Anxiety-like behavior during protracted morphine withdrawal is driven by gut microbial dysbiosis and attenuated with probiotic treatment

Abstract

The development of anxiety during protracted opioid withdrawal heightens the risk of relapse into the cycle of addiction. Understanding the mechanisms driving anxiety during opioid withdrawal could facilitate the development of therapeutics to prevent negative affect and promote continued abstinence. Our lab has previously established the gut microbiome as a driver of various side effects of opioid use, including analgesic tolerance and somatic withdrawal symptoms. We therefore hypothesized that the gut microbiome contributes to the development of anxiety-like behavior during protracted opioid withdrawal. In this study, we first established a mouse model of protracted morphine withdrawal, characterized by anxiety-like behavior and gut microbial dysbiosis. Next, we used fecal microbiota transplantation (FMT) to show that gut dysbiosis alone is sufficient to induce anxiety-like behavior. We further demonstrated that probiotic therapy during morphine withdrawal attenuated the onset of anxiety-like behavior, highlighting its therapeutic potential. Lastly, we examined transcriptional changes in the amygdala of morphine-withdrawn mice treated with probiotics to explore mechanisms by which the gut-brain axis mediates anxiety-like behavior. Our results support the use of probiotics as a promising therapeutic strategy to prevent gut dysbiosis and associated anxiety during opioid withdrawal, with potential implications for improving treatment outcomes in opioid recovery programs.

Keywords: Opioid withdrawal; amygdala; anxiety; gut-brain axis; probiotics; serotonin.

Figure 1.

Protracted morphine withdrawal is associated with elevated anxiety-like behavior, with sex-differences. (a) general experimental paradigm for morphine/placebo withdrawal and behavioral testing. (b) percent time spent in the open arms of the elevated plus maze for female and male mice. (c) number of entrances into the open arms of the elevated plus maze for female and male mice. (d) number of entrances into the closed arms of the elevated plus maze for female and male mice. (e) percent activity in the center of the open field test for female and male mice. (f) total activity measured by number of beam breaks in the open field test for female and male mice. Symbols represent individual mice; line and error bars represent mean and standard deviation; n_Female = 20 placebo, 16 morphine; n_Male = 12 placebo, 10 morphine;. ns = nonsignificant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using two-way ANOVA followed by Fisher’s least significant difference post hoc test.

Figure 2.

Gut microbial dysbiosis associated with protracted morphine withdrawal in female and male mice. (a) Principal coordinate analysis plot of Bray-Curtis distance (measure of β-diversity) for female mice withdrawn from morphine and placebo controls. (b) Principal coordinate analysis plot of Bray-Curtis distance (measure of β-diversity) for male mice withdrawn from morphine and placebo controls. (c) α-diversity plotted by Shannon index for female mice withdrawn from morphine and placebo controls. (d) α-diversity plotted by Shannon index for male mice withdrawn from morphine and placebo controls. (e) LEfSe plot for phyla enriched in morphine withdrawn-female mice. (f) LEfSe plot for genera enriched and depleted in morphine-withdrawn female mice. (g) LEfSe plot for species enriched and depleted in morphine-withdrawn female mice. (h) LEfSe plot for phyla enriched and depleted in morphine-withdrawn male mice. (i) LEfSe plot for genera enriched and depleted in morphine-withdrawn male mice. (j) LEfSe plot for species enriched in morphine-withdrawn male mice. Fem_PLA = female placebo controls (n=19), Fem_Mor = female morphine withdrawal (n=18), Mal_PLA = male placebo controls (n=8), Mal_Mor = male morphine withdrawal (n=8).

Figure 3.

Morphine withdrawal is associated with a distinct metabolomic profile characterized by alterations in tryptophan metabolism. (a) partial least squares discriminant analysis (PLS-DA) of colon metabolite profiles from morphine-withdrawn mice (morphine, red) and placebo controls (placebo, green). (b) volcano plot displaying differentially abundant metabolites in morphine withdrawal compared to placebo. (c) pathway enrichment analysis of differentially abundant metabolites between morphine withdrawal and placebo groups, highlighting significantly altered biochemical pathways. (d-g) relative abundance of key tryptophan pathway metabolites in colon contents from morphine withdrawn mice (morphine) and placebo controls (placebo), including kynurenic acid, 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, and serotonin sulfate. n=6/group; **p<0.01 using the Mann-Whitney test.

Figure 4.

Fecal microbiota transplantation (FMT) from mice withdrawn from morphine results in increased anxiety-like behavior in the elevated plus maze. (a) experimental paradigm for FMT and behavioral testing. (b) percent time spent in the open arms of the elevated plus maze for FMT donor mice withdrawn from morphine and placebo controls. (c) number of entrances into the open arms of the elevated plus maze for FMT donor mice withdrawn from morphine and placebo controls. (d) number of entrances into the closed arms of the elevated plus maze for FMT donor mice withdrawn from morphine and placebo controls. (e) percent time spent in the open arms of the elevated plus maze for mice who received FMT from morphine withdrawn mice and placebo controls. (f) number of entrances into the open arms of the elevated plus maze for mice who received FMT from morphine withdrawn mice and placebo controls. (g) number of entrances into the closed arms of the elevated plus maze for mice who received FMT from morphine withdrawn mice and placebo controls. n_Donors= 5 placebo, 5 morphine; n_Recipients= 9 placebo, 7 morphine; symbols represent individual mice; line and error bars represent mean and standard deviation; ns= nonsignificant, *p<0.05, **p<0.01 using unpaired t-test with Welch’s correction.

Figure 5.

Probiotic therapy during protracted withdrawal partially rescues the development of anxiety-like behavior during protracted morphine withdrawal. (a) experimental paradigm for probiotic therapy during protracted morphine withdrawal and behavioral testing. (b) percent time spent in the open arms of the elevated plus maze. (c) number of entrances into the open arms of the elevated plus maze. (d) number of entrances into the closed arms of the elevated plus maze. Symbols represent individual mice; line and error bars represent mean and standard deviation. n_{Placebo-Water} = 10, n_{Placebo-Probiotics} = 10, n_{Morphine-Water} = 10, n_{Morphine-Probiotics} = 12; *p < 0.05, **p < 0.01 using unpaired t-test with Welch’s correction.

Figure 6.

Transcriptional alterations to the amygdala associated with protracted morphine withdrawal in female and male mice. (a) differentially expressed gene (DEG) counts for Fem_Morph vs. Fem_Placebo and Male_Morph vs. Male_Placebo. (b) pathways identified by IPA as upregulated and downregulated in Fem_Morph vs. Fem_Placebo. (c) pathways identified by IPA as upregulated in Male_Morph vs. Male_Placebo. Fem_Morph = female morphine withdrawal (n=6), Fem_Placebo = female placebo controls (n=6), Male_Morph = male morphine withdrawal (n=6), Male_Placebo = male placebo controls (n=6).

Figure 7.

Transcriptional alterations in the amygdala during protracted morphine withdrawal and probiotic treatment. (a) differentially expressed gene (DEG) counts across all experimental comparisons. (b) Principal component analysis (PCA) of amygdala transcriptomes across all groups reveals clustering by treatment condition. (c) top differentially expressed pathways in morphine withdrawal identified using Ingenuity pathway analysis (IPA). (d) top differentially expressed pathways following probiotic treatment during withdrawal. (e) multiple linear regression model showing the relationship between altered serotonin signaling genes and percent time spent in the open arms of the elevated plus maze (EPM). (f) correlation matrix showing the relationship between serotonin signaling genes downregulated by probiotic treatment and EPM open arm time. MOR_Prob = morphine withdrawal + probiotic therapy (n=6); PLA_Prob = placebo + probiotic therapy (n=3); MOR_Water = morphine withdrawal + water control (n=6); PLA_Water = placebo + water control (n=3).

Figure 8.

Neurotransmitter gene expression changes in the amygdala across morphine withdrawal and probiotic treatment. (a, c, e) volcano plots show differentially expressed genes between treatment groups, highlighting key dopamine, serotonin, and tryptophan-related transcripts. (b, d, f) pathway-level summaries show directionality and magnitude of gene expression changes across dopaminergic, serotonergic, and tryptophan-related pathways. Values shown represent log₂ Fold changes for representative genes in each category; n=3/group.