Divergent outcomes of delta 9 - tetrahydrocannabinol (THC) in adolescence on mesocortical dopamine and cognitive development in male and female mice

Abstract

The increasing exposure to delta 9-tetrahydrocannabinol (THC) in youth sparks concerns about disruption of ongoing neurodevelopment. During adolescence, dopamine axons continue to grow from the striatum to the prefrontal cortex, promoting the refinement of inhibitory control. This process is coordinated by the Netrin-1 receptor, DCC, which is regulated by microRNA miR-218. In addition, microglial actions significantly influence adolescent cortical refinement. Here, we show that THC in adolescent mice has sex-specific effects on dopamine innervation in the adult prefrontal cortex. While females show no changes, in males, THC leads to a reduction in the volume occupied by dopamine axons in the medial prefrontal cortex and a decrease in the density of their presynaptic sites. However, it increases dopamine innervation in the orbitofrontal cortex. Assessment of the effects of THC in adolescence on impulse control in adulthood, using the Go-No/Go task, revealed male-specific alterations - THC increased premature responding but reduced the number of commission errors. Molecular analysis showed that, one week after adolescent THC, males display increased Dcc and decreased miR-218 levels. In contrast, females exhibit decreased Dcc levels without changes in miR-218. Furthermore, in the medial prefrontal cortex, females show smaller microglia soma size, potentially mitigating the impact of decreased Dcc on dopamine development. These findings suggest that in adolescent males, THC dysregulates the miR-218/DCC pathway, prompting mistargeting of dopamine axons and diverting their growth from medial to orbitofrontal regions. This work highlights the sex-specific impact of adolescent THC on dopamine and impulse control development and uncovers potential divergent molecular and epigenetic processes.

Keywords: Adolescence; Dcc; Dopamine; Impulsivity; MiR- 218; Mice; Microglia; Prefrontal cortex; THC.

Fig. 1

THC (5 mg/kg) in adolescence disrupts adult medial prefrontal cortex dopamine innervation in males but not females. A, Timeline of treatment and experimental procedures. B Left panel: photomicrograph of a coronal section of the pregenual medial prefrontal cortex at a low magnification (5X) showing an overlay of the contours traced to delineate the dopamine input to the subregions of interest. Scale bar = 500 mm. Right panel: photomicrograph of a coronal section of the pregenual medial prefrontal cortex at a high magnification (100 X) showing the tyrosine hydroxylase (TH)-positive (TH+) varicosities. Scale bar = 10 mm. C, Male mice treated with THC (5 mg/kg) during adolescence show decreased expanse of the dopamine input to the medial prefrontal cortex, but these differences are not observed in females. D, Reduced dopamine input to the medial prefrontal cortex in males administered THC in adolescence is associated with a concomitant decrease in total number of TH+ varicosity within the prelimbic (PrL) and infralimbic (IL) subregions of the medial prefrontal cortex. E, Males treated with THC in adolescence show an increase in the density of TH + varicosities in the orbitofrontal cortex, including dorsal agranular insular prefrontal cortex (daiPFC), ventral agranular insular prefrontal cortex (vaiPFC), ventral orbital prefrontal cortex (voPFC) subregions. ** significantly different, p < 0.01; * significantly different, p < 0.05. All data are shown as mean ± SEM (n = 4 per group)

Fig. 2

THC in early adolescent male mice improves action impulsivity in adulthood but impairs waiting impulsivity. A, Experimental timeline, and procedures B, Go/No-Go experimental design. C, Adult males that received THC in early adolescence have a lower proportion of commission errors compared to VEH. Area under the curve (AUC, inset) illustrates a lower proportion of commission errors overall in the THC compared to the VEH group. D, THC (5 mg/kg) does not affect the proportion of hits in Go trials. Area under the curve (AUC, inset) illustrates similar proportion of hits between treatment groups. E, Experimental design to capture premature responses. F, THC in early adolescence leads to increased proportion of premature responses in adulthood compared to VEH. Area under the curve (AUC, inset) illustrates a larger proportion of premature responses overall for the THC compared to VEH group. G, THC and VEH male groups show similar progressive ratio breakpoints in adulthood. H, Males that received THC in early adolescence show reduced body weight gain compared to VEH treated males across treatment days. Data are presented as the increment (Δ) in body weight (grams) per animal at each time point with their Day 1 body weight as the reference point. Area under the curve (AUC, right panel inset) illustrates lower Δ body weight (g)/animal gain in the THC group compared to VEH during treatment. The difference in body weight between treatment conditions does not persist into adulthood. ** significantly different, p < 0.01; * significantly different, p < 0.05. All data are shown as mean ± SEM (n = 9–11 per group)

Fig. 3

THC in early adolescence upregulates Dcc and downregulates miR-218 in VTA dopamine neurons of male mice. A, Experimental timeline and procedures. B, Tissue extraction and transcript amplification. C, THC upregulates Dcc mRNA expression in the VTA of males one week after last drug exposure in early adolescence. D, Male mice treated with THC (2.5 and 5 mg/kg) show a significant decrease in miR-218. E, Across treatment days, body weight gain is reduced in THC (5 mg/kg) groups compared to VEH controls. Data are presented as the increment (Δ) in body weight (grams) per animal at each time point with their Day 1 body weight as the reference point. Area under the curve (AUC) illustrates altered Δ body weight (g)/animal gain only between THC (5 mg/kg) and VEH groups. F, Experimental timeline and procedures (left), representative image of ex-vivo 2-photon microscopy of brain microglia coupled with a microtome and mechanized stage (right). G, THC (5 mg/kg) does not alter microglia density in the prefrontal cortex of males one week after last drug exposure; p-values of the difference between THC and VEH males are shown on 6 coronal sections of the average T2-weighted magnetic resonance image of the brain (left). H, Representative images of the mouse brain in males (left). THC (5 mg/kg) does not alter microglia soma size in the prefrontal cortex of males one week after last drug exposure; p-values of the difference between THC and VEH males are shown on 6 coronal sections of the average T2-weighted magnetic resonance image of the brain (left). ** significantly different, p < 0.01; * significantly different, p < 0.05. All data are shown as mean ± SEM (n = 6–8 per group)

Fig. 4

THC in early adolescence does not alter impulse control in adulthood in female mice. A, Experimental timeline, and procedures. B, Go/No-Go experimental design. C, THC and VEH female groups show similar proportion of commission errors in No-Go trials. Area under the curve (AUC, inset) illustrates similar proportion of commission errors between THC and VEH groups. D, Similar proportion of hits in Go trials between adult female mice administered THC (5 mg/kg) or VEH in early adolescence. Area under the curve (AUC, inset) illustrates similar proportion of hits between THC and VEH groups. E, Experimental design to capture premature responses. F, THC and VEH female groups show similar proportion of premature responses during the training phase. Area under the curve (AUC, inset) illustrates similar proportion of premature responses between THC and VEH groups. G, THC and VEH female groups show similar progressive ratio breakpoints in adulthood. H, Body weight gain is similar between THC and VEH treated females across treatment days. Data are presented as the increment (Δ) in body weight (grams) per animal at each time point with their Day 1 body weight as the reference point. Area under the curve (AUC, right panel inset) illustrates similar Δ body weight (g)/animal gain between THC and VEH groups during treatment. The lack of change in body weight between treatment conditions persists into adulthood. ** significantly different, p < 0.01; * significantly different, p < 0.05. All data are shown as mean ± SEM (n = 9–11 per group)

Fig. 5

In females, THC in early adolescence induces dose-dependent Dcc downregulation in dopamine neurons, without altering miR-218 levels. A, Experimental timeline and procedure. B, Tissue extraction and transcript amplification. C, Treatment with 5, but not 2.5 mg/kg THC in early adolescent female mice downregulates Dcc mRNA expression in the VTA, one week after the last drug exposure. D, THC (either 2.5 or 5 mg/kg) does not alter VTA miR-218 expression in females. E, Body weight gain across treatment days is similar between THC and VEH groups – data are presented as the increment (Δ) in body weight (grams) per animal at each time point with their Day 1 body weight as the reference point. Area under the curve (AUC) illustrates similar Δ body weight (g)/animals gain between THC and VEH groups. F, Experimental timeline and procedures (left), representative image of ex-vivo 2-photon microscopy of brain microglia coupled with a microtome and mechanized stage (right). G, THC does not alter microglia density in the prefrontal cortex of females one week after last drug exposure in early adolescence; p-values of the difference between THC and VEH females are shown on 6 coronal sections of the average T2-weighted magnetic resonance image of the brain (left). H, Female mice treated with THC (5 mg/ kg) show a significant decrease in microglia soma size in the prefrontal cortex; p-values of the difference between THC and VEH females are shown on 6 coronal sections of the average T2-weighted magnetic resonance image of the brain (left). ** significantly different, p < 0.01; * significantly different, p < 0.05. All data are shown as mean ± SEM (n = 11–12 per group)