Alterations of the Host Microbiome Affect Behavioral Responses to Cocaine.

Knockdown of intestinal bacteria using non-absorbable antibiotics.(a) Schematic representation of the experimental procedure. (b) Fluid intake did not differ between control (H2O) and antibiotic-treated (Abx) groups (n = 32 H2O, 39 Abx). (c) Mean body weight did not differ between groups and was not affected by antibiotic treatment (n = 32 H2O, 39 Abx). (d) Gross morphology of the cecum from untreated (left) and antibiotic-treated (right) mice with ruler shown for scale. (e) Quantitative measurement of cecal weights between control and antibiotic-treated mice demonstrates a marked increase in antibiotic-treated animals (***p < 0.0001; n = 24 H2O, 28 Abx). (f) qPCR analysis of total bacterial load between the two conditions demonstrates a decrease in bacterial load in antibiotic-treated mice (***p < 0.0001; n = 16 H2O, 14 Abx).

Knockdown of intestinal bacteria does not alter cocaine metabolism.(a) Serum levels of the cocaine metabolite benzoylecygonine 30, 60, or 90 minutes after a single high-dose injection of cocaine did not differ between control (H2O) and antibiotic-treated (Abx) mice (n = 4–5/group/timepoint). (b) Levels of benzoylecygonine 30 minutes after a 5 mg/kg injection of cocaine also did not differ between groups (n = 3 H2O, 5 Abx). (c) Levels of serum corticosterone in animals treated with antibiotics via the drinking water did not differ between groups (n = 10 H2O, 10 Abx).

Depletion of intestinal bacteria enhances behavioral responses to cocaine.(a) Control animals (H2O) and antibiotic-treated animals (Abx) exhibited no difference in place preference for cocaine at a dose of 10 mg/kg (n = 6 H2O, 8 Abx). (b) Conditioned place preference assay at a 5 mg/kg dose did not produce significant preference in control animals, but Abx-treated animals formed a robust preference for the cocaine-paired side (***p < 0.0001; n = 11 H2O, 22 Abx). (c) When both sides of the assay were paired with saline there was no detectable preference or group difference (n = 5/group). (d) Both control and antibiotic-treated mice developed locomotor sensitization in response to cocaine treatment, but there were no between group differences (n = 5/group). (e) Antibiotic-treated mice show an enhanced locomotor response to repeated doses of 5 mg/kg cocaine (*p < 0.05; n = 5/group).

Parenteral administration of antibiotics does not alter behavioral response.For these experiments animals were injected intraperitoneally with phosphate-buffered saline (PBS) or a cocktail of antibiotics (Inj-Abx) for 7 days prior to behavioral analysis. When the conditioned place preference test was performed, there were no group differences when measured at a dose of 10 mg/kg (a) or at a dose of 5 mg/kg (b). [n = 5/group].

Repletion of bacterial short-chain fatty acids reverses behavioral effect of antibiotics.To determine if the bacterial byproducts were responsible for the changes seen in conditioned place preference (CPP), we measured animals treated with antibiotics (Abx) and/or short-chain fatty acids (SCFA) via the drinking water. Animals were tested for CPP at 5 mg/kg as this was the dose at which a behavioral effect of Abx was seen. As above, Abx treated animals showed a robust preference for cocaine (blue bar). Treatment with SCFA alone did not alter CPP response (grey hatched bar). Animals treated with Abx and SCFA showed the same level of preference as the control animals (blue hatched bar) demonstrating that repletion of bacterial byproducts can reverse the behavioral effects of depleting gut microbiota [n = 5–22/group; **p < 0.01; ***p < 0.001].

Antibiotic-treated mice exhibit alterations in Bdnf and Ntrk2 transcripts in the NAc.(a) In mice treated with antibiotics and cocaine compared to controls levels of Bdnf were significantly elevated in antibiotic-treated (Abx) animals (*p < 0.05; n = 9–11/group); control animals, H2O. (b) Transcripts encoding the receptor for BDNF were also examined. Levels of Ntrk2 were significantly decreased specifically in antibiotic and cocaine-treated animals (**p < 0.01; n = 9–11/group).

Antibiotic-treated mice have altered expression of dopamine and glutamate-related transcripts in the NAc.Mice with antibiotic-induced reductions in gut bacteria (Abx) demonstrate increased levels of Drd1 (a) and Gria2 (c) in the NAc following treatment with cocaine; control animals, H2O. Levels of Drd2, Dlg4, Grin2a and Grin2b were unaltered. (*p < 0.05; **p < 0.01; n = 9–12/group).