N-Acetylcysteine Mitigates Social Dysfunction in a Rat Model of Autism Normalizing Glutathione Imbalance and the Altered Expression of Genes Related to Synaptic Function in Specific Brain Areas

Abstract

Prenatal exposure to valproic acid (VPA) is a risk factor for autism spectrum disorder (ASD) in humans and it induces autistic-like behaviors in rodents. Imbalances between GABAergic and glutamatergic neurotransmission and increased oxidative stress together with altered glutathione (GSH) metabolism have been hypothesized to play a role in both VPA-induced embriotoxicity and in human ASD. N-acetylcysteine (NAC) is an antioxidant precursor of glutathione and a modulator of glutamatergic neurotransmission that has been tested in ASD, although the clinical studies currently available provided controversial results. Here, we explored the effects of repeated NAC (150 mg/kg) administration on core autistic-like features and altered brain GSH metabolism in the VPA (500 mg/kg) rat model of ASD. Furthermore, we measured the mRNA expression of genes encoding for scaffolding and transcription regulation proteins, as well as the subunits of NMDA and AMPA receptors and metabotropic glutamate receptors mGLUR1 and mGLUR5 in brain areas that are relevant to ASD. NAC administration ameliorated the social deficit displayed by VPA-exposed rats in the three-chamber test, but not their stereotypic behavior in the hole board test. Furthermore, NAC normalized the altered GSH levels displayed by these animals in the hippocampus and nucleus accumbens, and it partially rescued the altered expression of post-synaptic terminal network genes found in VPA-exposed rats, such as NR2a, MGLUR5, GLUR1, and GLUR2 in nucleus accumbens, and CAMK2, NR1, and GLUR2 in cerebellum. These data indicate that NAC treatment selectively mitigates the social dysfunction displayed by VPA-exposed rats normalizing GSH imbalance and reestablishing the expression of genes related to synaptic function in a brain region-specific manner. Taken together, these data contribute to clarify the behavioral impact of NAC in ASD and the molecular mechanisms that underlie its effects.

Keywords: N-acetylcysteine; autism; glutathione; rats; valproic acid.

Figure 1

Timeline of the experiments.

Figure 2

N-acetylcysteine ameliorates the aberrant social phenotype but it does not affect the repetitive behaviors displayed by VPA-exposed animals. At PND 35, VPA-exposed rats showed reduced sociability in the three-chamber test, as they spent less time in the stimulus room (A) and sniffing the stimulus animal (B) compared to SAL-exposed rats. Treatment with NAC reversed the deficit displayed by VPA-exposed rats in this test (SAL-VEH, n = 7; SAL-NAC, n = 8; VPA-VEH, n = 6; VPA-NAC, n = 8). Moreover, VPA-exposed rats showed stereotypic behaviors in the hole-board test, as they made more head dippings (C) compared to SAL-exposed rats. Treatment with NAC was not able to rescue the repetitive behavior found in VPA-exposed rats (SAL-VEH, n = 6; SAL-NAC, n = 7; VPA-VEH, n = 10; VPA-NAC, n = 12). Data represent mean ± S.E.M. *p < 0.05, **p < 0.01 vs. SAL-VEH group; #p < 0.05, ##p < 0.01 vs. VPA-VEH group (Student's–Newman–Keuls post hoc test).

Figure 3

Brain region specific changes in total (A) and free (B) GSH levels. Total (A) and free (B) GSH levels in hippocampus (HIPP), amygdala (AMY), cerebellum (CER), dorsal striatum (DS), nucleus accumbens (NAc), and prefrontal cortex (PFC) of VPA-, SAL- and VPA-NAC-treated rats (n = 3 per group). Data represent mean ± SD. **p < 0.01, ***p < 0.001, vs. SAL-group TOTAL GSH; ^###p < 0.01 vs. SAL-group FREE GSH; °p < 0.05, °°p < 0.01, °°°p < 0.001 vs. VPA-group TOTAL GSH; ^§§§p < 0.001 vs. VPA-group FREE GSH (Tukey post-hoc test).

Figure 4

N-acetylcysteine partially rescues the altered expression of post-synaptic terminal network genes found in VPA-exposed rats. Relative mRNA levels of scaffolding (PSD95) and transcriptional regulators (CAMK2 and MeCP2), ionotropic NMDA receptor subunits (NR1, NR2a, and NR2b), ionotropic AMPA receptor subunits (GLUR1 and GLUR2) and metabotropic glutamate receptors MGLUR1 and MGLUR5, assessed in HIPP (A), AMY (B), CER (C), DS (D), NAc (E), and PFC (F) of rats prenatally exposed to either VPA or SAL and treated with NAC or its vehicle (VEH) (n = 3–4). Values represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. SAL/VEH group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. VPA/VEH group; °p < 0.05 vs. VPA/NAC group (Tukey post-hoc test).