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. Author manuscript; available in PMC: 2026 Jan 5.
Published in final edited form as: Behav Brain Res. 2024 Sep 21;476:115261. doi: 10.1016/j.bbr.2024.115261

Environmental enrichment and sex, but not n-acetylcysteine, alter extended-access amphetamine self-administration and cue-seeking

Troy D Fort 1, Miki C Azuma 1, Dylan A Laux 1, Mary E Cain 1,*
PMCID: PMC11513240  NIHMSID: NIHMS2025743  PMID: 39313073

Abstract

There are no approved therapeutics for psychostimulant use and recurrence of psychostimulant use. However, in preclinical rodent models environmental enrichment can decrease psychostimulant self-administration of low unit doses and cue-induced amphetamine seeking. We have previously demonstrated that glutamate-dependent therapeutics are able to alter amphetamine seeking to amphetamine-associated cues only in enriched rats. In the current experiment, we will determine if enrichment can attenuate responding and cue-induced amphetamine seeking during extended access to a high dose of intravenous amphetamine. We will also determine if N-acetylcysteine (NAC), a glutamate dependent therapeutic, can attenuate amphetamine seeking in differentially reared rats. Female and male Sprague-Dawley rats were reared in enriched, isolated, or standard conditions from postnatal day 21 to 51. Rats were trained to self-administer intravenous amphetamine (0.1 mg/kg/infusion) during twelve 6-hour sessions. During the abstinence period, NAC (100 mg/kg) or saline was administered daily. Following a cue-induced amphetamine-seeking test, astrocyte densities within regions of the medial prefrontal cortex (mPFC) and nucleus accumbens (ACb) were quantified using immunohistochemistry. Environmental enrichment decreased responding for amphetamine and during the cue-induced amphetamine-seeking test. NAC did not attenuate cue-induced amphetamine seeking or alter astrocyte density. Across all groups, female rats self-administered less amphetamine but responded more during cue-induced amphetamine seeking than male rats. While amphetamine increased astrocyte densities within the ACb and mPFC, it did not alter mPFC astrocyte densities in female rats. The results suggest that enrichment can attenuate responding during extended access to a high dose of amphetamine and the associated cues. Sex alters amphetamine-induced changes to astrocyte densities in a regionally specific matter.

Keywords: Amphetamine, differential rearing, environmental enrichment, GFAP, amphetamine seeking, sex differences

1. Introduction

Substance-use disorders (SUDs) are a significant issue, with age-adjusted overdose deaths increasing by 30% from 2019 to 2021. More specifically, psychostimulant overdose deaths have increased by 50% from 2019 to 2021 in the United States (Hedegaard et al., 2021). Therefore, evaluation of the neural and behavioral mechanisms perpetuating psychostimulant-use disorder is vital in helping to develop efficacious treatments and interventions. The importance of the development of such treatments is emphasized by the fact that there are currently no approved pharmacotherapies to treat psychostimulant-use disorder. Interestingly, preclinical rodent models suggest that a behavioral intervention, environmental enrichment, is effective at attenuating cue-induced amphetamine seeking (Chauvet et al., 2009; Garcia et al., 2015; Garcia & Cain, 2021; Thiel et al., 2010).

A widely used model to study the neurobiological and behavioral effects of environmental enrichment is the differential rearing paradigm (Rosenzweig & Bennett, 1969). In this paradigm, rats are reared through adolescence in different environmental conditions. In the enriched condition (EC), rats are group-housed in a large cage and are provided access to a variety of novel objects and handled daily during the post-weaning period. Exposure to all of these elements is essential in producing the most robust effect of environmental enrichment, as social interaction alone has not been shown to produce the neurobiological changes that occur in response to environmental enrichment (Renner & Rosenzweig, 1987; Rosenzweig et al., 1978). In the isolated condition (IC), rats are individually housed in hanging metal cages where the food, bedding, and water are changed without handling the animal during the post-weaning period. As such, EC and IC animals differ in the amount of physical activity, socialization, size of their homecage, experimenter and conspecific tactile interaction, and exposure to novel objects (Renner & Rosenzweig, 1987). In our paradigm, the effects of environmental enrichment and isolation are compared against a standard housing condition (SC) in which animals are pair-housed using the National Institute of Health standard housing guidelines (Council, 2011). The SC serves as a known laboratory standard and comparison point for rodent housing to compare EC and IC rearing. Animals are raised in these environments from postnatal day 21 (PND21) to approximately PND51. These dates are roughly analogous to childhood through late adolescence in humans (Spear, 2000a, 2000b).

Differential rearing has been shown to engender differences in behavioral responses to novel reinforcers and drugs of misuse. Environmental enrichment has been shown to decrease overall operant responding for multiple psychostimulants including amphetamine, cocaine, and methylphenidate when compared to animals reared in isolation (Arndt et al., 2019; Bardo et al., 1996; Bardo & Dwoskin, 2004; Bardo et al., 2001; Garcia & Cain, 2021; Malone et al., 2022; Stairs & Bardo, 2009; Wooters et al., 2011). It is well established that environmental enrichment beginning on PND 21 attenuates self-administration of low-unit doses of psychostimulants. However, enrichment does not attenuate self-administration of moderate to high doses of psychostimulants during short access sessions (Arndt et al., 2015; Arndt et al., 2019; Bardo & Dwoskin, 2004; Garcia et al., 2016; Green et al., 2002; Stairs & Bardo, 2009) or during extended access sessions for cocaine (Gipson et al., 2011). The current experiment will determine if environmental enrichment can attenuate self-administration during extended access for a high dose of amphetamine (0.1 mg/kg/infusion).

Environmental enrichment, when maintained throughout the experiment or used as an intervention during the abstinence period, decreases cue-induced seeking for psychostimulants (Chauvet et al., 2009; Garcia et al., 2015; Garcia & Cain, 2021; Thiel et al., 2010) when compared to isolated condition rats. We hypothesize that environmental enrichment decreases amphetamine seeking due to changes in glutamate homeostasis (Garcia et al., 2015; Garcia & Cain, 2020, 2021; Gill et al., 2012; Malone et al., 2022; Melendez et al., 2004). Disruptions in the balance and regulation of glutamate are characteristic of substance use and chronic cue-seeking. More specifically, the glutamate homeostasis theory of addiction posits that this homeostasis in extracellular glutamate levels between the synaptic and extrasynaptic spaces is maintained by a host of astrocytic and neuronal glutamate transporters (Kalivas, 2009). The proper release and elimination of glutamate in the extrasynaptic space is largely maintained by transporters predominately localized on the surface of astrocytes (Baker et al., 2002; Kalivas, 2009). Disruptions in glutamate homeostasis resulting from cocaine exposure are well-characterized. (Baker et al., 2003; Knackstedt et al., 2010; Sari et al., 2009).

While environmental enrichment can attenuate cue-induced seeking to psychostimulants, there are limited pharmacotherapies with similar efficacy. A therapeutic of interest to the treatment of psychostimulant seeking is the cysteine prodrug N-acetylcysteine (NAC) as it has shown potential efficacy in lowering cocaine seeking, both clinically and preclinically (Murray, Lacoste & Belin, 2014). NAC works to restore glutamate homeostasis by buffering decreases in extracellular glutamate (Baker et al., 2003). NAC has shown previous efficacy in lowering cue-induced seeking to cocaine (Frankowska et al., 2014; Murray et al., 2012; Reichel et al., 2011). However, the efficacy of NAC to lower seeking for other psychostimulants is mixed. Some reports have shown that NAC may be effective in lowering cue-induced reinstatement to methamphetamine (Frankowska et al., 2014; Siemsen et al., 2019), but other reports demonstrated that NAC was not able to attenuate cue-induced reinstatement to methamphetamine (Charntikov et al., 2018). We recently demonstrated that NAC does not alter amphetamine-induced cue-seeking following short-access amphetamine self-administration in standard housed rats (Fort & Cain, 2023). The varied efficacy of NAC may be due to the amount of psychostimulant exposure. Therefore, in the current experiment, we will determine the efficacy of NAC in reducing amphetamine seeking following extended access to a high dose of amphetamine. Unlike our previous work with NAC (Fort & Cain, 2023), in the current experiment we will test the efficacy of NAC in the differential rearing model because differential rearing alters glutamate homeostasis (Garcia et al., 2015; Garcia & Cain, 2020, 2021; Gill et al., 2012; Malone et al., 2022; Melendez et al., 2004), glial cell expression (Diniz et al., 2016; Diniz et al., 2010; Rahati et al., 2016; Szeligo & Leblond, 1977), and responding to low-unit doses of psychostimulants (Arndt et al., 2015; Arndt et al., 2019; Bardo & Dwoskin, 2004; Garcia et al., 2015; Green et al., 2002; Stairs & Bardo, 2009). We previously demonstrated that another potential therapeutic that alters glutamate function, Ceftriaxone (CTX), is effective in reducing cue-induced amphetamine-seeking only in enriched, but not standard-housed animals (Garcia et al., 2016). Therefore, we may observe that NAC is only efficacious in enriched rats.

In the current experiment, we tested the efficacy of NAC to reduce cue-induced amphetamine seeking following extended access to amphetamine (0.1 mg/kg/infusion; 6 hours per session). There is limited work examining sex differences in differentially reared rats. Given that cue-induced drug seeking is different for cocaine and methamphetamine in females (Frankowska et al., 2014; Fuchs et al., 2005; Kippin et al., 2005; Venniro et al., 2017), we powered the current experiment to test for sex differences. Specifically, we hypothesized that amphetamine-induced cue-seeking would be significantly higher in female rats when compared to male rats. We also hypothesized that differential rearing would alter the efficacy of NAC with NAC being most efficacious in enriched rats (Garcia et al., 2016). Lastly, we predicted that amphetamine-exposed rats would have increased astrocyte densities within the medial prefrontal cortex (mPFC; infralimbic and prelimbic cortices) and nucleus accumbens (ACb; core and shell) when compared to amphetamine-naive rats (Bowers & Kalivas, 2003; Fort & Cain, 2023).

2. Material and Methods

2.1. Animals

One hundred and forty-six male and female Sprague-Dawley rats (Charles River, Portage, MI, USA) arrived at Kansas State University on PND 21. Animals were maintained in one of three environmental housing conditions: enriched (EC), isolated (IC), and standard (SC) housing. All animals were given ad libitum access to food and water throughout the experiment except the water-deprived lever-press training (see method below). A 12-hr light-dark cycle was maintained in the animal colony room throughout the experiment. Additionally, the colony room temperature and humidity were maintained at approximately 22°C and 30-50%, respectively. All behavioral testing was conducted during the light portion of the animals’ light-dark cycles. All experimental procedures were conducted in accordance with the Institutional Animal Care and Use Committee at Kansas State University and the NIH guidelines for the care and use of laboratory animals.

2.2. Differential Rearing Environments

Rats arrived on PND21 and were randomly assigned to either the EC, IC, or SC conditions. Animals in the EC condition were housed with 8-10 other rats in a large metal cage (60 x 120 x 45 cm). The bottom of this large cage was lined with paper pulp bedding. Fourteen objects (consisting of children’s toys and PVC pipe) were re-arranged daily (7 new+7 old) in unique arrangements to maintain novelty. All objects were replaced with new objects weekly. Animals were handled daily while the novel objects were being rearranged. Animals in the IC condition were single-housed in hanging wire cages (17 x 24 x 20 cm). Hanging cages were composed of wire mesh on the front and bottom with solid sides. Importantly, IC rats were not handled throughout the rearing period and were not exposed to novel objects or bedding. Finally, SC rats were pair-housed in standard shoebox cages (20 x 43 x 20 cm). SC rats were given the same bedding as EC rats but were only handled once a week during cage changes. The inclusion of SCs provided a known laboratory standard of housing and a standard of comparison. All rats were maintained in their respective environmental conditions for 30 days before any experimental manipulation and remained in their respective conditions for the duration of the entire experiment. An overview of the experimental timeline is shown in Figure 1.

Figure 1.

Figure 1.

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Experimental timeline.

2.3. Apparatus

All training and testing were conducted in standard operant chambers (Med Associates). The standard operant chambers are enclosed within ventilated sound-attenuating chambers. Each chamber contained two levers, a house light, a tone generator, a tether (to administer amphetamine; chemical-resistant tubing covered by stainless steel extension spring; the spring prevents chewing of the tubing), lights above the lever, self-administration pump, and liquid presentation magazine (dipper 0.1 ml). The opening for the magazine is 5 x 4.2 cm and this allows the rat to head poke to drink from the dipper during lever press training.

2.4. Procedures

2.4.1. Lever-Press Training.

Please refer to Figure 1 for an overview of the experimental timeline. After rearing in their respective environments for 30 days, all animals underwent operant training sessions to facilitate the acquisition of the lever-reinforcer association. Experiments were conducted in the operant chambers in cohorts of 16 rats. Female and male rats from each condition were included in each cohort. To increase motivation for water reinforcement, all animals were water-restricted for 12-18 hours before each training session. The reinforcer for all lever press training sessions was a presentation of 0.1 ml of water. During the first session, water access was not contingent on lever pressing. During the second training session, only one lever was presented to the animal, and water reinforcement was contingent on active lever pressing. Following the successful acquisition of the lever-reinforcement contingency, all animals underwent three more fixed-ratio 1 (FR-1) water self-administration sessions. During the three FR-1 water training sessions, all animals were presented with both the active lever, which resulted in water reinforcement, and an inactive lever which had no programmed consequence. After completion of each session, rats were returned to their home cages and were given ad libitum access to water. We did not observe weight loss or signs of dehydration in the rats during the water deprivation and all rats readily learned to lever press.

2.4.2. Surgical Procedures.

Following acquisition of lever pressing, all animals were given 3-4 days of post-training recovery. Rats were deeply anesthetized with isoflurane (~ 5%) and implanted with indwelling jugular catheters using methods described previously (Fort & Cain, 2023; Garcia & Cain, 2021; Arndt, Dietz, & Cain, 2015). Polyurethane catheters (12 cm in length, 0.2 mm internal diameter: SAI Infusion Technologies; Lake Villa, IL ) were inserted through a dorsal incision in the animal’s back, tunneled under the skin, and into the animal’s right jugular vein. Catheter tubing was subcutaneously connected to a 22-gauge back-mounted cannula (Plastics One; Roanoke, VA) and sutured to surgical mesh (Biomedical Structures; Warwick, RI). To prevent animals from chewing or damaging back mounts, a stainless-steel bolt was threaded onto the end of the back mount. During the first 2 days of recovery rats were treated with Meloxicam (2.0 mg/kg after surgery;1.0 mg/kg thereafter) in order to alleviate pain. Baytril® (Enrofloxacin, 2.5% (25 mg/ml) diluted with sterile saline to a final concentration of 6-8 mg/ml administered in a 0.1 ml infusion volume) was used to prevent infection. Propofol (Methohexital 10 mg/ml @ 0.1 ml infusion) was used to check catheter patency. At this dose propofol administered intravenously causes rats to lose muscle tone and become lethargic within a few seconds of the infusion. Rats that did not display signs of a functional catheter were excluded from further analyses.

2.4.3. Amphetamine Self-Administration.

Following post-surgical recovery, all animals began 12, 6-hr fixed-ratio 1 (FR1) self-administration session. During FR1 self-administration, active lever pressing resulted in a 0.1 mg/kg infusion of amphetamine or the saline-vehicle administered over approximately 5.9 sec and the concomitant illumination of the cue light located above the active lever. This amphetamine dose was used during all self-administration sessions. This dose was selected as prior work indicates no difference in self-administration in differentially reared rats during short access amphetamine sessions (Arndt et al., 2015; Arndt et al., 2019; Bardo & Dwoskin, 2004; Garcia et al., 2016; Green et al., 2002; Stairs & Bardo, 2009). Illumination of the cue light terminated after the drug infusion. Following the infusion and cue-light presentation, the house light illuminated signaling a 20-second timeout period during which active lever presses were recorded but were inconsequential. Similarly, inactive lever presses were recorded but had no programmed consequence. Catheter patency was confirmed before the first and following the last self-administration session via propofol infusion (10 mg/ml; 0.1-0.15 ml, i.v.). To achieve stable responding, animals were required to average 10 or more amphetamine infusions across the last 6 self-administration sessions (Arndt, Johns & Cain, 2015; Cain, Denehy & Bardo, 2008). There were no stability criteria for saline self-administering animals. Data from animals in the amphetamine group with non-patent catheters or from animals that failed to meet the above stability criteria were excluded from all analyses. Six rats were excluded from the analysis due to a loss of catheter patency. The number of patent rats in each condition included in the analyses are detailed in Table 1.

Table 1.

Animal numbers for all experimental groups

EC-NAC IC-NAC SC-NAC EC-VEH IC-VEH SC-VEH
Male-Amp 6 6 6 6 7 6
Male-Saline 7 5 6 5 7 4
Female-Amp 7 7 8 6 5 8
Female-Saline 4 4 5 4 3 6
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Note: Group sizes reflect patent animals only.

Abbreviations: Enriched (EC), isolated (IC), standard (SC), N-acetylcysteine (NAC), vehicle (VEH), amphetamine (Amp)

2.4.4. NAC Treatment and Cue-Induced Amphetamine Seeking Testing.

Following 12 self-administration sessions, all animals entered a forced abstinence period. During the abstinence period, all animals remained in their respective environments and were not exposed to the drug, self-administration chamber, or drug-associated cues and received daily intraperitoneal injections of either NAC (100 mg/kg dissolved in sterile saline) or the saline vehicle. This dose of NAC was chosen due to its widespread use in the literature and our previous work (Fort & Cain, 2023; Frankowska et al., 2014; Murray et al., 2012; Reichel et al., 2011; Siemsen et al., 2019). Cue-induced craving was assessed at the cessation of the 14-day abstinence period. Animals received one final injection of NAC (100 mg/kg) or the saline vehicle two hours before cue-induced amphetamine-seeking testing. During the cue-induced amphetamine-seeking test on Withdrawal Day 14 (WD 14), the motivational impact of drug-associated cues was evaluated by reinforcing active lever presses with presentations of the previously associated drug cue but no delivery of the drug itself. The amphetamine-seeking test was 2 hours in duration and active lever presses resulted in the illumination of cue lights and house light. Inactive lever presses were recorded but had no programmed consequence. Given that no amphetamine was available during amphetamine-seeking testing, no timeout period was present during the amphetamine-seeking test.

2.4.5. Perfusions and Immunofluorescence.

Immediately following the amphetamine-seeking test, all animals were humanely euthanized via sodium pentobarbital (Sleep Away) overdose. Animals were transcardially perfused with 0.9% saline and 4% paraformaldehyde and brains were extracted for histochemical analysis. Following extraction, all specimens were transferred to sterilized scintillation vials containing 4% paraformaldehyde for a 12-hour post-fixation period. Following sufficient dehydration in 20% sucrose, all samples were maintained in 0.5M Sorenson’s buffer with 0.1% sodium azide added to prevent microbial contamination. Forty mm sections of the mPFC and ACb were collected from each specimen.

Immunoassays began by permeabilizing floating sections with 6, 5-minute rinses in PBS containing 0.2% Triton-X 100 (1X PBS-TX). Following permeabilization, free-floating sections were blocked in 1X PBS-TX 5% normal goat serum (Vector Laboratories: S-1000) for 30 minutes at room temperature. After blocking, sections were again subjected to 3, 5-minute rinses in PBS-TX. Free-floating sections were then incubated in 1:2,500 rabbit polyclonal anti-GFAP primary antibody (Abcam ab7260) to assess astrocytic densities in the mPFC and ACb. GFAP is a protein that stabilizes astrocyte processes and is widely used as a marker for astrocytes (Bushong et al., 2002; Kimelberg, 2004; Salois & Smith, 2016; Saur et al., 2014). Tissue was incubated in the 1:2,500 anti-GFAP primary (diluted in PBS-TX) for 18 hours at 4°C under gentle agitation on an orbital shaker. Following primary incubation, all tissue was again subjected to 3, 5-minute rinses in PBS-TX. Sections were then incubated in 1:200 diluted goat, anti-rabbit biotinylated secondary antibody (Vector Laboratories PK-4001) for three hours at room temperature under gentle agitation on an orbital shaker. Goat, anti-rabbit secondary antibody dilutions were diluted in 1X PBS-TX. Following three hours of secondary incubation, tissue was again rinsed with 3, 5 minute washes in 1X PBS-TX. Finally, tissue was incubated for 3 hours in 1:100 dilution of avidin-biotin (A and B reagents from Vectastain kit: Vector Laboratories) reagents, under gentle agitation at room temperature. Following three hours of incubation in the avidin-biotin solution, tissue was again rinsed 3 times with 1X PBS-TX. Tissue was then stained via brief submersion in 3-3’-diaminobenzidine (DAB) designed to enzymatically react with the horseradish peroxidase present in the ABC solution. Optimal GFAP detection was found to occur after approximately 45 seconds of reaction time in the DAB. The enzymatic reaction was stopped via rapid rinsing of the tissue with chilled 1X PBS. Free-floating sections were transferred to microscope slides and coverslipped using a Permount coverslipping medium (Fisher Scientific).

Images were acquired on an Olympus BX63F microscope located in the Behavioral Neuroscience core within the Department of Psychological Sciences at Kansas State University. All possible treatment group cells had between 3-7 samples per condition. Images were taken at 20X magnification at anterior, middle, and posterior regions of each area of interest. Mean GFAP cell densities were then derived by averaging cell counts across the three images and then dividing those counts by the total area of the 20X image. These aggregated densities were used in all statistical image analyses. All image analysis was conducted in Image J. Astrocyte densities were generated via hand counting of cells showing positive expression of GFAP. All image quantification was conducted by individuals blind to experimental conditions.

2.5. Data Analysis.

2.5.1. Self-Administration Responding

Active-lever responding during extended-access amphetamine self-administration was assessed using linear multilevel modeling. This analysis specified the animal’s rearing condition (EC, IC, SC), sex (male or female), self-administration session (repeated-measures variable), drug condition (amphetamine or saline), and all possible interactions as fixed effects. The intercept and subject slope of each session were specified as random effects. All alpha levels were set to .05 and main effects and interactions were probed using Tukey’s HSD and simple slopes tests where appropriate. All statistical analyses were conducted using JMP 16 Pro and figures were prepared using GraphPad Prism 9.

2.5.2. Cue-Induced Amphetamine Seeking

Data from the cue-induced amphetamine-seeking test sessions was analyzed using an ANCOVA approach in which the main effects of the rearing condition (EC, IC, SC), sex (male or female), treatment condition (NAC or vehicle), and all possible interactions were used to predict active-lever responding during the amphetamine seeking test. Additionally, average responding during the 12 self-administration sessions was added as a covariate to account for the predictive effects of self-administration responding on amphetamine seeking.

2.5.3. GFAP Immunohistochemistry

Data from the immunohistochemical analysis of GFAP+ cell densities in both regions of the mPFC (PL and IL) and ACb (core and shell) were analyzed in separate ANOVAs. Each ANOVA used the main effect of rearing condition (EC, IC, SC), sex (male or female), and the self-administered drug (AMP or SAL), as well as all possible two- and three-way interactions to predict GFAP+ cell densities. All alpha levels were set to .05 and significant main effects and interactions were probed using Tukey’s HSD and simple effects tests where appropriate.

3. Results

3.1. Extended-Access Amphetamine Self-Administration

As expected, there was a significant main effect of the drug condition (amphetamine or saline), such that active-lever responding was significantly higher for amphetamine when compared to saline, F(1, 131)= 304.27, p < .001. In addition, there was a significant main effect of environmental rearing conditions, F(2, 117) = 42.30, p < .001. Post-hoc probing of this effect via Tukey’s HSD indicated that EC animals had significantly lower overall responding for amphetamine when compared to IC (t(177)= −8.69, p < .001) and SC animals (t(177)= −7.11, p < .001) (see Figure 2). There was a significant two-way interaction between the drug self-administration condition and the self-administration session, F(11, 1336) = 18.35, p < .001, such that active lever-responding for amphetamine increased significantly during the course of self-administration but not in saline self-administering animals. There was also a significant two-way interaction between sex and the drug self-administration condition, F(1, 150) = 6.15, p = .01. Post-hoc probing of this interaction indicated that active-lever responding for amphetamine was significantly higher for males when compared to females (t(153) = 2.77, p = .01). There were no other significant interactions. Multilevel model results are detailed in Table 2.

Figure 2.

Figure 2.

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Mean (+/− SEM) active-lever responding for amphetamine (0.1 mg/kg/infusion) or saline in (A) enriched condition (EC), (B) isolated condition (IC), and (C) standard condition (SC) animals. The bar and asterisks indicate that mean active lever-responding for amphetamine significantly escalated over the course of self-administration, regardless of rearing condition or sex. Additionally, average selfadministration was significantly lower in males when compared to females and in EC rats when compared to both IC and SC rats. *** p < .001

Table 2.

Self-administration multilevel model fixed effects.

df F p
Rearing Condition 2 42.30 < .001
Sex 1 0.62 .43
Session 11 11.55 < .001
Drug Condition (AMP/SAL) 1 304.27 < .001
Rearing Condition*Sex 2 2.51 .08
Rearing Condition*Session 22 1.03 .43
Rearing Condition*Drug Condition 2 1.68 .19
Sex*Session 11 0.92 .52
Sex*Drug Condition 1 6.15 .01
Session*Drug Condition 11 18.35 < .001
Rearing Condition*Sex*Session 22 0.67 .87
Rearing Condition*Session*Drug Condition 22 1.52 .06
Rearing Condition*Sex*Drug Condition 2 2.83 .06
Sex*Session*Drug Condition 11 0.36 .97
Rearing Condition*Sex*Session*Drug Condition 22 0.76 .78
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Note: Significance is indicated by boldfaced font

3.2. Cue-Induced Amphetamine Seeking

Due to differences in self-administration as a result of environmental rearing conditions and sex, data from the cue-induced amphetamine-seeking test was analyzed using an ANCOVA approach. Omnibus results indicated significant group differences in cue-induced responding during the amphetamine cue-seeking test, F(12, 65) = 4.22, p < .001. Average responding during self-administration was a significant predictor of cue-induced responding during the amphetamine-seeking test on WD 14, F(1, 65) = 4.96, p = .03, B= 0.64, SE = 0.29. Rearing condition significantly impacted cue-induced amphetamine seeking, above and beyond the variance explained by self-administration responding, F(2, 65) = 3.68, p = .03. Post-hoc probing of this effect using Tukey’s HSD indicated that cue-induced amphetamine seeking was significantly lower in EC animals when compared to IC (t(65) = −2.15, p = .03) and SC animals (t(65) = −2.68, p = .01). Sex significantly impacted cue-induced amphetamine seeking beyond that which was predicted by self-administration responding, F(1, 65) = 16.34, p < .001. Specifically, amphetamine seeking was found to be significantly higher in females (M = 107.02, SE = 6.71) when compared to males (M = 67.84, SE = 6.98), p < .001. No other main effects or interactions, including treatment with NAC, were found to be significant predictors of cue-induced amphetamine seeking on WD 14 (Figure 3).

Figure 3.

Figure 3.

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A) Mean cue-induced active lever responding (+/− SEM) during the cue-induced amphetamine-seeking test for amphetamine self-administering animals. Given that there was no significant effect of NAC treatment on amphetamine-seeking responding, data are presented collapsed across NAC and vehicle treatment conditions. Results indicated that amphetamine seeking was significantly lower in males when compared to females, ( ### p < .001). Enriched rearing (EC) was shown to decrease cue-induced responding when compared to isolated (IC) or standard (SC) rearing, (* p < .05). B) Mean cue-induced active lever responding (+/− SEM) during the cue-seeking test for saline self-administering animals.

3.3. Astrocyte Density Within the Medial Prefrontal Cortex

Two separate three-way ANOVAs were conducted to assess astrocyte densities, measured via cells showing GFAP+ expression, within the infralimbic (IL) and prelimbic (PL) subregions. Within both regions, there were no significant main effects or interactions of NAC treatment, consistent with the behavioral data. Within the IL, results indicated a significant main effect of drug self-administration condition (amphetamine or saline), such that amphetamine self-administering animals showed greater astrocyte densities, F(1, 92) = 5.66, p = .02 (see Figure 4A). Additionally, analysis of astrocyte densities in the IL indicated a marginally significant two-way interaction between sex (male or female) and the drug self-administration condition (amphetamine or saline), F(1, 92) = 3.87, p = .05. This effect was probed via simple effects comparisons in which sex was specified as the moderator of the effect of amphetamine or saline self-administration on astrocyte density. These posthoc comparisons indicated that amphetamine self-administration increased astrocyte densities when compared to saline self-administration in male subjects, t(92) = −3.20, p = .002, but not in female subjects, t(92) = −0.28, p = .78 (Figure 4A).

Figure 4.

Figure 4.

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Data are presented collapsed across NAC and vehicle treatment conditions because there was no significant effect of NAC treatment on astrocyte density within the mPFC. A) Mean astrocyte densities (+/− SEM) in the IL cortex. In the IL, amphetamine significantly increased astrocyte densities relative to saline self-administration. Results also indicated that amphetamine-induced increases in astrocyte density primarily occurred in males when compared to females, * indicates p < .05, # indicates significant post-hoc comparison (p = .002) of marginally significant interaction between sex and drug self-administration condition (p = .05). B) Mean astrocyte densities (+/− SEM) in the PL cortex. In the PL, amphetamine significantly increased astrocyte densities relative to saline self-administration. Results also indicated that amphetamine-induced increases in astrocyte density primarily occurred in males when compared to females, * indicates p < .05, # indicates significant post-hoc comparison (p = .002) of marginally significant interaction between sex and drug self-administration condition (p = .07). C) Representative 20X image showing GFAP expression in the IL cortex. D) Representative 20X image showing GFAP expression in the PL cortex.

Within the PL, results again indicated a significant main effect of drug self-administration condition such that astrocyte densities were significantly greater in amphetamine self-administering animals when compared to saline self-administering animals, F(1, 85) = 6.41, p = .01 (see Figure 4B). Similar to the IL cortex, there was a marginally significant two-way interaction between sex and the drug self-administration condition, F(1, 85) = 3.33, p = .07. Again, this effect was probed via simple effects comparisons in which sex was specified as the moderator of the effect of self-administration condition on astrocyte densities. Similar to the IL cortex, posthoc comparisons showed that amphetamine exposure increased astrocyte densities in male subjects when compared to saline, t(85) = −3.24, p = .002, but did not increase astrocyte densities in female subjects, t(85) = −0.48, p = .63 (Figure 4B).

3.4. Astrocyte Density Within the Nucleus Accumbens

Two separate three-way ANOVAs were used to assess astrocyte densities in the ACb core and shell. In the ACb core, there was a significant effect of drug self-administration condition, such that animals that were previously exposed to amphetamine during self-administration showed greater astrocyte densities when compared to those that self-administered saline, F(1, 100) = 6.63, p = .01 (Figure 5A). No other main effects or interactions, including treatment with NAC, were found to be significant.

Figure 5.

Figure 5.

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Data are presented collapsed across NAC and vehicle treatment conditions because there was no significant effect of NAC treatment on astrocyte density within the nucleus accumbens. A) Mean astrocyte densities (+/− SEM) in the ACb core. Amphetamine significantly increased astrocyte densities relative to saline self-administration, * p < .05. B) Mean astrocyte densities (+/− SEM) in the ACb shell. Amphetamine significantly increased astrocyte densities relative to saline self-administration, * p < .05. C) Representative 20X image showing GFAP expression in the ACb core. D) Representative 20X image showing GFAP expression in the ACb shell.

Within the ACb shell, the same effect of drug self-administration condition was present. Again, animals that previously self-administered amphetamine showed significantly higher astrocyte densities when compared to saline self-administration, F(1, 99) = 4.41, p = .03 (Figure 5B). No other main effects or interactions, including treatment with NAC, were found to be significant.

4. Discussion

Environmental enrichment decreased self-administration of a high dose of amphetamine during extended access and also decreased responding to cues previously associated with amphetamine during a cue-induced amphetamine-seeking test. Across all groups, female rats self-administered less amphetamine but responded more during cue-induced amphetamine seeking than male rats. Amphetamine increased astrocyte density within the mPFC and ACb, consistent with past research (Armstrong et al., 2004; Bowers & Kalivas, 2003; Bridges et al., 2012; Fort & Cain, 2023; Haydon et al., 2009). However, amphetamine did not increase astrocyte density within the mPFC in female rats. This is similar to recent findings that cocaine does not alter astrocyte morphology in female rats in the ACb (Kim et al., 2022). This suggests the ability of amphetamine to alter astrocyte density varies as a function of sex. Our results suggest that NAC does not attenuate cue-induced amphetamine seeking following extended access to amphetamine self-administration. These results are consistent with our recent results that NAC was ineffective at attenuating cue-induced amphetamine seeking following short access to amphetamine (Fort & Cain, 2023).

4.1. Differential Rearing

It is well established that environmental enrichment beginning on PND 21 attenuates self-administration of low-unit doses of psychostimulants but does not attenuate self-administration of moderate to high doses of psychostimulants during short access sessions (Arndt et al., 2015; Arndt et al., 2019; Bardo & Dwoskin, 2004; Garcia et al., 2016; Green et al., 2002; Stairs & Bardo, 2009). To our knowledge, only one study has examined if environmental enrichment can decrease psychostimulant intake during extended access (Gipson et al., 2011). In this previous study, enrichment only decreased cocaine intake with low cocaine doses, similar to what is observed with short access sessions. Our current results suggest that enrichment can decrease amphetamine self-administration of a high amphetamine dose (0.1 mg/kg/infusion) during extended access. Interestingly, isolation did not increase self-administration during extended access when compared to SC rats.

In addition, environmental enrichment decreased responding during the cue-induced amphetamine-seeking test. Environmental enrichment during the abstinence period decreases the incubation of cocaine craving (Grimm et al., 2008; Solinas et al., 2008; Thiel et al., 2010; Thiel et al., 2009). When compared to an isolated group, enrichment that begins early in life (~PND21) decreases cue-induced amphetamine seeking (Garcia et al., 2019; Garcia & Cain, 2021; Hofford et al., 2014; Zhang et al., 2019). However, the majority of this previous work did not include a standard housed group. The current work demonstrates that enrichment reduces cue-induced amphetamine seeking even when compared to standard housed rats. Similar to the results obtained during self-administration, we again observed that isolated rats exhibited similar rates of cue-induced responding compared to standard housed rats. The current experiment only examined cue-induced amphetamine seeking following a 14-day abstinence period. Additional experiments are required to determine if isolation increases cue-induced amphetamine seeking following longer abstinence periods. To ensure the effects of enrichment were due to the environmental manipulation itself, and not the reduction in amphetamine self-administration during training, we used active lever responding during training as a covariate in our cue-induced amphetamine-seeking analysis (Frieman et al., 2018; Kieth, 2006; Rosenthal & Rosnow, 2008). While this does not account for the putative longer term pharmacological changes that may occur as a result of increased amphetamine exposure, it is a reliable approach for controlling for the variance that results from differences in self-administration responding.

4.2. Astrocyte Density

Consistent with past research, we observed that amphetamine increases astrocyte density, as measured by GFAP expression, within the mPFC (IL and PL) and ACb (core and shell) (Armstrong et al., 2004; Bowers & Kalivas, 2003; Bridges et al., 2012; Fort & Cain, 2023; Haydon et al., 2009). Importantly, there were two additional findings within these results. Differential rearing did not alter astrocyte densities in any of the brain regions measured. In addition, amphetamine did not increase astrocyte densities within the mPFC in female rats.

Previous research suggests that environmental enrichment increases the number of GFAP+ cells when compared to isolated or standard condition rats. This past work focused on the hippocampus or used a much longer enrichment length than the current experiment before quantifying GFAP+ cells (~60 days of enrichment) (Diniz et al., 2016; Diniz et al., 2010; Rahati et al., 2016; Szeligo & Leblond, 1977). While our current results suggest that 30 days of enrichment do not alter astrocyte densities within the mPFC and ACb, we did not include the hippocampus as a region of interest. Given the contributions of the mPFC and ACb to psychostimulant self-administration and cue-induced amphetamine seeking, our results suggest that astrocyte densities within these regions did not contribute to any of the behavioral differences observed.

Our results are similar to findings that in female rats, short access or long access to cocaine does not alter astrocyte morphology (e.g. surface area and volume) within the ACb (Kim et al., 2022). While this prior work did not quantify astrocyte density, together with the current results, it suggests that sex alters the impact of psychostimulants on astrocyte density and morphology. We did not observe an effect of sex on astrocyte density within the ACb. This suggess that sex may interact with the psychostimulant or may only impact morphology within the ACb. Kim et al. (2022) demonstrated that alterations in astrocyte morphology in male rats occur following 45 days of cocaine abstinence, but not 24 hours following cocaine self-administration. Our current experiment quantified astrocyte density following 14 days of abstinence. Together, the results suggest that psychostimulant-induced changes in astrocyte expression are ongoing during abstinence in male rats, but further work is needed to test additional abstinence periods and during psychostimulant self-administration. Overall, there is limited research examining sex effects on astrocytes and the current results support the need for additional research.

4.3. Sex Differences

In the current experiment, female rats self-administered less amphetamine but responded more during the cue-seeking test than male rats. Our findings are consistent with a growing body of evidence that suggests female rats have higher responding during cue-induced amphetamine seeking than males (Bender & Torregrossa, 2023; Corbett et al., 2023; Kerstetter et al., 2008; Nicolas et al., 2019). Some research suggests there are no sex differences in extended access to methamphetamine (Cox et al., 2013; Reichel et al., 2012; Roth & Carroll, 2004), while other research using higher doses of methamphetamine has observed sex differences in self-administration or cue-induced amphetamine seeking (Daiwile & Cadet, 2024; Daiwile et al., 2021; Daiwile et al., 2019; Funke et al., 2023; Ruda-Kucerova et al., 2015; Venniro et al., 2017). To our knowledge, no previous work has measured responding during extended access amphetamine self-administration or cue-induced amphetamine seeking in female rats. Our experiment uses a higher dose of amphetamine and therefore our results are somewhat unexpected when compared to the methamphetamine literature. Amphetamine and methamphetamine produce separable effects on dopamine release, dopamine clearance, and glutamate release within the mPFC and ACb (Goodwin et al., 2009; Shoblock et al., 2003). Amphetamine produces a larger increase in extracellular glutamate in ACb than methamphetamine. However, in the mPFC, methamphetamine produces a larger increase in extracellular glutamate when compared to amphetamine. Therefore, it is possible that the differential impact of sex between amphetamine and methamphetamine may be due to their differences in dopaminergic and glutamatergic function. Alternatively, given the mixed results of the impact of sex on methamphetamine in prior literature, the results may also be due to methodological differences between experiments.

4.4. N-acetylcysteine (NAC) Does Not Attenuate Cue-Induced Amphetamine Seeking

The current study aimed to assess whether NAC would be effective in decreasing cue-induced seeking following extended access to amphetamine. Ultimately, our results indicate that NAC was ineffective in attenuating amphetamine seeking regardless of environmental condition or sex. These results replicate our recent finding that NAC (100 mg/kg; ip) given daily during forced abstinence is unable to lower cue-induced amphetamine-seeking following short access to amphetamine (Fort & Cain, 2023). Our prior work demonstrated that the β-lactam antibiotic, Ceftriaxone (CTX), is effective in reducing cue-induced amphetamine-seeking only in enriched, but not standard-housed animals (Garcia et al., 2016). The current results suggest that the changes to glutamate homeostasis that result from environmental enrichment do not alter the efficacy of NAC (Garcia et al., 2015; Garcia & Cain, 2020, 2021; Gill et al., 2012; Malone et al., 2022; Melendez et al., 2004).

When designing the current study, we utilized methods consistent with previous reports of NAC’s efficacy in lowering cue-induced seeking to cocaine and cue-induced reinstatement to methamphetamine (Frankowska et al., 2014; Moussawi et al., 2009; Murray et al., 2012; Reichel et al., 2011; Siemsen et al., 2019). The current study used a single dose of NAC that has been widely used in the preclinical literature (Frankowska et al., 2014; Moussawi et al., 2009; Murray et al., 2012; Reichel et al., 2011; Siemsen et al., 2019). However, different doses of NAC may be needed to be efficacious at reducing amphetamine cue-seeking. As has been discussed, the discrepancy in effects across different psychostimulants may likely be due to the dissociable neurochemical effects occurring in response to cocaine, methamphetamine, and amphetamine (Goodwin et al., 2009; Shoblock et al., 2003). In addition to the inconsistencies occurring across psychostimulants, it is also worth noting inconsistencies exist in the clinical literature (Grant et al., 2010; Mousavi et al., 2015). Taken together, existing literature suggests that though NAC has shown efficacy in mitigating cue-induced craving for cocaine and methamphetamine, this effect likely does not extend to amphetamine. However, future experiments varying the dose, timing, and frequency of both NAC and amphetamine need to be conducted to fully determine if NAC can attenuate amphetamine cue-seeking.

Another point that has been raised in response to the inconsistencies in the results of NAC treatment is the presence of extinction training. Previous reports have suggested that the efficacy of NAC may be heightened when administered in conjunction with extinction training (Reichel et al., 2011). In a previous report, we demonstrated that NAC was ineffective in lowering cue-induced amphetamine-seeking following both abstinence-amphetamine-seeking and extinction reinstatement paradigms of drug-seeking (Fort & Cain, 2023). As such, it is likely that the efficacy of NAC for the treatment of cue-induced amphetamine seeking does not depend on the presence of extinction training.

4.5. Conclusion

Our results add to the literature supporting that a behavioral intervention, environmental enrichment, can attenuate both amphetamine intake and cue-induced seeking. The results extend the current literature by demonstrating that environmental enrichment can attenuate self-administration of a high amphetamine dose during extended access. While environment enrichment was effective at attenuating cue-induced amphetamine seeking, NAC was not. Taken together, our results suggest that environmental enrichment and sex alter extended-access amphetamine self-administration and cue-induced amphetamine seeking. Additionally, the effects of amphetamine exposure on astrocyte expression are both sex- and region-dependent.

Highlights.

  • Environmental enrichment decreases extended access high dose amphetamine self-administration

  • Environmental enrichment decreases cue-induced amphetamine seeking

  • Female rats respond more than male rats during a cue-induced amphetamine-seeking test

  • Amphetamine increases astrocyte density

  • n-acetylcysteine does not alter cue-induced amphetamine seeking

Acknowledgments:

MEC and TDF were supported during this experiment by DA035435 and P20GM113109. Special thanks to the following laboratory members that made this research possible: Mykenzie Allison, Joanne T. Gomendoza, Haley Green, Abbey Johnson, and Theodore J. Moser.

Footnotes

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Conflict of Interest:

On behalf of all authors, the corresponding author states that there is no conflict of interest.

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