Indoleamine-2,3-Dioxygenase Mediates Emotional Deficits by the Kynurenine/Tryptophan Pathway in the Ethanol Addiction/Withdrawal Mouse Model

Abstract

Objective: Our study was designed to investigate whether the indoleamine-2,3-dioxygenase (IDO)-mediated kynurenine/tryptophan (KYN/TRP) pathway participates in the development of emotional deficits from ethanol addiction/withdrawal mice.

Methods: The expression of proinflammatory factors, including tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), was tested by enzyme-linked immunosorbent assay (ELISA). The IDO levels in the hippocampus, cerebral cortex, and amygdala were measured by polymerase chain reaction (PCR) and western blot, and the neurotransmitters were tested by high performance liquid chromatography (HPLC). Emotional deficits of mice were evaluated by behavioral tests.

Results: Expression levels of inflammatory factors (TNF-α, IL-1β, and IL-6) were increased in mice after 4 weeks of alcohol exposure. As for indoleamine 2,3-dioxygenase (IDO) expression, only the subtype IDO1 was found to increase at both mRNA level and protein level in all the tested brain regions of ethanol addiction/withdrawal mice. In behavioral tests, mice exposed to alcohol showed gradually declined memory function accompanied by anxiety-like and depressive-like behaviors. Meanwhile, increased expression of KYN, decreased expression of 5-HT, and abnormal expression of 3-HK and KA were found in the hippocampus, cerebral cortex, and amygdala of ethanol addiction/withdrawal mice. Interestingly, the IDO1 inhibitor, 1-methyl-L-tryptophan (1-MT), reversed all above alterations induced by ethanol in mice.

Conclusion: Our results suggested that the TRP/KYN pathway, medicated by IDO1, in the hippocampus, cerebral cortex, and amygdala, plays an important role in the development of emotional deficits caused by ethanol addiction and withdrawal.

Keywords: 3-dioxygenase; anxiety; depression; ethanol addiction; indoleamine 2; memory.

FIGURE 1

Scheme of the experimental design. Mice received ethanol drinking for 8 weeks and received the first administration of 1-MT (0.1, 1, and 3 mg/kg, i.p.) or vehicle after 4 weeks of drinking; behavior tests were done at 0, 2, 4, 6, and 8 weeks after drinking. Each group contains five subgroups as follows: control, ethanol, and ethanol supplemented with different doses (0.1, 1, and 3 mg/kg body weight) of 1-MT. Each subgroup was divided into two sets (n = 8 per set). The mice in the 1st set received elevated plus maze test at 9:00, marble-burying test at 10:00, forced swimming test at 11:00, tail suspension test at 12:00, and ethanol preference test at 14:00. The mice in the 2nd set received a locomotor activity test at 9:00, Morris water maze test at 10:00–12:00, and ethanol preference test at 14:00.

FIGURE 2

Levels of TNF-α, IL-1β, and IL-6 in the hippocampus (A), cerebral cortex (B), amygdala (C) in drinking mice at 0, 2, and 4 weeks. n = 8 per group, and data were assessed by two-way ANOVA followed by a Duncan test. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with control group.

FIGURE 3

mRNA expressions of IDO1 (A1–C1), IDO2 (A2–C2) and TDO (A3–C3) in the hippocampus, cerebral cortex, and amygdala in drinking mice at 0, 2, and 4 weeks. n = 8 per group, and data were assessed by two-way ANOVA followed by a Duncan test. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with control group.

FIGURE 4

Protein expressions of IDO1 (A1–A4) and IDO2 (B1–B4) in the hippocampus, cerebral cortex, and amygdala in drinking mice at 0, 2, and 4 weeks. n = 8 per group, and data were assessed by two-way ANOVA followed by a Duncan test. *p < 0.05 and **p < 0.01 compared with control group. Con, control group; mod, model group (drinking group).

FIGURE 5

Effects of 1-MT on locomotor activity (A), ethanol preference test (B), forced swimming test (C), tail suspension test (D), marble-burying test (E), and elevated plus maze test (F–H) in drinking mice at week 0–8. n = 8 per group, and data were assessed by multi-way ANOVA followed by a Duncan test. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with control group; #p < 0.05, ##p < 0.01, and ###p < 0.001 compared with drinking group.

FIGURE 6

Effects of 1-MT on drinking-induced memory deficits in mice. Animal trajectory of control, model, and 3 mg/kg 1-MT groups are summarized in (A–C). The second row is the hotspots image of the first row. Latency to platform (D) and number of platform crossing (E) were tested at different weeks (0, 2, 4, 6, and 8 week). n = 8 per group, data were assessed by multi-way ANOVA followed by a Duncan test. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with control group; #p < 0.05, ##p < 0.01, and ###p < 0.001 compared with drinking group.

FIGURE 7

1-MT treatment decreased IDO1 expression in the hippocampus (B), cerebral cortex (C), and amygdala (D) in drinking mice at 4–8 weeks. Blots were summarized in (A). n = 8 per group, and data were assessed by multi-way ANOVA followed by Duncan test. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with control group; #p < 0.05, ##p < 0.01, and ###p < 0.001 compared with drinking group. Con, control group, mod, model group (drinking group).

FIGURE 8

Effects of 1-MT on KYN/TRP ratio (A–C) and 5-HT/TRP ratio (D–F) in the hippocampus, cerebral cortex, and amygdala in drinking mice. n = 8 per group, and data were assessed by multi-way ANOVA followed by a Duncan test. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with control group; #p < 0.05 and ##p < 0.01 compared with drinking group.

FIGURE 9

Effects of 1-MT on 5-HIAA/5-HT ratio (A–C), 3-HK/KA ratio (D–F) and QA level (G–I) in the hippocampus, cerebral cortex, and amygdala of drinking mice. n = 8 per group, and data were assessed by multi-way ANOVA followed by a Duncan test. *p < 0.05 and **p < 0.01 compared with control group; #p < 0.05 and ##p < 0.01 compared with drinking group.

FIGURE 10

Pathways linking IDO-mediated KYN/TRP mechanism to behavioral changes: ethanol induced overexpression of proinflammatory cytokines, which activated IDO, especially IDO1. TRP’s metabolic pathway was altered when IDO1 was activated, causing a decrease of 5-HT, which induced a decrease of the 5-HT/TRP ratio and 5-HIAA/5-HT ratio, ultimately leading to depressive- and anxiety-like behaviors. Meanwhile, most of the TRP was metabolized into KYN when IDO1 was activated, which broke the balance of KYN’s metabolic pathway. In the normal state, KYN’s metabolite is KA, which is an NMDA antagonist and is beneficial to memory function. When IDO1 was activated, most of KYN convert to QA, which is an NMDA receptor agonist and is a key contributor to increased neurotoxicity and cognitive deficits. These imbalances and behavioral deficits induced by drinking were improved by treatment of IDO1 inhibitor 1-MT.