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The aversive symptoms of withdrawal are a leading factor for drug relapses, yet there are limited therapeutic options to minimize or prevent withdrawal symptoms. The mechanism behind opioid withdrawal is still not fully understood, thus preventing the development of new therapeutics. This study is an extension of our previously proposed mechanism of a toll-like receptor 2 (TLR2) mediated withdrawal response as a result of morphine induced microbial change that occurs during morphine withdrawal. Transcriptome analysis of the pre-frontal cortex indicated that there was increased expression of genes related to TLR2 signaling in morphine withdrawal treated animals compared to placebo controls. Antibiotic treatment further altered TLR2 related genes, recovering some of the morphine induced effect and leading to additional suppression of some genes related to the TLR2 pathway. Morphine withdrawal induced gene expression was attenuated in a whole body TLR2 knockout model. 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