Fig. 5

Chemogenetic inhibition of the PKC-δ positive neurons in the CeA. A Schematic illustration of bilateral AAV5-DIO-mCherry (mCherry) or AAV5-DIO-hM4Di-mCherry (hM4Di) injection in the CeA of PKC-δ-Cre mouse. B Schematic illustration of experimental timeline. After 3 weeks of virus expression, PKC-δ-Cre mice were i.p. injected with either vehicle (Ctrl) or NTG (0.1 mg/kg) every other day to day 9 (totally 5 injections) and i.p. injected with CNO (5 mg/kg) on day 10 and 12. The mechanical threshold tested 2 hrs post-treatment after the NTG injection and 1 hr. post-treatment after the CNO injection. C Representative low (up) and high magnification (down) images of mCherry injection sites, confirming the correct injection of viral vectors or controls at bilateral CeA. Scale bar: 100 μm. D Behavioral consequences before and after CNO application PKC-δ-Cre mice pretreated with bilateral hM4Di virus injection in the CeA after chronic vehicle or NTG administration. Sustained mechanical hyperalgesia alleviated 1 hr. after the CNO application on day 10 and 12 (left, n = 4 per group; p < 0.05 *, and p < 0.01 **) while the marble burying test was unaffected (right, n = 4 per group) (E) Sustained basal mechanical hyperalgesia developed after repeated doses of NTG administration in both mCherry and hM4Di group (n = 6 per group). F Behavioral consequences before and after CNO application PKC-δ-Cre mice pretreated with bilateral mCherry and hM4Di virus injection in the CeA and chronic NTG administration. Sustained mechanical hyperalgesia alleviated 1 hr. after the CNO application on day 10 and 12 (left, n = 6 per group; p < 0.01 **, and p < 0.001 ***) while the marble burying test was unaffected (right, n = 6 per group). (G) Representative images of pERK positive neurons in the TG with or without CNO application (left). The numbers of pERK positive neurons in the TG were significantly decreased after the CNO application (right, n = 4 per group; p = 0.009). Scale bar: 100 μm. H The percentage of CGRP/pERK positive neurons in the TG were significantly decreased after the CNO application (n = 4 per group; p = 0.005). All data shown are mean ± SEM and analyzed by Friedman tests with Dunn’s post hoc test (D, F) or Mann-Whitney U test (G, H). Significance levels set at p < 0.05 *, p < 0.01 **, and p < 0.001 ***