Transgenerational transmission of aspartame-induced anxiety and changes in glutamate-GABA signaling and gene expression in the amygdala

Abstract

We report the effects of aspartame on anxiety-like behavior, neurotransmitter signaling and gene expression in the amygdala, a brain region associated with the regulation of anxiety and fear responses. C57BL/6 mice consumed drinking water containing 0.015% or 0.03% aspartame, a dose equivalent of 8 to 15% of the FDA recommended maximum human daily intake, or plain drinking water. Robust anxiety-like behavior (evaluated using open field test and elevated zero maze) was observed in male and female mice consuming the aspartame-containing water. Diazepam, an allosteric modulator of the GABA-A receptor, alleviated the anxiety-like behavior. RNA sequencing of the amygdala followed by KEGG biological pathway analysis of differentially expressed genes showed glutamatergic and GABAergic synapse pathways as significantly enriched. Quantitative PCR showed upregulation of mRNA for the glutamate NMDA receptor subunit 2D (Grin2d) and metabotropic receptor 4 (Grm4) and downregulation of the GABA-A receptor associated protein (Gabarap) mRNA. Thus, taken together, our diazepam and gene expression data show that aspartame consumption shifted the excitation-inhibition equilibrium in the amygdala toward excitation. Even more strikingly, the anxiety-like behavior, its response to diazepam, and changes in amygdala gene expression were transmitted to male and female offspring in two generations descending from the aspartame-exposed males. Extrapolation of the findings to humans suggests that aspartame consumption at doses below the FDA recommended maximum daily intake may produce neurobehavioral changes in aspartame-consuming individuals and their descendants. Thus, human population at risk of aspartame's potential mental health effects may be larger than current expectations, which only include aspartame-consuming individuals.

Keywords: artificial sweetener; emotional behavior; intergenerational transmission.

Fig. 1.

Anxiety and its response to diazepam in mice exposed to aspartame-containing drinking water. Anxiety-like responses were analyzed in male and female mice exposed daily to drinking water containing 0.03% aspartame, 0.015% aspartame or to plain drinking water for 12 wk using open field test (OFT; A and B) and elevated zero maze (EZM; D). In the OFT analysis (A and B), two-way ANOVA showed that male (A) and female (B) mice in the 0.03% aspartame group (blue line) and 0.015% aspartame (purple line) groups spent significantly shorter time in the center areas in the OFT compared to their counterparts in the plain drinking water (black) group (**** in A and B). Dunnett’s multiple comparisons test showed that significant differences emerged between 0.015% aspartame and plain water groups at 6 wk in males and females and persisted at 8 wk, 10 wk, and 12 wk (A and B). Significant differences between 0.03% aspartame and plain water groups emerged at 8 wk in males and females and persisted at 10 wk and at 12 wk (A and B). Typical tracks of open field exploration by one male mouse in each of the plain water, 0.03% aspartame and 0.015% aspartame groups showing differences in exploration of the center areas (C). The male and female mice in the 0.015% aspartame and plain water groups were examined in the EZM (D). Two-way ANOVA of the EZM data showed no significant effect of sex (E). Therefore, the data from male and female mice were analyzed together. The aspartame group spent significantly shorter time in the open areas of the EZM (E). Response of male and female mice in the 0.03% aspartame group to diazepam was analyzed in the OFT (E). Initially, baseline parameters were established 30 min following a single intraperitoneal administration of saline. Next, 48 h. later, the same mice received diazepam (3 mg/kg, i.p.) and 30 min following the diazepam administration, the mice were re-examined in the OFT (E). Repeated Measures ANOVA showed no significant effect of sex (E). Therefore, data from male and female mice were combined and analyzed. The time spent in the center areas was significantly increased following the diazepam administration compared to the saline administration at baseline (E). Typical tracks of open field exploration by one male mouse each in the saline and diazepam groups (F). Notes on symbols: # = comparison between 0.015% aspartame and plain water group; $ = comparison between 0.015% aspartame and plain water group. #; $ = P < 0.05; ##; $$ = P < 0.01; ###; $$$ = P < 0.001; ****, ####; $$$$ = P < 0.0001.

Fig. 2.

Transcriptome profiling of the amygdala by RNA sequencing in male mice exposed to 0.03% aspartame or plain drinking water for 12 wk. The top 20 most significantly enriched KEGG pathways are shown in order of lowest to highest adjusted P value, which is represented as the ^–log of the adjusted P value on the X-axis (A). Heat maps showing a comparison of normalized differential gene expression values of the 30 DEGs between the aspartame and plain water groups from the glutamatergic synapse pathway (B) and 20 DEGs from the GABAergic synapse pathway (C). Rows represent the gene symbol, and columns represent each replicate. The minimum to maximum expression is indicated by a gradient from dark blue to dark red, respectively.

Fig. 3.

Quantitative PCR analysis of expression of mRNAs for genes in the glutamatergic synapse and GABAergic synapse KEGG pathways in F0 (A–C) and F1 (D–F) generations in the amygdala of male and female mice exposed to 0.03% aspartame or plain water for 12 wk. In each analysis, expression of the mRNA of interest was normalized to that of ribosomal 18s RNA. Two-way ANOVA did not show significant effect of sex any of the analyses. Therefore, data from male and female mice were combined and analyzed. There was a significant upregulation of mRNA for glutamatergic genes Grin2d and Grm4 in the F0 (A and B) and F1 (D and E) generations and significant downregulation of the GABA-A receptor associate gene Gabarap in the F0 (C) and F1 (F) generations in the aspartame group. * = P < 0.05; ** = P < 0.01; **** = P < 0.0001.

Fig. 4.

Anxiety-like behavior in male and female mice in the F1 (A) and F2 (B) generations descending from the paternal aspartame lineage and response to diazepam (C and D) were analyzed using the open field test (OFT). Two-way ANOVA showed no significant effect of sex in F1 or F2 generations. Therefore, data from male and female mice were analyzed together. The mice from the paternal aspartame lineage spent significantly shorter time in the center areas of the open field compared to their counterparts from the plain water lineage in F1 (A) and F2 (B) generations (male + female combined). Male and female mice from F1 and F2 generations were examined in the OFT 30 min following a single intraperitoneal administration of saline (C and D). The same mice received diazepam (3 mg/kg, i.p.) 48 h. later and were re-examined in the OFT 30 min after the diazepam administration. Repeated Measures ANOVA did not show significant effect of sex. Therefore, data from male and female mice were combined and analyzed. The time spent in the center areas was significantly reduced following the diazepam administration compared to the earlier saline administration in F1 (C) and F2 (D) generations (male + female). *** = P < 0.001; **** = P < 0.0001.