GluA2-lacking AMPA receptors in the nucleus accumbens core and shell contribute to the incubation of oxycodone craving in male rats

Abstract

One of the most challenging issues in the treatment of substance use disorder, including misuse of opioids such as oxycodone, is persistent vulnerability to relapse, often triggered by cues or contexts previously associated with drug use. In rats, cue-induced craving progressively intensifies ('incubates') during withdrawal from extended-access self-administration of several classes of misused drugs, including the psychostimulants cocaine and methamphetamine. For these psychostimulants, incubation is associated with strengthening of excitatory synapses in the nucleus accumbens (NAc) through incorporation of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors that lack the GluA2 subunit and are therefore Ca²⁺ -permeable (CP-AMPARs). Once CP-AMPAR upregulation occurs, their stimulation is required for expression of incubation. It is not known if a similar mechanism contributes to incubation of oxycodone craving. Using male rats, we established that incubation occurs by withdrawal day (WD) 15 and persists through WD30. Then, using cell-surface biotinylation, we found that surface levels of the AMPAR subunit GluA1 but not GluA2 are elevated in NAc core and shell of oxycodone rats on WD15, although this wanes by WD30. Next, using intra-NAc injection of the selective CP-AMPAR antagonist Naspm before a seeking test, we demonstrate that CP-AMPAR blockade in either subregion decreases oxycodone seeking on WD15 or WD30 (after incubation), but not WD1, and has no effect in saline self-administering animals. The Naspm results suggest CP-AMPARs persist in synapses through WD30 even if total cell surface levels wane. These results suggest that a common neurobiological mechanism contributes to expression of incubation of craving for oxycodone and psychostimulants.

Keywords: incubation of craving; nucleus accumbens; oxycodone.

FIGURE 1

Oxycodone and saline self-administration training for rats used in biotinylation experiments. (A) Timeline of self-administration training (data shown here) and seeking tests (data shown in Figure 2). (B) Oxycodone self-administering (0.15 mg/kg/infusion) animals learn to discriminate between active and inactive nose ports during self-administration training (6 h/day × 10 days; n = 27 rats). (C) Active and inactive nose pokes do not differ during saline self-administration (n = 8 rats). (D) Oxycodone, but not saline, self-administering animals significantly increase their drug intake during the final 6 days of self-administration. *p < 0.05, **p < 0.01, ***p = 0.0001, ****p < 0.0001 versus days 1–4 of drug self-administration training. WD, withdrawal day

FIGURE 2

Incubation of oxycodone craving for rats used in biotinylation experiments. (A) Saline self-administering animals do not demonstrate a time-dependent change in active hole responding or inactive hole responding (withdrawal day 1 vs. withdrawal day 15), or a difference in active versus inactive hole responding. (B) Oxycodone self-administering animals demonstrate enhanced oxycodone seeking on withdrawal day 15 versus withdrawal day 1 (active hole responding) but no time-dependent change in inactive hole responding. (C) Enhanced oxycodone seeking persists on withdrawal day 30. Individual data points are shown for all groups (n values ranged from 8–10 rats/group). Data are mean ± SEM. **p < 0.01, ***p = 0.0001, ****p < 0.0001 versus withdrawal day 1. WD, withdrawal day; ns, non-significant

FIGURE 3

Cell surface levels of GluA1 but not GluA2 increase following withdrawal from extended-access oxycodone self-administration. Schematic showing nucleus accumbens core (A) and shell (B) subregions dissected for biotinylation experiments. Cell-surface (C) but not total (D) GluA1 protein levels in the nucleus accumbens core are significantly elevated relative to saline (Sal) self-administering controls on withdrawal day (WD) 15. Cell surface (E) but not total (F) GluA1 protein levels are significantly elevated in the nucleus accumbens shell on WD15 relative to WD1. Surface GluA2 surface and total protein levels are not changed in either subregion during withdrawal (G–J). Individual data points are shown for all groups (n values ranged from 8–10 rats/group). Data are mean ± SEM optical density expressed as percent of saline control values. *p < 0.05

FIGURE 4

Naspm injections have no impact on withdrawal day 15 responding in saline self-administering rats. (A) Timeline of experiment; all rats underwent two seeking tests (WD1 and WD15) and received aCSF/Naspm injections in a counterbalanced design. WD15 data are shown here, and WD1 data are provided in the results. (B) There is no difference between active and inactive lever presses during saline self-administration training (n = 24 rats). (C) Saline infusions over the 10 days of self-administration do not change. Naspm injection into the nucleus accumbens core (D) or shell (F) has no effect on saline seeking on withdrawal day 15 as compared to aCSF infusion (n = 6 rats/group). (E,G) Approximate placement of injection tips in the nucleus accumbens subregions. Data are ± SEM. WD, withdrawal day; aCSF, artificial cerebrospinal fluid

FIGURE 5

Naspm injections into the nucleus accumbens core or shell block expression of incubation of oxycodone craving on withdrawal days 15 and 30 but have no effect on withdrawal day 1. (A) Timeline. All rats underwent two seeking tests, either on WD1 and WD15 or on WD1 and WD30, and received both aCSF and Naspm treatments, in a counterbalanced design. WD, withdrawal day. Rats learn to discriminate between active and inactive levers (B) and oxycodone intake increases over the 10 days of self-administration (C). (D) Naspm injection into the nucleus accumbens core 15 min prior to a cue-induced relapse test on withdrawal day 1 does not alter oxycodone seeking. (E) Naspm injection into the nucleus accumbens core 15 min prior to a cue-induced seeking test on withdrawal day 15 significantly reduces oxycodone seeking. (F) Naspm injection into the nucleus accumbens core 15 min prior to a cue-induced seeking test on withdrawal day 30 significantly reduces oxycodone seeking. (G) Approximate placement and representative image of injection tips in the nucleus accumbens core. (H) Naspm injection into the nucleus accumbens shell 15 min prior to a cue-induced seeking test on withdrawal day 1 does not alter oxycodone seeking. (I) Naspm injection into the nucleus accumbens shell 15 min prior to a cue-induced seeking test on withdrawal day 15 significantly reduces oxycodone seeking. (J) Naspm injection into the nucleus accumbens shell 15 min prior to a cue-induced seeking test on withdrawal day 30 significantly reduces oxycodone seeking. (K) Approximate placement and representative image of injection tips in the nucleus accumbens shell. Data are ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. aCSF, artificial cerebrospinal fluid