FAAH Inhibition Restores Early Life Stress-Induced Alterations in PFC microRNAs Associated with Depressive-Like Behavior in Male and Female Rats

Abstract

Early life stress (ELS) increases predisposition to depression. We compared the effects of treatment with the fatty acid amide hydrolase (FAAH) inhibitor URB597, and the selective serotonin reuptake inhibitor paroxetine, on ELS-induced depressive-like behavior and the expression of microRNAs (miRs) associated with depression in the medial prefrontal cortex (mPFC), hippocampal CA1 area, lateral habenula and dorsal raphe in rats. We also examined the mRNA expression of serotonergic (htr1a and slc6a4) and endocannabinoid (cnr1, cnr2 and faah) targets in the mPFC following ELS and pharmacological treatment. Adult males and females exposed to the 'Limited Bedding and Nesting' ELS paradigm demonstrated a depressive-like phenotype and late-adolescence URB597 treatment, but not paroxetine, reversed this phenotype. In the mPFC, ELS downregulated miR-16 in males and miR-135a in females and URB597 treatment restored this effect. In ELS females, the increase in cnr2 and decrease in faah mRNAs in the mPFC were reversed by URB597 treatment. We show for the first time that URB597 reversed ELS-induced mPFC downregulation in specific miRs and stress-related behaviors, suggesting a novel mechanism for the beneficial effects of FAAH inhibition. The differential effects of ELS and URB597 on males and females highlight the importance of developing sex-specific treatment approaches.

Keywords: URB597; cannabinoids; depression; early life stress; microRNAs.

Figure 1

The effects of ELS and chronic late-adolescence treatment with URB or PAR on depressive-like behavior in adult males and females. (a) Male and female rats were exposed to early life stress (ELS; P7-14). Vehicle, URB597 (URB) or paroxetine (PAR) were injected during late adolescence (P45-60). Behavioral testing started on P90, and rats were sacrificed on P114 for molecular analysis (n = 7 for all groups). (b) In the OFT, ELS decreased the distance travelled in males, and URB treatment increased it compared to groups treated with vehicle and URB (left). PAR treatment decreased the distance travelled in NoELS females compared to females treated with vehicle and URB (right). (c) ELS increased freezing time in males; PAR treatment increased freezing time compared to URB treatment (left). In females, ELS increased freezing time (right). (d) PAR treatment decreased time spent in the center compared to vehicle and URB treatments in males (left). No significant differences were observed between the groups in females (right). (e) In the SP test, in males (left), URB treatment increased SP in the ELS groups compared to vehicle and PAR treatment; PAR treatment decreased SP compared to vehicle treatment in the NoELS groups (left). In ELS females (right), URB treatment increased SP compared to vehicle treatment; URB treatment decreased SP compared to vehicle treatment in NoELS females. (f) In the SR test, in ELS males (left), URB treatment increased the SR discrimination index compared to vehicle and PAR treatments; in NoELS males, URB and PAR treatment decreased SR compared to vehicle treatment (left). In ELS females (right), URB treatment increased the SR discrimination index compared to vehicle and PAR treatment; PAR treatment increased the SR discrimination index compared to the URB treatment in NoELS females. (g) In the FST, in ELS males (left), URB treatment decreased immobility compared to vehicle and PAR treatments; in NoELS males, vehicle treatment decreased immobility compared to URB and PAR treatments. In ELS females (right), URB treatment decreased immobility compared to the vehicle and PAR treatments; in NoELS females, PAR treatment increased immobility compared to vehicle treatment. ELS: early life stress; FST: forced swim test; miRNA: microRNA; mRNA: messenger RNA; OFT: open-field test; PND: post-natal day; SP: social preference; SR: social recognition. *, p < 0.05; **, p < 0.01; ***, p < 0.001 indicate statistically significant effects followed by post hoc comparisons; ##, p < 0.01; ###, p < 0.001 indicate statistical significance in main effects.

Figure 2

The effects of ELS and chronic late-adolescence treatment with URB or PAR on the expression of miR-135a-5p in adult males and females. (a) In the mPFC, PAR downregulated the expression of miR-135a in ELS males compared to ELS-Vehicle and ELS-URB (left). In NoELS males (left) and females (right), URB and PAR upregulated the expression compared to NoELS-Vehicle. In females (right), URB normalized the expression of miR-135a compared to ELS-Vehicle (n = 5–9). (b) In the CA1, in ELS males (left), PAR downregulated expression compared to ELS-Vehicle or ELS-URB; in NoELS males (left) both URB and PAR downregulated expression. In NoELS females (right), both URB and PAR upregulated expression (n = 5–9). (c) In the LHb, in ELS males (left) and females (right), PAR downregulated expression compared to ELS-Vehicle males and females or URB. In NoELS males and females, URB and PAR downregulated its expression (n = 5–9). (d) In the DR, in ELS males (left), PAR downregulated expression compared to ELS-Vehicle and ELS-URB. In NoELS males (left) and females (right), URB and PAR downregulated expression compared to NoELS-Vehicle males and females. In ELS females (right), URB and PAR downregulated its expression compared to ELS-Vehicle (n = 5–8). DR: dorsal raphe; ELS: early life stress; LHb: lateral habenula; mPFC: medial prefrontal cortex; PAR: paroxetine; URB: URB597. *, p < 0.05; **, p < 0.01; ***, p < 0.001 indicate statistically significant effects followed by post hoc comparisons. ##, p < 0.01; ###, p < 0.001 indicate statistical significance in main effects.

Figure 3

The effects of ELS and chronic late-adolescence treatment with URB or PAR on the expression of miR-16-5p in adult males and females. (a) In the mPFC, in ELS males, URB normalized the expression of miR-16 compared to ELS-Vehicle; in NoELS males, URB and PAR upregulated expression compared to NoELS-Vehicle. No significant differences were observed in females (n = 5–10). (b) In the CA1, in ELS males, PAR downregulated expression compared ELS-Vehicle or ELS-URB; in NoELS males PAR upregulated expression compared to NoELS-Vehicle; in NoELS females, URB and PAR upregulated expression compared to NoELS-Vehicle (n = 5–8). (c) In the LHb, in ELS males, URB and PAR upregulated expression compared to ELS-Vehicle; in NoELS males, PAR downregulated expression compared to NoELS-Vehicle. In NoELS females, URB and PAR downregulated expression compared to NoELS-Vehicle (n = 5–8). (d) In the DR, in ELS males, PAR upregulated expression compared to ELS-Vehicle or ELS-URB. In ELS females, URB and PAR upregulated expression compared to ELS-Vehicle (n = 5–8). DR: dorsal raphe; ELS: early life stress; LHb: lateral habenula; mPFC: medial prefrontal cortex; PAR: paroxetine; URB: URB597. *, p < 0.05; **, p < 0.01; ***, p < 0.001 indicate statistically significant effects followed by post hoc comparisons. #, p < 0.05; ###, p < 0.001 indicate statistical significance in main effects.

Figure 4

The effects of ELS and chronic late-adolescence treatment with URB or PAR on mRNA expression of serotonergic and endocannabinoid genes in the mPFC in adult males and females. (a) In ELS males, URB or vehicle downregulated the expression of htr1a compared to ELS-PAR; in NoELS males, URB and PAR downregulated its expression compared to NoELS-Vehicle. In females, URB upregulated htr1a expression compared to NoELS-Vehicle (n = 5–8). (b) In ELS males, vehicle and URB downregulated the expression of slc6a4 compared to ELS-PAR; in NoELS males, URB downregulated its expression compared to NoELS-Vehicle. In ELS and NoELS females, PAR upregulated the expression of slc6a4 compared to ELS- and NoELS-Vehicle or URB (n = 5–6). (c) In ELS males, URB or vehicle downregulated the expression of cnr1 compared to ELS-PAR; in NoELS males, URB downregulated its expression compared to NoELS-Vehicle or NoELS-PAR. In females, ELS downregulated cnr1 expression compared to the NoELS groups (n = 5–10). (d) In ELS males, URB or vehicle downregulated cnr2 expression compared to ELS-PAR; in NoELS males, URB or PAR downregulated its expression compared to NoELS-Vehicle. In females, ELS upregulated the expression of cnr2 and URB decreased cnr2 expression compared to ELS- and NoELS-Vehicle or PAR (n = 5–8). (e) In ELS males, URB or vehicle downregulated faah expression compared to ELS-PAR. In females, URB or PAR upregulated the expression of faah compared to ELS-Vehicle (n = 5–7). ELS: early life stress; PAR: paroxetine; URB: URB597. *, p < 0.05; **, p < 0.01; ***, p < 0.001 indicate statistically significant effects followed by posthoc comparisons; #, p < 0.05; ##, p < 0.01; ###, p < 0.001 indicate statistical significance in main effects.