Abstract
We previously demonstrated a role of piriform cortex (Pir) in relapse to fentanyl seeking after food choice-induced voluntary abstinence. Here, we used this model to further study the role of Pir and its afferent projections in fentanyl relapse. We trained male and female rats to self-administer palatable food pellets for 6 d (6 h/day) and fentanyl (2.5 µg/kg/infusion, i.v.) for 12 d (6 h/day). We assessed relapse to fentanyl seeking after 12 voluntary abstinence sessions, achieved through a discrete choice procedure between fentanyl and palatable food (20 trials/session). We determined projection-specific activation of Pir afferents during fentanyl relapse with Fos plus the retrograde tracer cholera toxin B (injected into Pir). Fentanyl relapse was associated with increased Fos expression in anterior insular cortex (AI) and prelimbic cortex (PL) neurons projecting to Pir. We next used an anatomical disconnection procedure to determine the causal role of these two projections (AI→Pir and PL→Pir) in fentanyl relapse. Contralateral but not ipsilateral disconnection of AI→Pir projections decreased fentanyl relapse but not reacquisition of fentanyl self-administration. In contrast, contralateral but not ipsilateral disconnection of PL→Pir projections modestly decreased reacquisition but not relapse. Fluorescence-activated cell sorting and quantitative PCR data showed molecular changes within Pir Fos-expressing neurons associated with fentanyl relapse. Finally, we found minimal or no sex differences in fentanyl self-administration, fentanyl versus food choice, and fentanyl relapse. Our results indicate that AI→Pir and PL→Pir projections play dissociable roles in nonreinforced relapse to fentanyl seeking versus reacquisition of fentanyl self-administration after food choice-induced voluntary abstinence.
SIGNIFICANCE STATEMENT We previously showed a role of Pir in fentanyl relapse after food choice-induced voluntary abstinence in rats, a procedure mimicking human abstinence or a significant reduction in drug self-administration because of the availability of alternative nondrug rewards. Here, we aimed to further characterize the role of Pir in fentanyl relapse by investigating the role of Pir afferent projections and analyzing molecular changes in relapse-activated Pir neurons. We identified dissociable roles of two Pir afferent projections (AI→Pir and PL→Pir) in relapse to fentanyl seeking versus reacquisition of fentanyl self-administration after voluntary abstinence. We also characterized molecular changes within Pir Fos-expressing neurons associated with fentanyl relapse.
Introduction
A significant barrier to treatment of drug addiction is high rates of relapse during abstinence (Hunt et al., 1971; Sinha, 2011). Over the past several decades, studies using animal relapse models have yet to identify medications approved by the Federal Drug Administration for relapse prevention (Venniro et al., 2020; Fredriksson et al., 2021). Traditionally, preclinical animal relapse models have used the extinction reinstatement (Shalev et al., 2002; Kalivas and McFarland, 2003) or homecage forced abstinence (Venniro et al., 2016) procedures. However, in these models, the abstinence period is experimenter imposed, which may limit translatability to humans. This is because the models do not incorporate a key feature of human addiction: voluntary abstinence because of either adverse consequences of drug use or availability of competing nondrug rewards (Epstein and Preston, 2003; Katz and Higgins, 2003).
To address this translatability issue, we developed a rat model of relapse after voluntary abstinence that is induced by presenting rats with a history of drug self-administration a series of discrete choices between a high-carbohydrate palatable food reward versus drug reward (Caprioli et al., 2015; Venniro et al., 2017a; Fredriksson et al., 2021); the choice procedure is based on previous studies (Lenoir et al., 2007, 2013; Ahmed, 2018). During the choice sessions, rats strongly prefer food over drug and either completely abstain or only self-administer very few drug infusions. We termed the discrete choice period “voluntary abstinence” (Caprioli et al., 2015).
Using this food choice-induced abstinence procedure, we investigated cortical mechanisms of relapse to fentanyl seeking (Reiner et al., 2020). We trained male and female rats to self-administer palatable food pellets (6 d) and fentanyl (12 d), followed by 13–14 food choice-induced voluntary abstinence days. We then assessed relapse to fentanyl seeking under extinction conditions. We found that relapse was associated with selective activation of anterior insular cortex (AI), orbitofrontal cortex (OFC), piriform cortex (Pir), and projections from Pir to OFC. Pharmacological inactivation of AI, OFC, and Pir with muscimol plus baclofen decreased relapse to fentanyl seeking. Next, we used an asymmetrical anatomical disconnection procedure (Gaffan et al., 1993; Setlow et al., 2002) to show that inhibition of projections between Pir and OFC decreased fentanyl relapse (Reiner et al., 2020). In this study, we also tested the behavioral specificity of the site-specific and projection-specific inhibition manipulations by testing their effect on a different measure of relapse: reacquisition of fentanyl self-administration. We found that the different manipulations had no effect on reacquisition (Reiner et al., 2020). Together, our data suggest that Pir, traditionally considered an olfaction-related region (Stettler and Axel, 2009), plays a role in opioid relapse-related behaviors.
The goal of our current study was to further investigate the role of Pir-related circuits in relapse to fentanyl seeking. We first used the retrograde tracer cholera toxin b (CTb), injected into Pir, to determine brain regions that project to Pir that are selectively activated (assessed by the activity marker Fos) during relapse. The afferent projections we studied were AI, prelimbic (PL) and infralimbic (IL) cortex, and basolateral amygdala (BLA). We chose these regions based on previous studies on their role in opioid relapse, as assessed in animal models (Reiner et al., 2019). We then followed up on the correlational results to determine the causal role of AI and PL projections to Pir in fentanyl relapse using the anatomical disconnection procedure.
Finally, we used fluorescence-activated cell sorting (FACS; Guez-Barber et al., 2011, 2012; Liu et al., 2014) and quantitative PCR (qPCR) to assess gene expression in Fos-positive Pir neurons that were activated during the relapse test and in Fos-negative neurons that represent the nonactivated majority of Pir neurons. In previous studies, we used FACS in combination with qPCR to characterize the molecular phenotype of activated neurons in our relapse-related studies (Guez-Barber et al., 2011; 2012; Fanous et al., 2013; Rubio et al., 2015; Li et al., 2015b; Fredriksson et al., 2023).
Materials and Methods
Subjects
We used 82 male and 82 female Sprague Dawley rats (body weight at the time of intravenous surgery, males, 250–410 g; females, 161–265 g; Charles River Laboratories). The rats were ∼8–10 weeks of age at the time of intravenous surgery. We housed the rats two per cage for 1–3 weeks and then individually after surgery to avoid potential damage to catheters and cannulae from social housing. We maintained the rats under a reverse 12:12 h light/dark cycle (lights off at 8:00 A.M.) with food and water freely available. We performed the experiments in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (eighth edition), under protocols approved by the local Animal Care and Use Committee. We excluded 65 rats because of catheter failure (n = 1), cannula misplacement (n = 33), CTb injection misplacement (n = 15), or illness (n = 16).
Drugs
We received fentanyl citrate (fentanyl) from the National Institute on Drug Abuse (NIDA) pharmacy and dissolved it in sterile saline. We chose a unit dose of 2.5 µg/kg/infusion for self-administration training based on our previous study (Reiner et al., 2020). In experiments (Exps.) 2–3, we dissolved muscimol and baclofen (Tocris Bioscience) in sterile saline and injected it intracranially at a dose of 50 + 50 ng in 0.5 μl/side (Stopper and Floresco, 2014; Venniro et al., 2017b; Reiner et al., 2020).
Intravenous surgery
We anesthetized the rats with isoflurane gas (5% induction, 2–3% maintenance; Covetrus) and inserted SILASTIC catheters into the jugular vein, as previously described (Venniro et al., 2017a, b). We injected the rats with ketoprofen (2.5 mg/kg, s.c., Covetrus) 1 h after surgery and the following day to relieve pain and inflammation. We allowed the rats to recover for 5–7 d before food self-administration training. During the recovery and all experimental phases, we flushed the catheters every 24–48 h with gentamicin (4.25–5 mg/ml; Fresenius Kabi) dissolved in sterile saline. If we suspected catheter failure during training, we tested patency with Diprivan (propofol, 10 mg/ml, 0.1–0.2 ml injection volume, i.v.; NIDA pharmacy), and if not patent, we catheterized the left jugular vein or eliminated the rat from the study.
CTb injection into Pir (Exp. 1)
After the last day of fentanyl self-administration training, we injected CTb (List Biological Laboratories) into Pir. We injected 40 nl of 1% CTb unilaterally into Pir with the needle left in place for an additional 5 min (Mahler and Aston-Jones, 2012; Marchant et al., 2014, 2016; Venniro et al., 2017b). We counterbalanced injections into either the left or right hemisphere and used a 1.0 µl, 32 gauge Neuros Syringe (Hamilton) attached to UltraMicroPump III with SYS-Micro4 controller (World Precision Instruments). The coordinates for Pir were AP, +3.4 mm; ML, ±4.3 mm (10° angle lateral to midline); DV, −6.7 mm. These coordinates are based on pilot studies to optimize spread of CTb within Pir and minimize spread to adjacent areas. The spread of the CTb injections was ∼20–30% of anterior Pir.
Intracranial surgery
We performed intracranial surgery in the same session as the intravenous surgery. For Exp. 2, using a stereotaxic instrument (Kopf), we implanted two guide cannulae (23 gauge, Plastics One) in each rat, 1 mm above Pir and above AI. We counterbalanced the location of the cannulae into either the left or right hemisphere in Pir and the ipsilateral or contralateral AI. We set the nose bar at −3.3 mm and used the following coordinates from bregma: AI, AP: +2.8 mm; ML, ±4.9 mm (10° angle lateral to midline); DV, −4.9 mm; Pir, contralateral to AI, AP, +3.4 mm; ML, ±3.9 mm (10° angle lateral to midline); DV, −6.2 mm; Pir, ipsilateral to AI, AP, +3.4 mm; ML, ± 2.5 mm (5° angle lateral to midline); DV, −6.6 mm. These coordinates are based on pilot and previous studies (Venniro et al., 2017b; Reiner et al., 2020).
For Exp. 3, we implanted two guide cannulae in each rat, one above Pir and one above PL. We used the following coordinates for Pir: AP, +3.4 mm; ML, ±3.9 mm (10° angle lateral to midline); DV, −6.2 mm, and the following coordinates for PL, contralateral to Pir, AP, +3.0 mm; ML, ±1.2 mm (10° angle lateral to midline); DV, −2.4 mm; PL, ipsilateral to Pir, AP, +3.0 mm; ML, ±0.2 mm (10° angle lateral to midline); DV, −2.4 mm. These coordinates are based on pilot and previous studies (Li et al., 2015a; Reiner et al., 2020). We anchored the cannulae to the skull with jeweler screws and dental cement.
Intracranial injections
In Exp. 2–3, we injected vehicle (saline) or muscimol and baclofen 15 min before starting the relapse or reacquisition test sessions at a rate of 0.5 μl/min and left the injectors (which extend 1.0 mm below the tips of the guide cannulae) in place for an additional minute to allow diffusion. We connected the syringe pump (Harvard Apparatus) to 10 μl Hamilton syringes attached to the 30 gauge injectors via polyethylene-50 tubing. We habituated rats to the injection procedure for 3 d before testing. We extracted the brains of the rats after testing and stored them in 10% formalin. We sectioned the rat brains (50 μm sections) using a Leica cryostat and stained the sections with cresyl violet. Finally, we verified cannula placements under a light microscope.
Immunohistochemistry
Immediately after the relapse test (Exp. 1), we anesthetized the rats with isoflurane and perfused them transcardially with ∼200 ml of 0.1 m PBS, pH 7.4, followed by ∼400 ml of 4% paraformaldehyde (PFA) in PBS. We removed the brains and postfixed them in 4% PFA for 2 h before transferring them to 30% sucrose in PBS for 48 h at 4°C. We froze the brains in dry ice and cut coronal sections (40 µm) of the different brain areas using a Leica cryostat. We collected the sections in PBS and stored them at 4°C until further processing.
CTb injection site verification (Exp. 1)
We selected a one-in-four series of 40 µm sections from Pir of each rat to determine CTb injection sites. We repeatedly rinsed free-floating sections in PBS (3 × 10 min) and incubated them for 1 h in 0.5% PBS-Tx with 10% normal horse serum (NHS). We then incubated sections overnight at 4°C in goat anti-CTb primary antibody (1:1000; catalog #703, List Biological Laboratories; RRID:AB_10013220) diluted in 0.5% PBS-Tx with 2% NHS. We rinsed the sections with PBS and incubated them for 2 h in Alexa Fluor 488 donkey anti-goat secondary antibody (1:500; catalog #705-546-147, Jackson ImmunoResearch; RRID:AB_2340430) diluted in 0.5% PBS-Tx with 2% NHS. We rinsed the sections again in PBS, mounted them onto gelatin-coated slides, air dried them, and coverslipped the slides with Vectashield Hard Set Mounting Medium with DAPI (catalog #H-1500, Vector Laboratories; RRID:AB_2336788). We only included rats in which the CTb injection site was restricted to Pir.
Fos and CTb double labeling (Exp. 1)
We processed a one-in-four series containing AI, PL, IL (4.2–3.0 mm anterior to bregma), and BLA (1.0–2.5 mm posterior to bregma) for immunohistochemical detection of Fos and CTb. We rinsed free-floating sections (3 × 10 min) and incubated them in 10% NHS with 0.5% PBS-Tx for 2 h. We then incubated the sections overnight at 4°C in 0.5% PBS-Tx containing 2% NHS with rabbit anti-c-fos primary antibody (1:1000; catalog #5348S, Cell Signaling Technology; RRID:AB_10557109) and goat anti-CTb primary antibody (1:1000; catalog #703, List Biological Laboratories; RRID:AB_10013220). We rinsed the sections in PBS and incubated them for 2 h in 0.5% PBS-Tx containing 2% NHS and donkey anti-rabbit Alexa Fluor 594 (1:500; catalog #711-585-152, Jackson ImmunoResearch; RRID:AB_2340621) and donkey anti-goat Alexa Fluor 488 (1:500; catalog #705-546-147, Jackson ImmunoResearch; RRID:AB_2340430). We rinsed the sections in PBS (3 × 10 min) and mounted them onto gelatin-coated slides, partially dried, and coverslipped the sections with Vectashield Hard Set Mounting Medium with DAPI (catalog #H-1500, Vector Laboratories, RRID:AB_2336788).
FACS (Exp. 4)
Immediately after the 90 min relapse test, we deeply anesthetized the rats with isoflurane for 90 s before decapitation. We extracted the brains, froze them in cold isopentane (approximately −40°C) for 20 s, and then stored them at −80°C. On a cryostat, we sliced 4 × 300 µm sections containing Pir (∼4.2-3.0 mm anterior to bregma) and used a 1 mm diameter micropunch to collect Pir tissue from each hemisphere. We stored the tissue in microcentrifuge tubes at −80°C until FACS processing.
We processed the tissue for FACS as described previously for fresh frozen tissue (Rubio et al., 2016; Fredriksson et al., 2023) with the following modifications: (1) We triturated the tissue with 1.2 ml of ice-cold Hibernate A (catalog #HA, Transnetyx) containing RNase inhibitor (1:200; catalog #30281-2, Lucigen); (2) we pooled two to three samples, matched to have similar active lever press responding during the relapse test, which allowed us to increase RNA yield from Fos-positive cells; (3) we fixed and permeabilized cells by adding the same volume of 100% of cold methanol (−20°C) for 15 min on ice, inverting the tubes every 5 min; and (4) after collecting the cells by centrifugation (1700 × g, 4 min, 4°C), we resuspended the cells in 0.6 ml of cold PBS and RNase inhibitor (1:200; catalog #30281-2, Lucigen) and then filtered the cells with 100 µm cell strainers (Falcon brand, BD Biosciences).
We incubated the cells with Phycoertyhrin (PE)-labeled anti-NeuN antibody (1:500; catalog #FCMAB317PE, Millipore; RRID:AB_10807694) and Alexa 647-labeled anti-phospho-Fos antibody (1:100; catalog #8677, Cell Signaling Technology; RRID:AB_11178518) for 30 min at 4°C and then washed the cells with 0.8 ml cold PBS. After collecting the cells by centrifugation (1300 × g, 3 min, 4°C), we washed the cells again with 1 ml cold PBS, followed by centrifugation (1300 × g, 3 min, 4°C), filtered with a 40 µm cell strainer, and resuspended the cells in 0.3 ml cold PBS plus RNase inhibitor for sorting in a BD FACSMelody Cell Sorter (BD Biosciences).
As we reported previously (Guez-Barber et al., 2011, 2012; Liu et al., 2014; Rubio et al., 2015, 2016; Li et al., 2015b), cells can be identified based on the distinct forward and side scatter properties. We excluded duplets based on the forward scatter height and width signal properties of the cell gate population. From the gate of the singlets, we sorted neurons according to PE (NeuN-immunopositive) and Alexa Fluor 647 (Fos-immunopositive) fluorescence signal. We set the threshold of Alexa Fluor 647 fluorescence signal based on background fluorescence signals of a naive homecage control group. Based on NeuN and Fos immunoreactivity, we sorted Fos-negative/NeuN-positive (Fos-negative) and Fos-positive/NeuN-positive (Fos-positive) events.
qPCR
We collected 5000 Fos-negative neurons and all Fos-positive neurons directly into 100 µl of the extraction buffer from the PicoPure RNA isolation kit (catalog #KIT0204, Applied Biosystems) and lysed the cells by pipetting up and down 10 times followed by incubation for 30 min at 42°C. We extracted RNA according to PicoPure RNA isolation protocol and synthesized single-strand cDNA using the Superscript III First-Strand cDNA synthesis kit (catalog #18080-051, Invitrogen) according to the protocol from the manufacturer.
We used gene-targeted preamplification of cDNA as described previously (Liu et al., 2014; Rubio et al., 2015; Li et al., 2015b). Briefly, we used a pooled primer solution of 0.2× concentration of TaqMan ABI primer/probes (20× TaqMan gene expression assay as the stock solution) and 80 nm of customized primer sets (Table 1). Each cDNA sample (7.5 µl) was mixed with the pooled primer solution (7.5 µl) and 15 µl of 2× TaqMan PreAmp Master Mix (catalog #4391128, Applied Biosystems). We preamplified cDNA in an ABI 9700 Thermal Cycler using the following program: 95°C hold for 10 min, denaturation at 90°C for 15 s, and annealing and extension at 60°C for 4 min (14 cycles).
We diluted the preamplified cDNA product seven times before proceeding to qPCR. We performed qPCR in duplicates with a FAM-labeled probe for each target gene and a VIC-labeled probe for the endogenous control gene (NeuN; Liu et al., 2014; Rubio et al., 2015, 2016; Li et al., 2015b). We used TaqMan Advanced Fast PCR Master Mix (catalog #4444557, Thermo Fisher Scientific) in a 7500 Fast TaqMan instrument, using the following program: 95°C hold for 20 s, then 40 cycles with denaturation at 95°C for 3 s, and annealing and extension at 60°C for 30 s. We analyzed reactions using the ΔΔCt method with NeuN as the housekeeping gene (Liu et al., 2014; Rubio et al., 2015, 2016; Li et al., 2015b; Fredriksson et al., 2023).
Self-administration apparatus
We trained rats to self-administer food and fentanyl in standard Med Associates self-administration chambers. We equipped each self-administration chamber with two operant panels with three levers located 7–8 cm above the stainless steel grid floor. We equipped the right panel of the chamber with a discriminative cue (white houselight, catalog #ENV-215m, Med Associates) that signaled the insertion and subsequent availability of the food-paired active (retractable) lever. We equipped the left panel of the chamber with a discriminative cue (red light, red lens, catalog #ENV-221m, Med Associates) that signaled the insertion and subsequent availability of the fentanyl-paired active (retractable) lever. We also equipped the right wall with an inactive (stationary) lever that had no reinforced consequences. We placed a bottle of water and a food hopper with standard laboratory chow on the transparent polycarbonate door of the chamber.
General procedure
The experiments generally consisted of three phases, food self-administration (6 sessions) and fentanyl self-administration (12 sessions), voluntary abstinence choice sessions (12 sessions), followed by a relapse test and for Exps. 2–3, a reacquisition test. We provide details of the phases below.
Food pellet self-administration
Before the first self-administration training session, we gave the rats a 1 h magazine training session which began with the presentation of the white house light, followed by the noncontingent delivery of 1 pellet every 3 min. We used 45 mg preferred or palatable food pellets described in our previous studies (12.7% fat, 66.7% carbohydrate, and 20.6% protein; catalog #1811155, TestDiet; Cifani et al., 2012; Pickens et al., 2012; Calu et al., 2014). We then trained the rats to lever press for food during six 1 h sessions that were separated by 10 min for six consecutive days. The sessions began with the presentation of the white house light, followed 10 s later by the insertion of the food-paired active lever (right panel). The white houselight remained on for the duration of the session and served as a discriminative cue for the palatable food. We trained the rats under a fixed-ratio-1 (FR1) reinforcement schedule, where one lever press resulted in the delivery of five 45 mg palatable food pellets and the presentation of a 20 s discrete tone cue (catalog #ENV-223AM, Med Associates), during which additional lever presses were not reinforced but still recorded. At the end of each 1 h session, the white houselight was turned off, and the active lever was retracted. To match the number of discrete cue presentations to that of fentanyl (see below), we limited the number of food-reinforced deliveries to 12 per hour. For two rats that were slow to acquire food self-administration, we restricted their homecage food intake to ∼85% of their daily intake overnight for one to two nights. After the rats increased their food-reinforced responding, they received free access to the homecage food for the rest of the experiment.
Fentanyl self-administration
We trained rats to self-administer fentanyl during six 1 h daily sessions that were separated by 10 min for 12 d. Fentanyl was infused at a dose of 2.5 µg/kg/infusion over 3.5 s (0.1 ml/infusion). Sessions began with the presentation of the red houselight for 10 s followed by the insertion of the fentanyl-paired active lever; the red houselight remained on for the duration of the session and served as a discriminative cue for fentanyl availability. We trained the rats under an FR1 reinforcement schedule, where one lever press resulted in the delivery of a drug infusion paired with the 20 s discrete white light cue above the fentanyl-paired active lever (white lens, catalog #ENV-221M, Med Associates). At the end of each 1 h session, the red houselight was turned off, and the active lever was retracted. To prevent overdose and decrease self-injurious biting and excessive grooming, we limited the number of infusions to 12 per hour. In addition, to decrease self-injurious biting, we provided Nylabones (Bio-Serv) in the homecage and in the operant chamber beginning with the first day of food self-administration and removed the Nylabones from the operant chamber for choice sessions and the relapse and reacquisition tests. For one rat that was slow to acquire fentanyl self-administration, we restricted its homecage food intake to ∼85% of its daily intake overnight for one to two nights. After this rat increased its fentanyl-reinforced responding, it received free access to the homecage food for the rest of the experiment.
Discrete choice-induced voluntary abstinence
We conducted 10–12 discrete choice sessions using the same parameters (dose of fentanyl, number of palatable food pellets per reinforcer delivery, stimuli associated with the two retractable levers) used during the training phases. We divided each 3 h choice session into 20 discrete trials that were separated by 9 min. Each trial began with the presentation of both discriminative cues previously associated with palatable food or fentanyl, followed 10 s later by the insertion of both the palatable food-paired and fentanyl-paired levers. Rats could then select one of the two levers. If the rats responded within 6 min, the reinforcer associated with the selected lever was delivered. Each reinforcer delivery was signaled by the fentanyl-associated or food-associated cue (white cue light or tone, respectively), retraction of both levers, and shutdown of the food and fentanyl discriminative cues. Thus, on a given trial, the rat could earn the drug or food reinforcer but not both. If a rat failed to respond on either active lever within 6 min, both levers were retracted, their related discriminative cues were turned off with no reinforcer delivery until onset of the next trial, and the trial was counted as an omission.
Relapse and reacquisition tests
The relapse test in the presence of the fentanyl-associated cues consisted of a single 90 min session on the day after the last voluntary abstinence session. The session began with the presentation of the red discriminative cue light, followed 10 s later by the insertion of the fentanyl-paired active lever; the red houselight remained on for the duration of the session. Active lever presses during testing resulted in contingent presentations of the light cue previously paired with fentanyl infusions but not an infusion of fentanyl. Based on our previous studies (Bossert et al., 2011; Li et al., 2015b; Caprioli et al., 2017; Venniro et al., 2017b) and the time course of Fos induction (Morgan and Curran, 1991), the relapse tests were 90 min. In Exps. 2–3, we tested the rats on reacquisition of fentanyl self-administration 1 d after the relapse test, using the same parameters as the fentanyl self-administration training.
Specific experiments
Exp, 1: Effect of relapse to fentanyl seeking on Fos expression in afferent projections to Pir (n = 11)
The goal of Exp. 1 was to characterize the circuitry of relapse to fentanyl seeking after food choice-induced voluntary abstinence by determining whether this relapse is associated with selective activation of one or more afferent projections into Pir. The experiment consisted of 3 phases (Fig. 1A), food and fentanyl self-administration training phase (3 weeks), choice-based voluntary abstinence phase (10 d), and a relapse test. We injected CTb into Pir 1 d after the last self-administration session and started the voluntary abstinence phase 3–4 d after these injections. We used two groups of rats (n = 5–6 per group; 3 males, 8 females) in an experimental design that included the between-subjects factor of Test Condition (No Test, Test). On test day, we brought the rats from the No Test group directly from their homecage and perfused them at the same time as the rats from the Test group.
Exp. 2: Effect of anatomical disconnection between AI and Pir on relapse to fentanyl seeking (n = 33)
The goal of Exp. 2 was to determine whether projections between AI and Pir play a causal role in relapse to fentanyl seeking. For this experiment, we used an anatomical disconnection procedure in which the role of a neuronal pathway or projection in a given behavior is inferred when behavior is disrupted by the contralateral but not by ipsilateral inactivation of two anatomically connected brain regions (Gaffan et al., 1993; Setlow et al., 2002).
The experiment consisted of 4 phases (Fig. 2A), food and fentanyl self-administration training phase (3 weeks), food choice-induced voluntary abstinence phase (12 sessions over 14 d), a relapse test, and a reacquisition test. We tested four groups of rats (n = 7–10 per group) using a two (Drug Condition, vehicle, muscimol plus baclofen) by two (Disconnection Manipulation, ipsilateral, contralateral injections) experimental design. Because we did not observe any differences in the vehicle ipsilateral and vehicle contralateral groups, we combined data from these two groups for a total of three groups, vehicle (n = 14, 7 males, 7 females), ipsilateral muscimol plus baclofen (n = 9, 4 males, 5 females), and contralateral muscimol plus baclofen (n = 10, 3 males, 7 females). We eliminated data from one vehicle group rat from the relapse test analysis because this rat was a statistical outlier according to the box plot generated with the descriptive statistics feature in IBM SPSS Statistics software. One rat became sick after the relapse test and did not undergo the reacquisition test.
Exp. 3: Effect of anatomical disconnection between PL and Pir on relapse to fentanyl seeking (n = 41)
The goal of Exp. 3 was to determine whether projections between PL and Pir play a causal role in relapse to fentanyl seeking. For this experiment, we again used the anatomical disconnection procedure. The experiment consisted of four phases (Fig. 3A), food and fentanyl self-administration training (3 weeks), food choice-induced voluntary abstinence (12 sessions over 14 d), a relapse test, and a reacquisition test. We tested four groups of rats (n = 7–13 per group) using a two (Drug Condition, vehicle, muscimol plus baclofen) by two (Disconnection Manipulation, ipsilateral, contralateral injections) experimental design. Because we did not observe any differences in the vehicle ipsilateral and vehicle contralateral groups, we combined data from these two groups for a total of three groups, vehicle (n = 15, 8 males, 7 females), ipsilateral muscimol plus baclofen (n = 13, 5 males, 8 females), and contralateral muscimol plus baclofen (n = 13, 8 males, 5 females). We eliminated data from three rats (two from the vehicle group and one from the contralateral muscimol plus baclofen group) from the relapse test analysis because these rats were statistical outliers according to the box plot generated with the descriptive statistics feature in IBM SPSS Statistics.
Exp. 4: Neuronal isolation and molecular phenotyping of Pir Fos-expressing neurons (n = 14)
In Exp. 4, we used FACS and qPCR to identify the molecular changes in Pir Fos-expressing neurons associated with relapse to fentanyl seeking after food choice-induced abstinence. The experiment consisted of three phases (Fig. 4A), food and fentanyl self-administration training (3 weeks), food choice-induced voluntary abstinence (12 sessions over 14 d), and a relapse test. We used one group of male and female rats (n = 14, 7 males, 7 females) in an experimental design that included the within-subjects factor of Lever (Active, Inactive). On abstinence day 15, the rats were exposed to the 90 min relapse test, and then we extracted all brains immediately after the relapse test. We processed Pir tissue for FACS and subsequent qPCR of selected genes, which were selected based in part on our previous FACS studies (Guez-Barber et al., 2011; Fanous et al., 2013; Rubio et al., 2015; Li et al., 2015b; Fredriksson et al., 2023).
Quantification and statistical analysis
Image acquisition and neuronal quantification
For cannula placement verification, we used a Dhyana 400DC (Tucsen) camera attached to a Zeiss Axioskop2 Plus microscope using Micro-Manager (Edelstein et al., 2014). For Exp. 1, we used an ORCA-Flash4.0 LT (Hamamatsu Photonics) attached to a Zeiss Axio Scope Imager M2 using Micro-Manager (Edelstein et al., 2014).
Exp. 1: Fos and CTb quantification
We captured immunofluorescent images for Fos and CTb immunoreactive cells in Pir using a 10× objective. We quantified cells in the same hemisphere as the CTb injection using the following bregma coordinates: AI, PL, IL (4.2–3.0 mm anterior to bregma) and BLA (1.0–2.5 mm posterior to bregma). We identified Fos-positive cells by a fluorescent stain in the nuclei and CTb-positive cells by a fluorescent stain in the cytoplasm. We semiautomatically quantified cells in the hemisphere ipsilateral to the injection site in four sections and computed the mean of these counts per area (i.e., each rat provides an individual data point for data analyses with data averaged from four images for each rat). We performed image-based quantification of Fos-positive, CTb-positive, and Fos plus CTb cells in ImageJ in a blind manner (inter-rater reliability between authors D.J.R. and S.M.C., r = 0.97, and between D.J.R. and S.B., r = 0.99).
Statistical analysis
We analyzed the data with repeated-measures or mixed-factorial ANOVAs using IBM SPSS Statistics (version 23, GLM procedure). We followed significant main effects and interactions (p < 0.05) with post hoc tests (univariate ANOVAs or Fisher's PLSD). We describe the different between- and within-subjects factors for the different statistical analyses below in Results. We only report significant effects that are critical for data interpretation as our multifactorial ANOVAs yielded multiple main and interaction effects. We indicate the results of post hoc analyses with asterisks in the figures but do not describe them in the text. Table 2 contains a complete report of the statistical analyses of the behavioral data. We analyzed the qPCR data with linear-mixed effects modeling (Yu et al., 2022) using JMP 16 software. Specifically, we used cell type (nominal, Fos-negative vs Fos-positive) as a fixed within-subject factor and sample number as a random factor. We corrected for multiple comparisons of the qPCR data using Benjamini–Hochberg's false discovery rate (FDR) correction method (implemented via the R p adjust function in IBM SPSS Statistics) and set the alpha significance level at 0.05, two tailed. Table 3 contains a complete report of the statistical analyses of the qPCR data and includes the uncorrected and FDR-adjusted (FDR-adj.) p values.
Results
Self-administration training and voluntary abstinence
In all experiments, the male and female rats reliably self-administered palatable food and fentanyl (Fig. 1B, 2B, 3B, and 4B) and strongly preferred palatable food over fentanyl during the abstinence sessions (Fig. 1C, 2C, 3C, and 4C). Table 2 contains a complete report of the statistical analyses. There were minimal or no sex differences in fentanyl self-administration, fentanyl versus food choice, and fentanyl relapse (data not shown).
Exp. 1: Effect of relapse to fentanyl seeking on Fos expression in afferent projections to Pir
The goal of Exp. 1 was to determine whether relapse to fentanyl seeking after food choice-induced voluntary abstinence is associated with selective activation of one or more afferent projections to Pir, identified using CTb as a retrograde tracer. The timeline of Exp. 1 is provided in Fig. 1A.
Relapse test
The number of lever presses on the active lever was greater than the number of lever presses on the inactive lever during relapse to fentanyl seeking (Fig. 1D, left). The repeated-measures ANOVA, which included the within-subjects factor of Lever (active, inactive), showed a significant effect of Lever (F(1,4) = 18.3, p = 0.01). For the time course of lever presses (Fig. 1D, right), the repeated-measures ANOVA, which included the within-subjects factors of Session Time (30, 60, 90 min) and Lever, showed a significant interaction between the two factors (F(2,8) = 11.9, p = 0.004).
Quantification for Fos and CTb
We quantified the number of cells in AI, PL, IL, and BLA that were Fos positive, CTb positive, and Fos plus CTb colabeled after the relapse test (Fig. 1F). The univariate ANOVA for each brain region included the between-subjects factor of Test Condition (Test, No Test). For the AI, the analysis showed a significant effect of Test Condition for Fos (F(1,9) = 6.8, p = 0.03) and Fos plus CTb colocalization (F(1,9) = 7.4, p = 0.02), but not CTb (F(1,9) = 1.3, p = 0.29). For the PL, the analysis showed a significant effect of Test Condition for Fos (F(1,9) = 6.6, p = 0.03) and Fos plus CTb colocalization (F(1,9) =11.0, p = 0.009), but not CTb (F(1,9) = 2.3, p = 0.16). For the IL, the analysis showed no significant effect of Test Condition for Fos (F(1,9) = 1.6, p = 0.24) or CTb (F(1,9) = 0.6, p = 0.47), but a significant effect for Fos plus CTb colocalization (F(1,9) = 6.0, p = 0.04). For the BLA, analysis showed no significant effect of Test Condition for Fos (F(1,9) = 0.7, p = 0.43), CTb (F(1,9) = 0.0, p = 0.86), or Fos plus CTb colocalization (F(1,9) = 0.1, p = 0.82).
Together, these data show that relapse to fentanyl seeking was associated with increased neuronal activity in AI and PL, and selective activation of AI→Pir and PL→Pir projections but not BLA→Pir projections. Fentanyl relapse was associated with selective activation of IL→Pir projections but not increased Fos expression in IL. Based on these data, in Exps. 2 and 3 we tested the causal role of AI→Pir and PL→Pir projections in fentanyl relapse.
Exp. 2: Effect of anatomical disconnection between AI and Pir on relapse to fentanyl seeking
In Exp. 1 we found that relapse to fentanyl seeking was associated with activation of neurons that project from AI to Pir. The goal of Exp. 2 was to determine whether this projection plays a causal role in relapse, using the anatomical disconnection procedure. As a control for the behavioral specificity of the anatomical disconnection procedure, we also tested the effect of this procedure on reacquisition of fentanyl self-administration in Exp. 2 and Exp. 3. We and other investigators have previously used reacquisition of drug self-administration after extinction as a relapse-related measure (Willcocks and McNally, 2013; Prasad and McNally, 2016; Khoo et al., 2017; Bossert et al., 2020; 2022), and in our previous study, we found that inactivation of Pir or the projections between Pir and OFC had no effect on reacquisition (Reiner et al., 2020). The timeline of Exp. 2 is provided in Fig. 2A.
Relapse test
Muscimol plus baclofen injections into AI and Pir decreased relapse to fentanyl seeking in the contralateral but not ipsilateral condition (Fig. 2D, left). The mixed ANOVA for total number of lever presses included the between-subjects factor of Drug Condition (vehicle, M + B ipsilateral, M + B contralateral) and the within-subjects factor of Lever. The analysis showed significant effects of Drug Condition (F(2,29) = 5.2, p = 0.01) and Lever (F(1,29) = 204.8, p < 0.001), and an interaction between the two factors (F(2,29) = 4.4, p = 0.02). For the time course of lever presses (Fig. 2D, right), the mixed ANOVA included the between-subjects factor of Drug Condition and the within-subjects factor of Session Time and Lever. The analysis showed significant effects of Drug Condition (F(2,29) = 5.2, p = 0.01), Session Time (F(2,58) = 85.6, p < 0.001), and Lever (F(1,29) = 204.8, p < 0.001), and interactions between Drug Condition and Lever (F(2,29) = 4.4, p = 0.02) and between Session Time and Lever (F(2,58) = 83.9, p < 0.001).
Reacquisition test
Muscimol plus baclofen injections into AI and Pir had no effect on reacquisition of fentanyl self-administration in both the ipsilateral and contralateral conditions (Fig. 2E). The mixed ANOVA included the between-subjects factor of Drug Condition and the within-subjects factor of Session Hour (1–6). The analysis showed a significant effect of Session Hour (F(5,145) = 10.7, p < 0.001), but not Drug Condition (F(2,29)=1.3, p = 0.28) or an interaction between the factors (F(10,145) = 0.6, p = 0.79).
Together, these data show that AI→Pir projections contribute to relapse to fentanyl seeking, but not reacquisition of fentanyl self-administration.
Exp. 3: Effect of anatomical disconnection between PL and Pir on relapse to fentanyl seeking
In Exp. 1 we found that relapse to fentanyl seeking was associated with activation of neurons that project from PL to Pir. The goal of Exp. 3 was to determine whether this projection plays a causal role in relapse to fentanyl seeking and reacquisition of fentanyl self-administration, again using the anatomical disconnection procedure. The timeline of Exp. 3 is provided in Fig. 3A.
Relapse test
Muscimol plus baclofen injections into PL and Pir had no effect on relapse to fentanyl seeking in the ipsilateral or contralateral conditions (Fig. 3D, left). The mixed ANOVA for total number of lever presses included the between-subjects factor of Drug Condition (vehicle, M + B ipsilateral, M + B contralateral) and the within-subjects factor of Lever. The analysis showed a significant effect of Lever (F(1,35) = 182.4, p < 0.001), but no significant effect of Drug Condition (F(2,35) = 0.2, p = 0.85) or an interaction between the two factors (F(2,35) = 0.0, p = 0.97). For the time course of lever presses (Fig. 3D, right), the mixed ANOVA included the between-subjects factor of Drug Condition and the within-subjects factors of Session Time and Lever. The analysis showed significant effects of Session Time (F(2,70) = 103.6, p < 0.001), Lever (F(1,35) = 182.4, p < 0.001), and an interaction between these two factors (F(2,70) = 96.3, p < 0.001). There was no significant effect of Drug Condition (F(2,35) = 0.2, p = 0.85) or interactions between Drug Condition and any of the other factors (p values > 0.05).
Reacquisition test
Muscimol plus baclofen injections into PL and Pir modestly decreased reacquisition of fentanyl self-administration in the contralateral but not ipsilateral condition (Fig. 3E). The mixed ANOVA included the between-subjects factor of Drug Condition and the within-subjects factor of Session Hour (1–6). The analysis showed significant effects of Session Hour (F(5,190) = 5.9, p < 0.001) and Drug Condition (F(2,38) = 3.5, p = 0.04), but no interaction between the two factors (F(10,190) = 1.3, p = 0.23).
Together, these data show that unexpectedly, PL→Pir projections may contribute to reacquisition of fentanyl self-administration but not relapse to fentanyl seeking.
Exp. 4: Neuronal isolation and molecular phenotyping of Pir Fos-expressing neurons
We previously found that fentanyl relapse is associated with increased Fos expression in Pir, and inactivation of Pir decreases fentanyl relapse (Reiner et al., 2020). The goal of Exp. 4 was to determine the molecular phenotype of Pir Fos-expressing neurons that are associated with fentanyl relapse using FACS and qPCR.
Relapse test
During the day 15 relapse test, the number of active lever presses was greater than the number of inactive lever presses (Fig. 4D). The repeated-measures ANOVA, which included the within-subjects factor of Lever (active, inactive), showed a significant effect of Lever (F(1,13) = 47.9, p < 0.001).
FACS
We isolated Fos-positive and Fos-negative Pir neurons by labeling the neurons with NeuN and Fos antibodies and sorted them as previously described (Guez-Barber et al., 2011, 2012; Liu et al., 2014; Rubio et al., 2015, 2016; Li et al., 2015b; Fredriksson et al., 2023). We first identified cells from debris based on the distinct forward and side scatter properties in the Cells gate (Fig. 4E, left). From the Cells gate, we then identified single cells in the Singlets gate (Fig. 4E, middle). From the Singlets gate, we identified activated neurons based on immunoreactivity for Fos and NeuN, thus sorting activated neurons (Fos-positive/NeuN-positive events) from nonactivated neurons (Fos-negative/NeuN-positive events; Fig. 4E, right, representative example of Fos-negative and Fos-positive gating from one sample).
qPCR
We used qPCR in our sorted neuronal samples to assess expression of molecular markers associated with neuronal activity (immediate early genes), neurotransmission (e.g., acetylcholine, epinephrine/norepinephrine, GABA, glutamate, and dopamine), epigenetic markers, or neuropeptides in Fos-positive and Fos-negative Pir neurons. We corrected for multiple comparisons using Benjamini–Hochberg's FDR correction method and set the alpha significance level at 0.05, two tailed. We report statistical analyses of the qPCR data below after FDR correction and show fold change and the uncorrected and FDR-adjusted p values in Table 3. We show the qPCR data in a heat map (Fig. 4F) and volcano plot (Fig. 4G) to summarize the main findings.
We detected significantly increased expression of the immediate early genes Arc, Egr1, FosB total, and BDNF exon IV (FDR-adj. p values < 0.05), increased expression of Fos that approaches significance (F(1,5.25) = 11.7, FDR-adj. p = 0.09), but not Homer 1 (F(1,5.13) = 1.2, FDR-adj. p = 0.41) in Fos-positive neurons compared with Fos-negative neurons (Fig. 4F). For adrenoreceptor- and acetylcholine receptor-related genes, we detected similar expression of Adra1a, Adra2a, Adrb1, Adrb2, Chrna4, and Chrm1,3-4 in Fos-negative and Fos-positive neurons (FDR-adj. p values > 0.3).
For BDNF-related genes, we detected significant increased expression of Trkb1 (full length; F(1,5.05) = 54.6, FDR-adj. p = 0.02), increased expression of Trkb2 (truncated isoform) that approaches significance (F(1,5.52) = 10.4, FDR-adj. p = 0.08), but not BDNF full coverage (F(1,5.18) = 4.5, FDR-adj. p = 0.22) in Fos-positive neurons. For dopamine-receptor-related genes, we detected similar expression of Drd1 and Drd2 in Fos-positive neurons (FDR-adj. p values > 0.2). For epigenetic-related genes, we detected similar expression of Dnmt3a, Hdac4, and Hdac5 in Fos-negative and Fos-positive neurons (FDR-adj. p values > 0.1).
For GABA-related genes, we detected increased expression of Gabra1 (F(1,4.32) = 52.9, FDR-adj. p = 0.02) in Fos-positive neurons and similar expression of Gabra5, Gabbr2, Gabrb3, and Gabrg2 (FDR-adj. p values > 0.2). For glutamate-related genes, we detected significant increased expression of Grin2a (F(1,5.09) = 19.5, FDR-adj. p = 0.05), increased expression of Gria4 that approaches significance (F(1,4.82) = 17.1, FDR-adj. p = 0.06), and similar expression of Gria1-2, Grin1a, Grin2b, and Grm1-5 (FDR-adj. p values > 0.1) in Fos-negative and Fos-positive neurons.
For neuropeptide-related genes, we detected similar expression of Cartpt, Cck, Cnr1, Npy, and Sst (FDR-adj. p values > 0.1) in Fos-negative and Fos-positive neurons. For opioid-related genes, we detected similar expression of Opcml, Oprd1, Oprk1, Pdyn, and Penk in Fos-negative and Fos-positive neurons (FDR-adj. p values > 0.2). We were not able to reliably detect Oprm1, Pvalb, Gabra3, Chrna2, and Chrna5 in Fos-positive neurons but could detect these genes in Fos-negative neurons.
These results indicate that relapse-activated Pir neurons are composed of cells with a diverse molecular phenotype, and differential changes in Fos-positive neurons might be because of a different proportion of cell types between Fos-negative and Fos-positive neurons and/or differential gene expression.
In summary, we found increased expression of immediate early genes in Fos-expressing Pir neurons, validating the FACS procedure. Additionally, fentanyl relapse was associated with statistically significant increased expression of Trkb1, Gabra1, and Grin2a (FDR-adj. p values < 0.05) and increased expression of Trkb2 (FDR-adj. p = 0.08) and Gria4 (FDR-adj. p = 0.06) in Pir Fos-positive neurons that approaches significance.
Discussion
We previously reported that Pir contributes to fentanyl relapse after food choice-induced voluntary abstinence (Reiner et al., 2020). Here, we determined the role of afferent projections to Pir in this relapse. We found that fentanyl relapse was associated with increased Fos expression in AI and PL and selective activation of AI and PL neurons that project to Pir (assessed by Fos plus CTb colocalization). Disconnection of AI and Pir decreased fentanyl relapse but not reacquisition of fentanyl self-administration. In contrast, disconnection of PL and Pir modestly decreased reacquisition but not relapse. Our FACS and qPCR data showed similarities and differences in the gene-expression pattern of a small population of relapse test-associated Fos-expressing Pir neurons versus Fos-negative neurons (which represent most Pir neurons).
Selective effect of anatomical disconnection of AI and Pir on fentanyl relapse
Our data showed that fentanyl relapse is associated with activation of AI→Pir projections. Based on these data, we hypothesized that disconnection of this projection would decrease relapse. Our hypothesis was also based on our previous studies showing that AI inactivation decreased relapse to fentanyl (Reiner et al., 2020) and methamphetamine (Venniro et al., 2017b) seeking after food choice-induced abstinence. We found that contralateral but not ipsilateral disconnection of AI and Pir decreased fentanyl relapse but not reacquisition, indicating a causal role of AI→Pir projections in relapse. In contrast and in agreement with our previous study (Reiner et al., 2020), disconnection of AI→Pir projections had no effect on reacquisition.
Previous studies indicate that the AI is critical for cue-induced reinstatement of cocaine and nicotine seeking after extinction (Di Pietro et al., 2006; Cosme et al., 2015; Pushparaj et al., 2015) and relapse to alcohol and nicotine seeking after punishment-induced abstinence (Campbell et al., 2019; Ghareh et al., 2022). These previous studies and our current results indicate a role of AI and its downstream projections in relapse across drug classes. Additionally, the role of AI and its projections in relapse to nonreinforced drug seeking appears independent of the method used to achieve abstinence (extinction, choice of nondrug alternative reward, punishment).
Selective effect of anatomical disconnection of PL and Pir on reacquisition of fentanyl self-administration
Our data showed that fentanyl relapse is associated with activation of PL→Pir projections. Based on these data, we hypothesized that disconnection of this projection would decrease nonreinforced fentanyl relapse, and as with the AI→Pir projection, that the disconnection would have no effect on reacquisition. We also based our hypothesis on a large literature on the role of PL in reinstatement of drug seeking across drug classes and reinstating stimuli (cue, stress, drug priming) and incubation of drug craving (Kalivas and McFarland, 2003; Peters et al., 2009; Bossert et al., 2013; Wolf, 2016; Dong et al., 2017; Khoo et al., 2017; Szumlinski and Shin, 2018).
Unexpectedly, we found that disconnection of PL→Pir projections had no effect on nonreinforced relapse to fentanyl seeking but modestly decreased reacquisition. Interpretation of the negative data for nonreinforced relapse is straightforward; to the degree that the disconnection manipulation inhibited activity of PL→Pir projections, relapse-associated activity of these projections does not contribute to fentanyl seeking during relapse testing. In contrast, the interpretation of the modest inhibitory effect of the disconnection manipulation on reacquisition is not straightforward because we only measured reacquisition using a single fentanyl unit dose located on either the descending arm (Martin and Ewan, 2008) or at the peak (Wade et al., 2015) of the dose–response curve. Consequently, without a full dose–response determination, decreased fentanyl self-administration can be because of either an increase or decrease in the reinforcing effects of fentanyl (Yokel, 1987; Piazza et al., 2000).
Our unexpected findings on the potential dissociable roles of AI→Pir versus PL→Pir in nonreinforced relapse to fentanyl seeking versus reinforced reacquisition of fentanyl self-administration have implications for preclinical and clinical research on brain circuits of relapse. One implication is that our findings support results from studies showing that mechanisms of reinforced drug self-administration versus nonreinforced drug reinstatement and relapse are often dissociable (Shalev et al., 2002; Everitt and Robbins, 2005; Kalivas et al., 2009; Venniro et al., 2020). Thus, it is important to incorporate measures of relapse that go beyond classical measures of nonreinforced drug seeking and include drug-taking measures (Willcocks and McNally, 2013; Khoo et al., 2017; Reiner et al., 2019; Bossert et al., 2022).
Another implication of the potential double dissociation in Pir-related mechanisms of relapse to fentanyl seeking versus reacquisition, and other examples discussed previously (Gibson et al., 2018; Fredriksson et al., 2021), is that they do not support the notion of a unitary relapse-related brain circuit. Instead, our data and other examples described in the references above, suggest that different circuits contribute to relapse based on the relapse-provoking stimulus, the method to achieve abstinence, the abstinence period, and the addictive drug (Badiani et al., 2011; Bossert et al., 2013; Marchant et al., 2019). A question for future research is the extent to which our Pir-related findings in the rat translate to humans and whether our findings generalize to other opioid drugs like heroin.
Finally, the notion that Pir contributes to fentanyl relapse is puzzling because of the primary role of this region in olfaction. We speculate that Pir may receive interoceptive signals from AI and communicate with higher-order cognitive processing areas, such as OFC and PL, to contribute to drug seeking and taking.
Gene-expression changes in Pir associated with fentanyl relapse
We used FACS and qPCR to compare gene-expression profiles of Fos-expressing neurons versus Fos-negative neurons. We identified expected increases in expression of immediate early genes (Arc, Egr1, and FosB and BDNF exon IV), which are rapidly induced by different stimuli. [Note: After FDR-correction, we detected increased expression of Fos that approached significance (FDR-adj. p = 0.09), likely because the 90 min brain dissection time point occurs after the peak time of Fos mRNA expression (∼15–30 min; Morgan and Curran, 1991)]. We also identified three constitutive genes—Grin2a, Trkb1, and Gabra1—whose expression was significantly higher in the Fos-positive versus Fos-negative neurons after FDR-correction (Table 3).
Our data suggest that the gene expression profile of the Fos-positive neurons, hypothesized to be part of behaviorally encoding neuronal ensembles (Cruz et al., 2013), is different from Fos-negative neurons. These data can be interpreted in the following different ways: (1) Fentanyl relapse is associated with an increased number of cells expressing these genes and/or (2) fentanyl relapse is associated with increased expression of these genes. Two questions for future research are (1) whether the differentially expressed immediate early and constitutive genes in the Fos-positive neurons are critical for fentanyl relapse, and (2) whether the gene expression changes are because of fentanyl self-administration, abstinence, the relapse test, or some combinations of these experiences.
Methodological considerations
Several methodological considerations are relevant to our study. First, we based our disconnection experiments on the Fos and CTb quantification data showing an association between fentanyl relapse and increased Fos plus CTb colocalization. Our data showed that Fos-positive cells coexpressing CTb are ∼5% in AI and ∼2% in PL. This small number of activated Pir-projecting neurons is consistent with the neuronal ensemble hypothesis, which proposes that learned behaviors are controlled by a small percentage of activated neurons (Hebb, 1949). Of note, to avoid spread to nearby regions, we limited the CTb injections within Pir. Therefore, the spread of CTb is ∼20–30% of Pir, and the number of activated afferent neurons identified by Fos-CTb double-labeling (Fig. 1F) is likely an underestimate.
Second, interpretation of the anatomical disconnection procedure relies on the assumption that projections are primarily ipsilateral. We observed minimal CTb labeling in contralateral AI or PL, indicating that their projections to Pir are primarily ipsilateral with minimal contralateral retrograde labeling. Additionally, we cannot infer directionality with the disconnection procedure, and previous studies have demonstrated Pir efferent projections to AI or PL (Chen et al., 2014; Bedwell et al., 2015; Diodato et al., 2016). Therefore, we cannot rule out that the projections in this direction contributed to the effects on fentanyl relapse or reacquisition.
Third, based on in vivo microdialysis studies measuring dopamine and its concentrations of metabolites in prefrontal cortex or nucleus accumbens after muscimol or baclofen infusions into ventral tegmental area, the inactivation time course should be at least 2 h (Yoshida et al., 1994; Westerink et al., 1998). This time course does not affect interpretation of our 90 min relapse tests but may confound interpretation of our 6 h reacquisition tests. Finally, our manipulations only partially decreased relapse (∼35%) or reacquisition (∼25%), indicating contributions of other regions and circuits to these behaviors.
Conclusions
We combined retrograde tracing, Fos immunohistochemistry, and anatomical disconnection to demonstrate a role of AI→Pir projections in relapse to fentanyl seeking after food choice-induced abstinence. Our results also suggest a potential modest role of PL→Pir projections in reacquisition of fentanyl self-administration. Additionally, we used FACS and qPCR to identify selective gene expression changes in Pir neurons activated during fentanyl relapse. Our results extend our previous results on the role of Pir in fentanyl relapse after voluntary abstinence (Reiner et al., 2020) and highlight the importance of testing the role of brain regions and their projections on both drug seeking and drug taking.
Footnotes
This work was supported by National Institutes of Health (NIH)–National Institute of General Medical Sciences Grants 1FI2GM128603 (D.J.R.), 1FI2GM142476 (J.J.C.), and NIH–National Institute on Drug Abuse Grants K99DA053211-01A1, 1ZIADA000467-18 (B.T.H.), 1ZIADA000434-22 (Y.S.), and 3R25DA051338. Support for a student internship (SB) was provided in part by an NIH-NIGMS Building Infrastructure Leading to Diversity (BUILD) Initiative Grant (5TL4GM118989) and the NIDA EDUCATE UMBC Research Training Program (3R25DA051338). We thank Dr. Katherine Savell for assistance in operating the BD FACSMelody Cell Sorter, Kayla Pitts for assistance in behavioral experiments, and Dr. Rajtarun Madangopal for assistance with ImageJ software and presentation and analyses of the qPCR data.
The authors declare no competing financial interests.
- Correspondence should be addressed to David J. Reiner at david.reiner{at}nih.gov or Yavin Shaham at Yshaham{at}intra.nida.nih.gov