2.1. miR-132 transgenic and miR-132/212 knockout mice

The CaMKII-Cre::miR-132/212^f/f conditional forebrain neuron knockout (referred to as ‘miR-132/212 cKO’) mouse line was previously described by our lab (Hansen et al., 2016). The miR-132/212^f/f animals were provided by Dr. J. Simon C. Arthur (University of Dundee, Scotland), and the CaMKII-Cre line (Tsien et al., 1996) was purchased from Jackson Labs (Stock number: 005359: Bar Harbor, ME, USA). Generation of the CaMKII-tTA::miR-132:CaMKII-Cre::miR-132/212^f/f mouse line (referred to as ‘miR-132 transgenic’) was recently reported in Aten et al. (2018a). In brief, miR-132 transgenic animals were created by breeding homozygous CaMKII-Cre::miR-132/212^f/f (miR-132/212 cKO) mice with a tetracycline-regulated bidirectional miR-132/cyan fluorescent protein (CFP) transgenic mouse line driven by CaMKII::tTA. Thus, miR-132 transgenic mice were homozygous for the miR-132/ 212^f/f locus and positive for Cre, tTA, and miR-132 transgenes. This ‘Tet-off’ transgenic animal model allowed for selective deletion of endogenous miR-132/212 and transgenic over-expression of miR-132 within the same population of excitatory forebrain neurons. For all experiments with transgenic and knockout mice, control (referred to as ‘WT’) animals were littermates that were negative for either the driver and/or responder genes. Genotyping for both the miR-132/212 cKO and miR-132 transgenic lines was previously described by our lab (Aten et al., 2018a; Hansen et al., 2016, 2010).

All mice utilized for experiments were bred, housed, and maintained under standard 12 h/12 h Light/Dark (LD) conditions and had ad libitum access to food and water. All behavioral and molecular experiments took place from the mid-to late-day light period. Experimental animals were screened, as described in Hansen et al. (2013), to ensure that they did not have any vision deficits. Of note, for experiments examining miR-132 and miR-212 expression after stress paradigms (Fig. 1; Supplementary Fig. 1–2), only male WT mice (8–12 weeks of age) were utilized to eliminate potential sex-specific effects. However, given the limited number of miR-132/212 cKO and miR-132 transgenic mice available, both males and females were used for behavioral and molecular experiments (Figs. ​(Figs.33–6); a sex-parsed presentation of data for WT, miR-132/212 cKO, and miR-132 transgenic anxiety behavioral experiments (Figs. ​(Figs.33 and ​and4)4) are provided in Supplementary figure 3. The Ohio State University Institutional Animal Care and Use Committee approved all protocols and methods, and all experiments were in accordance with the National Institutes of Health guide for the care and use of Laboratory animals.

(A) Acute multimodal stress experimental design: WT animals were subjected to 5 h of acute multimodal stress, wherein mice were placed in restraint tubes which rocked on a laboratory shaker for 5 h. Loud music was also played in the room. (B) RT-qPCR for miR-132 was performed on RNA samples isolated from the hippocampus (B) and amygdala (C) of control and acutely stressed mice. Similarly, hippocampal miR-212 (D) and amygdalar miR-212 (E) expression was probed via RT-qPCR. (F) Chronic restraint stress experimental design: WT animals were subjected to 2 h of restraint each day, throughout the 15 day paradigm. On day 16, RNA was isolated from both chronically stressed and control mice and hippocampal (G) and amygdalar (H) miR-132 expression was examined using RT-qPCR. Similarly, hippocampal (I) and amygdalar miR-212 (J) expression was probed via RT-qPCR. For both acute and chronic paradigms, miR-132 and miR-212 expression in control animals was set equal to a value of one, and miR-132 and miR-212 expression in stressed mice was normalized to this value. Significance was examined with Student’s t-test for each brain region, and data are presented as the mean ± SEM. *: p < 0.05; **: p < 0.01; ***: p < 0.001; n.s: not significant (p > 0.05). N = 9–10 mice per group.

(A) Representative elevated plus maze locomotor traces from WT (blue), miR-132/212 cKO (red), and miR-132 transgenic animals (green). (B) miR-132/212 cKO and miR-132 transgenic animals exhibited significantly fewer open arm transitions in the elevated plus maze compared to WT mice. Mean time spent in open arms (C) and mean latency to open arms (D) were significantly different between WT and miR-132 transgenic animals. (E) Representative open field test locomotor traces from WT, miR-132/212 cKO, and miR-132 transgenic animals. (F) Total duration in the center of the open field was significantly different between miR-132 transgenic and WT mice. (G) Number of crosses into the center was significantly reduced in miR-132 transgenic mice compared to WT mice. (H) Total immobility was significantly different between WT and miR-132 transgenic mice. Data were analyzed by one-way ANOVA with Bonferroni post-hoc tests. Data are presented as the mean ± SEM. *: p < 0.05; **: p < 0.01; n.s.: not significant (p > 0.05). N = 9–13 mice per group.

(A) Representative elevated plus maze locomotor traces from WT (blue) and miR-132 transgenic animals (green). Note that both WT and miR-132 transgenic animals were given a 0.4 μg doxycycline dose in their drinking water for three weeks prior to testing (and throughout the testing period). No differences in open arm transitions (B), open arm cumulative duration (C), or latency to open arm (D) were observed between WT and miR-132 transgenic animals in the elevated plus maze. (E) Representative open field locomotor traces from WT and miR-132 transgenic animals. No significant differences in duration in the center of the arena (F), center crossings (G), or time spent immobile (H) were observed between WT and miR-132 transgenic animals in the open field test. Data were analyzed by Student’s t-tests and are presented as the mean ± SEM. n.s.: not significant (p > 0.05). N = 15–20 mice per group.

(A) Representative immunohistochemical labeling for PTEN in the hippocampus and amygdala of WT (blue), miR-132/212 cKO (red), and miR-132 transgenic animals (green). The boxed regions in the low-magnification images approximate the locations of the regions that are depicted in the high-magnification panels (to the right for the hippocampus, and inset for the amygdala). (B) Quantification of PTEN immunolabeling in the hippocampus and amygdala. Note the increased expression of PTEN within the miR-132/212 cKO mice. Scale bar = 50 μm for the low magnification images (i.e., whole hippocampus and amygdala) and 30 μm for the high magnification images (i.e., CA1 cell layer and GCL-lower blade). Data were analyzed by two-way ANOVA with Bonferroni post-hoc correction and are presented as the mean ± SEM. **: p < 0.01; n.s.: not significant (p > 0.05); N = 3–6 mice per genotype.