AbstractVoltage gated sodium channels are key players in aberrant pain signaling and sensitization of nociceptors after peripheral nerve injury. The extent to which sodium channel activity after injury contributes to synaptic transmission at the first pain synapse however remains unclear. To investigate the effect of axotomy on synaptic transmission between dorsal root ganglia neurons and dorsal horn neurons, we reconstructed the first pain synapse in a novel microfluidic based compartmentalized cell culture system, which recapitulates the connectivity of peripheral pain signaling. We show that following axotomy of the distal axons, inhibition of Na_V1.7 and Na_V1.8 sodium channels in incoming presynaptic DRG axons is no longer sufficient to block activation of these axons and the resulting synaptic transmission to dorsal horn neurons. We found that blockade of Na_V1.6 activity is highly effective in reducing activation of incoming axons contributing to synaptic transmission after axotomy of DRG neurons. The microfluidic culture system described here offers an in vitro platform to recapitulate and study the first pain synapse.