Gut Microbiota Analysis in Rats with Methamphetamine-Induced Conditioned Place Preference

Abstract

Methamphetamine abuse is a major public health crisis. Because accumulating evidence supports the hypothesis that the gut microbiota plays an important role in central nervous system (CNS) function, and research on the roles of the microbiome in CNS disorders holds conceivable promise for developing novel therapeutic avenues for treating CNS disorders, we sought to determine whether administration of methamphetamine leads to alterations in the intestinal microbiota. In this study, the gut microbiota profiles of rats with methamphetamine-induced conditioned place preference (CPP) were analyzed through 16S rRNA gene sequencing. The fecal microbial diversity was slightly higher in the METH CPP group. The propionate-producing genus Phascolarctobacterium was attenuated in the METH CPP group, and the family Ruminococcaceae was elevated in the METH CPP group. Short chain fatty acid analysis revealed that the concentrations of propionate were decreased in the fecal matter of METH-administered rats. These findings provide direct evidence that administration of METH causes gut dysbiosis, enable a better understanding of the function of gut microbiota in the process of drug abuse, and provide a new paradigm for addiction treatment.

Keywords: 16S rRNA gene sequencing; gut microbiota; methamphetamine; propionates; short chain fatty acids.

FIGURE 1

Development of CPP induced by repeated administration of METH. Data are the mean ± SEM of time spent in the METH-paired chamber (Chamber A) during the CPP tests. Double asterisk indicates significant difference between the METH CPP group and the control group P < 0.01, n = 8 rats per group.

FIGURE 2

Rarefaction curve of 16S rDNA sequences of the 16 samples and principal coordinate analysis (PCoA) of the samples using Unweighted-UniFrac from pyrosequencing. (A) Rarefaction curves based on the 16S rRNA gene sequencing of the 16 samples from the METH CPP group and the control group (C represents the control group, CPP represents the METH CPP group). The rarefaction curves suggested that the bacterial community was represented well because the curves became relatively flat as the number of sequences analyzed increased. (B) Principal coordinate analysis (PCoA) of the samples using Unweighted-UniFrac from pyrosequencing. The red dots represent the control group, and the blue squares represent the METH-CPP group. The fecal microbiotas of the two groups could not be divided into clusters according to community composition using Unweighted UniFrac metrics and could not be separated clearly by PCoA analysis (ADONIS test, R² = 0.08276, p-value = 0.191).

FIGURE 3

Bacterial community structures in all samples at the genus level (A) and phylum level (B). The abundance is presented in terms of the percentage of the total effective bacterial sequences in the sample.

FIGURE 4

Linear Discriminant Analysis (LDA) shows distinct gut microbiome composition in the METH CPP group and the control group. (A) LDA scores, as calculated by the LEfSe of taxa differentially abundant in the two groups. (B) LEfSe cladogram representing differentially abundant taxa. Only taxa with LDA scores of more than 2 are presented.

FIGURE 5

Taxonomic differences of fecal microbiota between the control and METH CPP groups. Comparison of relative abundance at the bacterial family (A) and genus (B) levels between these two groups. Student’s t-test was used to determine whether differences existed between the two groups. ^∗p < 0.05.

FIGURE 6

Analysis of propionate and butyric acid in the fecal matter of the control group and the METH CPP group. (A) Representative GC-MS spectrum of the butanol derivative of propionate in the fecal sample. (B) Relative abundance of propionate and butyric acid in the fecal matter of the control and METH CPP group tested through GC-MS (Means ± SEM). Each sample was analyzed three times. ^∗Indicates difference between the control group and the METH CPP group (p < 0.05).