Consumption of the Prebiotic-Rich Chicory Taproot Contrasts the Cognitive and Motivational Consequences of Chronic Corticosterone Exposure and Modulates Gut Microbiota Composition in Mice

Abstract

Background: The human gastrointestinal tract harbors trillions of microbes that act in synergy with the brain to regulate its homeostasis and function. This interplay holds promise for innovative dietary-based interventions to support cognitive and motivational processes or contrast their decline in disease. While probiotics have traditionally been used for such interventions, several limitations have hampered their suitability and incited interest in prebiotics. Fructans represent a valid prebiotic whereby they are abundant in several vegetables (e.g., chicory taproots) and increase short-chain fatty acids (SCFAs) production via fermentation by gut microbes. SCFAs have been reported to modulate gene expression in the brain via epigenetic mechanisms. Here, we investigated whether chicory taproots may represent a strategy to contrast cognitive and motivational impairments induced by chronic corticosterone administration.

Methods: To test our hypothesis, we exposed C57BL/6 male mice (n = 18 per group) to corticosterone supplementation in drinking water and provided them with a fructan-rich diet (regular diet enriched with dried chicory taproots).

Results: Consistent with our hypothesis, chicory taproot consumption promoted the growth of selected microbial species and increased SCFA concentrations. To verify the functional role of these modulations, using a comprehensive behavioral test battery, we observed that chicory taproots contrasted the cognitive and motivational consequences of chronic corticosterone exposure. These behavioral modifications were associated with a modulation of gene expression and its epigenetic regulators in brain regions relevant for cognition and motivation.

Conclusions: These results highlight the role of prebiotics in preserving higher-order brain functions and offer insights into their therapeutic potential.

Keywords: Chronic stress; Epigenetic modulators; Executive functions; Fructans; Gut-brain axis; Short-chain fatty acids (SCFAs).

Figure 1

Timeline of the experiment and prebiotic administration’s effects on selected physiological parameters. (A) Timeline of the experiment. The behavioral and physiological tests were divided in 2 separate test batteries. Battery A included the FR test for associative learning, the PR test for motivation, the ASST for attentional capabilities and cognitive flexibility, and collection of biological samples (feces, brain areas, cecal content). Battery B included the Barnes maze test (Barnes) for short- and long-term spatial memory, the elevated ZM and the OF tests for anxiety-like behavior, the NOR test for short- and long-term recognition memory, and the response to restraint stress for physiological stress reactivity. The behavioral tests of batteries A and B were completed within the ninth and the eighth week of diet administration, respectively. The response to restraint stress (battery B) was evaluated during the 9th week of diet administration, while the biological samples (battery A) were collected during the 12th week. Body weight and food/liquid intake were monitored weekly throughout the duration of the experiment. Behavioral tests in battery A (FR, PR, ASST) required the application of a food restriction schedule aimed at maintaining the animals at 85% to 90% of their free feeding body weight (for details, see Supplemental Methods). Therefore, analyses of body weight variation and fructans intake involved only mice belonging to battery B, because they were not subjected to the food restriction schedule, while analysis of corticosterone intake comprised both test batteries. Food and liquid intakes were measured by weighing the food in the feeders and the bottles; therefore, because mice were housed in couples, for the analyses of fructans and CORT intake, the cage, instead of the single mouse, constituted the experimental unit. (B) Body weight gain (H₂O-CONTROL n = 18, H₂O-CHICORY n = 18, CORT-CONTROL n = 18, CORT-CHICORY n = 18). The chicory-enriched diet reversed the CORT-induced increase in body weight. (C) Food intake (H₂O-CONTROL n = 9, H₂O-CHICORY n = 9, CORT-CONTROL n = 9, CORT-CHICORY n = 9). Mice that were fed the prebiotic diet exhibited an increment in food intake compared with mice fed the standard diet. (D) Liquid intake (H₂O-CONTROL n = 18, H₂O-CHICORY n = 18, CORT-CONTROL n = 18, CORT-CHICORY n = 18). Mice fed the prebiotic diet exhibited an increment in liquid intake compared with mice fed the standard diet. (E) CORT intake (CORT-CONTROL n = 18, CORT-CHICORY n = 18). Mice exposed to the chicory-enriched diet showed increased CORT intake compared with mice exposed to the standard diet. ^£p < .05 H₂O-CONTROL vs. CORT-CONTROL, ^$p < .05 H₂O-CHICORY vs. CORT-CHICORY, ^€p < .05 H₂O-CONTROL vs. H₂O-CHICORY, ^§p < .05 CORT-CONTROL vs. CORT-CHICORY in Tukey’s post hoc test. ASST, attentional set-shifting task; CORT, corticosterone; FR, fixed ratio; OF, open field; NOR, novel object recognition; PR, progressive ratio; ZM, zero maze.

Figure 2

Prebiotic administration’s effects on plasma CORT concentrations and anxiety-related behaviors. (A) Plasma CORT concentrations at baseline and 25, 60, 120, and 240 minutes after restraint stress (H₂O-CONTROL n = 12, H₂O-CHICORY n = 13, CORT-CONTROL n = 15, CORT-CHICORY n = 12). The chicory-enriched diet reduced basal CORT concentrations in stressed mice. Prebiotic administration had no effect on the time spent in the open sectors of the elevated zero maze (H₂O-CONTROL n = 18, H₂O-CHICORY n = 17, CORT-CONTROL n = 18, CORT-CHICORY n = 18) (B), time spent in the center (C), or distance traveled (D) in the open field (H₂O-CONTROL n = 18, H₂O-CHICORY n = 18, CORT-CONTROL n = 18, CORT-CHICORY n = 18). ^£p < .05 H₂O-CONTROL vs. CORT-CONTROL, ^$p < .05 H₂O-CHICORY vs. CORT-CHICORY, ^§p < .05 CORT-CONTROL vs. CORT-CHICORY in Tukey’s post hoc test. ∗p < .05 main effect of treatment. CORT, corticosterone. The dashed line represents chance level.

Figure 3

Prebiotic administration’s effects on learning, memory, motivation, and executive functions. (A) Associative learning in the fixed ratio test (H₂O-CONTROL n = 14, H₂O-CHICORY n = 15, CORT-CONTROL n = 11, CORT-CHICORY n = 12). Mice receiving the chicory-enriched diet showed improved learning capabilities (increased nosepoke number) regardless of CORT treatment. (B) Motivation in the progressive ratio test (H₂O-CONTROL n = 14, H₂O-CHICORY n = 15, CORT-CONTROL n = 11, CORT-CHICORY n = 12). Chronically stressed mice exposed to the chicory-enriched diet showed increased motivation as indicated by a higher breaking point compared with their controls. Prebiotic administration improved attentional capabilities by reducing the trials to criterion in the simple discrimination (C) and intradimensional shift (D) stages of the attentional set-shifting task (H₂O-CONTROL n = 12, H₂O-CHICORY n = 12, CORT-CONTROL n = 12, CORT-CHICORY n = 12). Spatial learning (E) and memory in the Barnes maze test (H₂O-CONTROL n = 18, H₂O-CHICORY n = 18, CORT-CONTROL n = 18, CORT-CHICORY n = 18) (F, G, I, J). The chicory-enriched diet had no effect on either spatial learning (E) or memory (F, G, I, J). Recognition memory in the NOR test (H₂O-CONTROL n = 14, H₂O-CHICORY n = 17, CORT-CONTROL n = 13, CORT-CHICORY n = 18) (H–K). Prebiotic administration had no effect on the preference for the novel object either in the short-term (H) or the long-term (K) retention test. ^£p < .05 H₂O-CONTROL vs. CORT-CONTROL, ^$p < .05 H₂O-CHICORY vs. CORT-CHICORY, ^€p < .05 H₂O-CONTROL vs. H₂O-CHICORY, ^§p < .05 CORT-CONTROL vs. CORT-CHICORY in Tukey’s post hoc test. CORT, corticosterone; NOR, novel object recognition. Dashed lines represent chance level.

Figure 4

Prebiotic administration’s effects on gut microbiota composition (H₂O-CONTROL n = 11, H₂O-CHICORY n = 11, CORT-CONTROL n = 11, CORT-CHICORY n = 11). (A) Principal coordinate analysis showing a clear separation between mice exposed to the control diet and mice exposed to the chicory-enriched diet regardless of CORT treatment. (B) Shannon index indicating a significantly lower α diversity in mice exposed to the chicory-enriched diet compared with controls. (C) Microbial distribution at the phylum level. Relative abundances of phylum-level distributions of cecum microbiota are shown. Proteobacteria was significantly decreased while Tenericutes and Verrucomicrobia were significantly increased in the prebiotic groups compared with the corresponding control groups. (D) Microbial distribution at the family level. Relative abundances of family-level distributions of cecum microbiota are shown. The proportions of Coriobacteriaceae, Desulfovibrionaceae, and Lactobacillaceae were significantly decreased while those of Paraprevotellaceae, Prevotellaceae, and Verrucomicrobiaceae were significantly increased in the prebiotics groups compared with the corresponding control groups. All families comprising <0.01% of the total abundance were combined into the Other category. ^¶p < .05 main effect of diet. CORT, corticosterone.

Figure 5

Relative abundance of selected phyla, families, and genera with significant differences among the 4 experimental groups and SCFA concentrations (H₂O-CONTROL n = 11, H₂O-CHICORY n = 11, CORT-CONTROL n = 11, CORT-CHICORY n = 11). Relative abundance of Tenericutes (A), Verrucomicrobia (B), Proteobacteria (C), Prevotellaceae (D), Lactobacillaceae (E), Ruminococcus(F), Parabacteroides(G), and Allobaculum(H) are shown. Concentrations of cumulative SCFAs (I), acetate (J), propionate (K), and butyrate (L) are shown. ^£p < .05 H₂O-CONTROL vs. CORT-CONTROL, ^$p < .05 H₂O-CHICORY vs. CORT-CHICORY, ^€p < .05 H₂O-CONTROL vs. H₂O-CHICORY, ^§p < .05 CORT-CONTROL vs. CORT-CHICORY in Tukey’s post hoc test. ∗p < .05 main effect of treatment, ^¶p < .05 main effect of diet. CORT, corticosterone; DW, dry weight; SCFA, short-chain fatty acid.

Figure 6

Gene expression and epigenetic markers in selected brain areas. miRNA-29b-3p and miRNA-29c-3p expression in the prefrontal cortex (H₂O-CONTROL n = 11, H₂O-CHICORY n = 11, CORT-CONTROL n = 11, CORT-CHICORY n = 11) (A, C) and the hippocampus (H₂O-CONTROL n = 11, H₂O-CHICORY n = 11, CORT-CONTROL n = 12, CORT-CHICORY n = 12) (B, D), respectively. miRNA-132-3p and miRNA-212-3p expression in prefrontal cortex (H₂O-CONTROL n = 10–9, H₂O-CHICORY n = 7, CORT-CONTROL n = 11, CORT-CHICORY n = 9) (E, G) and the hippocampus (H₂O-CONTROL n = 9, H₂O-CHICORY n = 9–10, CORT-CONTROL n = 10–12, CORT-CHICORY n = 10–11) (F, H), respectively. Average DNA methylation of the Cnr1 gene in the prefrontal cortex and the hippocampus (H₂O-CONTROL n = 11, H₂O-CHICORY n = 11, CORT-CONTROL n = 11, CORT-CHICORY n = 11) (I, J). Cnr1 expression in the prefrontal cortex (H₂O-CONTROL n = 11, H₂O-CHICORY n = 11, CORT-CONTROL n = 11, CORT-CHICORY n = 11) (K) and the hippocampus (H₂O-CONTROL n = 10, H₂O-CHICORY n = 10, CORT-CONTROL n = 11, CORT-CHICORY n = 11) (L), respectively. CORT-treated mice receiving chicory-enriched diet showed increased Cnr1 expression in the hippocampus accompanied with increased expression of related miRNAs. ^$p < .05 H₂O-CHICORY vs. CORT-CHICORY, ^§p < .05 CORT-CONTROL vs. CORT-CHICORY in Tukey’s post hoc test. CORT, corticosterone; CTRL, control.