Interaction of stress and stimulants in female rats: Role of chronic stress on later reactivity to methamphetamine

Abstract

Previous research in humans and animals suggests that prior exposure to stress alters responsivity to drugs of abuse, including psychostimulants. Male rats show an augmented striatal dopamine response to methamphetamine following exposure to chronic unpredictable stress (CUS). Compared to males, female rats have been shown to be highly sensitive to the effects of stimulants and stress independently, however few studies have examined the interaction between stress and stimulants in female rats. Therefore, the current study investigated whether prior exposure to chronic stress potentiated the behavioral and neurochemical responses to an acute injection of methamphetamine in female rats. Adult female Sprague-Dawley rats were either exposed to CUS or left undisturbed (control) and then two weeks later received an injection of 1.0 or 7.5mg/kg methamphetamine. Based on open field findings, a subsequent group of rats were exposed to CUS or left undisturbed and then two weeks later received 7.5mg/kg methamphetamine and either dopamine efflux in the dorsal striatum or nucleus accumbens was measured or methamphetamine and amphetamine levels were measured in the brain and plasma. Female rats exposed to CUS traveled greater distances in the open field immediately following an injection of 7.5mg/kg, but not 1.0mg/kg, of methamphetamine and then showed high levels or stereotypy similar to control rats. Animals exposed to CUS had significantly greater increases in dorsal striatum dopamine following an acute injection of 7.5mg/kg methamphetamine compared to control rats, but not in the nucleus accumbens. These differences were not due to group differences in levels of methamphetamine or amphetamine in the brain or plasma. The current findings demonstrate stress-augmented neurochemical responses to a dose of methamphetamine, similar to that self-administered, which increases understanding of the cross-sensitization between stress and methamphetamine in females.

1.0. Introduction

The use of illicit drugs is a serious problem in the United States with 9.4% of Americans reporting that they are current drug users and over 1.4 million people trying non-medical stimulants in the previous year (Substance Abuse and Mental Health Services Administration, 2014). A growing literature suggests that there are subtle differences in the use and response of males and females to stimulants. National surveys report that among substance abuse treatment admissions, 8.6% of females chose methamphetamine (MA) or amphetamine as their primary substance abuse compared to 4.7% of males (Substance Abuse and Mental Health Services Administration, 2014). Women report an increased frequency of drug use and likelihood of dependence, suggesting that drug use in women escalates more quickly to addiction than in men. In addition, women have shorter drug-free abstinent time periods than their male counterparts (Kim and Fendrich, 2002; Becker and Hu, 2008; Dluzen and Liu, 2008). While clinical studies have demonstrated that females are vulnerable to MA dependence, the factors that contribute to MA responsivity in females remain to be elucidated. Given reports that the progression to dependence is accelerated in females, understanding factors that might influence the response to the first experience with the drug is critical as this often shapes subsequent use of the drug and the transition to dependence.

Stress responsivity may be a factor contributing to female vulnerability of stimulant use (for review see (Becker et al., 2017). Prior research has found that female rats are more sensitive to a variety of stressors as measured by increases in plasma corticosterone (Galea et al., 1997; Weinstock et al., 1998; Duchesne et al., 2009; Verma et al., 2010; McFadden et al., 2011; Zareian et al., 2011). Simpson and colleagues reported that females in MA treatment program report greater emotion and sexual trauma compared to their male counterparts, suggesting that stress may influence drug use behavior especially in females (Simpson et al., 2016). Stress can sensitize the nervous system to stimulant drugs, leading to an augmented response in individuals who are drug naive, a process referred to as cross-sensitization (Piazza and Le Moal, 1997, 1998; Marinelli and Piazza, 2002). For instance, male rats that received stress through food-restriction had significantly greater increases in accumbal dopamine following an injection of cocaine compared to ad libitum fed controls (Rouge-Pont et al., 1995). This increased sensitivity to cocaine has been observed following a variety of stressors including social stress and footshock (Sorg and Kalivas, 1991; Miczek et al., 2011). Prior exposure to chronic unpredictable stress (CUS) also leads to a sensitized dopamine increase in the dorsal striatum and nucleus accumbens core in male rats compared to non-stressed male rats following a single or repeated injections of 7.5mg/kg MA (Raudensky and Yamamoto, 2007; Matuszewich et al., 2014). Importantly, the dorsal striatum and nucleus accumbens play different roles in the addiction process. The nucleus accumbens contributes to incentive processes, while the dorsal striatum contributes to goal-directed behavior and habit formation (Smith et al., 2018). Despite multiple studies showing that a variety of stressors contribute to enhanced responsivity to stimulants in male rats, little research has investigated this cross-sensitization between stress and MA in female rats during the first exposure to the drug.

The sensitized dopamine response following chronic stress parallels an augmented behavioral response to a psychostimulant injection in drug naive rats. Exposure to a variety of chronic stress paradigms (food deprivation, restraint, unpredictable) results in a potentiated locomotor response after a cocaine injection in male rats (Bell et al., 1997; Prasad et al., 1998; Haile et al., 2001; Araujo et al., 2003). Likewise, male and female rats exposed to social or restraint stress exhibit potentiated behavioral responses to amphetamine and/or cocaine (Nikulina et al., 2004; Covington et al., 2005; Mathews et al., 2008; Doremus-Fitzwater and Spear, 2010; Holly et al., 2012; Yap et al., 2015; de Oliveira et al., 2016; Shimamoto, 2018). The increased sensitivity to stimulants following stress exposure may be due to the influence that stress has on the blood-brain-barrier. Prior research has shown that elevated corticosterone following chronic stress increases the breakdown of the blood-brain-barrier following experimenter administered MA (Northrop and Yamamoto, 2012). Following MA exposure, females have greater MA and amphetamine levels in brain and plasma compared to males (Rambousek et al., 2014), which may contribute to the long-lasting behavioral activation in females following an acute injection of MA (Milesi-Halle et al., 2005). Whether stress potentiates the concentration of MA in the brain, thereby contributing to MA-stimulated increases in dopamine and sustained behavioral effects is not known.

Based on prior research indicating that female rats have increased sensitivity to stress and stimulants alone, the current study sought to investigate the neurochemical cross-sensitization between chronic stress exposure and the first exposure to MA in female rats. The present study tested the hypothesis that drug naive female rats exposed to 10 days of CUS would show greater dopaminergic responses to an acute injection of MA due to greater MA levels and altered levels of its metabolite, amphetamine, in the brain and plasma, compared to non-stressed female control rats. This was accomplished by first utilizing locomotor response as a screening tool to identify a dose of MA that produced behavioral cross-sensitization and would likely produce neurochemical cross-sensitization in female rats. Next, neurochemical cross-sensitization between chronic stress and the first exposure to MA was investigated in the dorsal and ventral striatum - two areas of the brain that play different roles in behaviors associated with drug use. Lastly, to better understand a possible mechanism underlying neurochemical cross-sensitization, plasma and brain MA and amphetamine levels were assessed at a time point following MA injection when peak dopamine release occurred. Overall, these findings provide insight into the neurochemical changes induced by cross-sensitization in females that may influence subsequent behaviors associated with addictive-like behaviors.

2.0. Materials and methods

2.1. Animals

Adult female Sprague Dawley (Charles-River derived; Indianapolis, IN) rats from the animal colony at Northern Illinois University were used in these experiments. All rats were pair-housed throughout the experiment except where specified. Animal rooms were maintained on a 12 hr light/dark cycle (lights on at 6:00 hr) with temperature maintained at 22±2° C. Food and water were provided ad libitum except when manipulated as a stressor. The procedures in the current studies were approved by the local Institutional Animal Care and Use Committee and followed the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011).

2.2. Drug Injections

Rats were injected once with either 1.0 or 7.5mg/kg (+)-Methamphetamine HCl (i.p.) dissolved in 0.9% NaCl saline (Sigma Aldrich Laboratories, St. Louis, MO, USA) for testing in either the open field test, during microdialysis, or 30 minutes prior to trunk blood and brain collection.

2.3. Vaginal Smears

Clean 0.9% saline was taken up into an eyedropper for a total of 100 μl. The tip of the eyedropper was inserted into the vagina, the bulb of the eyedropper squeezed and then released so as to gently push saline into the vagina and then draw it back into the eyedropper. The fluid was placed on a slide and viewed under a microscope at l0x power. Diestrus was classified when the sample had a dominance of leukocytes and larger cells that were not nucleated. Proestrus was classified when the sample had a dominance of nucleated cells; and estrus classified when the sample had a dominance of cornified cells (Becker et al., 2005).

2.4. Procedures

Two weeks prior to testing, rats were handled for five minutes per day for five days, received vaginal smears daily (09:00) and weighed to monitor their overall health. Following acclimation, rats were assigned either to a non-stressed control group or to a chronic unpredictable stress group (CUS) modified from prior methods (Fitzgerald et al., 1996; Ortiz et al., 1996). For 10 days, rats in the CUS group received stressors applied randomly, twice a day as previously published (McFadden et al., 2011; Matuszewich et al., 2014). Briefly, stressors included 4 hours of wet bedding, 16 hours of food and water deprivation, lights on overnight (12 hours), 2 or 3 hours of lights off during the light cycle, 20, 30, or 50 minutes of rotating on a shaker table, 15 or 60 minutes in a cold room (2 °C) while isolated in mouse cage, isolation in a rat cage overnight, and 60 minutes of restraint stress in a Plexiglas restrainer (Harvard Apparatus, Hollison, MA). The stressors occurred consecutively or with a break of up to 22 hours (see Matuszewich et al., 2014 for schedule).

2.4. Experiment 1: Open Field

2.4.1. Distance

The open field test was conducted two weeks after the last stressor. On the first day of testing, the rat was individually placed into a 48 x 48 x 46 cm box with a camera overhead attached to a recorder. The rat was allowed to explore the box for 30 minutes, then injected with 0.9% saline i.p. and returned to explore the open field for another 90 minutes. On the second day of testing, similar procedures were followed, with a 30-minute habituation period and an injection of either 1.0 or 7.5mg/kg MA i.p. These doses of MA were chosen to be consistent with previous research demonstrating that 1.0 mg/kg induces conditioned place preference to MA in female rats and mice (Schindler et al., 2002; Chen et al., 2003; Hensleigh and Pritchard, 2014) and that 7.5 mg/kg is the approximate amount of MA female rats will self-administer on their first day when given extended access to the drug (Johansen and McFadden, 2017; McFadden et al., 2018; Cordie and McFadden, 2019). The rat was returned to the box for 90 minutes. After the MA injection, rats were isolated overnight to monitor their health and then placed in their home cage with their cage mate the following morning. Distance traveled was measured with five samples per second to track the path of the rat using Noldus EthoVision tracking software system (Noldus Information Technology, Wageningen, Netherlands).

2.4.2. Stereotypy

A quantitative scale based on (Ellinwood and Balster, 1974) was used to assess stereotypical behaviors following the 7.5mg/kg MA injection given prior research suggests that stereotypy is prevalent following similar doses of MA (Balsara et al., 1979). The predominant behavior in the open field was recorded during a 30 second observation every five minutes beginning five minutes after the MA injection until the end of the open field test. The rating scale used to assess rat behavior was as follows: 1= sleep, 2= awake but not moving, 3= normal exploration, 4= more active than normal but with normal movements, 5= running from edge to edge, 6= slow patterned at a normal level, 7= continuous rotational movements, 8= repetitive head movements.

2.5. Experiment 2: Microdialysis

2.5.1. Surgery and Microdialysis

A separate group of rats received intracranial cannula implants and then dopamine levels were measured through microdialysis in either the dorsal striatum or the nucleus accumbens. Each CUS rat were anesthetized eight days following the last stressor with xylazine (6mg/kg) and ketamine (70mg/kg i.p.) and placed into a Kopf stereotaxic frame. A 21G stainless steel guide cannula (11 mm in length, Small Parts, Inc.; Miami Lakes, FL) was implanted above the dorsal striatum (+0.50 mm anterior, +3.00 mm medial, and −0.10mm dorsal to bregma; CON n=10; CUS n=8) or nucleus accumbens (+1.50 mm anterior, +1.30 mm medial, and −0.10mm dorsal to bregma; CON n=6; CUS n=8) and secured by three metal screws and cranioplastic cement. A 27G stainless steel obturator was inserted into the cannula until the day of microdialysis. Antibiotic treatment was put on the incision site and 4.5mg/kg carprofen (Pfizer Animal Health; New York, NY, 50mg/ml diluted 1:10 in sterile water) was injected subcutaneously. Following surgery, the rat was monitored during recovery from anesthesia, administered carprofen the following day, weighed daily to assess health, and housed in isolation for the rest of the experiment.

Five days after surgery and the day before microdialysis data collection, the obturator was removed from the guide cannula while the rat was gently held. The rat was briefly anesthetized with an oxygen/isoflurane mixture and a microdialysis probe was slowly inserted through the cannula into the dorsal striatum (probe active membrane length 4.0mm) or nucleus accumbens (probe active membrane length 2.0mm). Microdialysis probes were constructed within our laboratory using methods previously described (Lowy et al., 1993; Matuszewich and Yamamoto, 2004; Matuszewich et al., 2014). Following probe insertion, the rat was returned to its Plexiglas cage and attached to a tether and swivel (Instech Laboratories, Inc.; Plymouth Meeting, PA). Dulbecco’s phosphate-buffered saline medium (138mM NaCl, 2.1mM KCl, 0.5mM MgCl₂, 1.5mM KH₂PO₄, 8.1mM NaH₂PO₄, 1.2mM CaCl₂, and 5mM d-glucose, pH 7.4; Fisher Scientific, Inc.; Pittsburg, PA) was perfused at a rate of 0.2 μl/min through the microdialysis probe overnight using a KD Scientific syringe infusion pump (Fisher Scientific, Inc.; Pittsburg, PA). The next morning, the rate of perfusion of the Dulbecco’s was increased to 2.0 μl/min for the dorsal striatum testing and 1.5 μl/min for the nucleus accumbens microdialysis testing. After a 2-hour equilibration period, the following 15-min samples were collected: 5 baseline samples and 8 post MA injection (7.5mg/kg i.p.) samples. The samples were immediately injected onto the high performance liquid chromatography with electrochemical detector (HPLC-EC) to assay for dopamine.

2.5.2. High Performance Liquid Chromatography

Microdialysis samples were analyzed for dopamine with HPLC-EC. A 8125 Rheodyne injector (Millipore Sigma, St. Louis, MO) with a 20 μl loop delivered the dialysis sample onto a reverse phase Synergi 4 μm C18 column 150 x 2 mm (Phenomenex; Torrance, CA). A Shimadzu 10ADVP pump continuously pumped mobile phase (32mM citric acid, 54.3mM sodium acetate, 0.074mM ethylenediaminetetraacetic acid, 0.32mM octyl sodium sulfate and 6% acetonitrile) at a flow rate of 0.25 ml/min. Compounds were detected with an LC-4B amperometric detector (Bioanalytical Systems; West Lafayette, IN), with a 3mm glassy carbon working electrode maintained at a potential of +0.5 V relative to an Ag/AgCl reference electrode. Data were collected using ChromPerfect Spirit Software (Justice Innovations, Inc., Denville, NJ).

2.5.3. Verification of probe placement

Following the microdialysis experiment, rats were overdosed with euthasol i.p. (Virbac Product# 710101; St. Louis, MO). Green McCormicks’ food coloring was perfused through the microdialysis probe, the rat was decapitated and the brain quickly removed and frozen. Forty-micron coronal sections were sliced. Microdialysis data was only used from rats when the probe was placed accurately in the dorsal striatum or within the nucleus accumbens (Figure 2A).

Figure 2.

(A) Approximate microdialysis probe locations in the dorsal striatum (CON: blue; CUS: red) and nucleus accumbens (CON: light blue; CUS: orange). (B) Percent change of dopamine in the dorsal striatum compared to baseline (BL) levels prior to 7.5mg/kg MA. * p<0.05 CUS compared to control rats; (C) Percent change of dopamine in the nucleus accumbens compared to baseline levels following 7.5mg/kg MA.

2.6. Experiment 3: Methamphetamine and Amphetamine Levels in Brain and Plasma

2.6.1. Sample Collection

Two weeks following the end of stress or an equivalent time frame in non-stressed controls (approximately 29 days from initial handling) rats were injected with 7.5mg/kg MA i.p. and returned to their home cage (CON n=6; CUS n=6). After 30 minutes, the time point with peak efflux in extracellular dorsal striatum dopamine (Figure 2a), rats were decapitated, whole brains were taken and immediately frozen on dry ice while trunk blood was collected (Vacuette Heparin Tubes, Greiner Bio-One; Radnor, PA). The blood was kept on wet ice until centrifuged at 2000g for 15 min, after which the plasma was pipetted off and stored at −20°C until analysis, while brains were stored at −80°C.

2.6.2. Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (LC-ESI-MS/MS)

The MA/amphetamine analysis used a sample volume of 0.5 mL. Plasma samples were diluted 10-fold and 20-fold in blank plasma samples for quantifying MA and amphetamine concentrations. For the analysis of the brain samples, tissue was homogenized and diluted 10-fold in blank brain homogenate to measure MA and amphetamine concentrations. The homogenates were then stored at −30° C until analysis. Dilution controls at 200 ng/mL MA and 100 ng/mL amphetamine were prepared in both plasma and blank rat brain homogenates. These dilution controls were diluted, processed, and analyzed in the same manner that the research samples were. Aliquots of study samples, calibrators and QCs were fortified with 0.025 mL of internal standard (0.1 μg/mLMA-d₈/amphetamine-d₅).

Chromatography utilized a Thermo Finnigan Surveyor LC pump (San Jose, CA) equipped with an inline solvent degasser, a thermo-statted autosampler, and a 100 x 3.0 mm, 3 μm MetaSil Basic column (MetaChem Technologies Inc.; Torrance, CA). The mass spectrometer was a Thermo Finnigan TSQ Quantum equipped with an Xcalibur (v 2.0) operating software with ThermoFinnigan LCquan software (v 2.0) for the quantitative calculations. The LC was interfaced to the MS by means of an ESI source with an injection volume of 10 μL. Isocratic separation was performed with 85% 0.1% formic acid in water and 15% acetonitrile, at a flow rate of 0.2 mL/min.

2.7. Statistical Analyses

All data was analyzed using SPSS 24.0 software (New York, NY, USA). Estrous stage (i.e. diestrus, proestrus, estrus) were used as a covariate for behavioral and microdialysis comparisons. When there were no significant covariate effects (i.e. open field measures or microdialysis in the dorsal striatum), the covariate was not used for further analyses. Greenhouse-Geisser correction was used to correct for sphericity assumption violations. Significant group differences (p<0.05) were further analyzed using Tukey’s HSD at each specific time point. All data are expressed as the mean±SEM.

To assess for differences in locomotion in the open field, a 7 (time- 15 minute blocks) x condition- CUS or CON) x 2 (injection- saline or MA) repeated-measures analysis of variance (ANOVA) was used for both the 7.5 and l.0mg/kg MA tests. Fifteen minutes prior to the injection of saline or MA was used as a baseline. For each injection day in the open field, a 7 (time- 15 minute blocks) x 2 (condition- CUS or CON) ANOVA was ran. To assess differences in stereotypy ratings during 7.5mg/kg MA open field test, a 17 (time- 5 minute blocks) x 2 (condition) Friedman’s Test was used. Tukey’s HSD was used for group comparisons at specific time points for post-hoc analyses.

Microdialysis data from female rats that had the probe placed in the correct region of interest with complete data samples was analyzed with a repeated-measures ANOVA. Separate analyses were used for the dorsal striatum and nucleus accumbens during which the two groups (CON, CUS) were compared over 9 dialysis samples. Microdialysis baseline data were averaged across the five baseline samples and analyzed between groups with an independent-samples t-test. The average baseline sample and the eight samples after the MA injection were compared with repeated-measures ANOVAs. Due to differences in baseline samples between CUS and control rats, the samples were converted to percent of the average baseline and compared with repeated-measures ANOVAs. Differences in MA and amphetamine levels in both the plasma and the brain tissue between CUS and control rats were compared using an unpaired t-test.

3.0. Results

3.1. Experiment 1: Open Field

3.1.1. Distance following 1.0mg/kg MA

Female rats were exposed to CUS or control conditions were tested in the open field with a saline or 1.0mg/kg MA challenge dose. The overall ANOVA indicated differences in distance traveled between the saline versus the 1.0mg/kg MA day of open field (F(1,44)=325.11, p<0.001) and a time by injection interaction (F(6,264)=82.27, p<0.001; Figure 1A). There was a significant effect of time following both injections but in the opposite direction: female rats significantly reduced distance traveled over the course of testing following a saline injection (F(6,132)=28.43, p<0.001) but increased the distance traveled following an injection of MA (F(6,132)=76.61, p<0.001; Figure 1A). No differences were observed between control and CUS groups in the distance traveled in the open field when females were given either the saline injection (Group: F(1,22)=1.71; Group x Time: F(6,132)=1.97) or the MA injection (Group: F(1,22)=0.01; Group x Time: F(6,132)= 0.84).

Figure 1.

(A) Average distance traveled in open field before (baseline; BL) and after an injection of saline or 1.0 mg/kg MA in female rats (CON n=12, CUS n=12). (B) Average distance traveled both before and after an injection of saline or 7.5mg/kg MA (CON n=23, CUS n=25). (C) Stereotypy ratings following an injection of 7.5mg/kg methamphetamine (CON n=23, CUS n=25). *p<0.05 comparing CUS females to CON females.; # p<0.05 main effect saline versus MA; Λ p<0.001 saline versus MA; + p<0.05 saline versus MA.

3.1.2. Distance following 7.5mg/kg MA

Given the lack of group differences following a low dose of 1.0mg/kg MA, a separate group of female rats was tested in the open field following a challenge injection of 7.5mg/kg MA. The overall ANOVA indicated significant differences in distance traveled between the saline versus the 7.5mg/kg MA day of open field (F(1,91 )=50.31, p<0.001) and a time by injection interaction (F(6,546)=45.32, p<0.001; Figure 1B). Following the saline injection, distance traveled in the open field decreased as time progressed (F(6,276)=46.52, p<0.001), but there was no difference between CUS and control rats overall (F(1,46)=0.77) or over time (F(6,276)=0.79). Following the MA injection, there was a significant effect of time (F(6,270)=63.56, p<0.001) and a significant time by condition interaction (F(6,270)=2.99, p<0.05) with CUS rats traveling a greater distance immediately after the MA injection compared to controls (15 min; Figure 1B), but not at any other time point and no main effect of condition (F(1,45)=0.35).

3.1.3. Stereotypy following 7.5mg/kg MA

From the open field recording, stereotypy was sampled at 5-minute intervals following the MA injection. There was a significant main effect of time (F(16,736)=84.01, p<0.001) with both groups showing stereotypy behaviors. There was no main effect of condition (F(1,46)=0.21) or a time by condition interaction (F(16,736)= 1.16), suggesting that all animals entered into stereotypy MA at a similar rate (Figure 1C).

3.2. Experiment 2: Microdialysis

3.2.1. Dopamine in the Dorsal Striatum following 7.5mg.kg MA

Prior exposure to CUS in female rats resulted in a greater increase in horizontal locomotion immediately following an injection of 7.5mg/kg MA whereas the low dose of 1.0mg/kg methamphetamine resulted in no group differences; therefore, dopamine efflux in the dorsal striatum and nucleus accumbens was measured only following 7.5mg/kg MA. In the dorsal striatum, the average baseline dopamine levels differed between groups (t(16)=2.58, p<0.05), with rats in the control group having overall higher levels $\left(\overline{X}=3.28\pm 0.31\text{pg}\right)$ compared to CUS rats $\left(\overline{X}=2.23\pm 0.23\text{pg}\right)$. As expected, MA increased raw dopamine levels in all groups (F(8,128)=77.75, p<0.001) however rats exposed to CUS show a greater increase in dopamine following the MA injection as evident by a significant time by condition interaction (F(8,128)=2.97, p<0.01). Due to the difference in baseline concentrations, the five baseline samples were averaged and change scores were also calculated for all samples and converted to a percentage with 100% equaling the average baseline. The injection of MA significantly increased the change from baseline dopamine for all groups (F(8,128)=77.11, p<0.01). Rats previously exposed to CUS showed a greater increase of dorsal striatum dopamine levels following the MA injection compared to control rats as evidenced by a significant time by condition interaction (F(8,128)=7.30, p<0.01), contributing to a main effect of condition (F(1,16)=7.22, p<0.05) with post-hoc analyses indicating a significant difference between CUS and CON at each time point following MA injection (p<0.05) except the last time point measured (Figure 2B).

3.2.2. Dopamine in the Nucleus Accumbens following 7.5mg/kg MA

Nucleus accumbens average baseline dopamine levels did not significantly differ in female rats that were previously exposed to CUS $\left(\overline{X}=1.59\pm 0.23\text{pg}\right)$ compared to control female rats $(\overline{X}=1.56\pm 0.26\text{pg}$; t(12)=−0.08). In comparing percent of baseline dopamine across samples, estrous stage was a significant covariate (time x estrous stage (F(8,88)=5.71, p<0.05); estrous (F(1,11)=5.28, p<0.05) and although only one rat was classified as being in proestrus on the day of testing, estrus-classified females had greater nucleus accumbens dopamine increases 30 and 45 minutes after MA (data not shown), which aligns with previous research findings that females in estrus have greater stereotyped behavior and amphetamine-induced dorsal striatum increases compared to females in metaestrus (Becker and Cha, 1989). When comparing raw dopamine levels, MA significantly increased dopamine in the nucleus accumbens (F(8,96)=44.39, p<0.001) however both CON and CUS rats had similar increases (interaction: F(8,96)= 0.41, p=0.91; main effect: (F(1,12)=0.01, p=0.92). Similar to raw scores, there was an effect of time, with MA significantly increasing percentage of baseline dopamine levels in the nucleus accumbens in both groups (F(8,96)=26.03, p<0.001). There was no difference between CUS exposed and control rats overall (F(1,12)=0.02) or across samples (F(8,96)=0.34), suggesting that both groups had similar nucleus accumbens dopaminergic changes from baseline following a systemic injection of MA (Figure 2C).

3.3. Experiment 3: Methamphetamine and Amphetamine Levels in Brain and Plasma

The different behavioral and dopaminergic responses to MA in rats exposed to CUS compared to control female rats could be due to differences in MA brain and plasma concentrations or its metabolism into amphetamine, the pharmacologically active metabolite. The brains and plasma were collected 30 minutes following a single 7.5mg/kg MA i.p. injection in both control and CUS rats. There were no differences in brain concentrations of MA, amphetamine, or the ratio of MA to amphetamine levels following an injection of MA in animals that were either previously stressed or not stressed (MA: t(10)=0.43, p=0.68), amphetamine: (t(10)=0.32, p=0.76), ratio: t(10)=0.40, p=0.67) female rats. There were also no differences on any measure in plasma levels (MA: (t(10)=0.43, p=0.67, amphetamine: (t(10)=1.19, p=0.26), ratio: t(10)=−1.39, p=0.20; Table 1).

Table 1.

Levels of MA and amphetamine in both brain and plasma 30 minutes after an injection of 7.5mg/kg MA in females.

	Brain			Plasma
	MA	AMPH	Ratio	MA	AMPH	Ratio
Female
CONTROL	8794.17 ± 891.00	1052.83 ± 104.19	8.34 ± 0.51	1003.33 ± 92.48	120.38 ± 12.44	8.40 ± 0.43
CUS	9196.17 ± 312.46	1098.67 ± 97.90	8.56 ± 1.09	1049.83 ± 54.20	140.17 ± 11.06	7.60 ± 0.38

4.0. Discussion

The present study examined cross-sensitization between chronic stress and MA in female rats. Female rats exposed to chronic stress two weeks earlier had increased dopamine efflux in the dorsal striatum following an acute injection of MA compared to control rats, which lasted through much of the sampling period following the MA injection (Figure 2B). Although CUS augmented the change in dopamine release within the dorsal striatum, no difference in dopamine release was observed in the nucleus accumbens at any time point. These differences in dopamine efflux during the first exposure to MA may have implications for the course of the development of addictive-like behaviors. Volkow and Morales (2015) purported that dopamine release in the ventral striatum alone is insufficient to account for addiction-like behavior. Rather, a shift from involvement of the ventral striatum to the dorsal striatum likely contributes to the addiction-like phenotype (Everitt and Robbins, 2013; Volkow and Morales, 2015; Smith and Laiks, 2018). The dorsal striatum is thought to play an important role in goal directed behaviors and habit formation (Everitt and Robbins, 2013; Volkow and Morales, 2015; Smith and Laiks, 2018). Thus, applied to the current experiment, control and stress exposed females may experience similar rewarding properties of MA, but the greater increase in dopamine in the dorsal striatum may increase the probability of further engaging in drug-seeking and drug-related habitual behaviors that are associated with addiction. Indeed, prior studies have found that women transition from the recreation use of MA to addiction more quickly than their male counterparts (Dluzen & Liu, 2008).

The cross-sensitization observed in the current study supports the body of research in male rats, suggesting that stress may render an organism more sensitive to the effects of psychostimulants (Prasad et al., 1998; Covington and Miczek, 2001; Haile et al., 2001; Araujo et al., 2003; Nikulina et al., 2004; Holly et al., 2012; Matuszewich et al., 2014). Previous research has shown that female rats also have increased sensitivity to cocaine or amphetamine following chronic stress exposure (Bisagno et al., 2004; Doremus-Fitzwater and Spear, 2010; Holly et al., 2012), and the current study extends those findings to include cross-sensitivity between chronic stress and methamphetamine as measured through dorsal striatum dopamine efflux in female rats. Of interest, in comparison to a previous study using male rats and the current methods (see Matuszewich et al., 2014), females appear to exhibit greater locomotor behavior and striatal dopamine release in response to the 7.5mg/kg challenge compared to male rats. However, both sexes exhibited CUS cross-sensitization.

Female rats exposed to CUS showed a greater dopamine response in the dorsal striatum to the MA challenge injection compared to control rats, however, this potentiation was not observed in the nucleus accumbens. Our finding in the nucleus accumbens differs from prior research in male rats following a stimulant challenge (Raudensky and Yamamoto, 2007). Several aspects of the research design may account for these differences, such as the specific region sampled within the brain. For example, the prior studies finding potentiated dopamine following stress exposure were within the nucleus accumbens shell, whereas dopamine in the core is unaltered following footshock or immobilization stress alone (Deutch and Cameron, 1992; Kalivas and Duffy, 1995). Other studies have focused only on the nucleus accumbens shell, observing a potentiated response in dopamine to cocaine following social defeat stress in both sexes (Holly et al., 2012) and potentiated dopamine response to d-amphetamine following social defeat stress in male mice (Han et al., 2015). In the current study, the microdialysis probe extended through both the nucleus accumbens core and shell, such that opposing differences in the nucleus accumbens core may have offset any changes in the shell. Future studies could further refine the subregions within the nucleus accumbens to assess the specificity of the dopaminergic cross-sensitization, but it is possible that the CUS procedures target specifically dopamine input to the dorsal striatum and goal-directed / habit formation behavior in female rats.

One mechanism through which stress may potentiate the stimulant-induced increase in locomotion and dopamine in the dorsal striatum is by affecting the blood brain barrier and concentration of MA within the brain (Northrop and Yamamoto, 2012, 2015). Although synergistic effects of CUS and high doses of MA have been reported previously on the blood brain barrier in male rats administered 7.5mg/kg MA every two hours totaling four injections (Northrop and Yamamoto, 2012), the current study found no differences between control and female rats exposed to CUS following an acute injection of MA (Table 1). We intentionally collected brain and plasma levels at 30 minutes following the MA injection to parallel the peak increases of dopamine efflux in female rats, however the half-life of MA is 70 minutes and the peak amount of MA crossing the blood brain barrier may differ at later time points (Cho et al., 2001b; Cho et al., 2001a). Although no differences were observed in brain methamphetamine levels following an acute injection of the drug, future studies will investigate if differences exist following chronic neurotoxic injections of methamphetamine.

Another brain mechanism that may mediate the CUS-induced sensitization to MA exposure is through organic cation transporter 3 (OCT3). OCT3 is a low-affinity, high-capacity monoamine transporter and is expressed in both the dorsal striatum and the nucleus accumbens (Koepsell et al., 2007; Gasser et al., 2009), can be blocked by corticosterone (Gasser et al., 2006), and expression inversely relates to MA hyperlocomotion and dopamine efflux (Kitaichi et al., 2003; Kitaichi et al., 2005; Fujimoto et al., 2007; Nakayama et al., 2007). It would be interesting to measure OCT3 expression in the dorsal striatum following CUS exposure in female rats to determine if the stress-induced increases in dopamine efflux are due to a reduction in OCT3.

In the present study, some of the females received a 1 mg/kg injection of MA, which is commonly used to induce conditioned-place preference (Schindler et al., 2002; Chen et al., 2003; Hensleigh and Pritchard, 2014). However, no differences in locomotor responses were observed between the CUS and control animals. A separate cohort of animals was exposed to 7.5 mg/kg injection of MA, which is similar to the amount of MA female rats will self-administer on their first day when given extended access to the drug (Johansen et al., 2017; McFadden et al., 2018; Cordie et al., 2019). At this self-admini strati on based dose, behavioral cross-sensitization was observed between CUS and MA 15 minutes after the injection with females exposed to CUS exhibiting greater locomotion compared to control animals. This behavioral sensitivity was specific to locomotor behaviors as both groups displayed robust stereotypical behaviors after the first 15 min, which most likely masked any further locomotor differences at later time points. The time course of the locomotor cross-sensitization observed in this study and switch to stereotypy is similar to male rats, where locomotion was potentiated immediately following the stimulant injection (i.e. 15 min) in the males exposed to CUS, but then returned to levels comparable to controls as stereotypy became the dominant behavior (Matuszewich et al., 2014). A moderate dose of 2 or 3 mg/kg may be useful to further characterize the behavioral profiles of CUS and control females as those have been shown to enhance locomotion in female rats (Shoblock et al., 2003; Milesi-Hallé et al., 2007), although the time frame may have to be extended as 3 mg/kg dose of methamphetamine also enhanced stereotypy and decreased locomotion at a comparable time to what was observed in the current study (Milesi-Hallé, 2007). While future studies are necessary to determine the best dose to maximize locomotor differences while minimizing stereotypy, our findings suggest that open field behavior may be a useful tool for predicting differences in dopamine release in the dorsal striatum.

The cross-sensitization observed in rodent studies is also present in humans. In a study of women and men, exposure to social stress, life event stress, or perceived stress was positively correlated with dopamine release when exposed to a low dose (0.3mg/kg) of amphetamine as measured through PET (Oswald et al., 2007; Wand et al., 2007). These clinical findings indicate that stress, whether actual or perceived, modulates the effects of stimulants on the brain. Understanding stress as a risk factor for drug use is critical to improving treatment approaches, especially for women who report higher levels of stress (Matud, 2004) and differing responsivity to MA compared to men (Dluzen and Liu, 2008). The current findings have implications for understanding the complex interactions between stress and stimulants and can help establish the etiology and treatment of drug use in females.