Lactobacillus reuteri ATG-F4 Alleviates Chronic Stress-induced Anhedonia by Modulating the Prefrontal Serotonergic System

Abstract

Mental health is influenced by the gut-brain axis; for example, gut dysbiosis has been observed in patients with major depressive disorder (MDD). Gut microbial changes by fecal microbiota transplantation or probiotics treatment reportedly modulates depressive symptoms. However, it remains unclear how gut dysbiosis contributes to mental dysfunction, and how correction of the gut microbiota alleviates neuropsychiatric disorders. Our previous study showed that chronic consumption of Lactobacillus reuteri ATG-F4 (F4) induced neurometabolic alterations in healthy mice. Here, we investigated whether F4 exerted therapeutic effects on depressive-like behavior by influencing the central nervous system. Using chronic unpredictable stress (CUS) to induce anhedonia, a key symptom of MDD, we found that chronic F4 consumption alleviated CUS-induced anhedonic behaviors, accompanied by biochemical changes in the gut, serum, and brain. Serum and brain metabolite concentrations involved in tryptophan metabolism were regulated by CUS and F4. F4 consumption reduced the elevated levels of serotonin (5-HT) in the brain observed in the CUS group. Additionally, the increased expression of Htr1a, a subtype of the 5-HT receptor, in the medial prefrontal cortex (mPFC) of stressed mice was restored to levels observed in stress-naïve mice following F4 supplementation. We further demonstrated the role of Htr1a using AAV-shRNA to downregulate Htr1a in the mPFC of CUS mice, effectively reversing CUS-induced anhedonic behavior. Together, our findings suggest F4 as a potential therapeutic approach for relieving some depressive symptoms and highlight the involvement of the tryptophan metabolism in mitigating CUS-induced depressive-like behaviors through the action of this bacterium.

Keywords: Anhedonia; Lactobacillus reuteri; Prefrontal cortex; Serotonin.

Fig. 1

CUS-induced anhedonic behaviors were recovered by chronic Lactobacillus reuteri ATG-F4 consumption. (A) Experimental schedule. (B) Sucrose preference after CUS±F4. Uncorrected Fisher’s LSD; Control (n=7), CUS+Veh (n=9), CUS+F4 (n=10). (C) Body weight after CUS±F4. Uncorrected Fisher’s LSD; Control (n=7), CUS+Veh (n=9), CUS+F4 (n=10). *p<0.05, **p<0.01, ns (not significant).

Fig. 2

CUS and F4 consumption changed 5-HT and tryptophan metabolic pathways in the serum and brain. (A) A simplified schematic diagram of tryptophan metabolic pathways. (B~H) Concentrations of tryptophan metabolites in serum. (B) Tryptophan. Unpaired Welch’s t-test; Control (n=16), CUS+Veh (n=9), CUS+F4 (n=8). (C) 5-HTP. Uncorrected Fisher’s LSD; Control (n=16), CUS+Veh (n=9), CUS+F4 (n=8). (D) 5-HT. Uncorrected Fisher’s LSD; Control (n=16), CUS+Veh (n=9), CUS+F4 (n=8). (E) 5-HIAA. Uncorrected Fisher’s LSD; Control (n=16), CUS+Veh (n=9), CUS+F4 (n=8). (F) Kynurenine. Uncorrected Fisher’s LSD; Control (n=16), CUS+Veh (n=9), CUS+F4 (n=8). (G) Kynurenic acid. Uncorrected Fisher’s LSD; Control (n=16), CUS+Veh (n=9), CUS+F4 (n=8). (H) Tryptamine. Unpaired Welch’s t-test; Control (n=16), CUS+Veh (n=9), CUS+F4 (n=8). (I~O) Concentrations of tryptophan metabolites in brain. (I) Tryptophan. Unpaired Welch’s t-test; Control (n=16), CUS+Veh (n=7), CUS+F4 (n=8). (J) 5-HTP. Uncorrected Fisher’s LSD; Control (n=16), CUS+Veh (n=7), CUS+F4 (n=8). (K) 5-HT. Uncorrected Fisher’s LSD; Control (n=16), CUS+Veh (n=7), CUS+F4 (n=8). (L) 5-HIAA. Unpaired Welch’s t-test; Control (n=16), CUS+Veh (n=7), CUS+F4 (n=8). (M) Kynurenine. Uncorrected Dunn’s test. Control (n=16), CUS+Veh (n=7), CUS+F4 (n=8). (N) Kynurenic acid. Uncorrected Dunn’s test; Control (n=16), CUS+Veh (n=7), CUS+F4 (n=8). (O) Tryptamine. Unpaired Welch’s t-test; Control (n=16), CUS+Veh (n=7), CUS+F4 (n=8); 5-HTP, 5-hydroxytryptophan; 5-HT, Serotonin; 5-HIAA, 5-Hydroxydindoleacetic acid. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Fig. 3

CUS-induced upregulation of Htr1a in the mPFC was recovered by F4 treatment. (A~C) mRNA expression levels of 5-HT receptor types in the mPFC. (A) Htr1a. Tukey’s test; Control (n=4), CUS+Veh (n=5), CUS+F4 (n=4). (B) Htr1b. Tukey’s test; Control (n=4), CUS+Veh (n=5), CUS+F4 (n=7). (C) Htr2a. Tukey’s test; Control (n=4), CUS+Veh (n=5), CUS+F4 (n=7). (D~F) mRNA expression levels of 5-HT receptor types in the vHIP. (D) Htr1a. Tukey’s test; Control (n=3), CUS+Veh (n=6), CUS+F4 (n=8). (E) Htr1b. Tukey’s test; Control (n=3), CUS+Veh (n=6), CUS+F4 (n=8). (F) Htr2a. Dunn’s test; Control (n=3), CUS+Veh (n=6), CUS+F4 (n=8). (G~I) mRNA expression levels of 5-HT receptor types in the BLA. (G) Htr1a. Tukey’s test; Control (n=4), CUS+Veh (n=5), CUS+F4 (n=6). (H) Htr1b. Tukey’s test; Control (n=4), CUS+Veh (n=5), CUS+F4 (n=6). (I) Htr2a. Tukey’s test; Control (n=4), CUS+Veh (n=5), CUS+F4 (n=6). *p<0.05, ns (not significant). mPFC, medial prefrontal cortex; vHIP, ventral hippocampus; BLA, basolateral amygdala.

Fig. 4

CUS-induced anhedonic behaviors were alleviated by Htr1a knockdown in the mPFC. (A) Experimental schedule. (B) Representative images of AAV injection site in the mPFC. Scale bar=1,000 μm (C) Efficiency of Htr1a knockdown (KD) by AAV expression in the mPFC. Mann-Whitney U test; Scramble (n=6), Htr1a KD (n=6). (D) Sucrose preference after CUS±Htr1a KD. Tukey’s test; No CUS+AAV-scramble (n=8), CUS+AAV-scramble (n=8), CUS+AAV-shHtr1a (n=8). (E) Body weight after CUS±Htr1a KD. Tukey’s test; No CUS+AAV-scramble (n=8), CUS+AAV-scramble (n=8), CUS+AAV-shHtr1a (n=8). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns (not significant).