Role of Projections between Piriform Cortex and Orbitofrontal Cortex in Relapse to Fentanyl Seeking after Palatable Food Choice-Induced Voluntary Abstinence

Abstract

We recently developed a rat model of relapse to drug seeking after food choice-induced voluntary abstinence. Here, we used this model to study the role of the orbitofrontal cortex (OFC) and its afferent projections in relapse to fentanyl seeking. We trained male and female rats to self-administer palatable food pellets for 6 d (6 h/d) and intravenous fentanyl (2.5 μg/kg/infusion) for 12 d (6 h/d). We assessed relapse to fentanyl seeking after 13-14 voluntary abstinence days, achieved through a discrete choice procedure between fentanyl infusions and palatable food (20 trials/d). In both sexes, relapse after food choice-induced abstinence was associated with increased expression of the activity marker Fos in the OFC. Pharmacological inactivation of the OFC with muscimol plus baclofen (50 + 50 ng/side) decreased relapse to fentanyl seeking. We then determined projection-specific activation of OFC afferents during the relapse test by using Fos plus the retrograde tracer cholera toxin B (injected into the OFC). Relapse to fentanyl seeking was associated with increased Fos expression in the piriform cortex (Pir) neurons projecting to the OFC, but not in projections from the basolateral amygdala and thalamus. Pharmacological inactivation of the Pir with muscimol plus baclofen decreased relapse to fentanyl seeking after voluntary abstinence. Next, we used an anatomical disconnection procedure to determine whether projections between the Pir and OFC are critical for relapse to fentanyl seeking. Unilateral muscimol plus baclofen injections into the Pir in one hemisphere plus unilateral muscimol plus baclofen injections into the OFC in the contralateral, but not ipsilateral, hemisphere decreased relapse. Our results identify Pir-OFC projections as a new motivation-related pathway critical to relapse to opioid seeking after voluntary abstinence.SIGNIFICANCE STATEMENT There are few preclinical studies of fentanyl relapse, and these studies have used experimenter-imposed extinction or forced abstinence procedures. In humans, however, abstinence is often voluntary, with drug available in the drug environment but forgone in favor of nondrug alternative reinforcers. We recently developed a rat model of drug relapse after palatable food choice-induced voluntary abstinence. Here, we used classical pharmacology, immunohistochemistry, and retrograde tracing to demonstrate a critical role of the piriform and orbitofrontal cortices in relapse to opioid seeking after voluntary abstinence.

Keywords: addiction; anterior insular cortex; disconnection; opioid; orbitofrontal cortex; self-administration.

Figure 1.

Effect of relapse to fentanyl seeking after voluntary abstinence on Fos expression. A, Timeline of exp. 1. B, Self-administration: number of reinforced responses (food, 5 pellets/reinforcer delivery; fentanyl, 2.5 μg/kg/infusion) during the 6 h sessions. C, Choice during training: number of food-reinforced responses and fentanyl infusions earned during the 3 h choice sessions (20 trials/session), denoted by C1, C2, and C3 in B. D, Voluntary abstinence: number of food-reinforced responses and fentanyl infusions earned during the 3 h choice sessions (20 trials/session). E, Relapse test: number of active and inactive lever presses during 60 min test session (left) and the 20 min time course (right; n = 22 males, 20 females, between-subjects design). *p < 0.05, different from the inactive lever. F, Representative images of Fos+ cells in orbitofrontal and anterior insular cortex, at 10× magnification. Scale bar, 100 μm. G, Fos-IR quantification: number of Fos+ cells (counts/mm²). *p < 0.05, different from the no test group (n = 7/group, between-subjects design). Data are the mean ± SEM. For individual data, see Extended Data Figure 1-1.

Figure 2.

Effect of OFC inactivation on relapse to fentanyl seeking after voluntary abstinence. A, Timeline of exp. 2. B, Self-administration: number of reinforced responses (food, 5 pellets/reinforcer; fentanyl, 2.5 μg/kg/infusion) during the 6 h sessions. C, Choice during training: number of food-reinforced responses and fentanyl infusions earned during the 3 h choice sessions (20 trials/session), denoted by C1, C2, and C3 in B. D, Voluntary abstinence: number of food-reinforced responses and fentanyl infusions earned during the 3 h choice sessions (20 trials/session). E, Relapse test: number of active and inactive lever presses during the 3 h test session (left) and 1 h time course (right) after vehicle or muscimol plus baclofen (M+B) injections. *p < 0.05, different from vehicle group. F, Reacquisition test: number of fentanyl infusions (2.5 μg/kg/infusion) during the 1 h time course of the 6 h session after vehicle or muscimol plus baclofen injection (n = 10–17/group, between-subjects design). Data are the mean ± SEM. G, Images showing placement of cannula into the OFC at 2.5× magnification. Scale bar, 500 μm. Placements for lateral OFC are shown in red, and placements for ventral OFC are shown in black. Vehicle placements are shown with open circles, and muscimol plus baclofen placements are shown with closed circles. For individual data, see Extended Data Figure 2-1. For data from cannula misplacements, see Extended Data Figure 2-2.

Figure 3.

Effect of AIV inactivation on relapse to fentanyl seeking after voluntary abstinence. A, Timeline of exp. 3. B, Self-administration: number of reinforced responses (food, 5 pellets/reinforcer; fentanyl, 2.5 μg/kg/infusion) during the 6 h sessions. C, Choice during training: number of food-reinforced responses and fentanyl infusions earned during the 3 h choice sessions (20 trials/session), denoted by C1, C2, and C3 in B. D, Voluntary abstinence: number of food-reinforced responses and fentanyl infusions earned during the 3 h choice sessions (20 trials/session). E, Relapse test: number of active and inactive lever presses during the 3 h test session (left) and 1 h time course (right) after vehicle or muscimol plus baclofen (M+B) injections. *p < 0.05, different from vehicle group. F, Reacquisition test: number of fentanyl infusions (2.5 μg/kg/infusion) during the 1 h time course of the 6 h session after vehicle or muscimol plus baclofen injections (n = 13/group, between-subjects design). Data are the mean ± SEM. G, Images showing placement of cannula into AIV at 2.5× magnification. Scale bar, 500 μm. Vehicle placements are shown with open circles, and muscimol plus baclofen placements are shown with closed circles. For individual data, see Extended Data Figure 3-1. For data from cannula misplacements, see Extended Data Figure 2-2.

Figure 4.

Effect of relapse to fentanyl seeking on Fos expression in afferent projections to the OFC. A, Timeline of exp. 4. B, Self-administration: number of reinforced responses (food, 5 pellets/reinforcer; fentanyl, 2.5 μg/kg/infusion) during the 6 h sessions. C, Choice during training: number of food-reinforced responses and fentanyl infusions earned during the 3 h choice sessions (20 trials/session), denoted by C1, C2, and C3 in B. D, Voluntary abstinence: number of food-reinforced responses and fentanyl infusions earned during the 3 h choice sessions (20 trials/session). E, Relapse tests: number of active and inactive lever presses during the 90 min test session (left) and the 30 min time course (right). *p < 0.05, different from inactive lever. F, Placement of CTb injection into the OFC and representative image captured at 2.5× magnification (scale bar, 500 μm). G, From left to right: number of Fos+ cells (counts/mm²) in the Pir, BLA, and thalamus; number of CTb+ cells per mm² in all 4 regions; and number of CTb+ cells that colabel with Fos in all four regions. *p < 0.05, different from no test group. Representative images showing Fos (red) or CTb (green; 40× magnification). Scale bar, 100 μm. Red arrows denote Fos+ cell, and green arrows denote CTb+ cell (n = 5–7/group, between-subjects design). Data are the mean ± SEM. For individual data, see Extended Data Figure 4-1.

Figure 5.

Effect of Pir inactivation on relapse to fentanyl seeking after voluntary abstinence. A, Timeline of exp. 5. B, Self-administration: number of reinforced responses (food, 5 pellets/reinforcer; fentanyl, 2.5 μg/kg/infusion) during the 6 h sessions. C, Choice during training: number of food-reinforced responses and fentanyl infusions earned during the 3 h choice sessions (20 trials/session), denoted by C1, C2, and C3 in B. D, Voluntary abstinence: number of food-reinforced responses and fentanyl infusions earned during the 3 h choice sessions (20 trials/session). E, Relapse test: number of active and inactive lever presses during the 3 h test session (left) and 1 h time course (right) after vehicle or muscimol plus baclofen (M+B) injections. *p < 0.05, different from vehicle group. F, Reacquisition test: number of fentanyl infusions (2.5 μg/kg/infusion) during the 1 h time course of the 6 h session after vehicle or muscimol plus baclofen injections (n = 14–15/group, between-subjects design). Data are the mean ± SEM. G, Images showing placement of cannula into the Pir at 2.5× magnification. Scale bar, 500 μm. Vehicle placements are shown with open circles, and muscimol plus baclofen placements are shown with closed circles. For individual data, see Extended Data Figure 5-1. For data from cannula misplacements, see Extended Data Figure 2-2.

Figure 6.

Effect of anatomical disconnection between the Pir and OFC on relapse to fentanyl seeking. A, Timeline of exp. 6. B, Self-administration: number of reinforced responses (food, 5 pellets/reinforcer; fentanyl, 2.5 μg/kg/infusion) during the 6 h sessions. C, Choice during training: number of food-reinforced responses and fentanyl infusions earned during the 3 h choice sessions (20 trials/session), denoted by C1, C2, and C3 in B. D, Voluntary abstinence: number of food-reinforced responses and fentanyl infusions earned during the 3 h choice sessions (20 trials/session). E, Relapse test: number of active and inactive lever presses during the 3 h test session (left) and 1 h time course (right) after unilateral Pir and ipsilateral or contralateral OFC vehicle or muscimol plus baclofen (M+B) injections. *p < 0.05, different from vehicle and ipsilateral groups (n = 9–12/group, between-subjects design). F, Reacquisition test: number of fentanyl infusions (2.5 μg/kg/infusion) during the 1 h time course of the 6 h session after unilateral Pir and contralateral OFC vehicle or muscimol plus baclofen injections (n = 5/group, between-subjects design). Data are the mean ± SEM. G, Images showing placement of cannulas in the OFC and Pir at 2.5× magnification. Scale bar, 500 μm. Placements in the ipsilateral condition are shown in red, and placements in the contralateral condition are shown in black. Vehicle placements are shown with open circles, and muscimol plus baclofen placements are shown with closed circles. For individual data, see Extended Data Figure 6-1. For data from cannula misplacements, see Extended Data Figure 2-2.