Depletion of the Microbiome Alters the Recruitment of Neuronal Ensembles of Oxycodone Intoxication and Withdrawal

Validation of precipitated withdrawal and intoxication in passively injected rats. A, B, The von Frey test was used to confirm that oxycodone withdrawal decreased pain thresholds and oxycodone intoxication increased pain thresholds. No significant difference in pain thresholds was found between the antibiotic-treated and untreated groups. Therefore, each state was pooled. Intoxicated animals exhibited higher pain thresholds compared with the saline and withdrawal groups. The withdrawal group exhibited lower pain thresholds compared with the saline group (***p < 0.001) and intoxication group (###p < 0.001). C, The total withdrawal score was calculated as the sum of all individual withdrawal scores. No difference in withdrawal scores was observed between antibiotic-depleted animals and untreated animals, so each state was pooled (D). The withdrawal animals exhibited higher withdrawal scores compared with intoxicated animals (p < 0.001).

Antibiotic depletion reduces α-diversity. A, Antibiotic depletion was measured using both the Shannon diversity index and Choa1 index. No difference in either index was found between groups for the initial baseline (BSL) time point. The final time point was taken following both antibiotic depletion and the final oxycodone exposure (posttreatment). In the posttreatment conditions, the OXY+ABX group exhibited a significant decrease in α diversity compared with both the SAL and OXY groups, indicated by the Shannon diversity index (p < 0.001) and Chao1 index (p < 0.008). B, A biplot of the PCoA by Bray–Curtis similarity clustering indicated that the SAL and OXY groups clustered together, whereas the antibiotic-treated animals clustered separately. C, Taxonomic plots that show the relative abundance of phyla across different groups at BSL and posttreatment. D, E, The OXY+ABX group exhibited a significant decrease in both Bacteroidetes (p < 0.003) and Firmicutes (p < 0.001) at the phylum level. Each animal is represented at both timepoints at the phylum level in the relative abundance plots. F, Caecal weights were measured at the time of killing. A significant increase in caecal weights was observed in antibiotic-treated rats (p < 0.001); *p < 0.05, **p < 0.002, ***p < 0.001, significant difference from saline; #p < 0.05, ##p < 0.002, ###p < 0.001 significant difference from OXY group.

Quantification of Fos+ neuronal ensembles during oxycodone intoxication and withdrawal. Ensembles that were recruited during both intoxication and withdrawal were quantified by counting Fos+ neurons that were active 90 min before killing. Saline, saline + naloxone group; OXY, oxycodone + saline; WD, oxycodone + naloxone. Coronal slices (40 μM) were stained for Fos and then visualized using DAB enhanced with nickel to obtain representative images. The representative regions are outlined with dashes; *p < 0.05, **p < 0.002, ***p < 0.001, significant difference from saline; #p < 0.05, ##p < 0.002, ###p < 0.001 significant difference from OXY group.

Quantification of Fos+ neuronal ensembles during oxycodone intoxication with antibiotic treatment. A, Differences in the number of Fos+ neurons were evaluated during oxycodone intoxication between antibiotic-treated and untreated rats. The regions that were altered during intoxication are shown in green (OXY) and green with black stripes (OXY+ABX). Coronal slices (40 μM) were stained for Fos and then visualized using DAB enhanced with nickel to obtain representative images. Representative regions are outlined with dashes. B, Example images of each region that was altered by antibiotic depletion; *p < 0.05, **p < 0.002, ***p < 0.001, significant difference from saline; #p < 0.05, ##p < 0.002, ###p < 0.001 significant difference from OXY group.

Quantification of Fos+ neuronal ensembles during oxycodone withdrawal with antibiotic treatment. A, Differences in the number of Fos+ neurons were evaluated during oxycodone withdrawal between antibiotic-treated and untreated rats. The regions that were altered during withdrawal are represented in purple (WD) and purple with black stripes (WD+ABX). B, Example images of each region that was altered by antibiotic depletion; *p < 0.05, **p < 0.002, ***p < 0.001, significant difference from saline; #p < 0.05, ##p < 0.002, ###p < 0.001 significant difference from non-depleted withdrawal group (WD).