Sensitivity to Morphine Reward Associates With Gut Dysbiosis in Rats With Morphine-Induced Conditioned Place Preference

Abstract

Gut microbiota has been found to establish a bidirectional relationship with the central nervous system. Variations of the gut microbiota has been implicated in various mental disorders, including opioid use disorders. Morphine exposure has been repeatedly found to disrupt the gut microbiota, but association between the gut microbiota and the sensitivity to morphine reward remains unknown. In this study the conditioned place preference (CPP) paradigm was used for morphine-treated rats and saline-treated rats. After the CPP procedure, the morphine-treated rats were divided equally into the low and high CPP (L- and H-CPP) groups according to the CPP scores. We adopted 16S rRNA sequencing for the fecal bacterial communities at baseline and post-conditioning. By comparing the morphine-treated group with saline-treated group, we found alterations of microbial composition in the morphine-treated group, but no significant differences in alpha diversity. The L-CPP group and H-CPP group differed in microbial composition both before and after morphine treatment. The relative abundance of certain taxa was correlated to the CPP scores, such as Alloprevotella and Romboutsia. This study provides direct evidence that morphine exposure alters the composition of the gut microbiota in rats and that microbial alterations are correlated to the sensitivity to morphine reward. These findings may help develop novel therapeutic and preventive strategies for opioid use disorder.

Keywords: conditioned place preference; gut dysbiosis; gut microbiota; morphine; rat model; sensitivity to morphine reward.

Figure 1

(A) The conditional place preference (CPP) score of rats were compared between the morphine and saline group in the preconditioning phase, and there was slight difference appeared in the CPP score between the morphine group and saline group. (B) The CPP score of rats were compared between the morphine and saline group in the postconditioning phase, and there was slight difference appeared in the CPP score between the morphine group and saline group. (C) The weights of rats were compared between the morphine and saline group in the preconditioning phase. There was no significant difference between the weight of posttreatment for morphine and saline group. (D) The CPP score of rats were compared among the saline, L-CPP and H-CPP group in the postconditioning phase. The CPP score of H-CPP is significantly higher than L-CPP and saline group, and there was no difference between the CPP score of L-CPP and saline group. The CPP score means the difference between time spent in the drug-paired side after CPP training and time at baselinSe.t udent’s t test was used to analyze the data. The central line shown in each box plot indicates the median of data. Whiskers extend to cover the whole range of values. Statistical significance was accepted at p<0.05. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Figure 2

Rarefaction curve. Rarefaction curves were based on the 16S rRNA gene sequencing of the all samples from baseline group and the post-treatment for morphine and saline group. The rarefaction curve suggested that the sequencing depth was satisfactory and represented the majority of bacterial species, because the curves became relatively flat as the number of sequences analyzed increased.

Figure 3

Comparison of the community alpha diversity. Alpha diversity was measured by ACE (A), Chao (B), Shannon (C) and Simpson (D). A Mann-Whitney test or student’s t test was used to analyze the data. The central line shown in each box plot indicates the median of data. Whiskers extend to cover the whole range of values. Statistical significance was accepted at p < 0.05. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4

Comparison of the gut microbiota composition among the baseline and post-treatment for the morphine and saline group. Comparison of the relative abundance of gut microbiota at the family and genus levels among the saline-baseline, saline post-treatment, morphine-baseline and morphine post-treatment groups. (A) The relative abundance of Parasutterella, Coriobacteriaceae, Desulfovibrio and Peptococcaceae_1 were shown. (B) The relative abundance of Allobaculum, Alloprevotella, Rikenella and Corynebacterium were shown. (C) The relative abundance of Clostridium_XlVa, Enterococcaceae, Staphylococcaceae and Streptococcaceae were shown. A Mann-Whitney test and Wilcoxon signed rank test were used to analyze the data. A student’s t test was used for the data with Gaussian distribution. The central line shown in each box plot indicates the median of data. Whiskers extend to cover the whole range of values.Statistical significance was accepted at p < 0.05. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 5

Comparison of the gut microbiota composition between the low and high conditional place preference group (L-CPP versus H-CPP) after the morphine-induced CPP training. (A) The relative abundance of Alloprevotella, Romboutsia, Roseburia, Clostridium_IV and Schwartzia, were found to be different between L-CPP and HCPP. (B) The relative abundance of Catabacter, Elusimmicrobium, Dorea, Christensenella and Anaeroflium were found to be different between L-CPP and H-CPP. A Mann-Whitney test was used to analyze the data. The central line shown in each box plot indicates the median of data. Whiskers extend to cover the whole range of values. A student’s t test was used for the data with Gaussian distribution. Statistical significance was accepted at p < 0.05. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 6

Correlation between specific taxa and the CPP scores after the morphine-induced CPP training. The relative abundance of Alloprevotella (A) and Romboutsia (B) were positively correlated with the CPP score, while the relative abundance of Roseburia (C) and Elusimicrobium (D) were negatively correlated with the CPP score. A Spearman correlation was performed for Romboutsia, Roseburia and Elusimicrobium, and a Pearson correlation was performed for Alloprevotella. The r value and p value were used to evaluate statistical significance.

Figure 7

Comparison of the gut microbiota composition between L-CPP and H-CPP groups at baseline and the correlation between the relative abundance of Rothia at baseline and CPP score. (A) The significant differences among Helicobacter, Olsenella, and Rothia were noted. A Mann-Whitney test was used to analyze the data. A Wilcoxon signed rank test was used to analyzed the data. A student’s t test was used for data with Gaussian distribution. The central line shown in each box plot indicates the median of data. Whiskers extend to cover the whole range of values. Statistical significance was accepted at p<0.05. (B) The relative abundance of Rothia was negatively correlated with the CPP score. Spearman correlation was performed for Rothia. The r value and p value were used to evaluate statistical significance. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.