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. Author manuscript; available in PMC: 2022 Nov 1.
Published in final edited form as: Drug Alcohol Depend. 2021 Sep 2;228:109033. doi: 10.1016/j.drugalcdep.2021.109033

An opposing role for prelimbic cortical projections to the nucleus accumbens core in incubation of craving for cocaine versus water

Travis M Moschak 1,2, Regina M Carelli 1
PMCID: PMC8595637  NIHMSID: NIHMS1739731  PMID: 34500244

Abstract

Background:

Both drug and natural reward-seeking have been shown to increase following an extended period of abstinence, a phenomenon termed ‘incubation of craving’. Although this phenomenon involves many brain regions, the projections from the prelimbic cortex (PrL) to the nucleus accumbens (NAc) core have been strongly implicated in incubation of cocaine-seeking. However, this circuit has not been investigated in the context of incubation of craving for natural rewards.

Methods:

Male Long Evans rats were trained to self-administer cocaine or water/saline 6 hr/d for 14 days and subsequently entered 1 month of experimenter-imposed abstinence. Rats then underwent an optogenetic stimulation protocol used to induce long term depression in the PrL terminals to the NAc core immediately before beginning an extinction test used to assess incubation of craving.

Results:

Control cocaine rats showed heightened drug-seeking on day 30 when compared to day 1 of abstinence, demonstrating incubation of craving. Notably, optogenetic stimulation of the PrL to NAc core pathway blocked this behavior in cocaine rats. In contrast, optogenetic stimulation of the PrL to NAc core pathway induced incubation of craving in water/saline rats.

Conclusions:

These findings suggest that neuroadaptations in the PrL to NAc core pathway play opposing roles in the incubation of craving for cocaine versus water.

Keywords: cocaine, abstinence, prelimbic, nucleus accumbens, rats, craving

1. Introduction

Drug-seeking behavior increases following extended abstinence, a phenomenon termed ‘incubation of craving’ (Pickens et al., 2011). This incubation is associated with increased synaptic plasticity in several brain regions, including the nucleus accumbens (NAc) core. For example, neural activity in the NAc core in response to drug cues is heightened following extended abstinence from cocaine (Hollander & Carelli, 2005, 2007). Additionally, extended cocaine abstinence is accompanied by an increased AMPA/NMDA ratio (Kourrich et al., 2007) and incubation of cocaine craving is mediated by an upregulation of calcium-permeable AMPA receptors in the NAc core (Conrad et al., 2008).

Incubation of craving has also been shown for natural rewards (Grimm, 2020; Grimm et al., 2012; Dingess et al., 2017; Counotte et al., 2014; Roura-Marinez et al., 2019; although see Jones et al., 2008). The molecular underpinnings of this phenomenon appear partially shared, yet distinct from cocaine. For example, incubation of natural reward craving also results in an increased AMPA/NMDA ratio driven by an upregulation of calcium-permeable AMPA receptors (Dingess et al., 2017; but see Counotte et al., 2014). However, extended abstinence from cocaine and natural rewards both yield distinct neuroadaptations in the NAc core not shared by the other (cocaine: increased BDNF, Grimm et al., 2003; sucrose: decreased L-aspartate, Roura-Martinez et al., 2019).

Importantly, the NAc core does not act in isolation, and several projections to this region play a role in incubation of craving (Wolf, 2016). One such projection, the prelimbic cortex (PrL), shows heightened reactivity to cocaine cues following extended abstinence (West et al., 2014), and glutamatergic activity within the PrL is required for incubation of cocaine craving (Shin et al., 2018). Furthermore, inducing long-term depression (LTD) in PrL projections to the NAc core blocks the incubation of cocaine-craving (Ma et al., 2014). However, a comparison of the role of this circuit in the incubation of craving for cocaine versus natural rewards has yet to be investigated. To this end, we investigated the effect of an optogenetic LTD-induction protocol on incubation of craving in animals trained to self-administer either cocaine or water.

2. Material and methods

2.1. Subjects

Twenty-six, male Long Evans rats (Cocaine ChR2: n=6, Cocaine mCherry: n=6, Water/Saline ChR2: n=7, Water/Saline mCherry: n=7, Charles River, Raleigh, NC) initially aged 8 −10 weeks and weighing 300–325 g were housed in an animal vivarium at the University of North Carolina at Chapel Hill with a 12h/12h reverse light cycle (lights on at 8 PM). All procedures were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and in accordance with the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee.

2.2. Apparatus

Behavioral data were collected using 8 behavioral chambers (43 × 43 × 53 cm) housed in sound-attenuating cubicles (Med Associates, St. Albans, VT). One wall of the chamber contained a nosepoke device with a houselight and speakers for tones and white noise mounted above it. On the opposite wall was a food/water receptacle flanked by two retractable levers with cue lights mounted above them. Cocaine or saline infusion was driven by a motorized pump (Med Associates) through tubing that was fed through a counterweighted arm to provide mobility to the animal. Operant chamber input and output was controlled by MED-PC (Med Associates).

2.3. Surgery

For all surgeries, rats were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine (i.p.). Initially, rats were infused with 600 nl of channelrhodpsin (AAV5-CamKIIa-hchR2(h134R)-mCherry) or mCherry control (AAV5-CamKIIa-mCherry) virus under the CAMKIIa promotor at a concentration of 3.2 × 1012 viral genomes/ml bilaterally into the PrL (+2.7 mm AP, ±0.6 mm ML, −3.8 mm DV from bregma). Four weeks later, rats were implanted with a jugular catheter and bilateral optical fibers (200 μm diameter, 0.22 numerical aperture) in the NAc core (+1.5 mm AP, ±2.5 mm ML, −6.9 mm DV from Bregma, lowered at 10° angle). Rats were given a week of recovery and virus was given 8 weeks to express before optical manipulations.

2.4. Self-administration

A timeline of the experiment is shown in Figure 1A. In the self-administration task, a nosepoke led to either an intravenous 0.2 ml infusion of cocaine (0.33 mg/infusion) or an equivalent volume of both saline (i.v) and water (delivered to the water receptacle) coupled with a 30 s tone/houselight compound stimulus. Nosepokes during this 30 s period did not result in additional reinforcement. Animals self-administered 6 hr/day for 14 days. In addition to self-administration, animals trained in a contextually distinct set of operant behaviors for sucrose pellet reward for an unrelated experiment (see Moschak et al., 2018 for details on these procedures). Rats were water restricted (cocaine rats: ~30 ml/day; water/saline rats: ~20 ml/day) during self-administration and extinction procedures.

Figure 1.

Figure 1.

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A. Experimental Design. Rats self-administered cocaine or water/saline for 6 hr/day for 14 days before entering abstinence. On day 1 of abstinence, rats were given a 2 hr extinction test. On day 30 abstinence, rats were given 10 min of 1 Hz stimulation followed by a 2 hr extinction test. B. Cocaine self-administration. Rats significantly escalated cocaine intake across the 14 days of drug access. C. Water/saline intake. Rats significantly decrease water/saline intake initially, but stabilized intake thereafter.

2.5. Extinction

Following 14 days of self-administration, animals underwent 30 days of experimenter-imposed abstinence. On day 1 and day 30 of abstinence, animals had a 1 hour extinction test. This test was identical to the self-administration task above, but no reinforcers were delivered.

2.6. Optogenetics

On day 30 of abstinence, animals were given optical stimulation of the NAc core fibers in their homecages for 10 min (10mW, 473 λ laser at a frequency of 1 Hz), which has been shown to induce LTD in the PrL to NAc core pathway (Ma et al., 2014). Immediately thereafter, animals began the day 30 extinction session.

2.7. Data Analysis

A one-way ANOVA (Day) was used to analyze reinforcement across the 14 days of self-administration. A 2 × 2 × 2 ANOVA (Reinforcer x Virus x Abstinence) was used to assess the effect of optogenetic stimulation on extinction. All individual comparisons were done with Bonferonni corrections.

2.8. Histology

Upon completing the experiment, animals were anesthetized and perfused with phosphate buffered saline followed by 4% paraformaldehyde (Millipore Sigma, St. Louis, MO). Brains were removed and stored in 4% paraformaldehyde for 24 hrs followed by 20% sucrose in 0.1 M phosphate buffer. Brains were then sectioned, mounted on slides, and imaged with a Leica 217 DFC 450C microscope. Virus expression and optical fiber placement were mapped on to an atlas (Paxinos & Watson, 2007).

3. Results

3.1. Self-Administration

Consistent with previous studies (Ahmed & Koob, 1998), 6hr access to cocaine resulted in a significant escalation of intake over time (F(13,143) = 3.54, p < 0.001; Fig. 1B, left). Conversely, animals decreased water/saline intake initially, but stabilized responding thereafter (F(13,169) = 4.39, p < 0.001; Fig. 1B, right).

3.2. Extinction

An ‘incubation of craving’ effect was observed in control cocaine rats, whereby rats increased drug-seeking following extended abstinence (Cocaine: Virus x Abstinence interaction: F(1,10) = 8.11, p = 0.017; Bonferonni post hocs showed that in mCherry Cocaine rats, Day 30 nosepokes were significantly higher than Day 1; Fig. 2A left). Furthermore, this was blocked by 10 min of 1 Hz stimulation of PrL terminals in the NAc core (Cocaine: Virus x Abstinence interaction: F(1,10) = 8.11, p = 0.017; Bonferonni post hocs showed that Cocaine ChR2 rats had significantly lower nosepokes on Day 30 than Cocaine mCherry rats Figs. 2A right, 2B), replicating previous work by Ma et al. (2014).

Figure 2.

Figure 2.

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A. Cocaine seeking behavior. Following 30 days of abstinence, cocaine mCherry rats significantly increased extinction nosepokes (left). This effect was blocked following optogenetic stimulation in cocaine ChR2 rats (right). * p < .05 comparing D1 mCherry to D30 mCherry. # p < .05 comparing D30 mCherry to D30 ChR2. B. Comparison of mCherry and ChR2 cocaine rats’ extinction nosepokes over the course of 1 hr. * p < 0.05. C. Water/saline seeking behavior. Following 30 days of abstinence, water/saline mCherry rats did not increase extinction nosepokes (left). However, optogenetic stimulation in water/saline ChR2 rats resulted in significantly increased extinction nosepokes after 30 days (right). # p < .05 comparing D30 mCherry to D30 ChR2. D. Comparison of mCherry and ChR2 water rats’ extinction nosepokes over the course of 1 hr. * p < 0.05 E. Sample image of cell body fluorescence in the PrL. F. Sample image of terminal fluorescence in the NAc core. G. Location of optical fibers in the NAc core. ○ = Cocaine, mCherry; ● = Cocaine, ChR2; □ = Water/saline, mCherry; ■ = Water/saline, ChR2

However, we found the opposite effect in water/saline rats (Reinforcer x Virus X Abstinence interaction: F(1,22) = 15.40, p = 0.001). Here, there was no ‘incubation of craving’ in control water/saline rats (Bonferonni post hocs found no differences between Day 1 and Day 30 nosepokes in Water/Saline mCherry rats; Fig. 2C left). However, 10 min of 1 Hz stimulation of PrL terminals in the NAc core significantly increased extinction responding in water/saline rats (Water/Saline: Virus x Abstinence interaction: F(1,12) = 6.86, p = 0.022; Bonferonni post hocs showed that in ChR2 Water/Saline rats, Day 30 nosepokes were significantly higher than Day 1; Figs. 2C right, 2D).

3.3. Histology

Virus expression was observed in the PrL (Fig. 2E) and NAc Core (Fig. 2F). All fibers were in the NAc core (Fig. 2G).

4. Discussion

Here, we demonstrate that the PrL to NAc core pathway plays an opposing role in enhanced extinction responding following an extended period of abstinence, termed ‘incubation of craving’, for cocaine versus water reinforcers. Specifically, we found that an optogenetic LTD-induction protocol in the PrL to NAc core circuit blocked enhanced extinction responding following prolonged abstinence from cocaine self-administration, replicating prior work (Ma et al., 2014). However, while we did not observe an incubation of craving for water reward, we observed that the same optogenetic protocol in the PrL to NAc core circuit increased extinction responding following abstinence in water/saline animals. Together, this suggests that neuroadaptations in the PrL to NAc core circuit following extended abstinence promote incubation of cocaine craving but may suppress incubation of water craving. However, while we believe our findings are suggestive of neural adaptations in the PrL to NAc core circuit following abstinence from water, additional work directly measuring neural adaptations is required to establish this possibility.

The fact that optogenetic stimulation selectively enhanced extinction responding for water after abstinence suggests that abstinence-induced neural adaptations in the PrL to NAc core circuit may act to suppress incubation of water craving. This is similar to findings in animals with a history of cocaine following LTD induction in the infralimbic to NAc shell circuit (Ma et al., 2014). There, LTD induction also enhanced incubation of cocaine craving, which the authors took as evidence that the neuroadaptations in the infralimbic to NAc shell circuit developed to suppress incubation of cocaine craving. However, our findings suggest that neuroadaptations in the same circuit (PrL to NAc core) following extended abstinence may yield opposing results depending on the type of reward.

These opposing effects on cocaine and water/saline ‘incubation of craving’ may be the result of differences in the neural adaptations in this circuit. Abstinence from both cocaine and natural rewards yield some similar neuroadaptations in the NAc core. Both result in an increased AMPA/NMDA ratio (Kourrich et al., 2007; Dingess et al., 2017; but see Counotte et al., 2014) driven by an upregulation of calcium-permeable AMPA receptors (Conrad et al., 2008; Dingess et al., 2017). However, there are also several differences in neuroadaptations following cocaine or natural reward abstinence. Heightened BDNF in the NAc core and decreased 5-HT2C receptors in the prefrontal cortex are both seen following extended abstinence from cocaine, but not sucrose (Grimm et al., 2003; Swinford-Jackson et al., 2016). Additionally, gene expression in PrL projections to the NAc core was weakened following extended abstinence from sucrose, but not cocaine (Roura-Martinez et al., 2019). These studies demonstrate that different neuroadaptations occur in the PrL and NAc core following extended abstinence from cocaine or natural reward, which may be sufficient to drive the opposing behavioral effects.

The opposing behavioral effects following identical LTD induction protocols may be related to modulation of distinct neuronal ‘ensembles’ in the NAc core. Specifically, discrete subcircuits exist in the NAc that selectively process drug versus ‘natural’ reward (Carelli et al., 2000; Carelli & Wondolowski, 2003; Carelli & Ijames, 2001). These findings are consistent with the view that the NAc is composed of neuronal ‘ensembles’ or groups of cells with different functional properties (Pennartz et al., 1994; Carelli and Wightman, 2004). A particular ensemble is temporarily and synchronously activated by a specific set of excitatory inputs (e.g. from the PrL) that in turn can activate distinct outputs and influence behavior. Thus, the same mechanism of neuroadaptation could result in opposing downstream effect driven by distinct neural ensembles.

In conclusion, our findings suggest that neuroadaptations in the PrL to NAc core pathway may play opposing roles in incubation of craving for either cocaine or water. Future studies are needed to further investigate the similarities and differences in neuroadaptations in this circuit following abstinence from both drugs of abuse and natural rewards, as well as how generalizable the findings with water are to other natural rewards. Finally, it is important to determine if a similar oppositional role applies to other relevant circuits, such as the infralimbic to NAc shell pathway and the basolateral amygdala to NAc core circuit, both implicated in reward seeking behaviors.

Highlights.

  • Drug and natural reward-seeking show ‘incubation of craving’

  • Prelimbic cortex (PrL)-nucleus accumbens (NAc) core linked to this process

  • Here, we used optogenetics to investigate role of PrL-NAc core pathway

  • Optogenetic stimulation had opposing roles on incubated cocaine/water seeking

Acknowledgments

Funding for this research was provided by the National Institute of Health/National Institute on Drug Abuse DA034021 to R.M.C. and DA045764 to T.M.M. Funding sources had no role in study-design; data collection, analysis, or interpretation; writing; or decision to submit article for publication.

Role of Funding Source

Nothing declared

Footnotes

Declaration of Competing Interest

None

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