A limited access oral oxycodone paradigm produces physical dependence and mesocorticolimbic region-dependent increases in DeltaFosB expression without preference

Abstract

The abuse of oral formulations of prescription opioids has precipitated the current opioid epidemic. We developed an oral oxycodone consumption model consisting of a limited access (4 h) two-bottle choice drinking in the dark (TBC-DID) paradigm and quantified dependence with naloxone challenge using mice of both sexes. We also assessed neurobiological correlates of withdrawal and dependence elicited via oral oxycodone consumption using immunohistochemistry for DeltaFosB (ΔFosB), a transcription factor described as a molecular marker for drug addiction. Neither sex developed a preference for the oxycodone bottle, irrespective of oxycodone concentration, bottle position or prior water restriction. Mice that volitionally consumed oxycodone exhibited hyperlocomotion in an open field test and supraspinal but not spinally-mediated antinociception. Both sexes also developed robust, dose-dependent levels of opioid withdrawal that was precipitated by the opioid antagonist naloxone. Oral oxycodone consumption followed by naloxone challenge led to mesocorticolimbic region-dependent increases in the number of ΔFosB expressing cells. Naloxone-precipitated withdrawal jumps, but not the oxycodone bottle % preference, was positively correlated with the number of ΔFosB expressing cells specifically in the nucleus accumbens shell. Thus, limited access oral consumption of oxycodone produced physical dependence and increased ΔFosB expression despite the absence of opioid preference. Our TBC-DID paradigm allows for the study of oral opioid consumption in a simple, high-throughput manner and elucidates the underlying neurobiological substrates that accompany opioid-induced physical dependence.

Keywords: Dependence; Mouse; Oral self-administration; Oxycodone; Prescription opioid; Withdrawal.

Fig. 1.. A limited access oxycodone TBC-DID paradigm produces naloxone-precipitated withdrawal without the development preference.

A) The schematic shows the experimental timeline. B) An increasing oxycodone concentration did not impact the % preference for the treated bottle in mice subject to a six-day TBC-DID paradigm. C) The daily oxycodone dose (mg/kg) consumed increased as a function of oxycodone in a session-dependent manner. D) An increasing oxycodone concentration caused a dose-dependent increase in the number of naloxone-precipitated jumps. E) The number of naloxone-precipitated withdrawal jumps was positively correlated with the average daily dose (mg/kg) of oxycodone consumed during the TBC-DID sessions. Data are expressed as mean ± S.E.M. (n = 10 per group). “*” indicates high (1 mg/ml) concentration group vs. water (0 mg/ml) group where****p < 0.0001, **p < 0.01, *p < 0.05, “+”indicates high (1 mg/ml) vs. middle (0.5 mg/ml) concentration group, “X” indicates high (1 mg/ml) vs. low (0.1 mg/ml) concentration group, “#” indicates middle (0.5 mg/ml) concentration group vs. water (0 mg/ml) group, “$” indicates middle (0.5 mg/ml) vs. low (0.1 mg/ml) concentration group, and “^’indicates vs. Session 1 withing the same concentration group with the same symbol indications.

Fig. 2.. Both male and female mice show similar levels of oral oxycodone preference, consumption and precipitated withdrawal.

A) The schematic shows the experimental timeline. B) No significant alterations in the % preference for oxycodone-treated bottle were seen in a sex or concentration or session-dependent manner. C) Both male and female oxycodone consuming mice consumed a higher dose compared to water consuming mice overall. D) Both male and female oxycodone consuming mice exhibited more naloxone-precipitated withdrawal jumps compared to water consuming mice overall. E) Naloxone-induced body weight loss trended to differ as a function of the treated bottle but was not altered by sex and the interaction was not significant. Data are expressed as mean ± S.E.M. (n = 8 per group). “#” indicates middle (0.5 mg/ml) concentration oxycodone group vs. water group where # # # #p < 0.0001.

Fig. 3.. Escalating dose forced choice oxycodone prior to oxycodone TBC-DID does not lead to oxycodone bottle preference.

A) The schematic shows the experimental timeline with “TF” indicating days when mice were tested on the tail-flick test. B) The % preference for oxycodone-treated bottle did not differ as a function of sex, treated bottle or session. C) While across groups, oxycodone consuming mice consumed a higher dose compared to water consuming mice overall, across sexes, male mice consumed a lower dosage compared to female mice overall. D) Oxycodone consuming mice exhibited more naloxone-precipitated withdrawal jumps compared to water consuming mice overall. E) Naloxone-induced body weight loss did not differ as a function of the treated bottle or sex and the interaction was not significant. Data are expressed as mean ± S.E.M. (n = 7-8 per group). “*” indicates high (1 mg/ml) concentration oxycodone group vs. water group where ****p < 0.0001 “+” indicates male mice vs. female mice.

Fig. 4.. Fixed dose forced choice oxycodone prior to oxycodone TBC-DID leads to oxycodone bottle aversion.

A) The schematic shows the experimental timeline with “HP” and “OF” indicating days when mice were tested on the hot plate and open field activity meter test respectively. B) Oxycodone consuming mice had a lower % preference compared to water consuming mice overall. C) Oxycodone consuming mice consumed a higher dose compared to water consuming mice overall. Data are expressed as mean ± S.E.M. (n = 4-6 per group). “*” indicates high (1 mg/ml) concentration oxycodone group vs. water group where ****p < 0.0001.

Figure 5:. Oral oxycodone at behaviorally active doses produces hyperlocomotion and preferentially produces hot plate antinociception compared to tail-flick antinociception.

A) Hot water tail-flick latencies showed modest but reliable alterations as a function of the treated bottle and differed by sex. B) Hot plate latencies differed as a function of the treated bottle and session and the interaction between treated bottle and session and sex was significant. Both male and female mice showed longer withdrawal latencies in the hot plate test post-oxycodone compared to their water baseline levels. In male mice, oxycodone consumption increased response latencies in the hot plate test post-oxycodone compared to water consumption. C) Oxycodone consuming mice travelled a greater distance compared to water consuming mice overall in the open field activity meter test. Data are expressed as mean ± S.E.M. (n = 4-6 per group). “*” indicates high (1 mg/ml) concentration oxycodone group vs. water group where ****p < 0.0001 and **p < 0.01, “+” indicates oxycodone consuming male mice vs. water consuming male mice with the same symbol indications.

Fig. 6.. Representative photomicrographs show impact of oxycodone consumption and naloxone challenge on ΔFosB-expressing cells in the NAc, amygdala and VTA.

Representative images show distribution of ΔFosB staining in coronal sections at the level of the NAc, amygdala and VTA derived from A, C, E) water- and B, D, F) oxycodone-consuming mice respectively. The scale bar equals 500 μm. (Nucleus accumbens – NAc, Basolateral amygdalar nucleus - BLA, Central amygdalar nucleus capsular part – CeC, Ventral tegmental area - VTA)

Fig. 7.. Naloxone challenge following oral oxycodone consumption leads to greater numbers of ΔFosB expressing cells in the NAc core, shell, CeC, and VTA.

Following naloxone challenge, oxycodone consuming mice showed higher numbers of ΔFosB expressing cells in the A) NAc core, B) NAc shell, E) CeC, and F) VTA but not the D) BLA when compared to water consuming mice. C) The number of naloxone-precipitated withdrawal jumps was positively correlated with the number of ΔFosB-expressing cells in the NAc shell (left panel). In contrast, no significant correlations was observed between the % preference and the number of ΔFosB-expressing cells in the NAc shell (right panel). (Nucleus accumbens – NAc, Basolateral amygdalar nucleus - BLA, Central amygdalar nucleus capsular part – CeC, Ventral tegmental area - VTA) Data are expressed as mean ± S.E.M. (n = 5-6 per group). *p < 0.05 vs. water consuming mice.

Fig. 8.. Naloxone challenge increases the number of ΔFosB-expressing cells preferentially in oxycodone consuming but not water consuming mice.

A) Male and female mice subjected to either the escalating (Fig. 3) or fixed (Fig. 4) forced choice regimen followed by a six-day oxycodone TBC-DID sessions consumed similar doses of oxycodone. Oxycodone consumption followed by naloxone challenge led to higher numbers of ΔFosB expressing cells in the B) NAc core, C) NAc shell, E) CeC, and F) VTA but not the D) BLA compared to oxycodone consumption without naloxone challenge. In contrast, water consumption followed by naloxone challenge led to higher numbers of ΔFosB expressing cells in the E) CeC alone. (Nucleus accumbens – NAc, Basolateral amygdalar nucleus - BLA, Central amygdalar nucleus capsular part – CeC, Ventral tegmental area - VTA) Data are expressed as mean ± S.E.M. (n = 5-11 per group). “*” indicates vs. corresponding no naloxone group where ****p < 0.0001, ***p < 0.001, and *p < 0.05, “#” indicates vs. corresponding naloxone challenge group, and “+” indicates main effect of oxycodone consumption with the same symbol indications. n.s. - non-significant.