Dietary polyphenols drive dose-dependent behavioral and molecular alterations to repeated morphine

Abstract

Opioid Use Disorder (OUD) is associated with tremendous morbidity and mortality. Despite this burden, current pharmacotherapies for OUD are ineffective or intolerable for many patients. As such, interventions aimed at promoting resilience against OUD are of immense clinical interest. Treatment with a Bioactive Dietary Polyphenol Preparation (BDPP) promotes resilience and adaptive neuroplasticity in multiple models of neuropsychiatric disease. Here, we assessed effects of BDPP treatment on behavioral and molecular responses to repeated morphine treatment in male mice. BDPP pre-treatment alters responses for both locomotor sensitization and conditioned place preference. Most notably, polyphenol treatment consistently reduced formation of preference at low dose (5 mg/kg) morphine but enhanced it at high dose (15 mg/kg). In parallel, we performed transcriptomic profiling of the nucleus accumbens, which again showed a dose × polyphenol interaction. We also profiled microbiome composition and function, as polyphenols are metabolized by the microbiome and can act as prebiotics. The profile revealed polyphenol treatment markedly altered microbiome composition and function. Finally, we investigated involvement of the SIRT1 deacetylase, and the role of polyphenol metabolites in behavioral responses. These results demonstrate polyphenols have robust dose-dependent effects on behavioral and physiological responses to morphine and lay the foundation for future translational work.

Figure 1

Baseline effects of BDPP polyphenols and locomotor response to morphine. (A) Graphical timeline of the associated studies. (B) To ensure that treated water did not affect drinking we measured consumption in a subset of animals during the first two weeks. (C) Body weight change over the course of the experiment was also measured. (D) Repeated injections of saline did not affect locomotor activity in either treatment group. (E) Repeated 5 mg/kg injections of morphine resulted in a significant time x treatment interaction with polyphenol animals having increased locomotion on day 3. (F) Polyphenol treatment resulted in decreased development of locomotor sensitization to 15 mg/kg morphine. Data presented as means ± SEM. *p < 0.05; **p < 0.01. N: For panels B&C = 16–20/group; For panels D-F = 6–8/group.

Figure 2

Effects of BDPP Polyphenols on formation of morphine conditioned place preference. (A) After two weeks of BDPP or control pretreatment, mice were tested on a conditioned place preference assay using a five-day protocol. (B) CPP results across a dose range showed a robust dose x treatment interaction with BDPP-treated mice showing decreased preferences at lower doses of morphine, but increased preference for the higher 15 mg/kg dose. (C) In a subsequent experiment, mice were pretreated with morphine prior to polyphenol treatment and CPP testing. (D) While morphine pretreatment enhanced subsequent formation of CPP, BDPP polyphenol pretreatment still resulted in reduced formation of preference at 5 mg/kg morphine. Data presented as means ± SEM. *p < 0.05; **p < 0.01. N = 7–29/group with individual points on graphs.

Figure 3

Effects of BDPP polyphenol treatment on transcriptional response to morphine in the nucleus accumbens. (Top) Timeline for sequencing experiments. Mice were pretreated for two weeks prior to CPP training and were sacrificed one hour after the post test session. (A–E) Volcano plots of all treatment groups relative to Control Saline group. (F) Gene ontology analysis of synapse related genes in H2O and BDPP 15 mg/kg groups. (G) GO terms uniquely regulated in BDPP 15 mg/kg and Ctrl 15 mg/kg groups compared to Ctrl Saline. (H) Transcription factor enrichment analysis of top enriched treatment groups in Ctrl and BDPP 15 mg/kg groups. (I) Venn diagram off all genes statistically significant in Ctrl and BDPP 15 mg/kg groups relative to Ctrl Saline. (J) Heatmap of fold change expression of all genes from the previous panel. (K) Volcano plot of all significantly different genes between the two morphine 15 mg/kg groups. Fold change is relative to Ctrl 15 mg/kg. (L) Gene ontology analysis of top terms regulated up or down between the two 15 mg/kg morphine groups.

Figure 4

Effects of BDPP treatment on cecal microbial composition in morphine treated mice. (A) Timeline for 16 s sequencing experiment. Mice were pre-treated for two weeks prior to CPP training and were sacrificed 24 h after the post test session. (B) Alpha diversity of the gut microbiomes were calculated using Chao1 diversity metric, and shows reduced Alpha diversity in 5 mg/kg BDPP treated mice (C) Beta diversity measured using the unweighted Unifrac distance metrics and shows BDPP treated mice possess a unique microbiome from control treated mice in 5 mg/kg morphine group, but overlap in 15 mg/kg morphine group (D) Stacked bar chart showing the relative phylum abundance in mice from all treatment groups, each phyla represented in a different color (E) Stacked bar chart showing phyla expression levels of the low abundance phyla (this chart corresponds to the top section of the graphs from panel D). (F) Heatmap showing changes in phylum diversities with BDPP treatment relative to controls at each dose. Asterisks represent p values from Wilcoxon text. (G) BDPP treated mice show a significant decrease in Firmicutes to Bacteroidetes Ratio compared to controls in the 5 mg/kg morphine treatment group (H) Heatmap displaying the log2 Fold Change (FC) of selected altered bacterial genera in BDPP treated animals relative to respective control counterparts in 5 mg/kg and 15 mg/kg morphine group. Asterisks represent p values from Wilcoxon text. (I) Cladogram representing taxonomic biomarkers characterizing the differences between BDPP and control treated mice in the 5 mg/kg morphine group (J) Cladogram with taxonomic biomarkers characterizing the differences between BDPP and control treated mice in the 5 mg/kg and 15 mg/kg morphine groups (Full Key for figures I and J found in Tables S13). *p < 0.05 **p < 0.01 ***p < 0.001. N = 8/group.

Figure 5

Correlations between Phyla and Genus Total Abundance and CPP Score. Mice that were pre-treated with BDPP for two weeks followed by CPP testing had cecal content collected 24 h after test day for 16 s analysis (A) Correlation heatmap of select phyla (columns) with CPP preference, across all treatment groups or within individual treatment groups (rows). Exact r values for each phylum and exact p values are available in Supplementary Table S14. (B) Correlation heatmap of individual select genus (columns) with CPP preference across all treatment groups or within individual treatment groups (rows). Exact r values for each genus and exact p values are available in Supplementary Table S15.

Figure 6

Mechanistic studies of polyphenol effects. (A) To test the contribution of SIRT1 activation to the behavioral effects of BDPP treatment a SIRT1 inhibitor was infused into the NAc during CPP. (B) CPP at 5 mg/kg morphine again showed a main effect of BDPP treatment, but no effect of SIRT1 inhibition. (C) Effects of treatment with two key metabolites from the BDPP cocktail was assessed. (D) Treatment with BDPP metabolites did not result in significant changes in morphine preference at either dose. N = 4–8/group with individual points on graphs.