Article Figures & Data
Figures
- Figure 1.
Experimental design and targeting of PV INs in the PFC using DREADDs. A, Experimental design and timeline. C57BL/6J PV-Cre mice ∼7.5 months of age underwent surgery to inject AAV2-hM4Di-mCherry (inhibitory DREADD) or AAV2-mCherry (control virus). Mice were allowed three weeks to recover to enable sufficient time for DREADD expression. Animals were then subjected to CVS procedure twice a day for 14 d or served as controls. The first stressor was a TST to determine acute effects of PV IN inhibition in animals within the CVS group. Animals were dosed with 1 mg/kg CNO before each stressor to inhibit PV INs during the CVS procedure; 24 h after the end of CVS, animals were subjected to FST, following which mice were euthanized, body was perfused, and brains and organs were collected. No CNO was administered during the testing phase in FST. B, Design of AAV2-hSyn-hM4Di-mCherry (top) and AAV2-hSyn-mCherry (bottom) vectors employing the DIO strategy. Two pairs of heterotypic, antiparallel loxP recombination sites (blue triangles) achieve Cre-mediated transgenes inversion and expression under the control of hSyn promoter. ITR, left-inverted terminal repeat; hSyn, human synapsin; WPRE, woodchuck hepatitis DREADD posttranscriptional regulatory element. C, Successful Cre-mediated recombination of DREADDs demonstrated by presence of red mCherry (left); green fluorescence identifies PV INs (right); hM4Di receptors selectively expressed in PV INs as illustrated by mCherry (red) and PV (green) co-expression (merged, yellow, bottom middle image). Scale bar: 100 μm.
- Figure 2.
Injection site, viral spread. and targeting of IL. A, Injection site and viral spread mapped onto respective mouse bregma coordinates, following bilateral injection of hM4Di DREADD into the IL of PV-Cre mice (n = 9). Light green rectangular box depicts the IL region with the injection sites restricted to the region. Each animal’s injection site and viral spread has been represented by a unique color. B, Representative image from one animal demonstrating the viral spread in PFC detected by red mCherry fluorescence. Image shows the spread was restricted to the IL with the white dashed lines outlining the IL cortex region of the PFC. Scale bars: 1000 and 500 μm. C, Percentage of DREADD transfected PV INs in the IL of PV-Cre mice. The percentage of infected cells in IL was calculated by dividing the number of mCherry-positive cells in IL by the total number of mCherry-positive cells in a specific focal plane and bregma coordinate. Brain bregma coordinates used for IL cytoarchitecture were taken from Franklin and Paxinos (2007). *indicates significant effect p < 0.05 compared to outside IL group. Values represent mean ± SEM, n = 9 per group.
- Figure 3.
Impact of acute chemogenetic inhibition of PV IN in the IL mPFC on coping behavior in the TST. Acute inhibition of PV INs in the IL mPFC during TST, reduced total time spent struggling (A), increased total time spent immobile (B), and reduced latency to immobility (C). All mice were treated with CNO (1 mg/kg, i.p.) 30 min before TST. Behaviors were analyzed for a total time of 6 min. Values represent mean ± SEM, n = 9–10 per group; * indicates significant effect p < 0.05 versus corresponding control group.
- Figure 4.
Effects of chronic chemogenetic inhibition of PV INs during CVS on coping behavior in the FST following CVS. Chronic inhibition of PV INs in the IL mPFC during CVS, resulted in increased total time spent swimming (A) and decreased total time spent immobile (C) in the FST. B, D, Changes in swimming and immobility behavior, respectively, over the 10 min of FST. Values represent mean ± SEM, n = 9–10 per group; * indicates planned comparison significant effect p < 0.05 versus corresponding No CVS hM4Di group; # indicates planned comparison significant effect p < 0.05 versus corresponding CVS Control group. Extended Data Figure 4-1 demonstrates chronic inhibition of PV INs did not affect locomotor activity. Extended Data Figure 4-2 demonstrates chronic CNO administration did not lead to any changes in FST behavior following CVS or control. Extended Data Figure 4-3 demonstrates CVS effects on FST did not depend on the age of the animal and also shows chronic injection stress and housing conditions had no effect on FST behavior in control animals.
- Figure 5.
Fos immunoreactivity in the PrL, IL, BLA, and vlPAG. Chronic inhibition of PV INs in the IL mPFC during CVS, attenuated CVS-mediated reduction in Fos expression in the PrL, BLA, and vlPAG, respectively (A, C, D) but did not prevent CVS-mediated reduction in Fos expression in the IL cortex (B) following FST. Values are presented as mean ± SEM; n = 7–10 per group; * indicates significant result p < 0.05 post hoc (A) and planned comparisons (C, D) compared with respective No CVS Control groups. PrL, IL, BLA, and vlPAG stands for prelimbic, infralimbic, basolateral amygdala and ventrolateral periaqueductal grey, respectively. Extended Data Figure 5-1 demonstrates Fos expression in various other brain regions where no significant effect of PV IN modulation was observed.
- Figure 6.
Impact of chronic stress on organ and body weights. Chronic inhibition of PV INs in the IL mPFC during CVS, attenuated CVS mediated increases in adrenal gland weight (A) and attenuated CVS mediated decreases in body weight (C, D). No change in CVS-induced decreases in thymus weight was observed following PV IN inhibition (B). Data are presented as absolute organ and body weights. Values represent mean ± SEM; n = 9–10 per group; * indicates planned comparisons significant effect p < 0.05 versus corresponding No CVS Control groups.
Tables
- Table 1
Data structure, type of test to analyze the data, and observed power of key results
Data structure Type of test Power a Normal distribution Unpaired sample
t test (struggling)Main effect hM4Di DREADD :
Cohen’s d = 1.2b Normal distribution Unpaired sample
t test (immobility)Main effect hM4Di DREADD:
Cohen’s d = 1.3c Normal distribution Unpaired sample
t test (latency to immobility)Main effect hM4Di DREADD:
Cohen’s d = 1.3d Normal distribution Two-way ANOVA
(swimming duration)Main effect of stress: 0.91
Main effect of hM4Di DREADD: 0.55e Normal distribution Two-way repeated measures ANOVA
(swimming duration over time)Main effect stress:
Power = 0.91Main effect hM4Di DREADD:
Power = 0.55
Main effect of time:
Power = 1f Normal distribution Two-way ANOVA
(immobility duration)Main effect stress:
Power = 0.85g Normal distribution Two-way repeated measures ANOVA
(immobility duration over time)Main effect stress:
Power = 0.85
Main effect time:
Power = 1h Normal distribution Two-way ANOVA
(PrL)Main effect stress:
Power = 0.98
Interaction (stress × hM4Di DREADD):
Power = 0.76i Normal distribution Two-way ANOVA
(IL)Main effect stress:
Power = 0.99j Normal distribution Two-way ANOVA
(BLA)Main effect stress:
Power = 0.89k Normal distribution Two-way ANOVA
(vlPAG)Main effect stress:
Power = 0.94l Normal distribution Two-way ANOVA
(adrenal weight)Main effect stress:
Power = 0.90
Interaction (stress × hM4Di DREADD):
Power = 0.57m Normal distribution Two-way ANOVA
(thymus weight)Main effect stress:
Power = 1n Normal distribution Two-way ANOVA
(final body weight)Main effect stress:
Power = 0.700 Normal distribution Two-way repeated measures ANOVA
(body weight over time)Main effect stress:
Power = 0.6
Main effect time:
Power = 0.9p Normal distribution Unpaired sample
t test (viral spread in IL)Main effect hM4Di DREADD in IL:
Cohen’s d = 1.2Table depicts the data structure, type of statistical test used, and power of key results for each of the statistical tests used in the manuscript. Each analysis includes a letter indicator linking the test in the table to the analysis in the text.
Extended Data Figure 4-1
Effect of chronic inhibition of PV IN on locomotor activity. Chronic inhibition of PV INs had no effect on locomotor activity as demonstrated by no change in distance travelled (A) or velocity (B) in hM4Di group compared with control group in a Y maze task. Values represent mean ± SEM, n = 9–10 per group (p > 0.05). Download Figure 4-1, TIF file.
Extended Data Figure 4-2
Effect of chronic dosing of CNO and saline in control and CVS animals in FST. Chronic CNO administration had no effect on immobility (A) or swimming duration (B) in FST and also did not have any effect on body weight (C) in either CVS or Control groups. Values represent mean ± SEM; n = 8 per group (p > 0.05); * indicate planned comparisons significant effect p < 0.05 versus corresponding No CVS Control groups. Download Figure 4-2, TIF file.
Extended Data Figure 4-3
Effect of age, chronic injection, and housing condition in FST. A, B, Effect of age on FST immobility duration. Analysis of z scores showed that CVS had no effect on FST behavior in different age groups (t = 0.4, df = 16, p = 0.7; B). Younger and older animals were run in separate experiments. Z score calculation was done as described previously using formula z = (X-μ)/σ to indicate how many SDs (σ) each CVS immobility duration value (X) was from the mean of control group (μ) for each age (Guilloux et al., 2011). C, D, Effect of chronic injection stress and housing conditions on immobility duration in FST in control animals. There was no significant effect of chronic injection stress (t = 0.8, df = 14, p = 0.4; C) or housing conditions (t = 0.2, df = 14, p = 0.9; D) in FST behavior. Values represent mean ± SEM; n = 8–10 per group; * indicates significant effect p < 0.05 versus corresponding control group. Download Figure 4-3, TIF file.
Extended Data Figure 5-1
Fos protein expression in several brain regions following CVS. Figure depicts Fos protein expression in lateral septum, anterior and posterior ventral BNST, and dlPAG. No significant treatment effects of PV IN inhibition was observed in any of the above-mentioned brain regions. Values represent mean ± SEM, n = 7–10 per group. Download Figure 5-1, TIF file.
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