Impact of Morphine withdrawal on genes related to the TLR2 pathway expressed in the Prefrontal Cortex

Abstract

Millions of people suffer from opioid use disorder, because of the ongoing opioid epidemic. 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. These results provide more support that TLR2 plays an integral role in morphine withdrawal mechanisms and could be a potential therapeutic target to minimize opioid withdrawal associated co-morbidities.

Introduction

The opioid epidemic has caused over 600,000 deaths and left millions of people struggling with opioid use disorder. ¹ Substance withdrawal, and even the anxiety associated with anticipating withdrawal symptoms, is a leading cause of relapses, however there are few therapeutic options to combat withdrawal severity. ² With opioid use disorder, withdrawal treatment is often necessary to safely cease substance use, but the accessibility and difficulty of these treatments result in many unsuccessful attempts at drug abstinence.^3–6 A better understanding of the mechanism driving withdrawal is needed to create more effective treatment options.

Current research is focused on determining the mechanisms of drug actions, and transcriptomic approaches are being used to identify downstream mechanisms that can be used as drug targets for all stages of drug use. ⁷ Morphine treatment has been associated with alterations in gene expression that persists through morphine withdrawal.^8–11 These changes can be observed in numerous brain regions and are thought to contribute to drug related behaviors mediated by each brain region. ⁸ A brain region that shows opioid mediated transcriptional changes is the pre-frontal cortex (PFC), which is involved in cross talk to other brain regions during various stages of drug use.^9,12–14 The PFC is primarily involved in goal-oriented behaviors and communicates these behaviors with the amygdala and basal ganglia.^15,16 Addiction leads to changes in limbic and prefrontal signaling that result in alterations to reward cues, increased withdrawal symptoms, and decreased function of prefrontal signaling needed for inhibitory responses and impulse control, important behaviors for sustained drug abstinence. ¹⁴ The numerous behaviors mediated by the PFC at various stages of drug use, and the transcriptional changes observed during opioid treatment, make the PFC an interesting area to further investigate during morphine withdrawal related transcriptional profiles. Additional research into the pathways being altered in the PFC may reveal a withdrawal related mechanism that may be therapeutically targeted to attenuate withdrawal severity.

Additionally, during morphine treatment, there is an increase in pathways associated with toll-like receptor (TLR) activation in the prefrontal cortex, resulting in proinflammatory cytokine release, thus altering the neural signaling and plasticity.^17,18 Increased inflammation has been found to contribute to drug related consequences such as, hyperalgesia, tolerance, dependence, addiction, and withdrawal.^17–21 Increased TLR signaling in the PFC has also been associated with drug related anxiety-like behavior following morphine treatment. ²² TLR inhibition can decrease the development of morphine tolerance and improve the analgesic properties of morphine.^18,23 A TLR2 knockout can attenuate somatic symptoms of morphine withdrawal and is also associated with other behavioral alterations outside of morphine treatment, such as hyperactivity, decreased sociability, and impaired cognition.^24,25 Increased TLR2 signaling in the PFC is also correlated with other behavioral changes, such as cognitive impairment associated with aging. ²⁶ There is a clear relationship between morphine treatment and TLR activity in the PFC, but understanding the behavioral implication of this activity and how to therapeutically intervene is yet to be understood.

This study is a continuation of our previous research that proposed a potential gram-positive, TLR2 mediated mechanism contributing to the somatic symptoms of morphine withdrawal. ²⁴ Previous data showed that morphine withdrawal resulted in an increase in gram-positive bacteria, and antibiotic treatment altered the withdrawal response. There was also an attenuation of withdrawal severity in a TLR2 whole body knock-out model, suggesting the behavioral effects of the morphine withdrawal induced changes in the microbiome are mediated through the TLR2 pathway. This study aims to further investigate the TLR2 pathway during morphine withdrawal by performing RNA sequencing on the PFC of the mice from our previous studies. We hypothesize that we will observe a similar pattern of morphine induced changes that will be altered by antibiotic treatment and rescued in a TLR2 knockout. We will be focusing on the transcriptional changes in the PFC, and genes related to the TLR2 pathway, to determine if our previously proposed mechanism is supported by transcriptional changes in the brain.

Methodology

Animals

Adult 12–15-week-old male C57BL/6 wild-type mice from Jackson Laboratory were used in this study. As reported in Truitt et al., 2023, animals were housed with unrestricted access to food and water on a 12-h light dark cycle and received fresh bedding weekly. Mice were also monitored for general health and signs of distress during morphine withdrawal. Only minor weight loss was observed during treatment, as expected, and mice sustained an average weight of 25–30 g. The whole body TLR2 knockout animals were bred in house and received the same housing conditions as the wild-type animals. For each treatment group there was an n = 5–6 mice used, resulting in 62 total mice used for this project. The samples collected in these experiments are from the mice used in our previous study, Truitt et al., 2023, where the number of animals needed for a sufficient n in the behavior measurements was calculated with a power analysis. At the end of treatment, mice were sacrificed using cervical dislocation and tissue samples were collected.

Statement of ethics

All animal procedures and research techniques were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) and the Institution Biosafety Committee (IBC) of the University of Miami (IACUC protocol number: [23-105] IBC protocol number: [21-157]).

Drug treatment

Mice received a 75 mg slow-release morphine pellet for a 72-h period to develop morphine dependence. Control mice were treated with placebo pellets that breaks down at the same rate as the morphine pellets. After 72 h, the pellets were surgically removed to initiate spontaneous withdrawal. Grouping of mice into either morphine or placebo treated groups was randomized based on cage number. Treatment of mice was described in Truitt et al., 2023, including the pellet implantation, pellet removal, verification of dependence and withdrawal, and prevention of confounding variables. In our previous research, we verified that the mice used in this experiment were dependent on morphine, and elicited a somatic withdrawal response once the morphine pellet was removed. All procedures and treatments were done in the hood located in the animal housing facility.

Antibiotic treatment

To determine if the microbiome alters morphine induced gene changes in the PFC, the microbiome was altered using an antibiotic cocktail for seven days prior to morphine treatment. The antibiotic cocktail consisted of 10 mg/mL bacitracin, 10 mg/mL metronidazole, 40 mg/mL neomycin sulfate, 4 mg/mL Vancomycin, and 24 ug/mL pimaricin dissolved in water daily. Mice received 0.2 mL of the antibiotic cocktail each day (control mice received a daily gavage of their autoclaved drinking water), and their bedding was changed daily to minimize reconstitution of the microbiome from coprophagic behavior. Antibiotics were maintained in the drinking water once morphine treatment began with 0.5 mg/mL bacitracin, 2 mg/mL neomycin, 0.2 mg/mL vancomycin, 1.2 µg/mL pimaricin and this was replaced daily.

Tissue collection

To collect the PFC, the head of the animal was isolated, and a midline cut was made in the skin to expose the skull. Scissors were carefully used to cut down the midline of the skull, ensuring that the brain was not damaged underneath. Pieces of the skull were removed to expose the brain, and once enough bone was removed, the brain was removed in one piece and place in a shallow dish of ice-cold phosphate buffered saline. A razor was used to separate the left and right hemisphere of the brain. On each hemisphere, the olfactory bulb was removed, and the meninges were removed to minimize blood contamination of samples. The prefrontal cortex was then removed and placed in a 1.5 mL tube and flash frozen. Samples were stored in a −80° freezer until processed.

Transcriptome analysis

Sequencing of the PFC tissue samples was done by Novogene Co., and raw reads (Fastq files) were received for analysis. Analysis of results was done blinded to treatment group. All raw reads were processed with Pegasus Cluster (CentOS 7) hosted by Institute for Data Science & Computing at University of Miami. Raw reads of fastq format were first processed through fastp (v0.22.0) software. ²⁷ In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing poly-N and low-quality reads from raw data. All the downstream analyses were based on the clean data with high quality. Reference genome and gene model annotation files were downloaded from genome website directly (GRCm39 from ensembl http://useast.ensembl.org/index.html). Index of the reference genome was built using Hisat2 (v2.2.1) ²⁸ and paired-end clean reads were aligned to the reference genome using Hisat2. featureCounts function ²⁹ from Subread package (2.0.6) was used to count the reads numbers mapped to each gene. Differential expression analysis of two conditions/groups was performed using the DESeq2 R package ³⁰ (1.40.2) implemented in R (4.2.3) and R studio (Build 353). The resulting p-values were adjusted using the Benjamini and Hochberg approach for controlling the false discovery rate. Genes with an adjusted p-value ≤ 0.05 found by DESeq2 were assigned as differentially expressed. This sequencing ensures a high accuracy reading with a rigorous quality control of samples. This results in a quality score (Q30 score) of more than 85%, insuring high reliability of data. We also used a large N per group for sequencing to ensure reproducibility.

Results

Morphine withdrawal induces transcriptional changes in the PFC

To investigate how the TLR2 pathways is altered during morphine withdrawal in the prefrontal cortex, transcriptional analysis between morphine and placebo treated animals was done with tissues collected at different timepoints of withdrawal: 2, 12, and 24 h post pellet removal (Figure 1). Volcano plots of results revealed at 2 h post pellet removal, there were 149 genes significantly downregulated in morphine treated mice compared to placebo, and 180 significantly upregulated genes (Figure 1(A)). At 12 h post pellet removal, there were the highest number of dysregulated genes during withdrawal with 261 significantly downregulated and 199 significantly upregulated genes in the morphine treated animals (Figure 1(B)). By 24 h post pellet removal, there were still 118 significantly downregulated genes and 100 significantly upregulated genes (Figure 1(C)). This shows that morphine withdrawal produces gene changes in the PFC as early as 2 h post pellet removal and through the first 24 h of withdrawal. Focusing on genes relating to the TLR2 pathway, we observed an increase of the TLR2 gene at 24 h post pellet removal, log2(FC) = 1.3953, q = 0.032 (Figure 1(D)). We also saw genes downstream of TLR2 altered during morphine withdrawal: MAPk3 at 2 h (log2(FC) = 0.46, q = 0.015) and 12 h post pellet removal (log2(FC) = 0.3751, q = 0.0411), and a TNF-a related gene, TNFsf10, was upregulated at 24 h post pellet removal (log2(FC) = 1.1194, q = 0.0192). These results show that genes related to the TLR2 pathway are significantly upregulated in morphine withdrawal treated animals compared to placebo treated controls.

Figure 1.

Volcano plots representing significantly upregulated genes (red) and significantly downregulated genes (blue) in morphine treated animals compared to placebo controls at 2 (A), 12 h (B), and 24 h (C) after pellet removal. A heat map shows the log2 fold change of genes related to the TLR2 pathway at each timepoint of withdrawal (D). Fold change is plotted on a color gradient in which increased expression is represented by coral and decreased expression is purple. Gene expression that was significantly altered is represented by *q < 0.05, **q < 0.01. A schematic of the genes in TLR2 pathway at homeostatic conditions (E). There is an n = 5 per group in each comparison.

Antibiotic treatment further alters the withdrawal induced transcriptional changes

There was a global impact of antibiotic treatment on morphine withdrawal induced transcriptional changes, with antibiotic resulting in increased gene upregulation and more alteration to TLR2 related genes observed at all timepoints of withdrawal. First to compare the effect of antibiotics on morphine withdrawal transcriptional change, RNA sequencing was done on the PFC of animals treated with either antibiotics and morphine or water and morphine (ABX + MORPH vs H2O + MORPH). This first comparison compares the effect of antibiotic treatment in morphine treated animals, to determine if antibiotics can reduce the changes caused by morphine treatment. At 2 h post pellet removal, there were 1228 significantly downregulated genes in the ABX + MORPH mice compared to the H2O + MORPH mice and 1247 upregulated genes (Figure 2(A)). At 12 h post pellet removal, 973 downregulated and 964 upregulated genes were observed in the ABX + MORPH versus H2O + MORPH (Figure 2(B)). These results implicate that during morphine withdrawal, antibiotic treatment can further alter gene expression.

Figure 2.

Volcano plots representing significantly upregulated genes (red) and significantly downregulated genes (blue) in: ABX + MOR compared to H2O + MOR (A & B), ABX + MORPH compared to ABX + PLCB (D & E), and ABX + PLCB compared to H2O + PLCB (G & H) at 2 h and 12 h post pellet removal. Heat maps for each comparison show log2 fold change of genes related to the TLR2 pathway with increased expression represented by coral and decreased expression represented purple on a gradient scale (C, F, & I). Gene expression that was significantly altered is represented by *q < 0.05, **q < 0.01, ***q < 0.001, ****q < 0.0001. There is an n = 5 per group in each comparison.

There was also dysregulation of genes related to the TLR2 pathway in ABX + MORPH versus H2O + MORPH (Figure 2(C)). There was both upregulation and downregulation of MapK related genes in the ABX + MORPH group at 2 h post pellet removal. MAPk3 (log2(FC) = −0.77, q < 0.0001) and Mapk8ip3 (log2(FC) = −0.23, q = 0.01) were both downregulated in the ABX + MORPH compared to H2O + MORPH, and Mapk11 (log2(FC) = 0.49, q < 0.0001) was upregulated in the ABX + MORPH group. At 12 h post pellet removal Mapk3 (log2(FC) = 0.454, q = 0.0005) was upregulated while Mapk11 (log2(FC) = −0.503, q < 0.0001) was downregulated in the ABX + MORPH group. The same trend was seen in Nfkb related genes being both upregulated and downregulated in the ABX + MORPH compared to H2O + MOPRH at different timepoints. At 2 h post pellet removal Nfkbia (log2(FC) = −0.36, q = 0.022) and Nfkbib (log2(FC) = −0.36, q = 0.019) were both significantly downregulated, but both upregulated at 12 h post pellet removal: Nfkbia (log2(FC) = 0.628, q = 0.0002) and Nfkbib (log2(FC) = 0.29, q = 0.05). Lastly there was a downregulation of Tnf related genes at 12 h post pellet removal in the ABX + MORPH group: Tnfaip8l1(log2(FC) = −0.582, q = 0.004) and Traf3 (log2(FC) = −0.41, q = 0.02).

To determine if antibiotic treatment could recover the gene alteration during morphine withdrawal, RNA sequencing of the PFC was compared between morphine and placebo treated animals that both received an antibiotic cocktail prior to pellet implantation (ABX + MORPH vs ABX + PLCB). This comparison is done to determine if, in the context of antibiotic treatment, there will still be differential gene expression in the morphine treated animals compared to placebo. At 2 h post pellet removal ABX + MORPH treated animals had 257 significantly downregulated genes and 502 upregulated genes compared to ABX + PLCB treated animals (Figure 2(D)). The number of significantly altered genes increases to 2238 downregulated and 2187 upregulated genes at 12 h post pellet removal (Figure 2(E)). The transcriptome effects of morphine withdrawal are larger in the context of antibiotic treatment than with morphine treatment alone. There is even more alteration in genes related to the TLR2 signaling pathway (Figure 2(F)). In antibiotic treated animals, there is still a significant increase in the TLR2 gene, log2(FC) = 1.03, q = 0.017, at 12 h post pellet removal. There is both an increase and decrease in MapK related genes in morphine treated antibiotic animals with MAPk3 significantly upregulated (log2(FC) = 0.78, q < 0.0001) and Mapk11 significantly downregulated (log2(FC) = −0.5, q = 0.0004) at 12 h post pellet removal. A similar pattern is seen with Nfkb related genes with Nfkbia upregulated at 12 h post pellet removal (log2(FC) = 0.56, q = 0.0002) in ABX + MORPH animals, and Nfkbib downregulated at 2 h post pellet removal (log2(FC) = −0.35, q = 0.05). There was also a significant downregulation in Tnfaip8l1 gene (log2(FC) = −0.68, q = 0.0001) at 12 h post pellet removal. These results show that with antibiotic treatment, there are still many morphine withdrawal induced transcriptional changes, and the TLR2 related genes showed even more dysregulation with antibiotic treatment.

As a control, RNA sequencing of antibiotic placebo and water placebo treated animals were compared (ABX + PLCB vs H2O + PLCB). This comparison was made to determine the effects of antibiotics alone on gene expression, independently of morphine treatment. At 2 h post pellet removal there were 156 genes significantly downregulated and 169 upregulated by antibiotics alone (Figure 2(G)). There were few genes significantly altered by 12 h post pellet removal with only 17 downregulated and 43 upregulated genes (Figure 2(H)). There was also little difference in the genes related to the TLR2 pathway with the only significant gene being Mapk11 upregulated at 2 h post pellet removal, which could explain why it was upregulated when comparing the ABX MORPH and H2O MORPH (Figure 2(I)). This shows that antibiotics independently of morphine do not elicit the same impact that antibiotics do during morphine withdrawal.

Morphine withdrawal induced transcriptional changes are rescued in a TLR2 knockout animal

To investigate if our proposed mechanism of TLR mediating somatic morphine withdrawal also impacts morphine withdrawal induced transcriptional changes, RNA sequencing of the PFC from whole body TLR2 knockout animals treated with morphine and placebo were analyzed at 24 h post pellet removal. When comparing the TLR2 K/O morphine and placebo treated animals, there were zero significantly upregulated or downregulated genes (Figure 3(A)). This suggests that the knockout of TLR2 recovers the transcriptional effects of morphine withdrawal, providing more evidence of the role of TLR2 in morphine withdrawal. Comparing the TLR2 K/O MORPH and the WT MORPH, there were only nine significantly downregulated and seven significantly upregulated genes (Figure 3(B)). The low number of altered genes in this comparison indicates that the knockout of TLR2 does not fully recover the effects of morphine treatment on genes, as this comparison would have many more altered genes if TLR2 K/O produced a full recovery. Lastly, the TLR2 K/O PLCB and WT PLCB showed the highest amount of significantly altered genes, with 21 downregulated and 37 upregulated genes, showing that the TLR2 knockout model slightly altered gene expression independently of morphine treatment (Figure 3(C)). Genes related to the TLR2 pathway were not significantly different between any compassion, again showing that the TLR2 knockout did not increase these genes during morphine treatment compared to the TLR2 PLCB, but also did not fully recover them, as they are not decreased when compared to the WT MORPH group (Figure 3(D)). These genes were also not altered in the TLR2 K/O PLCB compared to WT PLCB, verifying that the baseline gene differences did not confound the results of the TLR2 pathway.

Figure 3.

Volcano plots representing significantly upregulated genes (red) and significantly downregulated genes (blue) between: TLR2 K/O MORPH compared to TLR2 K/O PLCB (A), TLR2 K/O MORPH compared to WT MORPH (B), and TLR2 K/O PLCB compared to WT MORPH (C). A color gradient heat map shows log2 fold change of genes related to the TLR2 pathway with increased expression represented by coral and decreased expression represented by purple. In these comparisons, no genes were significantly altered between groups. A summary schematic representing the effects of morphine, antibiotics, and the TLR2 K/O model on the TLR2 pathway (E). There is an n = 6 per group in each comparison.

In summary, we found that morphine withdrawal treatment resulted in a significant upregulation of genes relating to the TLR2 pathway (Figure 3(E)). Antibiotic treatment resulted in downregulation of several of the genes in this pathway, however depending on the timepoint of withdrawal, some of the genes remained upregulated. Morphine withdrawal induced gene expression was abolished in a whole body TLR2 knockout model, suggesting that the role of TLR2 morphine withdrawal driven transcriptional changes.

Discussion

Our results show that morphine withdrawal elicits changes in gene expression in the PFC. This effect was observed through the first 24 h of withdrawal and likely continues past that, as many genes were still dysregulated after 24 h of withdrawal. While analysis of additional timepoints would determine how long these effects lasts, the focus of this study was to observe the short-term consequences of morphine withdrawal, and the genes that may be contributing to the somatic effects of withdrawal. Additionally, the overview of bulk RNA sequencing does not specify in which cell types these changes are occurring, and additional sequencing at the single cell level would reveal what cell types are involved in these changes and shed more light on the overall mechanism of withdrawal. Our previous studies proposed a gram-positive, TLR2 mediated mechanism of somatic withdrawal, and results from the current report on transcriptome analysis showed that genes related to the TLR2 pathway were elevated during morphine withdrawal, providing more support for this proposed mechanism.

Additionally, when treated with antibiotics, there were even more differentially expressed genes, especially in the TLR2 pathway. Other studies have shown that changes to the microbiome can result in transcriptomic changes in the brain, that results in behavioral and biophysical alterations. ³¹ Morphine treatment specifically has been shown to upregulate TLR mediated inflammatory pathways, and this upregulation is not observed in the absence of the microbiome. ³² In our study, we see that antibiotic treatment leads to more differentially expressed genes during morphine treatment but a decrease in many of the genes in the TLR2 pathway. There is not a full recovery of this pathway with antibiotic treatment, but it is clear that the alteration to the microbiome has an impact on these genes during morphine withdrawal. There was less impact on gene expression and the TLR2 pathway with antibiotics in the placebo treated animals, indicating that antibiotics alone did not have as much of an impact on the transcriptome, but still can alter morphine withdrawal induced gene changes that in the context of morphine withdrawal. This is potentially due to the fact that morphine treatment after antibiotic manipulation of the gut microbiome results in a distinct composition of the microbiome, that could result in unique gene expression profile compared to antibiotics or morphine treatment alone. ²⁴ In summary, the antibiotic results confirmed that there is a relation between the gut biome manipulations and differential gene expression.

A potential mechanism by which the microbiome can modulate systemic changes is through activation of TLRs by bacterial products. Thus, depletion of these bacteria in the antibiotic model would lead to decreased TLR signaling. Morphine withdrawal specifically causes an increase in gram-positive bacteria, and therefore the deletion of TLR2, the receptor for gram-positive bacterial products, results in a decrease of somatic withdrawal behavior. ²⁴ Our study further showed that a TLR2 knockout also results in a partial recovery of morphine withdrawal induced transcriptional changes. The lack of differentially expressed genes in morphine treated TLR2 knockout animals compared to the placebo treated controls, suggest that TLR2 is necessary for morphine withdrawal induced transcriptional changes in the PFC. This also indicates that the expansion of gram-positive bacteria drives the changes, since both antibiotic depletion and TLR2 knockout alter morphine withdrawal induced transcriptional changes. The attenuation of transcriptome changes in the TLR2 knockout was likely not a total recovery of morphine's impact since the knockout morphine treated mice did not differ much from the wildtype morphine treated animals. However, even a partial recovery in the TLR2 knockout animals provides further evidence that the somatic morphine withdrawal response may be mediated through a TLR2 mechanism.

This study highlights the gut microbiome as a potential therapeutic target for treating the consequences of somatic morphine withdrawal. We show that treatment of antibiotics prior to morphine exposure resulted in the recovery of several of the TLR2 related genes that were altered in morphine withdrawal. However, it is important to note the potential difficulties of therapeutic antibiotic treatment, as antibiotic resistance is a growing concern in the population, especially with gram-positive bacteria. ³³ A broad-spectrum antibiotic like used in this study, would not be the ideal treatment option, and previous research shows this antibiotic treatment does not deplete all the bacterial community in morphine conditions, as it does in placebo treatment, indicating some resistance to the antibiotics may have resulted with chronic opioid use. ²⁴ Further research targeting specific pathogenetic bacterial taxa that are contributing to morphine withdrawal may lead to more conclusive results and better treatment options. Alternatively, therapeutic research could focus on the TLR2 pathway, which proved to have a more robust recovery in morphine withdrawal induced transcriptional changes. A TLR2 inhibitor or cell-specific knockouts could be used to further investigate this pathway during morphine withdrawal. Lastly, gut-mediated TLR2 signaling is likely one part of a much larger mechanism, as there are other factors that can also contribute to morphine withdrawal. Dopamine signaling, for example is decreased in the PFC during morphine treatment yet increased during morphine abstinence, and changes in dopamine signaling can also have an impact on morphine withdrawal. ³⁴

Conclusion

In conclusion, this study set out to further investigate the role of TLR2 in morphine withdrawal by analyzing gene expression in the prefrontal cortex under different conditions. Morphine withdrawal resulted in distinct transcriptional changes in the prefrontal cortex when compared to placebo treatment, including the upregulation of TLR2 related genes. Antibiotic treatment prior to morphine treatment caused further alteration in gene expression in the PFC and resulted in the downregulation of TLR2 related genes when compared to morphine treatment alone. In a TLR2 knockout model, morphine withdrawal induced transcriptional changes are no longer observed, implicating the importance of TLR2 in morphine withdrawal induced gene expression. This study is a preliminary look into the changes of transcriptomic profiling during morphine withdrawal, and future work should investigate the cell types associated with these gene changes and if these changes at the transcriptional level also have functional significance.