Pseudorabies virus(PRV),a neurotropic virus perfectly suited for neuronal tracing,which can be transported in neuronal processesand acrosssynaptic gaps,and thus is able to spread in neuronal circuits.Previous work has shown that PRV can travel from postsynaptic sites to axon termini,buthow PRV transport in neurons and cross the synapses are still unclear.Here,we use optical correlative imaging method based on super-resolution microscopy imaging and single-particle tracking to study the transport of PRV in cultured hippocampal neurons,especially the retrograde trans-synaptic movements.We mapped the trajectories of PRV in regard to the locations of microtubules and synaptic sites.The virions could travel along a microtubule toward plus-or minus-end,switch to another microtubule and cross synapses.Furthermore,the velocity of PRV in neuronsdecreasedafter chronic down-regulation of neural activity with TTX-treatment,but did not change by acute blocking of activity.These results suggest that neuronal activity may play an important role in the spread of PRV.