<i>In vivo</i> two‐photon imaging of structural dynamics in the spinal dorsal horn in an inflammatory pain model

Abstract Two‐photon microscopy imaging has recently been applied to the brain to clarify functional and structural synaptic plasticity in adult neural circuits. Whereas the pain system in the spinal cord is phylogenetically primitive and easily exhibits behavioral changes such as hyperalgesia in response to inflammation, the structural dynamics of dendrites has not been analysed in the spinal cord mainly due to tissue movements associated with breathing and heart beats. Here we present experimental procedures to prepare the spinal cord sufficiently to follow morphological changes of neuronal processes in vivo by using two‐photon microscopy and transgenic mice expressing fluorescent protein specific to the nervous system. Structural changes such as the formation of spine‐like structures and swelling of dendrites were observed in the spinal dorsal horn within 30 min after the multiple‐site injections of complete Freund's adjuvant (a chemical irritant) to a leg, and these changes continued for 5 h. Both AMPA and N ‐methyl‐D‐aspartate receptor antagonists, and gabapentin, a presynaptic Ca 2+ channel blocker, completely suppressed the inflammation‐induced structural changes in the dendrites in the spinal dorsal horn. The present study first demonstrated by in vivo two‐photon microscopy imaging that structural synaptic plasticity occurred in the spinal dorsal horn immediately after the injection of complete Freund's adjuvant and may be involved in inflammatory pain. Furthermore, acute inflammation‐associated structural changes in the spinal dorsal horn were shown to be mediated by glutamate receptor activation.