Cocaine and Chronic Stress Exposure Produce an Additive Increase in Neuronal Activity in the Basolateral Amygdala

Abstract

Cocaine addiction is a chronic, relapsing disorder. Stress and cues related to cocaine are two common relapse triggers. We have recently shown that exposure to repeated restraint stress during early withdrawal accelerates the time-dependent intensification or “incubation” of cue-induced cocaine craving that occurs during the first month of withdrawal, although craving ultimately plateaus at the same level observed in controls. These data indicate that chronic stress exposure during early withdrawal may result in increased vulnerability to cue-induced relapse during this period. Previous studies have shown that chronic stress exposure in drug-naïve rats increases neuronal activity in the basolateral amygdala (BLA), a region critical for behavioral responses to stress. Given that glutamatergic projections from the BLA to the nucleus accumbens are critical for the incubation of cue-induced cocaine craving, we hypothesized that cocaine withdrawal and chronic stress exposure produce separate increases that additively increase BLA neuronal activity. To assess this, we conducted in vivo extracellular single-unit recordings from the BLA of anesthetized adult male rats following cocaine or saline self-administration (6 h/day for 10 days) and repeated restraint stress or control conditions on withdrawal days (WD) 6–14. Recordings were conducted from WD15 to WD20. Interestingly, cocaine exposure alone increased the spontaneous firing rate in the BLA to levels observed following chronic stress exposure in drug-naïve rats. Chronic stress exposure during cocaine withdrawal further increased firing rate. These studies may identify a potential mechanism by which both cocaine and chronic stress exposure drive cue-induced relapse vulnerability during abstinence.

INTRODUCTION

Cocaine addiction continues to pose a major public health concern, and one of the challenges for treatment is the propensity for abstinent users to relapse. Stress is one of the most common reasons addicts cite for relapsing and studies in humans suggest a link between chronic stress exposure and increased relapse vulnerability^1,2. However, less is known about exactly how chronic stress exposure impacts relapse vulnerability during abstinence, including the neural mechanisms driving these effects. Recent studies from our lab have investigated how chronic stress exposure influences cocaine craving and relapse vulnerability over time³. These studies were conducted using the incubation model of craving and relapse in which cue-induced drug seeking progressively intensifies (“incubates”) during withdrawal or forced abstinence from extended-access cocaine self-administration^4,5,6,7. Incubation provides an animal model for a common human scenario in which abstinence is imposed by incarceration or hospitalization⁸ and has been demonstrated in abstinent human cocaine users⁹. We have found that exposure to repeated but not acute restraint stress during the first two weeks of withdrawal accelerates incubation of cue-induced cocaine seeking in adult male rats, which is thought to increase relapse vulnerability during this time period³.

To examine the neurobiological mechanisms driving chronic stress induced-acceleration of incubation of craving, we focused on the basolateral amygdala (BLA), a region critical for behavioral responding to stress¹⁰. Previous studies have shown that repeated restraint stress exposure increases neuronal excitability in the BLA of adult male rats¹¹. Notably, repeated restraint stress increases the spontaneous firing rate of BLA projection neurons¹², which are glutamatergic and make up ~80% of the neurons in the BLA¹³. Given that glutamatergic projections from the BLA to the nucleus accumbens (NAc) are critical for incubation of cocaine craving¹⁴, we tested the hypothesis that cocaine and chronic stress exposure produce an additive increase in BLA neuronal activity, which may contribute to the enhanced seeking behavior observed in these animals. To investigate this, we conducted in vivo extracellular single-unit recordings from anesthetized adult male rats during withdrawal from saline or cocaine self-administration and following exposure to repeated restraint stress or control conditions. These studies are the first to assess the effects of cocaine alone and cocaine plus chronic stress exposure on in vivo BLA neuronal activity and may identify mechanisms driving chronic stress- and cue-induced enhancement of cocaine seeking.

MATERIALS AND METHODS

Subjects and Surgery

All procedures were approved by the Rosalind Franklin University Institutional Animal Care and Use Committee, and conducted in accordance with the USPHS Guide for Care and Use of Laboratory Animals. As described previously³, male Sprague-Dawley rats (Envigo, Indianapolis, IN) weighing 250–275g upon arrival were housed on a reverse light cycle with food and water freely available. Briefly, after an initial acclimation period (5–7 days), rats were anesthetized with ketamine/xylazine (80 mg/kg; 10 mg/kg, i.p.) and a silastic catheter (Plastics One, Roanoke, VA) was inserted into the right jugular vein and passed subcutaneously to the mid-scapular region. Aside from the initial acclimation period, rats were singly housed for the entirety of the experiment.

Cocaine self-administration

After 5–7 days of recovery, rats underwent saline or cocaine self-administration for 6 h/day for 10 days under a fixed-ratio-1 (FR1) reinforcement schedule. As described previously^3,15, cocaine was self-administered at a dose of 0.5 mg/kg/infusion. Self-administration was conducted in operant chambers (MED Associates, St. Albans, VT) equipped with two nose-poke holes (active and inactive). Active hole responses turned on the infusion pump and led to the delivery of a 20-sec light cue and a 20-sec timeout period. During this timeout period, nose pokes in the active hole were recorded but did not result in an infusion. Nose poking in the inactive hole had no consequences. Sessions started at the onset of the dark cycle (~9:00 AM). Any rats that did not learn to self-administer cocaine and/or had faulty catheters were euthanized.

Tests for Cue-Induced Cocaine Seeking & Restraint Stress

As described previously³, seeking tests were conducted using a within-subjects design in which time-dependent changes in cue-induced responding were assessed by comparing the number of active hole responses on WD1 and WD15. On each test day, rats received a cue-induced seeking test (30 min) at the onset of the dark cycle, during which nose-pokes in the active hole resulted in presentation of the light cue previously paired with cocaine or saline but no infusion. From WD6-WD14, rats underwent repeated restraint stress or control conditions (between 2:00 and 4:00 PM). Rats in the repeated restraint stress group were placed in a restraint hemicylinder for 20 min once a day for 5 days, followed by 2 days off and then an additional 2 days of restraint (total of 7 sessions over a 9-day period). Controls were placed in a transparent cage on the same schedule. This pattern of intermittent stress exposure was chosen because it has been shown to decrease inter-session habituation and produce more robust changes in corticosterone and anxiety-like behavior compared to administering the restraint sessions daily¹⁶.

On WD15, rats underwent a second cue-induced seeking test (30 min) and recordings were then conducted from WD15 to WD20. In addition to saline controls, an additional untreated control group was generated to ensure that behavioral testing and cue-exposure was not affecting BLA neuronal activity. This group received IV catheter surgery and was handled regularly and maintained over the same number of weeks as the other animals but never exposed to the self-administration chambers. A total of 33 rats and five groups were included in this study: Untreated Control (n=4), Saline/Control (n=9), Saline/Stress (n=9), Cocaine/Control (n=5), Cocaine/Stress (n=6). Two of the rats in the Cocaine/Stress group and 2 of the rats in the Cocaine/Control group were included in the behavioral studies in our previously published paper (first figure in reference 3). They were the first set of rats used to conduct in vivo recordings (described below). Additional rats were then generated for all subsequent behavioral testing and electrophysiological recordings.

In vivo Extracellular Single-Unit Electrophysiological Recordings

Because chronic stress-induced acceleration of incubation of cocaine craving can be observed on WD15 but not WD48³, recordings were conducted on or close to WD15. Because only one or two rats can be recorded per day, recordings were conducted on WD15-WD20, i.e., 1–6 days after the last restraint stress or control session on WD14. As described previously^12,17, rats were anesthetized with urethane (1.5 g/kg, i.p.) and placed in a stereotaxic apparatus (Stoelting, Wood Dale, IL). Briefly, burr holes were drilled in the skull and a concentric bipolar electrode probe (0.25 mm outer diameter, Rhodes Medical Instrument, Summerland, CA) was lowered into the medial prefrontal cortex (+2.7 to +3.2 mm anterior and −0.5 to −0.7 mm lateral from bregma, −4.2 to −5.4 mm ventral from the surface of the skull¹⁸) to monitor the local field potential oscillatory activity. Appropriate depth of anesthesia was confirmed by slow rhythmic waveforms of approximately 1 Hz frequency. Next, burr holes were drilled over the BLA (−2.5 to −3.6 mm caudal and −4.4 to −5.1 mm lateral from bregma). Single-barrel glass electrodes were prepared from glass pipettes (World Precision Instruments, Sarasota, FL) using a vertical microelectrode puller (PE-2, Narishige, Tokyo, Japan). The pipettes were gently broken under a microscope to produce a tip of 1–2 μm in diameter (10–20 MΩ; Rosenkranz & Grace 1999) and were filled with 2% Pontamine Sky Blue in 2 M sodium chloride solution. The recording electrode was then slowly lowered into the amygdala using a hydraulic microdrive (Model MO-10, Narishige, East Meadow, NY).

Signals collected by the recording electrode were amplified by a head-stage connected to a preamplifier (Dagan, Minneapolis, MN) with frequencies filtered at 0.3 Hz (low cut-off frequency) and 3 kHz (high cut-off frequency). The output signal was connected to an audio monitor (Model AM8 Grass Instruments, West Warwick, RI) and the amplified outputs were digitized through an interface (5–10 kHz; Model ITC-18, HEKA, Bellmore, NY). The outputs were fed to a computer (Mac Pro/2.8 Apple, Cupertino, CA), monitored using AxoGraph X software version 1.6.4 (Axon Instruments, Inc., Foster City, CA) and stored for off-line analysis. At the end of each recording, Pontamine Sky Blue was ejected (−29 μA, ~45 min) to mark the recording site. As described previously¹⁷, brains were sliced and stained and all recording sites were reconstructed in atlas representations¹⁸.

Data Analysis

BLA neurons were included if they fulfilled the following 3 criteria: (i) Their location was within the basal or the lateral nuclei of amygdala. (ii) Their action potentials (APs) showed a clear signal to noise ratio (> 3:1). (iii) They were recorded for at least 3 minutes. The activity of neurons in the BLA was assessed by measuring the spontaneous firing rate (average number of spikes per second, Hz), following previously published methods^12,17,19. BLA activity was also assessed by quantifying the number of spontaneously active neurons recorded per electrode track, an estimation of the relative number of active neurons¹².

Behavioral data were analyzed using a repeated measures ANOVA with treatment group as the between factor and withdrawal test day as the within factor. Electrophysiological data was analyzed using one-way ANOVA. When a significant effect was observed, Holm-Sidak post-hoc analyses were conducted. AP half-width distribution was analyzed by frequency histogram. Analysis of data was performed using Statistica (Tibco; Palo Alto, CA) and GraphPad Prism (La Jolla, CA). Statistical significance was set at p < 0.05. All data are expressed as mean ± standard error of the mean (SEM).

RESULTS

Chronic Stress Exposure Does Not Affect Cue-Induced Responding and Increases Firing Rate of BLA Neurons in Saline-Treated Controls

Previous studies have shown that repeated restraint stress increases the spontaneous firing rate of BLA projection neurons in adult drug-naïve male rats¹², but these studies were conducted in animals that had not received surgery or operant training. We wanted to confirm that catheter implantation, saline self-administration and cue-induced seeking tests did not disrupt these effects. We also wanted to determine whether chronic stress exposure has any impact on responding to a cue that has not been paired with cocaine. To do this, three groups of animals were generated: untreated controls and saline-treated rats exposed to control conditions (Saline/Control) or repeated restraint stress (Saline/Stress). Untreated controls underwent IV catheter surgery and were handled regularly but never exposed to the self-administration chambers. Saline-treated rats underwent IV catheter surgery and saline self-administration (6 h/day for 10 days) followed by a cue-induced seeking test on WD1 (30 min) where poking in the active hole (previously paired with saline and a cue-light) delivered only the cue. Rats were then divided into two equal groups (based on the number of saline infusions obtained and active and inactive hole responding during self-administration, data not shown) and exposed to repeated restraint stress (Saline/Stress) or control conditions (Saline/Control) from WD6-WD14. All rats underwent a second cue-induced seeking test on WD15. BLA neuronal activity was recorded between WD15 and WD20 (or the equivalent amount of time in the untreated control group) in all 3 groups (Fig. 1A).

Figure 1. Chronic Stress Exposure Does Not Affect Cue-Induced Responding but Increases Firing Rate of BLA Neurons in Saline Self-Administering Controls.

(A) Experimental Timeline: SA, self-administration; WD, withdrawal day. (B) Seeking Tests: Nose-pokes in the previously active hole (AH) and the inactive hole (IH) during the cue-induced seeking tests on WD1 and WD15. During the seeking tests, AH nose pokes led to presentation of a light-cue previously paired with saline delivery while responding on the IH was without consequence. Data are expressed as mean ± SEM number of AH and IH nose pokes. Cue-induced responding did not differ between saline rats subjected to stress (Sal/Stress) and those that were not (Sal/Con). However, both groups showed an overall increase in responding from WD1 to WD15 (in both the AH and IH) that was not directed selectively to the active, cue-paired hole and that likely reflects an overall increase in exploratory behavior. #p<0.05, WD1 vs WD15 collapsed across both groups. (C) Firing Rate: Representative traces are shown from each group and data are expressed as average firing rate (± SEM). The graph on the left shows both individual data points and group averages, while the graph on the right shows only group averages and statistical findings. Saline-treated rats exposed to repeated restraint stress (Sal/Stress) showed an increase in neuronal firing rate in the BLA compared to both unstressed saline rats (Sal/Con) and untreated controls (Untrt Con). **p<0.01, Sal/Stress vs. Sal/Con and Sal/Stress vs. Untrt Con. (D) Neurons/Track: Data are expressed as the average number (±SEM) of spontaneously active neurons per electrode track, an estimation of the relative number of active neurons. No group differences were observed.

We found that repeated restraint stress exposure (Saline/Stress group, n=9/group) had no effect on cue-induced seeking compared to Saline/Controls (n=9/group). However, rats in both groups showed a significant increase in responding on both the active (cue-light) and inactive (without consequence) hole from WD1 to WD15 (Fig. 1B). For active hole responding, a significant effect of test day was observed (F_1,16=19.28, p<0.001) but no treatment group effect (F_1,16=0.36, p=0.55) or group by test day interaction (F_1,16=0.83, p=0.38). Similarly, for inactive hole responding, a significant effect of test day was observed (F_1,16=4.77, p=0.04) but no treatment group effect (F_1,16=0.46, p=0.51) or group by test day interaction (F_1,16=0.31, p=0.59). These data show that overall responding increased in both saline groups from WD1 to WD15 but this was not directed selectively to the active, cue-paired hole. Therefore, we interpret these data as indicating a small but significant change in overall exploratory behavior from WD1 to WD15 in all saline-treated animals.

In vivo recordings were conducted from the BLA on WD15–20. As expected¹², we found a robust increase in spontaneous firing rate in the Saline/Stress group (n=39 neurons/9 rats) compared to both control groups (untreated, n=25 neurons/4 rats; saline, n=43 neurons/9 rats) (Fig. 1C). A significant treatment group effect was observed (F_2,104=10.5, p<0.001) and post-hoc analyses revealed a significant increase in firing rate in the Saline/Stress group compared to both Untreated (p=0.006) and Saline/Controls p<0.001). No significant difference in firing rate was observed between the two control groups (Untreated Control vs Saline/Control, p=0.44), so these groups were combined for future analyses and will be referred to as the drug-naïve control group in subsequent text and figures (n=68 neurons/13 rats).

To sample the relative number of active neurons throughout the BLA, the electrode was lowered through the BLA at predefined coordinates that remained the same across all treatment groups. Consistent with previous findings¹², no difference in the number of spontaneously firing neurons throughout the BLA was observed in the Saline/Stress group (n=154 tracks) compared to both Untreated (n=70 tracks) and Saline/Controls (n=152 tracks) (F_2,373=0.16, p=0.85; Fig. 1D). As described above for firing rate, both control groups were combined for future analyses (drug-naïve, n=222 tracks).

Taken together, these data replicate previous findings showing that repeated restraint stress exposure in drug-naïve adult male rats increases the firing rate of spontaneously active neurons in the BLA, but has no effect on the relative number of active neurons¹². These data also indicate that exposure to operant chambers and cue lights does not influence BLA activity (Untreated vs. Saline/Controls, Fig. 1C,D). In addition, while repeated stress exposure does not influence behavioral responding as measured by active hole and inactive hole responding (Fig. 1B, Saline/Control vs. Saline/Stress), it has a robust effect on the firing rate of BLA neurons (Fig. 1C), indicating that chronic stress exposure alone is driving the observed changes in BLA activity.

Chronic Stress Exposure During Withdrawal Enhances Cue-Induced Cocaine Seeking and Produces an Additive Increase in the Firing Rate of BLA Neurons

To assess the effects of cocaine and chronic stress exposure on neuronal activity in the BLA, rats were trained to self-administer cocaine (0.5 mg/kg/infusion) under extended-access conditions (6 h/day for 10 days). Identical to the paradigm described in Figure 1, rats were divided into two equal groups based on intake and active and inactive hole responding during self-administration (Cocaine/Control, n=5/group; Cocaine/Stress, n=6/group; self-administration data not shown). All rats underwent two seeking tests (WD1, W15) and exposure to repeated restraint stress or control conditions from WD6-WD14 (Fig. 2A). Consistent with our previous findings³, rats exposed to repeated restraint stress showed enhanced cue-induced cocaine seeking on WD15 compared to those exposed to control conditions (Fig. 2B). There was no treatment group effect (F_1,9=2.27, p=0.17) but there was a significant effect of test day (F_1,9=31.31, p<0.001) and a significant treatment group by test day interaction (F_1,9=7.997, p=0.0198). Post-hoc analyses revealed a significant difference between seeking behavior on WD15 in the Cocaine/Stress group compared to the Cocaine/Control group (p=0.04) (Fig. 2B). A paired t-test (t₄=2.55, p=0.03, 1-tailed) confirmed a time-dependent increase in active hole responding in the Cocaine/Control group. No significant effects were observed for inactive hole responding between groups or across test days (treatment group: F_1,9=3.2, p=0.11; test day: F_1,9=0.004, p=0.95; treatment group by test day: F_1,9=0.42, p=0.53; Fig. 2B). Taken together, these data replicate previous findings showing that chronic stress exposure during early withdrawal accelerates the time-dependent intensification or incubation of cue-induced cocaine craving by increasing active hole responding on WD15 to levels typically observed at later withdrawal time points³.

Figure 2. Chronic Stress Exposure During Withdrawal Enhances Cue-Induced Cocaine Seeking and Produces an Additive Increase in the Firing Rate of BLA Neurons.

(A) Experimental Timeline: SA, self-administration; WD, withdrawal day. (B) Seeking Tests: Nose pokes in the previously active hole (AH) and the inactive hole (IH) during the cue-induced seeking tests on WD1 and WD15. Data are expressed as mean ± SEM number of AH and IH nose pokes. Chronic stress exposure during withdrawal from cocaine self-administration (Coc/Stress) enhanced cue-induced cocaine seeking (AH nose-pokes) on WD15 compared to cocaine rats that were not subjected to stress (Coc/Con), while no group difference in IH responding was observed. *p<0.05, Coc/Stress vs. Coc/Con on WD15. #p<0.05, WD1 vs WD15 collapsed across both groups. (C) Firing Rate: Representative traces are shown from each group and data are expressed as average firing rate (± SEM) as described in Fig. 1C. Cocaine exposure alone increased the neuronal firing rate in the BLA compared to the combined drug-naïve control group (Untrt Con + Sal/Con rats from Fig. 1C, dashed line) and chronic stress exposure during withdrawal (Coc/Stress) produced a further increase in firing rate. *p<0.05, vs. drug-naïve control group; #p<0.05, Coc/Stress vs. Coc/Con. (D) Neurons/Track: Data are expressed as the average number of spontaneously active neurons per electrode track, an estimation of the relative number of active neurons. Cocaine exposure followed by either control conditions (Coc/Con) or chronic stress exposure (Coc/Stress) produced an increase in the number of spontaneously active neurons/track compared to the combined drug-naïve control group (from Fig. 1D) (*p<0.05, vs. drug-naïve control group).

In vivo recordings were conducted from the BLA on WD15–20 (Fig. 2A). Interestingly, cocaine exposure alone (Cocaine/Control, n=37 neurons/5 rats) produced a robust increase in spontaneous firing rate in the BLA compared to the combined drug-naive control group (n=68 neurons/13 rats) (Fig. 2C). The magnitude of the increase in firing rate in the Cocaine/Control group (1.55 ± 0.26 Hz) was very similar to that observed for the Saline/Stress group (1.71 ± 0.32 Hz, Fig. 1C), indicating that cocaine exposure alone increases neuronal activity to levels observed following chronic stress exposure in saline-treated rats. In addition, chronic stress exposure during withdrawal from cocaine self-administration (Cocaine/Stress, n=68 neurons/6 rats) produced a further enhancement in neuronal activity (Fig. 2C). A significant treatment group effect was observed (F_2,170=18.27, p<0.0001) and post-hoc analyses revealed a significant increase in firing rate in the Cocaine/Control group compared to the drug-naïve control group (p=0.02) and a further increase in firing rate in the Cocaine/Stress group compared to Cocaine/Controls (p=0.02). Together, these data indicate that cocaine exposure alone enhances BLA neuronal activity and that cocaine plus chronic stress exposure produces an additive increase in activity (Fig. 2C), which may contribute to the enhanced cue-induced seeking behavior observed in these animals (Fig. 2B).

The relative number of spontaneously active neurons throughout the BLA was also assessed in cocaine-treated animals exposed to chronic stress or control conditions. Interestingly, regardless of stress exposure, all animals that self-administered cocaine (Cocaine/Control, n=75 tracks; Cocaine/Stress, n=107 tracks) displayed a greater number of spontaneously active neurons in the BLA compared to the combined drug-naïve control group (n=222 tracks) (Fig. 2D). A significant treatment effect was observed (F_2,401=8.18, p<0.001) and post-hoc analyses revealed a significant increase in the number of spontaneously active BLA neurons in the Cocaine/Control and Cocaine/Stress groups compared to drug-naïve controls (p=0.035, p=0.0005, respectively) but no difference between the two cocaine-treated groups (p=0.38). Taken together, these data indicate cocaine exposure alone increases BLA activity and that chronic stress exposure during withdrawal produces an additive increase in activity by increasing the firing rate of neurons that are already active (Fig. 2C) without changing the relative number of spontaneously firing neurons (Fig. 2D).

BLA Neuronal Recordings

The recording sites for all treatment groups were spread throughout the BLA (Fig. 3). The BLA is comprised of projection neurons (~80%) and interneurons (~15–20%), which can be tentatively differentiated by their action potential (AP) half-widths, which are of longer duration in BLA projection neurons^19,20,21. Only neurons with an AP half-width above 200 μs were included in the present study. To further investigate whether different types of neurons were being sampled, AP half-width from all recorded BLA neurons across all 5 treatment groups (212 neurons from 33 rats) was plotted as a frequency distribution and fit tested to a single polynomial or two polynomials. The distribution was best fit with one polynomial curve rather than two, consistent with a single population (Fig. 4A). The AP half-width of neurons from the five treatment groups were also similar (F_4,207=1.53, p=0.19; Fig. 4B). In addition, the mean AP half-width of neurons in each group was >300 μs, which is in the range of BLA projection neuron half-widths. Together, these findings suggest that the analyzed data were sampled from a similar population of neurons across treatment groups and that these were likely BLA projection neurons.

Figure 3. Location of BLA Recording Sites.

Recording sites in the BLA were reconstructed from brain sections¹⁸ after localization of the Pontamine Sky Blue Dye ejected after each recording (example shown in 4D). (A) Recording sites from the drug-naïve control groups (Untrt Con and Sal/Con) and saline-treated rats exposed to chronic stress (Sal/Stress) groups are shown in grey, yellow, and red circles, respectively. (B) Recording sites from the Cocaine-treated rats exposed to control conditions (Coc/Con) or chronic stress (Coc/Stress) are shown in blue and black squares, respectively. The numbers above each section indicate distance (in mm) from bregma.

Figure 4. BLA neuronal recordings.

(A) AP Half-Width Distribution: BLA neurons can be tentatively separated into projection neurons and interneurons based on AP half-width. The frequency distribution of the AP half-width (μs) from all recorded neurons is shown here. The data distribution is best fit with one polynomial curve instead of two, indicating that all recordings were conducted from a single population of BLA neurons and that these were most likely projection neurons. (B) AP Half-Width: AP half-width was similar across all treatment groups (see group descriptions and abbreviations in the legends for Figs. 1 & 2). Data are shown as group average (±SEM) AP Half-Width (μs). (C) Firing Pattern: Changes in the firing pattern of BLA neurons were assessed by quantifying the coefficient of variation (CV) of the inter-event interval (IEI). No change was observed across treatment groups (group average ± SEM). These data indicate that there was no effect of drug treatment or stress exposure on the neuronal firing pattern in the BLA. (D) Histology: Histological section showing Pontamine Sky Blue Dye in the BLA (example shown is −3.12 mm from bregma). Dye ejected at the end of each recording was used to reconstruct and confirm the location of all recorded neurons (recording sites shown in Fig. 3).

Changes in the firing pattern of BLA neurons can also reflect functional neuronal changes, even in the absence of changes in firing rate. This can be quantified as the coefficient of variation (CV) of the inter-event interval (IEI). Analyzing this measure allows us to determine whether drug treatment or stress exposure produced any significant changes in firing pattern, such as a shift towards irregular firing or burst firing. As shown in Fig. 4C, there was no difference in the CV of IEI across the five treatment groups in our study (F_4,207=0.39, p=0.82), which suggests that neither drug nor stress treatments had a significant effect on neuronal firing patterns in the BLA.

DISCUSSION

This study investigated the effects of cocaine treatment and stress exposure on BLA neuronal activity in adult male rats. As expected from previous results¹², repeated restraint stress increased the firing rate of BLA neurons but did not increase the relative number of spontaneously active BLA neurons. Rats that self-administered cocaine and underwent 2–3 weeks withdrawal showed an increase in spontaneous firing rate in the BLA and an increase in the number of spontaneously active neurons per track, indicating an overall increase in BLA population activity following cocaine treatment. When chronic stress exposure was combined with cocaine withdrawal, there was a further increase in neuronal firing rate in the BLA but no further change in the number of spontaneously active neurons. Together, these data suggest that cocaine and chronic stress exposure produces an additive increase in BLA activity, which could contribute to the enhanced cue-induced cocaine seeking behavior observed in these animals (reference 3, Fig. 2B). These data are the first to report the effects of cocaine self-administration on in vivo BLA neuronal activity and the first to assess the effects of cocaine plus chronic stress exposure on these measures.

Stress Exposure and Cue-Induced Relapse Vulnerability

Over the past few decades, the majority of studies investigating the effects of stress on relapse vulnerability have studied acute stress-induced relapse using the reinstatement model. In these studies, an acute stressor such as foot shock is given to reinstate previously extinguished drug-seeking behavior (for review see reference 22). However, to the best of our knowledge, only two studies to date have investigated the effects of chronic stress exposure during withdrawal on cue-induced cocaine seeking behavior^3,23. Both studies, along with the current study, were conducted using adult male rats. Our previous findings (Fig. 2B in reference 3) indicated that repeated restraint stress (20 min/session) exposure during early withdrawal (WD6-WD14) from extended-access cocaine self-administration (6 h/day for 10 days) increases cue-induced seeking behavior on WD15 (1 day after the last stress or control session). WD15 is a time period when, following extended-access (6 h) cocaine self-administration, incubation of cue-induced craving is on the rising phase and has not yet plateaued^4,24. However, using a separate group of rats, we found that there was no long-lasting effect of this same stress regimen (WD6-WD14) when cue-induced seeking was tested weeks later at WD48³, a time point when cue-induced cocaine seeking has plateaued^4,24. These results indicate that repeated stress in early withdrawal accelerates incubation of cocaine craving, although craving plateaus at the same level observed in rats not exposed to stress.

A recent study from Ball and colleagues found that a longer repeated restraint stress exposure (3 h/day) during the first week of withdrawal (WD2-WD8) following short-access cocaine self-administration (3 h/day for ~10 days) did not affect cue-induced cocaine seeking on WD15 (7 days after the last stress or control session)²³. Similarly, this same repeated stress regimen did not affect cue-induced reinstatement on WD14 in a separate group of rats that underwent extinction training instead of forced abstinence. However, this same stress regimen enhanced cocaine-primed reinstatement of drug seeking on WD14, and this was blocked by daily administration of the D1-like receptor antagonist SCH-23390 during restraint stress exposure. There are several important differences between this study and our findings. These include different behavioral testing paradigms, timing and duration of the restraint stress exposure, and timing of seeking tests relative to the last stress exposure. Another important difference is that our studies use repeated interrupted restraint stress, while the study by Ball and colleagues used repeated daily restraint stress²³. Compared to daily repeated restraint stress sessions, interrupted restraint stress has been shown to decrease inter-session habituation and enhance anxiety-like behavior. This was demonstrated by lower body weight, greater corticosterone production and adrenal gland weight, and enhanced anxiety-like behavior in the elevated plus maze in the interrupted stress group compared to the daily stress group¹⁶. This raises the possibility that differences in the magnitude of the stress response across studies may contribute to different behavioral effects. In any case, future studies are needed to further characterize these effects and parse apart which variable(s) are important for differential behavioral responding following chronic stress exposure.

As the incubation phenomenon and the role of stress in relapse generalizes across classes of addictive drugs^1,6, the importance of studying the interaction of chronic stress and abstinence obviously extends beyond cocaine. Interestingly, the Shalev laboratory has shown that chronic but not acute mild food restriction stress during the first two weeks of withdrawal from extended-access heroin self-administration leads to a robust increase in cue-induced seeking in both male and female rats on WD15^25,26,27. Future studies are needed to assess how different chronic stress regimens affect cue-induced seeking behavior across various drugs of abuse in order to move towards improving treatment strategies to promote abstinence in recovering addicts.

Although one study has investigated the effects of chronic stress exposure on relapse vulnerability in both male and female rats²⁷, all others discussed here have used male rats. Although clinical studies indicate sex differences in both cocaine addiction²⁸ and stress and anxiety disorders²⁹, few preclinical studies have examined interactive effects of cocaine and stress exposure on relapse vulnerability in both male and female rodents. In addition, the majority of studies investigating BLA physiology have also been conducted in male rats. However, a recent study found that drug-naïve females show enhanced spontaneous firing rate in the BLA compared to males and this changes across the estrous cycle³⁰. How BLA activity is affected by chronic stress and/or cocaine exposure in adult female rats has yet to be assessed. Extending these studies to female rats is essential in order to work towards developing treatment strategies effective for preventing relapse in both male and female patients.

Underlying Mechanisms of Enhanced BLA Activity following Cocaine and Stress Exposure

There are several factors that could contribute to the enhanced firing rate of BLA projection neurons we have observed following chronic stress exposure and drug treatment (Figs. 1 & 2). The majority of these factors have been investigated following chronic stress exposure alone (in drug-naïve rats), while little is known about changes in BLA activity following cocaine or cocaine plus chronic stress exposure. First of all, studies examining the effects of acute versus repeated restraint stress exposure on corticosterone levels and BLA activity have shown that, although a single restraint stress session leads to acute activation of the hypothalamic pituitary adrenal (HPA) axis as indicated by an acute increase in plasma corticosterone levels¹⁶, it does not increase the spontaneous firing rate of BLA neurons when recorded one day after the single stressor¹². Therefore, it is likely that the change in BLA firing rate observed following repeated restraint stress is due to a cumulative effect of repeated HPA axis activation. In addition, exposure to repeated restraint stress increases glutamatergic drive in the BLA³¹ and the lateral nucleus of the amygdala (LAT)³² of adult male rats, which is likely to contribute to the increased BLA firing rate observed in these animals. Previous studies have also shown that repeated restraint stress in adult male rats increases neuronal membrane excitability of BLA principal neurons^11,33, which may also contribute to the observed increase in firing rate. Another factor that could be contributing is changes in glutamate receptor expression in BLA projection neurons, as repeated stress exposure^34,35 and cocaine self-administration³⁶ have both been shown to increase glutamate receptor expression in the BLA. Finally, changes in GABAergic transmission in the BLA could be contributing to increased firing rate. GABAergic interneurons make up ~15–20% of the neurons in the BLA¹³ and play an important role in regulating the activity of projection neurons. For example, targeted lesions of a small subset of rat BLA GABA interneurons have been shown to enhance BLA neuronal activity³⁷, indicating that even small changes in GABAergic transmission within the BLA could contribute to significant changes in activity, such as those observed here. However, a more recent study found that an overall change in GABAergic inhibition in the lateral amygdala of adult male rats is unlikely to be driving the increased firing rate observed in this region following repeated restraint stress³². The role these mechanisms or others play in driving the effects of cocaine and cocaine plus chronic stress exposure on changes in BLA activity remain an important focus for future studies. It will also be important to better understand the relationship between electrophysiological measures of BLA activation and results obtained with other indices such as c-fos activation.

Regardless of the mechanism, our data clearly indicate that, similar to repeated restraint stress exposure in drug-naïve rats (i.e., reference 12, Fig. 1), extended-access cocaine self-administration alone enhances activity in the BLA compared to drug-naïve controls (Fig. 2). However, unlike repeated restraint stress, it does so by increasing both spontaneous firing rate and the number of spontaneously active neurons per track, which could indicate that there are distinct mechanisms driving these effects. While this is the first report to assess changes in in vivo BLA activity following extended-access cocaine self-administration, our findings are consistent with an in vitro patch clamp study showing that a non-contingent binge regimen of cocaine (3 i.p. injections/d for 7 d) increased glutamatergic synaptic transmission in the lateral amygdala of adult male rats³⁸.

Our findings also indicate that, while cocaine exposure alone is enhancing BLA activity, chronic stress exposure during withdrawal produces an even greater increase in BLA activity. The BLA projects directly to the NAc and this pathway has been shown to play a critical role in driving various motivated behaviors³⁹, including the enhanced nicotine self-administration observed following exposure to repeated intermittent restraint stress during abstinence⁴⁰. Interestingly, this pathway has been shown to play an important role both in mediating structural and behavioral changes induced by a sensitizing cocaine regimen⁴¹ and in the expression of incubated cue-induced cocaine seeking¹⁴. One possibility is that chronic stress exposure during cocaine withdrawal is strengthening the BLA to NAc pathway, which could be contributing to the enhanced seeking behavior observed in these animals. Data from human studies supports the idea that cocaine and stress exposure enhance activity in the amygdala and in the amygdala to striatal pathway. For instance, a history of prior trauma/stress is associated with amygdala hyperactivity⁴². In addition, stronger connectivity between the amygdala and limbic circuitry, including the striatum, is observed in patients with cocaine use disorder^43,44. Finally, prior trauma in individuals with a cocaine use disorder has been found to enhance amygdala-striatal connectivity both at resting state⁴⁵ and in response to cocaine cues⁴⁶. An important focus of future studies will be to further dissect the specific neural circuitry driving cue- and chronic stress-induced changes in cocaine seeking and relapse vulnerability.