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. 2017 Jul 25;4(4):ENEURO.0087-17.2017. doi: 10.1523/ENEURO.0087-17.2017

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Copyright © 2017 Smith et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Figure 2. — Inhibition of ACC, but not somatosensory cortex reverses hyperalgesia in primary and bystander mice. A, Timeline of data collection and experimental manipulation: Sfx refers to surgery, which took place 7-14 d before beginning of experiments. Blue bar represents bystander and control mice, black bar represents primary mice, with corresponding EtOH % (v/v); VF and orange arrows represent von Frey testing at end of 24-h abstinence; WD represents withdrawal from alcohol; black syringe represents CNO injection (20 min before the second mechanical test). B, C, Overlapping pattern of viral expression (blue) for each treatment group in the ACC and SI. Representative photomicrographs of hM4Di viral expression, within the (D) ACC (orange) with DAPI in blue (E) SI (orange) and DAPI in blue. Left panels: scale bars, 100 μm; right panels: scale bars, 20 μm. F, Animals in which DREADDs were expressed in ACC and given Veh (n = 5, primary; n = 5, bystander) showed a significant decrease in threshold compared to Veh-treated separately housed control animals (n = 8). By contrast, animals given CNO before testing (n = 6, primary; n = 6, bystander) showed no hyperalgesia compared to CNO-treated controls (n = 6). According to ANOVA, this led to a significant difference of treatment (F_(1,30) = 4.79, p = 0.037), as well as a significant interaction (F_(2,30) = 3.37, p = 0.048). CNO groups were no longer significantly different from separately housed controls according to Fishers LSD. G, Bystander (n = 5-7/group) and primary (n = 6/group) mice bilaterally transfected with the hM4Di DREADD virus in SI demonstrated significant decreases in mechanical thresholds on the second WD session compared to separately housed controls (n = 5-6), leading to significant differences between groups (F_(2,29) = 14.88, p < 0.0001), but no significant effects of CNO treatment or an interaction, indicating that inactivation of SI had no effect on hypersensitivity. Mean basal responses of all groups represented by dotted line (—). *p < 0.05 compared to controls receiving the same treatment, according to Fishers LSD.