Sex-specific maladaptive responses to acute stress upon in utero THC exposure are mediated by dopamine

Abstract

Cannabis remains by far the most consumed illicit drug in Europe. The availability of more potent cannabis has raised concerns regarding the enhanced health risks associated with its use, particularly among pregnant women. Growing evidence shows that cannabis use during pregnancy increases the risks of child psychopathology. We have previously shown that only male rat offspring prenatally exposed to Δ⁹-tetrahydrocannabinol (THC), a rat model of prenatal cannabinoid exposure (PCE), display a hyperdopaminergic phenotype associated with a differential susceptibility to acute THC- and stress-mediated effects on sensorimotor gating functions. Here, we explore the contribution of the hypothalamic-pituitary-adrenal (HPA) axis, key regulator of body adaptive stress responses, to the detrimental effects of acute stress on ventral tegmental area (VTA) dopamine neurons and sensorimotor gating function of PCE rats. We report a sex-dependent compromised balance in mRNA levels of genes encoding mineralocorticoid and glucocorticoid receptors in the VTA, alongside with stress-induced pre-pulse inhibition (PPI) impairment. Notably, VTA dopamine neuronal activity is causally linked to the manifestation of stress-dependent deterioration of PPI. Finally, pharmacological manipulations targeting glycogen-synthase-kinase-3-β signaling during postnatal development correct these stress-induced, sex-specific and dopamine-dependent disruption of PPI. Collectively, these results highlight the critical sex-dependent interplay between HPA axis and dopamine system in the regulation of sensorimotor gating functions in rats.

Keywords: Cannabis; DREADD; Dopamine; HPA axis; Neurodevelopment; Psychopathology; Stress; vulnerability.

Fig. 1. Sex-specific effects of acute stress on sensorimotor gating function.

A) Timeline of experimental procedures for pre-pulse inhibition (PPI) experiments (top) and blood collection (bottom). B) Forced swim test (FST) differentially impacts on sensorimotor gating functions in male and female rats (*p = 0.036 CTRL males vs. PCE males-shaded gray area; **p = 0.003 CTRL males vs. CTRL females; Sidak’s test; n_rats = 7 CTRL females, 9 PCE females, 15 CTRL males, 16 PCE males). C) Corticosterone and D) adrenocorticotropic hormone (ACTH) concentrations were measured from plasma samples collected from unstressed rats and from stressed rats after 30 min from the beginning of the FST: CORT: *p = 0.01 stressed CTRL males vs. stressed CTRL females; **p = 0.003 stressed PCE males vs. stressed PCE females. ACTH: **p = 0.006 stressed CTRL males vs. stressed CTRL females; Sidak’s test. Data are expressed as ng/ml of plasma and obtained from 7 unstressed and 8 stressed CTRL males, 6 unstressed and 7 stressed CTRL females,12 unstressed and stressed PCE males, 7 unstressed and 9 stressed PCE females. Unless otherwise indicated, data are represented with scatter plots with mean and ± SEM. Figure created with BioRender.com.

Fig. 2. Feedback sensitivity to acute stress is reduced in PCE males.

A) Timeline of experimental procedures. The quantitative real-time PCR analysis of gene expression levels of MR (Nr3c2) and GR (Nr3c1) were measured from VTA tissue punches collected from unstressed rats and from stressed rats after 30 min from the beginning of the forced swim test (FST). B) Fold change ratio of mRNA expression levels of Nr3c2 in VTA of males at preadolescence from unstressed rats and from stressed rats after the FST (*p = 0.0113 main effect of PCE; n_rats = 9 naïve (unstressed) and 6 stressed CTRL males, 13 unstressed and 9 stressed PCE males). C) Graphs show the mRNA expression levels of Nr3c1 in VTA as a fold change ratio in PCE male rats compared with their corresponding control group, in both unstressed or stressed rats (**p = 0.0054 main effect of PCE; *p = 0,04 main effect of stress- shaded gray area; n_rats = 10 unstressed and 7 stressed CTRL males, 17 unstressed and 14 stressed PCE males). D) Fold change ratio of mRNA expression levels of Nr3c2 in VTA of unstressed and stressed females at preadolescence (*p < 0.05 stressed PCE vs. stressed CTRL- shaded gray area; Sidak’s test; n_rats = 9 unstressed and 6 stressed CTRL females, 10 unstressed and 4 stressed PCE females). E) Graphs show the mRNA expression levels of Nr3c1 in VTA as a fold change ratio in stressed and unstressed PCE female rats compared with their corresponding control group (n_rats = 9 unstressed and 6 stressed CTRL females, 10 unstressed and 8 stressed PCE females). Unless otherwise indicated, data are represented with scatter plots with mean and ± SEM. Figure created with BioRender.com.

Fig. 3. Maladaptive responses to acute stress are mediated by VTA dopaminergic activity.

A) Schematic timeline of PCE treatment and behavioral experiments. TH:: Cre positive male offspring rats were bilaterally injected with AAV5-hSyn-DIO-hM4D(Gi)-mCherry or control virus (AAV5-hSyn-DIO-mCherry) in the VTA at PND7. After 3 weeks, male offspring were injected with clozapine N-Oxide (CNO), then after an acute stress rats were exposed to pre pulse inhibition test (PPI). CNO administration occurred 30 min before the PPI. B) Representative fluorescent images of Gi-mCherry and TH positive cells in coronal brain sections containing the VTA, counterstained for DAPI (left panel). Yellow square indicates one example of ROI used for cell count (left panel). Scale bar, 250 μM. High magnification of yellow ROI for labeling quantification in VTA (top right): arrows indicate cells double positive for TH (green) and Gi-mCherry (red). Scale bar, 25 μM. Pie charts showing the proportions of Gi-mCherry positive (red) and TH/Gi-mCherry positive cells (yellow) within sampled ROIs in CTRL and PCE animals (bottom right). C) Forced swim test (FST) does not affect the startle amplitude. Startle amplitude values are represented as arbitrary units (AU). (n_rats = 7 PCE-control; 10 PCE-Gi; 11 CTRL- control; 13 CTRL-Gi). D) Gi-DREADD activation prevents FST-induced impairment of sensorimotor gating functions in PCE males (**p = 0.001 CTRL-control vs PCE-control-shaded gray area; Sidak’s test; n_rats = 10 PCE-Gi; 16 CTRL- control and PCE-control; 21 CTRL-Gi). E) Effect of acute restraint stress (RS) in the startle amplitude after Gi-DREADD activation in PCE male offspring (n_rats = 10 CTRL-control and PCE-Gi; 13 CTRL-Gi; 7 PCE-control). F) Gi-DREADD activation prevents PPI impairment exhibited by PCE males (*p = 0.04 CTRL-control vs PCE-control- shaded gray area; Sidak’s test; n_rats = 9 PCE-Gi; 10 CTRL- control and PCE-control; 12 CTRL-Gi). Unless otherwise indicated, data are represented with scatter plots with mean and ± SEM. Figure created with BioRender.com.

Fig. 4. Chemogenetic activation of VTA DA neurons induces maladaptive responses to acute stress in female rats.

A) Schematic timeline of PCE treatment and behavioral experiments. TH::Cre positive female offspring rats were bilaterally injected with AAV5-hSyn-DIO-hM3D(Gq)-mCherry or control virus (AAV5-hSyn-DIO-mCherry) in the VTA at PND7. After 3 weeks, female offspring were injected with clozapine N-Oxide (CNO), then after an acute stress rats were exposed to pre pulse inhibition test (PPI). CNO administration occurred 30 min before the PPI. B) Representative fluorescent images of Gq-mCherry and TH positive cells in coronal brain sections containing the VTA, counterstained for DAPI (left panel). Yellow square indicates one example ROI used for cell count (left panel). Scale bar, 250 µM. High magnification of yellow ROI for labeling quantification in VTA (top right): arrows indicate cells double positive for TH (green) and Gq-mCherry (red). Pie charts showing the proportions of Gq-mCherry positive (red) and TH/Gq-mCherry positive cells (yellow) within sampled ROIs in CTRL and PCE animals (bottom right). Scale bar, 25 µM. C) Gq-DREADD activation affects the startle amplitude in control female offspring after FST. Startle amplitude values are represented as arbitrary units (AU) (*p = 0019 main effect of Gq; n_rats = 16 CTRL-control; 21 PCE-control; 17 CTRL-Gq; 16 PCE-Gq). D) Effect of Gq-DREADD activation in the startle amplitude after the exposure to an acute restraint stress (RS) in female rats (****p < 0.0001 main effect of Gq; n_rats = 16 CTRL-control, PCE-control and PCE-Gq; 17 CTRL-Gq). E) Gq-DREADD activation impairs sensorimotor gating functions following FST in female rats (*p = 0,04 main effect of Gq; n_rats = 16 CTRL-control and PCE-Gq; 21 PCE-control; CTRL-Gq). F) Gq-DREADD activation induces PPI impairment in response to RS in female rats (****p < 0.0001 main effect of Gq; n_rats = 16 CTRL-control and PCE-control; 17 CTRL-Gq; 16 PCE-Gq). Unless otherwise indicated, data are represented with scatter plots with mean and ± SEM. Figure created with BioRender.com.

Fig. 5. Pharmacological manipulations with pregnenolone or lithium prevent stress-induced impairment of gating functions in PCE male progeny.

A) Schematic timeline of PCE treatment and subchronic treatment with pregnenolone (PREG). B) Effect of PREG on the startle amplitude after forced swim test (FST) in PCE male offspring. Startle amplitude values are represented as arbitrary units (AU) (n_rats = 10–13 CTRL/PCE-PREG; 16 CTRL/PCE-VEH). C) PREG prevents forced swim test (FST)-induced impairment of pre-pulse inhibition (PPI) in male PCE offspring (*p = 0.02 PCE-VEH vs. CTRL-VEH; *p = 0.01 PCE-VEH vs. PCE-PREG; Tukey’s test; n_rats = 9 CTRL/PCE-PREG; 16−17 CTRL/PCE-VEH). D) Schematic timeline of PCE treatment and subchronic treatment with lithium. E) Effect of lithium on the startle amplitude after forced swim test (FST) in PCE male offspring. Startle amplitude values are represented as arbitrary units (AU) (n_rats = 7−8 CTRL/PCE-Lithium; 8−9 CTRL/PCE-VEH). F) Lithium prevents FST-induced impairment of PPI in male PCE offspring (**p = 0.003 PCE-VEH vs. CTRL-VEH; Tukey’s test; n_rats = 7−8 CTRL/ PCE-Lithium; 8−9 CTRL/PCE-VEH). Unless otherwise indicated, data are represented with scatter plots with mean and ± SEM. Figure created with BioRender.com.