In Vivo Ca²⁺ Imaging of the Insular Cortex during Experimental Tooth Movement

Pain and discomfort are common problems for patients undergoing orthodontic treatment. We have demonstrated that cortical excitation propagation in the somatosensory and insular cortices (IC) induced by electrical stimulation of the periodontal ligament (PDL) is facilitated 1 d after experimental tooth movement (ETM). However, it is necessary to examine ETM-induced changes in neural responses at a single-cell level to understand the mechanisms of cortical plastic changes, in which excitatory glutamatergic and inhibitory GABAergic neurons are intermingled to form cortical local circuits. We performed in vivo 2-photon Ca²⁺ imaging by loading the Ca²⁺ indicator Oregon Green BAPTA with the astrocyte marker sulforhodamine. We focused on the IC region that exhibited the largest neural response to maxillary PDL (mxPDL) stimulation using a VGAT-Venus transgenic rat that expresses venus fluorescent protein in GABAergic neurons and discerned changes in the neural activities of each cortical neuronal subtype before and during ETM treatment of the maxillary incisor and first molar. Notably, 1 d after ETM treatment (1d-ETM), the number of neurons responding to mxPDL stimulation increased from 47.6% to 64.2% in excitatory neurons and from 44.5% to 66.2% in inhibitory neurons. On the other hand, only 3% to 4% of excitatory and inhibitory neurons responded to mandibular molar PDL (mbPDL) stimulation in control rats, and the 1d-ETM group showed significant increases in excitatory (14.0%) and inhibitory neurons (22.5%) responding to mbPDL stimulation. Interestingly, most mbPDL-responding neurons also responded to mxPDL stimulation. The population of excitatory and inhibitory neurons that responded only to mxPDL stimulation was comparable between the control and 1d-ETM groups. The facilitative responses in the 1d-ETM group had almost recovered 7 d after ETM treatment. These results suggest that ETM induces parallel increases in PDL-responding neurons and changes some insensitive neurons to respond to both mxPDL and mbPDL stimulation.