The Impacts of Adolescent Cannabinoid Exposure on Striatal Anxiety- and Depressive-Like Pathophysiology Are Prevented by the Antioxidant N-Acetylcysteine

Abstract

Background: Exposure to Δ⁹-tetrahydrocannabinol (THC) is an established risk factor for later-life neuropsychiatric vulnerability, including mood- and anxiety-related symptoms. The psychotropic effects of THC on affect and anxiogenic behavioral phenomena are known to target the striatal network, particularly the nucleus accumbens, a neural region linked to mood and anxiety disorder pathophysiology. THC may increase neuroinflammatory responses via the redox system and dysregulate inhibitory and excitatory neural balance in various brain circuits, including the striatum. Thus, interventions that can induce antioxidant effects may counteract the neurodevelopmental impacts of THC exposure.

Methods: In the current study, we used an established preclinical adolescent rat model to examine the impacts of adolescent THC exposure on various behavioral, molecular, and neuronal biomarkers associated with increased mood and anxiety disorder vulnerability. Moreover, we investigated the protective properties of the antioxidant N-acetylcysteine against THC-related pathology.

Results: We demonstrated that adolescent THC exposure induced long-lasting anxiety- and depressive-like phenotypes concomitant with differential neuronal and molecular abnormalities in the two subregions of the nucleus accumbens, the shell and the core. In addition, we report for the first time that N-acetylcysteine can prevent THC-induced accumbal pathophysiology and associated behavioral abnormalities.

Conclusions: The preventive effects of this antioxidant intervention highlight the critical role of redox mechanisms underlying cannabinoid-induced neurodevelopmental pathology and identify a potential intervention strategy for the prevention and/or reversal of these pathophysiological sequelae.

Keywords: Accumbens; Antioxidant; Anxiety; Cannabinoids; Depression; Neurodevelopment.

Figure 1

Effects of NAC on anxiety- and depressive-like manifestations induced by THC exposure during adolescence. (A) Schematic representation of the elevated plus maze apparatus. (B, C) THC-treated rats made fewer entries and spent less time in the open arms of the apparatus. NAC administration did not prevent these effects (veh, n = 13; THC, n = 13; NAC, n = 11; THC/NAC, n = 12). (D) Schematic representation of the forced swimming test apparatus. (E) NAC administration prevented the increased immobility induced by chronic THC exposure (veh, n = 14; THC, n = 16; NAC, n = 12; THC/NAC, n = 12). (F) Schematic representation of the novelty-suppressed feeding test. (G) No differences between groups were observed in the latency to approach the food during the task. (H) THC-treated rats exhibited a longer latency to start feeding during the test, while NAC administration normalized this effect. (I) The rats exposed to THC and NAC consumed more food than the THC- and NAC-treated groups (veh, n = 13; THC, n = 13; NAC, n = 10; THC/NAC, n = 10). (J) Schematic representation of the contextual fear conditioning box. (K–M) Chronic THC exposure induced an increase in freezing time while reducing the number of rearings and the time spent on rearing behavior. NAC administration prevented the THC-related effects on freezing and number of rearings but was ineffective in restoring the rearing behavior time (veh, n = 12; THC, n = 13; NAC, n = 10; THC/NAC, n = 8). ∗p < .05, ∗∗p < .01, ∗∗∗p < .001. NAC, N-acetylcysteine; THC, Δ⁹-tetrahydrocannabinol; veh, vehicle.

Figure 2

Effects of NAC on THC-induced dysregulations of neurotransmitter levels. (A) MALDI-IMS images of a representative rat brain section including AcbSh and AcbC from each group. (B) Representative localizations of the AcbSh and AcbC on rat brain coronal sections adapted from (76). The numbers indicate the distance from bregma. (C, D) Adolescent THC exposure increased GLUT levels (m/z = 146.95; veh, n = 14; THC, n = 12; NAC, n = 14; THC/NAC, n = 12) and decreased ARG levels (m/z = 213.30; veh, n = 14; THC, n = 10; NAC, n = 14; THC/NAC, n = 11) in AcbSh, while the concomitant administration of NAC prevented these THC-related dysregulations. (E) NAA concentrations (m/z = 198.13; veh, n = 14; THC, n = 12; NAC, n = 14; THC/NAC, n = 12) in the AcbSh were reduced by administration of NAC. (F, G) No differences between groups were observed in GABA (m/z = 353.16; veh, n = 16; THC, n = 16; NAC, n = 16; THC/NAC, n = 16) and HVA (m/z = 450.17; veh, n = 16; THC, n = 14; NAC, n = 15; THC/NAC, n = 15) relative quantifications in the AcbSh. (H) Administration of NAC alone reduced GLUT levels (m/z = 146.95; veh, n = 14; THC, n = 14; NAC, n = 14; THC/NAC, n = 14) in the AcbC compared with the veh and THC groups. (I) ARG concentration (m/z = 213.30; veh, n = 14; THC, n = 13; NAC, n = 14; THC/NAC, n = 14) in the AcbC was higher in the THC/NAC group than the NAC- and THC-treated rats. (J) No differences between groups were detected in NAA levels (m/z = 198.13; veh, n = 14; THC, n = 14; NAC, n = 14; THC/NAC, n = 14) in the AcbC. (K, L) Both adolescent THC exposure and NAC administration decreased GABA levels (m/z = 353.16; veh, n = 16; THC, n = 14; NAC, n = 16; THC/NAC, n = 16) and HVA levels (m/z = 450.17; veh, n = 16; THC, n = 13; NAC, n = 15; THC/NAC, n = 15) in AcbC. ∗p < .05, ∗∗p < .01, ∗∗∗p < .001. AcbC, nucleus accumbens core; AcbSh, nucleus accumbens shell; ARG, arginine; GABA, gamma-aminobutyric acid; GLUT, glutamate; HVA, homovanillic acid; MALDI-IMS, matrix-assisted laser desorption/ionization imaging mass spectrometry; NAA, N-acetylaspartate; NAC, N-acetylcysteine; THC, Δ⁹-tetrahydrocannabinol; veh, vehicle.

Figure 3

Effect of NAC on molecular adaptations induced by THC in the AcbSh. (A–H) Insets on the top of the bar graphs are representative Western blots for p-mTOR, t-mTOR, p-Akt (Thr308), t-Akt, D1R, D2R, GAD65, BDNF, mGluR2/3, p-ERK1/2, and t-ERK1/2 in AcbSh. (A) p-mTOR and t-mTOR expression levels were increased by NAC administration (veh, n = 6; THC, n = 6; NAC, n = 4; THC/NAC, n = 4). (B) The NAC group showed higher t-Akt levels than the veh, THC, and THC/NAC groups, while the p-Akt:t-Akt ratio was decreased following NAC and THC/NAC compared with THC (veh, n = 6; THC, n = 6; NAC, n = 4; THC/NAC, n = 4). (C) D1R expression was increased by NAC administration (veh, n = 6; THC, n = 6; NAC, n = 4; THC/NAC, n = 4). (D) THC/NAC induced a reduction in D2R level (veh, n = 6; THC, n = 6; NAC, n = 4; THC/NAC; n = 4). (E) Adolescent THC exposure increased GAD65 levels, while the concomitant administration of THC and NAC prevented this effect (veh, n = 6; THC, n = 6; NAC, n = 4; THC/NAC, n = 4). (F) BDNF expression was increased by NAC administration (veh, n = 6; THC, n = 5; NAC, n = 4; THC/NAC, n = 4). (G) The NAC group showed higher mGluR2/3 levels than the THC and THC/NAC groups (veh, n = 5; THC, n = 6; NAC, n = 4; THC/NAC, n = 3). (H) p-ERK1 expression levels were increased by NAC administration, while t-ERK1 was reduced by THC/NAC compared with the NAC group. Moreover, adolescent THC exposure increased t-ERK2 levels, while the concomitant administration of THC and NAC prevented this effect (veh, n = 6; THC, n = 6; NAC, n = 4; THC/NAC, n = 4). ∗p < .05, ∗∗p < .01, ∗∗∗p < .001. AcbSh, nucleus accumbens shell; BDNF, brain-derived neurotrophic factor; D1R, D₁ receptor; D2R, D₂ receptor; ERK, extracellular signal-regulated kinase; mGluR, metabotropic glutamate receptor; mTOR, mechanistic target of rapamycin; NAA, N-acetylaspartate; NAC, N-acetylcysteine; p, phosphorylated; THC, Δ⁹-tetrahydrocannabinol; t, total; veh, vehicle.

Figure 4

Effect of NAC on molecular adaptations induced by THC in the AcbC. (A–H) Insets on the top of the bar graphs are representative Western blots for p-mTOR, t-mTOR, p-Akt (Thr308), t-Akt, D1R, D2R, GAD65, BDNF, mGluR2/3, p-ERK1/2 and t-ERK1/2 in AcbC. (A) Adolescent THC exposure decreased t-mTOR expression levels, while the concomitant administration of THC and NAC prevented this dysregulation (veh, n = 7; THC, n = 6; NAC, n = 7; THC/NAC, n = 6). No differences between groups were observed in p-mTOR (veh, n = 6; THC, n = 6; NAC, n = 6; THC/NAC, n = 6) and p-mTOR:t-mTOR ratio (veh, n = 6; THC, n = 6; NAC, n = 5; THC/NAC, n = 5). (B–D) No effects were found in the expression levels of p-Akt (veh, n = 7; THC, n = 6; NAC, n = 7; THC/NAC, n = 7), t-Akt (veh, n = 7; THC, n = 7; NAC, n = 6; THC/NAC, n = 6), p-Akt:t-Akt ratio (veh, n = 7; THC, n = 6; NAC, n = 7; THC/NAC, n = 7), D1R (veh, n = 6; THC, n = 6; NAC, n = 7; THC/NAC, n = 7), and D2R (veh, n = 7; THC, n = 7; NAC, n = 6; THC/NAC, n = 6). (E) GAD65 levels were reduced by NAC administration (veh, n = 7; THC, n = 7; NAC, n = 7; THC/NAC, n = 7). (F, G) No differences between groups were observed in the expression of BDNF (veh, n = 7; THC, n = 6; NAC, n = 7; THC/NAC, n = 6) and mGluR2/3 (veh, n = 7; THC, n = 6; NAC, n = 7; THC/NAC, n = 7). (H) Concomitant administration of THC and NAC increased the levels of pERK1 (veh, n = 7; THC, n = 7; NAC, n = 7; THC/NAC, n = 7), p-ERK2 (veh, n = 7; THC, n = 7; NAC, n = 7; THC/NAC, n = 7), and p-ERK2:t-ERK2 ratio (veh, n = 7; THC, n = 6; NAC, n = 7; THC/NAC, n = 7). No changes were found in t-ERK1 (veh, n = 7; THC, n = 6; NAC, n = 6; THC/NAC, n = 7), t-ERK2 (veh, n = 7; THC, n = 6; NAC, n = 6; THC/NAC, n = 7), and p-ERK1:t-ERK1 (veh, n = 7; THC, n = 6; NAC, n = 6; THC/NAC, n = 7). ∗p < .05. AcbC, nucleus accumbens core; BDNF, brain-derived neurotrophic factor; D1R, D₁ receptor; D2R, D₂ receptor; ERK, extracellular signal-regulated kinase; mGluR, metabotropic glutamate receptor; mTOR, mechanistic target of rapamycin; NAA, N-acetylaspartate; NAC, N-acetylcysteine; p, phosphorylated; THC, Δ⁹-tetrahydrocannabinol; t, total; veh, vehicle.

Figure 5

Effects of NAC on neuronal and oscillatory patterns induced by adolescent THC exposure in the AcbS vs. the AcbC. (A, C) Representative rate histograms of putative GABA cells in the AcbSh and AcbC recorded from each group. (B, D) No differences between groups were found in the firing frequency of AcbSh GABA neurons (veh, n = 32 cells/9 rats; THC, n = 49 cells/15 rats; NAC, n = 27 cells/8 rats; THC/NAC, n = 27 cells/5 rats), while the administration of NAC alone decreased the firing rate of GABA neurons in the AcbC (veh, n = 46 cells/16 rats; THC, n = 55 cells/15 rats; NAC, n = 44 cells/6 rats; THC/NAC, n = 44 cells/8 rats). (E) Representative spectrogram of a 5-minute recording. (F, K) Average normalized LFP power spectra in AcbSh and AcbC. (G) Adolescent THC exposure reduced delta waves in the AcbSh, while this effect was prevented by the concomitant administration of THC and NAC (veh, n = 20 recording sites/8 rats; THC, n = 25 recording sites/9 rats; NAC, n = 33 recording sites/8 rats; THC/NAC, n = 19 recording sites/6 rats). (H, I) THC-treated rats exhibited higher AcbSh beta and low gamma oscillations than the veh group, and these effects were prevented by NAC administration (veh, n = 20 recording sites/8 rats; THC, n = 25 recording sites/9 rats; NAC, n = 33 recording sites/8 rats; THC/NAC, n = 19 recording sites/6 rats). (J) NAC administration reduced high gamma waves in the AchSh (veh, n = 20 recording sites/8 rats; THC, n = 25 recording sites/9 rats; NAC, n = 33 recording sites/8 rats; THC/NAC, n = 19 recording sites/6 rats). (L) No differences between groups were found in delta oscillations in the AcbC (veh, n = 41 recording sites/15 rats; THC, n = 32 recording sites/11 rats; NAC, n = 41 recording sites/7 rats; THC/NAC, n = 30 recording sites/7 rats). (M–O) NAC administration decreased beta, low gamma, and high gamma waves in the AcbC (veh, n = 41 recording sites/15 rats; THC, n = 32 recording sites/11 rats; NAC, n = 41 recording sites/7 rats; THC/NAC, n = 30 recording sites/7 rats). ∗p < .05, ∗∗p < .01, ∗∗∗p < .001. AcbC, nucleus accumbens core; AcbSh, nucleus accumbens shell; GABA, gamma-aminobutyric acid; LFP, local field potential; NAC, N-acetylcysteine; THC, Δ⁹-tetrahydrocannabinol; veh, vehicle.