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. 2022 Feb 14;9(1):ENEURO.0381-21.2021. doi: 10.1523/ENEURO.0381-21.2021

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Copyright © 2022 Tiwari 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 3. — Chronic hM4Di-DREADD-mediated inhibition of CaMKIIα-positive forebrain excitatory neurons during the early postnatal window does not influence anxiety-like behavior in adulthood in CaMKIIα-tTA::TRE-hM4Di bigenic mice. A, Shown is a schematic for the experimental paradigm for vehicle (5% sucrose) or CNO (5 mg/kg) administration in the early postnatal window (P2 to P14) to CaMKIIα-tTA::TRE-hM4Di bigenic pups, which were then assessed for anxiety-like behaviors 3 months postcessation of CNO treatment in adulthood. B, Shown are representative tracks for vehicle-treated (top panels) and PNCNO-treated (bottom panels) CaMKIIα-tTA::TRE-hM4Di bigenic male and female mice in the open field arena. C, Two-way ANOVA indicated a PNCNO × sex interaction for total distance moved in the OFT arena, with post hoc Tukey’s analysis revealing a significant difference between the vehicle-treated female and male CaMKIIα-tTA::TRE-hM4Di bigenic mice. D–F, We noted significant main effects of sex for the percentage of time spent in the center (D), the percentage of the distance traveled in the center (E), and the total number of entries to the center of the open field arena (F). n = 10 (males) and n = 10 (females) for vehicle-treated CaMKIIα-tTA::TRE-hM4Di bigenic mice; n = 12 (males), n = 12 (females) for PNCNO-treated CaMKIIα-tTA::TRE-hM4Di bigenic mice. G, Shown are representative tracks for vehicle-treated (top panels) and PNCNO-treated (bottom panels) CaMKIIα-tTA::TRE-hM4Di bigenic male and female mice in the elevated plus maze. H–M, We noted a significant main effect of sex for the percentage of time in the closed (H) and open (I) arms of the plus maze, and for the number of entries to the closed (L) and open (M) arms, but not for the percentage of distance traveled in closed (J) or open (K) arms. n = 14 (males) and n = 10 (females) for vehicle-treated CaMKIIα-tTA::TRE-hM4Di bigenic mice; n = 12 (males) and n = 12 (females) for PNCNO-treated CaMKIIα-tTA::TRE-hM4Di bigenic mice. N, Shown are representative tracks for vehicle-treated (top panels) and PNCNO-treated (bottom panels) CaMKIIα-tTA::TRE-hM4Di bigenic mice in the LD box. No significant interaction of PNCNO × sex was noted in the LD box. O, P, However, we did observe a significant main effect of sex in the total time spent in the light box (O), and a significant main effect of PNCNO treatment on the total number of entries into the light chamber of the LD box (P). n = 14 (males) and n = 10 (females) for vehicle-treated CaMKIIα-tTA::TRE-hM4Di bigenic mice; n = 12 (males) and n = 12 (females) PNCNO-treated CaMKIIα-tTA::TRE-hM4Di bigenic mice. Results are expressed as the mean ± SEM, and groups are compared using two-way ANOVA, followed by the Tukey’s post hoc comparison test when a significant PNCNO × sex interaction was noted (*p < 0.05). The main effects of sex are indicated as ^$p < 0.05; the main effects of CNO treatment are indicated as ^@p < 0.05.