GFP+ axonal collaterals were restricted to the dorsal spinal cord, with only a few collaterals located medially and ventrally in the spinal cord (Figure 1A-C). AAV8-Sema3A and AAV8-LacZ viruses were then coinjected into the L2 and L4 regions of the lumbar spinal cord of P0 Emx1-Cre;ccGFP mice. The spread of AAV8 infection was assessed by LacZ immunoreactivity detected throughout the lumbar (L2-L4) spinal cord (Figure 1D-F). Emx1-Cre;ccGFP mice that received AAV8-LacZ injection alone were used as controls (Figure 1A-C). Researchers observed a significant reduction in CS axon collaterals in the dorsal and medial regions of the lumbar spinal cord in mice injected with AAV8-Sema3A/AAV8-LacZ virus compared with control mice (Figure 1G–L, S). No significant reduction was observed in the ventral horn of the lumbar spinal cord of mice injected with AAV8-Sema3A/AAV8-LacZ (Figure 1K, L, Q-S), consistent with the observation that endogenous expression of Sema3A is enriched in the ventral spinal cord. These results indicate that elevated Sema3A expression is sufficient to reduce axon collateral formation of CSNs in the medial and dorsal spinal cord.

Figure 1. Sema3A negatively regulates CS collateral formation in vivo. (Gu Z, et al., 2019)