Article Figures & Data
Figures
- Figure 1.
Selective expression of hM4Di-DREADD in CaMKIIα-positive forebrain excitatory neurons in CaMKIIα-tTA::TRE-hM4Di bigenic mice. A, Shown are representative confocal images indicating expression of the HA-tagged hM4Di-DREADD in the hippocampus as identified by HA/CaMKIIα double immunofluorescence. B, C, HA-tagged hM4Di-DREADD expression was not observed in either PV-positive inhibitory interneurons (B) or GFAP-positive astrocytes (C). D, HA-tagged hM4Di-DREADD in the cortex was also observed in the CaMKIIα-positive neurons as identified with HA/CaMKIIα double immunofluorescence. E, F, Immunofluorescence experiments indicate the absence of expression of HA-tagged hM4Di-DREADD in subcortical brain regions, namely the periaqueductal gray (E) and pallidum (F). G, Shown is a schematic of the experimental paradigm for harvesting cortex and hippocampus at P7 for Western blotting analysis. H, I, HA expression was clearly noted in the cortex (H) as well as the hippocampus (I) in Western blots from CaMKIIα-tTA::TRE-hM4Di bigenic pups (P7). J, Shown are representative Western blots for c-Fos along with their respective actin loading controls at P7 half an hour after vehicle (Veh) or CNO treatment for cortex (top panel) and hippocampus (bottom panel). K, L, Quantitative densitometry indicated a significant reduction in c-Fos protein levels in the cortex (K) as well as the hippocampus (L) of PNCNO-treated bigenic pups at P7 compared with their vehicle-treated controls. M, Shown is a schematic of the experimental paradigm for harvesting cortex and hippocampus at P35 in the juvenile window for Western blotting analysis. N, O, HA expression was noted in the cortex (N) as well as the hippocampus (O) of CaMKIIα-tTA::TRE-hM4Di bigenic juvenile mice (P35). P, Shown are representative Western blots for c-Fos along with their respective actin loading controls at P35 half an hour after vehicle (Veh) or CNO treatment for cortex (top panel) and hippocampus (bottom panel). Q, R, Quantitative densitometry indicated a significant reduction in c-Fos protein levels in the cortex (Q) but not in the hippocampus (R) of JCNO-treated bigenic mice at P35 compared with their vehicle-treated controls. All immunofluorescence experiments and Western blotting experiments were performed on n = 3–5/group. Results are expressed as the mean ± SEM. *p < 0.05, compared with vehicle-treated controls using the two-tailed, unpaired Student’s t test.
- Figure 2.
Influence of chronic hM4Di-DREADD-mediated inhibition of CaMKIIα-positive forebrain neurons in the early postnatal or juvenile windows on weight and reflex development. 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) in CaMKIIα-tTA::TRE-hM4Di bigenic pups. Pups were assessed for weight gain across the postnatal developmental window and for reflex behaviors on postnatal days 9 and 12. B, No significant change was observed in the weight profile of CNO-administered pups compared with their vehicle-treated age-matched controls across the duration of CNO treatment from P2 to P14 (n = 6). C–E, Reflex behaviors were not altered in PNCNO-treated CaMKIIα-tTA::TRE-hM4Di bigenic pups compared with vehicle-treated controls at P9 or P12 as assessed by determining the number of correct landings for air righting (C), and the time taken for reorientation in both negative geotaxis (D) and surface righting (E) assays. F, Shown is a schematic for the experimental paradigm for vehicle (5% sucrose) or CNO (5 mg/kg) administration in the early juvenile window (P28 to P40) to CaMKIIα-tTA::TRE-hM4Di bigenic male mice. G, No significant change was noted in the weight profile of animals fed with CNO (5 mg/kg) once daily from P28 to P40 compared with their vehicle-treated controls across the duration of drug treatment (n = 5–6/group). Results are expressed as the mean ± SEM, and groups are compared using the two-tailed, unpaired Student’s t test.
- 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.
- Figure 4.
Chronic chemogenetic inhibition of CaMKIIα-positive forebrain excitatory neurons during the early postnatal window does not influence despair-like behavior or sensorimotor gating responses in adult 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 despair-like behavior in adulthood using the FST and sensorimotor gating responses (PPI). B, Shown is a schematic for the FST tank. C, D, We observed no significant PNCNO × sex interactions for either the percentage of immobility time (C) or the total number of immobility events [D; n = 14 (males), n = 10 (females) for vehicle-treated CaMKIIα-tTA::TRE-hM4Di bigenic mice; n = 12 (males), n = 10 (females) for PNCNO-treated CaMKIIα-tTA::TRE-hM4Di bigenic mice]. D, We did note a significant main effect of sex for the number of immobility events. E, Shown is a schematic for the protocol used for PPI to assess sensorimotor gating responses in adult male mice. PPI testing was conducted as described in Materials and Methods with basal startle determined following habituation, and PPI determined for +4 dB (69 dB), +8 dB (73 dB), and +16 dB (81 dB) above background noise (65 dB), followed by exposure to 120 dB for final startle. F, PNCNO-treated adult CaMKIIα-tTA::TRE-hM4Di bigenic male mice show a significant increase in basal startle response compared with vehicle-treated controls. G, No significant differences were noted in sensorimotor gating between vehicle- and PNCNO-treated CaMKIIα-tTA::TRE-hM4Di bigenic male mice (n = 10/group). Results were subjected to two-way ANOVA, followed by the Tukey’s post hoc comparisons test for experiments with four treatment groups (main effects of sex are indicated as $p < 0.05), and by the two-tailed, unpaired Student’s t test for experiments with two treatment groups. Results are expressed as the mean ± SEM. *p < 0.05 compared with the vehicle-treated controls.
- Figure 5.
Chronic hM4Di-DREADD-mediated inhibition of CaMKIIα-positive forebrain excitatory neurons during the juvenile window does not influence anxiety, despair, or sensorimotor gating behavior in adulthood in CaMKIIα-tTA::TRE-hM4Di bigenic male mice. A, Shown is a schematic for the experimental paradigm for vehicle (5% sucrose) or CNO (5 mg/kg) administration in the juvenile window (P28 to P40) to CaMKIIα-tTA::TRE-hM4Di bigenic mice, which were then assessed for anxiety-like behaviors 2 months postcessation of CNO treatment in adulthood in the male cohort. B, Shown are representative tracks for vehicle-treated (top panel) and JCNO-treated CaMKIIα-tTA::TRE-hM4Di bigenic male mice (bottom panel) in the open field arena. C–F, No significant difference was noted between vehicle- and JCNO-treated male mice in the total distance traveled in the arena (C), the percentage of time spent in the center (D), the percentage of distance traveled in the center (E), or the total number of entries to the center of the open field arena (F; n = 18 for vehicle-treated male mice; n = 16 for JCNO-treated male mice). G, Shown are representative tracks for vehicle-treated (top panel) and JCNO-treated CaMKIIα-tTA::TRE-hM4Di bigenic male mice (bottom panel) in the elevated plus maze. H–M, No significant difference was observed between vehicle- and JCNO-treated male mice in the percentage of time spent in the closed (H) or open (I) arms of the EPM, as well as the percentage of distance traveled in closed (J) or open (K) arms, and for the number of entries to the closed (L) or open (M) arms (n = 18 for vehicle-treated male mice; n = 16 for JCNO-treated male mice). N, Shown are representative tracks for vehicle-treated (top panel) and JCNO-treated (bottom panel) CaMKIIα-tTA::TRE-hM4Di bigenic male mice in the LD box. O, P, No significant difference was noted between vehicle- and JCNO-treated male mice in either the total time spent in (O) or the total number of entries (P) into the light chamber of the LD box (n = 18 for vehicle-treated male mice; n = 16 for JCNO-treated male mice). Q, Shown is a schematic for the FST tank. R, S, No significant difference was noted between vehicle- and PNCNO-treated male mice for the percentage of immobility time (R) or for the total number of immobility events (S; n = 11 for vehicle-treated male mice; n = 11 for JCNO-treated male mice). T, Shown is a schematic for the protocol used for PPI to assess sensorimotor gating responses in adult male mice. PPI testing was conducted as described in Materials and Methods with basal startle determined following habituation, and PPI determined for +4 dB (69 dB), +8 dB (73 dB), and +16 dB (81 dB) above background noise (65 dB), followed by exposure to 120 dB for final startle. U, Basal startle response in JCNO-treated CaMKIIα-tTA::TRE-hM4Di bigenic male mice was unaltered compared with vehicle-treated CaMKIIα-tTA::TRE-hM4Di bigenic male controls. V, CaMKIIα-tTA::TRE-hM4Di bigenic male mice with a history of JCNO treatment did not show any change in PPI compared with the vehicle-treated controls (n = 10 for vehicle; n = 9 for JCNO male mice). Results are expressed as the mean ± SEM, and groups are compared using the two-tailed, unpaired Student’s t test.
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