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. Author manuscript; available in PMC: 2020 Dec 9.
Published in final edited form as: Brain Res. 2019 Feb 1;1712:63–72. doi: 10.1016/j.brainres.2019.01.044

Dynamic Interactions of Ceftriaxone and Environmental Variables Suppress Amphetamine Seeking

Erik J Garcia 1, David L Arndt 1, Mary E Cain 1
PMCID: PMC7724651  NIHMSID: NIHMS1521328  PMID: 30716289

Abstract

Extrasynaptic glutamate within the nucleus accumbens is a driver of relapse. Cocaine, ethanol, and methamphetamine reduce the expression of cystine-glutamate antiporter (xCT) and primary glial glutamate transporter 1 (GLT1) leading to increased extrasynaptic glutamate. Ceftriaxone (CTX) restores xCT and GLT1 expression and effectively suppresses cocaine and ethanol reinstatement, however, the effects of CTX on amphetamine (AMP) reinstatement are not determined. Rodents were reared in an enriched condition (EC), isolated (IC), or standard conditions (SC) and trained in an AMP self-administration (0.1 mg/kg/infusion). EC, IC, and SC rats received injections of SAL or CTX (200 mg/kg) after daily extinction sessions. Then rats were tested in cue and AMP-induced reinstatement tests. We hypothesized that EC rearing would reduce reinstatement by altering GLT1 or xCT expression in the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC). In Experiment 2, pair-housed rats received once-daily AMP (1.0 mg/kg i.p.) or SAL for eight days followed by once-daily CTX (200 mg/kg i.p.) or SAL injections for 10 days. CTX treatment reduced cue-induced drug seeking in EC rats but not IC or SC rats. In a AMP-induced reinstatement test, CTX reduced AMP-induced drug seeking in EC and SC rats, but not IC rats. Western blot analyses revealed that AMP self-administration and non-contingent repeated AMP exposure did not downregulate GLT1 or xCT in the NAc or mPFC. Therefore, the ability for EC housing to reduce amphetamine seeking may work through other mechanisms.

Keywords: Environmental Enrichment, Amphetamine Reinstatement, Ceftriaxone, Glutamate transporter GLT1 and cystine-glutamate antiporter xCT, Nucleus accumbens (NAc), Medial prefrontal cortex (mPFC)

1. Introduction

Substance Use Disorders (SUDs) are characterized by chronic cyclical processes of abstinence and drug taking (American Psychiatric Association 2013). Despite the effort of many psychostimulant users, many have recurring relapse episodes (Koob and Volkow 2010; Piazza and Deroche Gamonet 2013; Venniro et al., 2016). Extinguished drug seeking can be initiated by re-exposure to drugs, stress, or drug-associated cues (Epstein and Preston 2003; Shalev et al., 2001; Venniro et al., 2016). Rodents also initiate drug seeking upon exposure to drug-associated cues indicating they are a valid model of human relapse (Kalivas and McFarland 2003; Shaham et al., 2003; Venniro et al., 2016). Glutamatergic signaling and homeostasis within the mesocorticolimbic pathway is integral to SUDs (Kalivas 2009; Schmidt and Pierce 2010). Therefore, protecting the mechanisms responsible for maintaining optimal glutamate can reduce SUDs.

Regulating synaptic and extrasynaptic glutamate is regulated by glutamate transporters. The cystine-glutamate antiporter (xCT) regulates basal glutamate in the nucleus accumbens (NAc), and when it is inhibited basal glutamate levels fall by 50-70% (Baker et al., 2002; McBean 2002). GLT1, another glutamate transporter, removes upwards of 90% of excess extrasynaptic glutamate. Together these mechanisms maintain glutamate homeostasis (Bjørnsen et al., 2014; Tanaka et al., 1997) and recent evidence indicates xCT and GLT1 are downregulated after chronic cocaine self-administration, neurotoxic doses of methamphetamine, and ethanol, resulting in disruptions in glutamate homeostasis (Althobaiti et al., 2016; Das et al., 2015; Knackstedt et al., 2010; Reissner et al., 2015; Trantham-Davidson et al., 2012).

Ceftriaxone (CTX), a β-lactam antibiotic, restores the function and expression of GLT1 and xCT, potentially restoring optimal glutamate levels (Das et al., 2015; Knackstedt et al., 2010; Trantham-Davidson et al., 2012). Chronic treatment with CTX (200 mg/kg) reduces cocaine self-administration, and cocaine reinstatement without affecting palatable food self-administration (Ward et al., 2011). Similarly, CTX administration suppresses cocaine induced locomotor activity (Tallarida et al., 2013) and ethanol self-administration (Das et al., 2015). These experiments illustrate a role for CTX to suppress behaviors associated with preclinical addiction models possibly by restoring GLT1 and/or xCT. Surprisingly, subtoxic doses of methamphetamine do not alter GLT-1 (Szumlinski et al., 2017) or xCT expression suggesting that illicit drug exposure has divergent effects on the transporters responsible for maintaining glutamate homeostasis (Abulseoud et al., 2012; Alshehri et al., 2017; Althobaiti et al., 2016; Rasmussen et al., 2011). Amphetamine is more potent at stimulating locomotor activity, evokes more dopamine and glutamate release in the NAc when compared to methamphetamine, and less glutamate release in the PFC when compared to methamphetamine (Shoblock et al., 2003). Yet, in vitro models indicate that amphetamine application does not affect GLT1 expression (Underhill et al., 2014). Therefore, it is unclear if amphetamine in vivo has any effect of glutamate transporter expression.

Early life experiences and adverse events change the probability to develop SUDs. In rats, enrichment condition (EC) rearing reduces amphetamine self-administration, enhances extinction learning and suppresses cue-induced reinstatement when compared to isolated condition (IC) rearing (Bardo et al., 2013; Stairs et al., 2006). During cue-induced reinstatement of cocaine seeking, drug-cue presentations increase synaptic glutamate release and cause a spillover of glutamate in extrasynaptic regions where metabotropic glutamate receptors (mGlur) are preferentially expressed. Despite no basal differences in glutamate levels, subcutaneous amphetamine evokes more glutamate efflux in the NAc in EC rats when compared to IC rats (Rahman and Bardo 2008). Interestingly, homodimer metabotropic glutamate receptor 2/3 (mGluR2/3) expression in the mPFC and striatum are comparable across rearing groups, but blocking mGluR2/3—a presynaptic autoreceptor—elevates elevates extracellular glutamate in EC and SC rats, but not in IC rats (Bardo and Hammer 1991; Bardo et al., 1990; Bardo et al., 1995; Heidbreder et al., 2001; Melendez et al., 2004; Rahman and Bardo 2008). While the source of this glutamate efflux is not determined in differentially reared rodents, mGluR2/3 regulates prelimbic PFC afferents to NAc core synaptic glutamate release after cocaine self-administration (Smith et al., 2017). Therefore, we hypothesize that amphetamine disrupts glutamate homeostasis by reducing the expression of GLT1 in the NAc which reduces clearance of glutamate from the extracellular space (Danbolt 2001).

Here, we address a gap in the literature by examining the effects of amphetamine self-administration and amphetamine exposure on glutamate transporters. We aimed to determine if amphetamine alters GLT-1 and xCT expression in the NAc or medial prefrontal cortex (mPFC) in independent rodent amphetamine sensitization and self-administration experiments. While in vitro evidence indicates amphetamine application does not downregulate GLT1, we hypothesized that like cocaine, in vivo amphetamine exposure would downregulate GLT1 and xCT in the NAc and mPFC. Second, we hypothesized that EC rearing protects against downregulation of glial transporters and maintains homeostasis to curtail cue-induced reinstatement when compared to IC and SC-reared rats. Finally, to restore glial transporter expression, we employed CTX prior to reinstatement tests to determine if it could mitigate cue or amphetamine-induced reinstatement.

2. Results

2.1. Experiment 1: Effects of CTX on extinction, cue-induced, and amphetamine-induced reinstatement

2.1.1. Stable amphetamine self-administration

To assess the differences in number of infusions earned a repeated measures ANOVA was conducted. During the 11 amphetamine self-administration sessions, there were main effects of environmental condition, F(2,24) = 6.07, p<0.01, and session, F(10,240) = 12.44, p <0.01 (Figure 1B) on number of amphetamine infusions earned. The interaction between environmental condition and session was not significant, F(20,240) = 1.12, p >0.05 . Importantly, during the last five sessions of amphetamine self-administration, there was no main effect of session, or significant interactions with environmental condition on the number of amphetamine infusions, indicating that average amphetamine intake was stable within an environmental condition with EC rats averaging less amphetamine intake. A similar ANOVA model was used to determine differences in total active lever presses, which includes presses on the active lever during the timeout period when responses are not reinforced. These analyses revealed that there were main effects of environmental condition, F(2,24) = 3.90, p <0.05, and session, F(10,240) = 35.50, p <0.001, but no interaction between environmental condition and session, F(20,240) = 0.52, p >0.05 (data not shown). There were no significant differences in inactive lever responses between the environmental conditions, across session, and there was no significant interaction between environmental condition and session, all p’s >0.05 (Figure 1C).

Figure 1:

Figure 1:

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(A) Experiment 1 Timeline. All rats were reared in EC, IC, or SC conditions for 30 days, briefly trained to lever press, and them implanted with indwelling jugular catheters prior to the start of amphetamine (0.1 mg/kg/infusion) self-administration sessions. Each line represents a 1-hr session conducted daily. All sessions after the vertical arrow were followed by CTX (200 mg/kg) or saline (i.p).(B) amphetamine infusions (0.1 mg/kg/infusion) and (C) inactive lever presses between EC, IC, and SC rats during FR-1 self-administration. EC rats responded less than IC (*) and SC (Λ) rats, p < .01. SC rats responded less than IC rats (#) during the first session, p <.01. Lever presses during extinction sessions on the previously (D) active and (E) inactive lever during between EC, IC, and SC rats. No treatment was administered following sessions 1-5. CTX (200 mg/kg) or saline was administered immediately after sessions 6-11. Regardless of CTX treatment group, EC rats responded less on the previously active lever than IC (*) and SC (Λ) rats. EC rats responded less than IC (*) and SC (Λ) rats on the previously inactive lever. Number sign (#) indicates that IC rats responded less than SC rats, p < .01. All values in figures B and C represent the mean ± SEM. (F) Cue-induced reinstatement (n=4-5/condition). Previously active lever presses during extinction session 11 and the cue-induced reinstatement test between EC, IC, and SC rats. Asterisk (*) indicates significant increase in previously active lever responding when compared to previous day extinction session 11, p < .01. (G) Amphetamine-induced reinstatement (n=4-5/condition). Previously active lever during extinction session 15 and the amphetamine-induced reinstatement test between EC, IC, and SC rats. Asterisk (*) indicates significant increase in previously active lever responding during the amphetamine-induced reinstatement test when compared to previous day extinction session 15, p < .01. Data in panels F and F are presented as box and whisker plots with symbols representing individual rats. ‘Box’ expands the interquartile range 25th to 75th percentile and the median is the horizontal line in the box. The ‘whiskers’ extend to minimum and maximum observed values.

Furthermore, ANOVA analyses confirmed no baseline differences in amphetamine infusions, total active lever presses and inactive lever presses between those rats that would be assigned to CTX or saline. For infusions, there were no differences between future CTX vs saline-treated rats, F(1,21) = 0.49, p >0.05, and no interactions between future treatment assignment and session, F(10,210) = 0.56, p >0.05 or environmental condition, F(2,21) = 0.45, p >0.05. For total active lever presses, there were no differences between future CTX or saline-treated rats, F(1,21) = 4.21, p >0.05, and no interaction between future treatment assignment and environmental condition, F(2,21) = 0.28, p >0.05. There was an interaction between future treatment and session on total active levers, F(10,210) = 2.29, p >0.05, However, there was no significant difference between future CTX vs saline rats on total active lever presses for any day (all p’s >0.05). For inactive lever presses, there were no differences between future CTX vs saline-treated rats, F(1,21) = 1.92, p >0.05, and no interactions between future treatment assignment and session, F(10,210) = 1.32, p >0.05 or environmental condition, F(2,21) = 0.17, p >0.05.

2.1.2. EC enhances extinction learning compared to SC rats

During the first 5 sessions of extinction, rats did not receive CTX treatment. ANOVA revealed no differences between those rats that would be assigned to CTX treatment vs saline, F(1,21) = 2.46, p = 0.13, and no significant interactions with their future group assignments and session, F<1, all p’s >.05 or environmental condition, F<1, all p’s >.05. These analyses confirm that there were no differences prior to CTX treatment. During early extinction training, there was a main effect of environmental condition, F(2,21) = 11.87, p <.001, session, F(4,84) = 31.08, p <.001, and a significant interaction between session and environmental condition, F(8,84) = 2.98, p<.01. Enriched rats exhibited less drug seeking behavior as evidenced by reduced responses on the previously active lever when compared to IC rats during extinction sessions 1 and 3, F’s(1,84) = 8.42 and 19.74, p <.01, and SC rats during sessions 1, 2, and 3, F’s(1,84) = 9.66-42.21, p <.01 (Figure 1D). Time course analysis of extinction Day 1 revealed a significant main effect of group F(2,24) = 6.64, p < 0.05, time bin F(11,264) = 30.24, p <.001, and a group by time bin interaction F(22,264) = 1.63, p <0.05. SC rats had more active lever responses when compared to EC or IC rats in the first 10 minutes F’s > 10.09, p <.01. There were no differences between EC and IC rats in an individual time bin but generally IC rats had more average responses when compared to EC rats. However, it is important to note that EC rats responded less during the amphetamine training sessions, which could have moderated responses early in extinction training. Importantly, there was no difference between the environmental conditions during sessions 4 and 5, indicating that responding was similar between the environmental conditions before CTX treatment began. Environmental condition also altered responding on the inactive lever during extinction sessions 1-5 (Figure 1E). The ANOVA revealed a main effect of session, F(4, 84) = 15.38, p <.01, and environmental condition, F(2,21) = 4.75, p <0.05. EC rats responded less than IC rats during session 1, F(1,84) = 7.85, p <.01, and SC rats on the previously inactive lever during sessions 1-3 and 5, F’s(1,84) = 12.54-24.68, p <.01. IC rats also responded less than SC rats during sessions 2 and 5, F(1,84) = 6.98 and 9.40, , p <.01.

2.1.3. CTX treatment does not enhance late extinction learning

All rats continued to decrease active lever responses across the late extinction sessions as evidenced by a main effect of session, F(5,105) = 3.18, p=.01. There were no main effects or interactions with CTX treatment or environmental condition. There were no significant differences in inactive lever responding during this phase (Figures 1D and E).

2.1.4. Enrichment and CTX suppress cue-induced reinstatement

We compared all active lever responding during the last extinction session to the active lever responding during the cue-induced reinstatement session to determine if CTX attenuated the expression of cue-induced reinstatement. The ANOVA revealed main effects of session, F(1,21) = 140.97, p <.001, environmental condition, F(2,21) = 6.35, p <.01, and CTX treatment, F(1,21) = 6.14, p <.05. The interactions between session and environmental condition, session and CTX treatment, and environmental condition and CTX treatment were all significant (all p’s<.05). In addition, the three-way interaction between session, environmental condition, and CTX treatment was significant, F(2,21) = 7.92, p <0.01, suggesting that the ability of CTX treatment to reduce cue-induced reinstatement depended on environmental condition (Figure 1F). Probing the three-way interaction revealed that EC, IC, and SC rats treated with saline all reinstated amphetamine seeking, F’s(1,21) = 24.95-39.42, p <0.01. While IC rats, F(1,21) = 28.15, p <.01, and SC rats, F(1,21) = 47.01, p <.01 treated with CTX reinstated amphetamine seeking, the EC rats did not, F(1,21) = .001, p > .05, suggesting that CTX blocked cue-induced reinstatement in EC rats. Analyses on the inactive lever revealed a main effect of environmental condition, F(2,21) = 4.25, p <0.05, such that EC had less inactive lever presses during the cue-induced reinstatement test. No other effects or interactions were statistically significant (data not shown).

2.1.5. CTX suppresses amphetamine-induced reinstatement in SC rats

Following the cue-induced reinstatement test, rats continued to receive their respective CTX or saline treatment and additional extinction sessions, each training session followed by CTX or saline treatment. Once responding was similar to the end of the first extinction phase, rats were tested for amphetamine-induced reinstatement. The ANOVA revealed main effects of session, F(1,21) = 37.64, p <.001, environmental condition, F(2, 21) = 5.74, p =.01, and CTX treatment, F(1,21) = 5.39, p <0.05. There was a significant interaction between session and CTX treatment, F(1,21) = 4.41, p = .05, and a significant interaction between environmental condition and CTX treatment, F(1,21) = 4.33, p <.05(Figure 1G). Simple effects indicated that SC, F(1,21) = 130.05, p <.01, and EC, F(1,21) = 9.03, p<.01, rats treated with saline reinstated amphetamine-seeking, while CTX blocked amphetamine-induced reinstatement in EC, F(1,21) = 3.37, p >.05, and SC, F(1,21) =2.60, p >.05, rats. CTX treatment did not alter responding on the active lever during the reinstatement session in IC rats, F(1,21) = 3.21, p>.05, but IC rats did not express reinstatement, F(1,21) = 1.83, p >.05, when compared to the last extinction session. Analyses indicated that there were no main effects or interactions for inactive lever presses, suggesting all groups responded similarly on the inactive lever (data not shown).

2.1.6. GLT1 is not altered by differential rearing or amphetamine

Separate factorial ANOVAs revealed no differences in GLT1 expression in the NAc (Figure 2A) or mPFC (Figure 2B), suggesting that GLT1 expression in the NAc or mPFC was not affected by environmental condition or chronic CTX treatment (all F’s <1, p>.05).

Figure 2:

Figure 2:

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Normalized GLT1 expression in the (A) NAc or (B) mPFC following the amphetamine-induced reinstatement test in EC, IC, and SC rats (n=4-5/condition). The expression of GLT1 did not differ between environmental conditions or CTX treatment groups within the NAc of the mPFC.Representative western blot images (insets). Representative western blot image labels: ‘I’ denotes IC, ‘E’ denotes EC, and ‘S’ denotes SC. ‘Sal’ denotes saline, and ‘CTX’ denotes ceftriaxone. All values normalized to calnexin 80kD band. Data are represented as box and whisker plots with symbols representing individual rats. ‘Box’ expands the interquartile range 25th to 75th percentile and the median is the horizontal line in the box. The ‘whiskers’ extend to minimum and maximum observed values.

2.2. Experiment 2: Amphetamine injections induce behavioral sensitization without altering GLT1 or xCT expression in the NAc or mPFC

2.2.1. Locomotor response to amphetamine

The omnibus ANOVA revealed a main effect of drug, F(1,20) = 296.64, p <.001, such that amphetamine increased the average locomotor response when compared to saline, and a main effect of session such that there was greater locomotor activity after repeated amphetamine administrations, F(1,20) = 41.64, p <.001. There was a significant interaction of drug and session, F(1,20) = 30.78, p <.001. Simple effects indicated that the amphetamine treated rats but not saline rats showed behavioral sensitization as evidenced by the increased locomotor activity on the eighth exposure, F(1,20) = 72.01, p <.001. In summary, the amphetamine treated rats to be assigned to saline or CTX treatment each developed behavioral sensitization (Figure 3A).

Figure 3:

Figure 3:

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Locomotor response to amphetamine (AMP) or saline (SAL) (n=6/condition). (A)Total distance traveled in response to AMP or SAL on session 1 and session 8. Asterisk (*) indicates that AMP increased the total distance traveled when compared to SAL. Caret (Λ) indicates that repeated AMP resulted in an elevated total distance traveled on session 8 when compared to session 1 in AMP-treated rats. Expression of normalized (B) GLT1 or (C) xCT in the NAc following repeated AMP and CTX treatments in SC rats. Expression of normalized (D) GLT1 or (E) xCT in the mPFC following repeated AMP and CTX treatments in SC rats. The expression of GLT1 or xCT in the NAc or mPFC was not changed by any treatment. Representative western blot images (insets). S denotes saline, and A denotes amphetamine. Sal denotes saline, and CTX denotes ceftriaxone. All values normalized to calnexin 80kD band. Data are represented as box and whisker plots with symbols representing individual rats. ‘Box’ expands the interquartile range 25th to 75th percentile and the median is the horizontal line in the box. The ‘whiskers’ extend to minimum and maximum observed values.

2.2.2. GLT1 and xCT expression in the NAc

ANOVA analyses were used to determine average differences in NAc GLT1 and xCT transporter expression. The ANOVA indicated that there was no effect of amphetamine, CTX treatment, or an interaction on GLT1 or xCT expression in the NAc, F’s <1, p>.05 (Figure 3B and 3C).

2.2.4. GLT1 and xCT expression in the mPFC

ANOVA analyses indicated that within the mPFC, there were no main effects of amphetamine, CTX treatment F’s <1, or an interaction F(1,20) = 1.61, p = .22 on GLT1 or xCT expression, F’s <1, p>.05 (Figure 3D and 3E).

3. Discussion

The current experiments determined that the efficacy of chronic CTX treatment to reduce amphetamine seeking is moderated by environmental condition and that amphetamine, either self-administered or experimenter administered, does not affect GLT1 expression. Additionally, xCT expression in the NAc or mPFC is not affected by repeated experimenter administered amphetamine injections. Repeated treatments of CTX blocked cue-induced reinstatement in EC rats, and blocked amphetamine-induced reinstatement in EC and SC rats. Therefore, CTX treatment is capable to suppress amphetamine relapse-like behavior. Surprisingly, protein analyses determined that neither amphetamine or CTX altered GLT1 expression in the NAc or the mPFC in any of the environmental housing conditions. Furthermore, amphetamine sensitization was not accompanied by downregulation of GLT1 or xCT in either the NAc or the mPFC providing more evidence that amphetamine alone does not change GLT1 or xCT in these aforementioned brain regions.

Environmental enrichment (EC) reliably reduces cue-induced reinstatement after cocaine (Chauvet et al., 2009), methamphetamine (Hofford et al., 2014), and sucrose reinstatement (Grimm et al., 2008). In the present experiment, all rats reinstated amphetamine seeking in the cue-induced reinstatement test. However, the amphetamine seeking response was suppressed when CTX treatment was paired with enrichment housing. Thus, in EC rats, CTX prevented cue-induced amphetamine seeking. This result suggests that CTX may enhance resilience to cue-induced drug seeking commonly observed in EC rats. This exciting result is highlighted by the complimentary finding that EC housing facilitates extinction learning (Stairs et al., 2006). Together, these therapeutic approaches have the capability to reduce drug taking faster and reduce the potential for relapse behavior that results from drug-associated cues. Although CTX did not change GLT1 or xCT expression in the NAc, other research indicates that CTX has the capability to recruit and reorganize astrocytes to increase astrocyte surface area and volume in the NAc. Importantly, these increases are associated with increased colocalization between astroglial and synapsin, indicating a greater potential for astrocytes to contact and restore optimal glutamate levels in this region (Scofield et al., 2016). Therefore, we hypothesize that EC rearing in combination with chronic CTX treatment could have resulted in a significant increase in the number of contacts between astrocytes and synapses in the NAc, however, future experiments to determine the validity of this hypothesis are needed. Alternatively, environmental housing conditions also moderate the efficacy of Type I and Type II/III metabotropic glutamate receptor ligands, which are important for maintaining basal non-vesicular glutamate levels (Arndt et al., 2014; Arndt et al., 2015; Baker et al., 2003; Gill et al., 2012; Gill et al., 2012; Kalivas 2009). With this in mind, the results suggest that the synergistic CTX+EC protective effect could be dependent on the degree of colocalization between astrocytes and synapse which ultimately influence presynaptic glutamate release (Smith et al., 2017) via mGluR2/3. However, this has not been explicitly tested in differentially reared rodents after amphetamine self-administration.

CTX treatment blocks cocaine primed reinstatement when CTX is supplemented with extinction training (Knackstedt et al., 2010; Lacrosse et al., 2016; Scofield et al., 2016), yet, CTX has no effect extinction learning (Knackstedt et al., 2010). Here, the EC and SC rats treated with saline reinstated amphetamine seeking after the amphetamine injection, suggesting that extinction training alone is unable to completely mitigate amphetamine seeking. Surprisingly, the IC rats treated with saline did not demonstrate amphetamine-induced reinstatement, which may be at odds with previously published literature (Stairs et al., 2006). However, there are critical differences in experimental procedures that may account for these disparate results. In our procedure, the rats had more extinction sessions, a cue-induced reinstatement procedure, and no cues present during the amphetamine-induced reinstatement test. Given the dynamic interactions that extinction training and differential rearing have on subsequent seeking behavior (Grimm et al., 2008; Thiel et al., 2011) it is not surprising that amphetamine-induced reinstatement is sensitive to these procedures. Another possible explanation is that the rearing groups respond differently to the amphetamine challenge after abstinence/extinction training. The difference in amphetamine-induced reinstatement could be attributed to differences in the development of tolerance or equally as likely, sensitization, however these have not been explicitly tested after self-administration. Future research examining CTX treatment, extinction learning and environmental manipulations are needed because many effective strategies employ concurrent behavioral and pharmacological interventions to reduce relapse vulnerability.

Our data indicate that short access amphetamine self-administration does not alter GLT1 in the NAc or mPFC. Currently, we cannot rule out the possibility that long access amphetamine self-administration could impact GLT1 or xCT expression, because long access (6h) cocaine self-administration downregulates GLT1 in the NAc core and shell to a greater extent when compared to short access (Fischer-Smith et al., 2012). However, in vitro models suggest that amphetamine does not alter GLT1 (Underhill et al., 2014). Application of amphetamine internalizes EAAT3 but not GLT1 in dopamine neurons, suggesting that amphetamine-induced transporter internalization processes are localized to neuronal glutamate transporters (EAAT3) and not astrocyte-expressed transporters or antiporters (Underhill et al., 2014). Importantly, the mechanism that is responsible for the rapid upregulation and then downregulation after chronic exposure to cocaine, methamphetamine, or ethanol is not currently known, but protein kinase C (PKC) activation decreases GLT1 membrane expression through a serine 486-mediated residue (Kalandadze et al., 2002). Amphetamine dose-dependently recruits PKC from membrane and intracellular pools, but whether these different pools contribute to GLT1 downregulation after self-administration is not known (Giambalvo 1992a; Giambalvo 1992b).

CTX treatment reduces locomotor activity to acute and chronic cocaine injections (Tallarida et al., 2013) and also mitigates amphetamine-induced stereotypy and total ambulations, but CTX fails to attenuate the development of amphetamine sensitization (Rasmussen et al., 2011). In the present experiment, repeated amphetamine injections (1.0 mg/kg) induced behavioral sensitization but did not affect GLT1 or xCT expression in the NAc or mPFC when compared to control animals. Our results are in agreement with other literature (Sidiropoulou et al., 2001) and suggest that doses as high as 5 mg/kg of amphetamine do not change GLT1 expression in the NAc or PFC. Therefore, higher repeated dosing regimens of amphetamine are unlikely to result in significant adaptations to GLT1, and again illustrate the divergent effects of different psychomotor stimulants and GLT1 expression in the NAc and PFC.

Similarly, it appears that the role of xCT varies as a function of the psychomotor stimulant. XCT is also downregulated in the NAc following cocaine self-administration (Knackstedt et al., 2010) and experimenter administered cocaine injections (Madayag et al., 2007). The reduction of xCT antiporter significantly impairs glutamate homeostasis by altering mGluR2/3 tone and contributes to elevations in extrasynaptic glutamate and spillover in the NAc during reinstatement tests (Baker et al., 2003; Baker et al., 2002; Kalivas 2009). However, route of administration can change the adaptations to this antiporter. Nicotine self-administration downregulates xCT, but nicotine fails to affect xCT expression when delivered through osmotic minipumps (Knackstedt et al., 2009). Similar differences have been observed when comparing the effects of methamphetamine on xCT protein expression to cocaine, because methamphetamine alone does not result in downregulation of xCT in the NAc or mPFC (Althobaiti et al., 2016). These results are similar to the results of the present experiment in that repeated amphetamine administrations do not alter xCT expression in the NAc or mPFC, and further suggest that different psychomotor stimulants have divergent effects on GLT1 and xCT expression in the NAc and mPFC.

The divergent effects of psychomotor stimulants on GLT1 or xCT expression may be mediated by an interaction between dopamine and glutamate in mPFC and NAc. Methamphetamine is less effective at releasing dopamine in the PFC compared to amphetamine, which may increase glutamate in the PFC because dopamine inhibits glutamate release from the PFC (Abekawa 2000). Therefore, the increased dopamine resulting from amphetamine results in greater inhibition of glutamate release from the PFC, and accounts for the differences between methamphetamine and amphetamine in glutamate release observed in the NAc and PFC (Shoblock 2003). How these potential interactions affect GLT1 or xCT expression in the NAc or mPFC is not yet determined but suggest divergent interactions that contribute to the mechanisms responsible for the drug-induced reductions in glutamate transporters or antiporters. Importantly, the role that these interactions are involved or even regulated by cue vs drug induced reinstatement is not determined and calls for additional research to determine these separate mechanisms across broad types of drugs of abuse.

Chronic amphetamine exposure—either contingent or noncontingent—has little, if any effect on GLT1 or xCT expression in the NAc or mPFC. Yet, EC rearing and chronic CTX treatment reduce cue-induced reinstatement. Further, CTX was effective in suppressing amphetamine-induced reinstatement in EC and SC rats. The dynamic interactions of CTX treatment with behavioral interventions highlight the importance of future research with CTX across different drug classes to understand the mechanisms by which CTX alters reinstatement.

4. Methods and materials

4.1. Animals

Male Sprague-Dawley rats (Charles River, Wilmington, MA, USA) were housed in one of three environmental rearing conditions: enriched (EC), isolated (IC), or standard (SC). Rats had ad libitum access to food and water for the duration of the experiment, with the exception of lever press training. The rats were housed in a temperature (~22°C) and humidity controlled (30-45%) colony room that was maintained on a 12-h light-dark cycle. All behavioral tests were conducted in the light cycle. Tests, procedures, and reports were in accordance with the Institutional Animal Care and Use Committee at Kansas State University, and in agreement with the NIH guidelines for animal experimentation (National Research Council 2010; National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals 2011).

4.2. Environmental housing conditions

Rats arrived at the research lab at exactly 21 days of age and were randomly assigned to either the EC, IC, or SC housing conditions using previously described procedures (Bardo et al., 1995; Stairs et al., 2006). EC rats lived with 8-12 other cohorts and were housed in a large metal cage (60×120×45 cm) that was lined with pine chip bedding. To maintain enrichment and novelty, 14 objects (small children’s toys, and PVC pipe) were rotated and/or replaced daily for novel arrangements. In addition, during the rearing process, each EC rat was handled for approximately one minute each day. IC rats were reared individually and were housed in hanging wire cages (17×24×20 cm). IC cages had wire mesh on the front and bottom and solid stainless steel sides and rear. The IC rats were not exposed to novel objects and were not handled during the rearing period. SC rats were housed in pairs in standard opaque shoebox cages (20×43×20 cm). SC rats were handled minimally during the weekly cage change throughout the rearing period and did not have novel objects. The cages of the SC rats were lined with pine chip bedding. SC rats were included to provide a known laboratory standard for comparison between the EC and IC rats. All rats were housed in their respective housing conditions for 30 days before experimentation commenced and remained in their housing environment for the duration of the study.

4.3. Apparatus

4.3.1. Operant chambers

Lever press training and amphetamine self-administration were conducted inside operant conditioning chambers (ENV-001, Med Associates, St. Albans, VT). Each chamber was enclosed in a sound-attenuating compartment and operated by a computer interface. During lever press training, 20% sucrose was presented in a recessed food receptacle after the active lever was pressed. The two metal response levers were located on either side of the food tray 7.3 cm above the metal grid floor. A 28-V, 3-cm diameter white cue light was centered above each response lever. A Cage Tweeter tone generator (Med Associates) was centered on the back wall. Drug infusions were administered via a syringe pump (PHM-100, Med Associates) connected to a 10-ml syringe holding the correct amphetamine concentration based on each rat’s body weight.

4.3.2. Locomotor activity chambers

The locomotor response to amphetamine or saline was automatically measured using activity chambers (46.6 × 46.6 × 46.6 cm; Coulbourn Instruments). Each activity chamber was constructed of transparent plexiglass walls and plastic flooring covered with pine shavings. A 16 × 16 photocell array surrounded the locomotor chamber and was 2.54 cm above the flooring. Each photocell was spaced 2.54 cm apart and automatically measured the horizontal distance movement of the rat in centimeters. The horizontal movement was saved after each 5-min block and summed to yield the total horizontal distance in the 60-min test sessions. A white noise generator was used to create background noise (70 dB) to mask external sounds.

4.4. Drugs

Ceftriaxone (Sandoz) was dissolved in 0.9% saline (200 mg/kg; 1.0 mg/ml) and stored at 20° C. Ceftriaxone (CTX) or saline was injected intraperitoneally (i.p.) immediately after the operant sessions. D-amphetamine (Sigma Aldrich, TX, USA) was dissolved in 0.9% sterile saline (0.1 mg/kg/infusion) and was self-administered intravenously.

4.5. Behavioral procedures

4.5.1. Lever training, surgical procedures, and amphetamine self-administration (n = 27)

At 52 days of age, rats were food restricted to 85% of their free-feeding weights and trained to lever press on a single lever for 20% sucrose solution (dissolved in distilled water). Rats were trained on a fixed-ratio 1 (FR-1) schedule of reinforcement. Responses made on the active lever resulted in sucrose reinforcement; inactive lever led to no programmed response. Data for both active and inactive lever presses were recorded. The same active lever (left or right) was maintained for each rat for both the sucrose training and amphetamine self-administration phases of the experiment.

Following sucrose training, rats were returned to free-feeding weights for the remainder of the experiment. Three days later rats were deeply anesthetized with ketamine (80 mg/kg; 1 mg/ml, i.p.) and diazepam (5 mg/kg; 1 mg/ml, i.p.) prior to jugular catheter implantation. Polyurethane catheters measured approximately 12 cm. in length, 0.2 mm internal diameter (SAI Infusion Technologies) were inserted through a dorsal incision and were tunneled under the skin and into the rat’s left jugular vein. Catheter tubing from the jugular vein was connected subcutaneously to a 22-gauge back-mounted cannula (Plastics One; Roanoke, VA) and sutured to surgical mesh (Biomedical Structures; Warwick, RI). A stainless-steel bolt covered the catheter cannula cap to prevent chewing/damage to the back mount. To maintain patency and to protect against infection, catheters were flushed daily with heparinized saline (10-30 IU/ml; 0.1 ml before self-administration and 0.1 ml after self-administration) and cefazolin (50 mg/ml; 0.1 ml intravenous (i.v.) infusion).

Rats recovered for 7-10 days before amphetamine self-administration sessions (Figure 1A). Amphetamine self-administration sessions were 1h. Active lever responses resulted in a 100 μl infusion over 5.9-sec of 0.1 mg/kg/infusion d-amphetamine sulfate (Sigma Aldrich; dissolved in 0.9% sterile NaCl), and cue complex cue light illumination above active lever, and a tone (3000 Hz, 80 dB). When the infusion was over, the cue complex was terminated and the house light illuminated to signal a 20-sec time out period. Lever pressing during the time out period was recorded but did not result in amphetamine reinforcement. Inactive lever responding was recorded but had no programmed consequence. Rats remained on an FR-1 schedule during amphetamine self-administration until stable responding was achieved. The following criteria were used to determine stable responding: (1) 20% or less variability in active lever presses across three sessions; (2) greater than a 2:1 ratio of active: inactive lever presses across the three sessions; (3) at least ten infusions per session (Bastle et al., 2012). After 11 sessions, responding was stable for each environmental group and extinction commenced. During extinction sessions, all cues were off and therefore responding on the active lever was recorded but had no programmed consequence. Rats were injected with CTX (200 mg/kg, ip) or saline daily following the 6th extinction session (Weiland et al., 2015). When rats met the extinction criterion (Bastle et al., 2012) cue-induced reinstatement was tested. During the first 3-seconds of the cue-induced reinstatement session, there was a non-contingent illumination of the cue complex. Thereafter, responding on the active lever resulted in the presentation of the cue complex followed by the house light for 20-sec. CTX or saline continued to be administered throughout the next extinction phase (4d) and subsequent amphetamine-induced reinstatement test. For the amphetamine-induced reinstatement session, rats were pretreated (15min) with amphetamine (0.25 mg/kg, sc) or saline and placed in the operant chamber. Responses on the active or inactive levers had no programmed consequence. Five days after the amphetamine-induced reinstatement test, rats were sacrificed and brains were removed for western blots (see below) (Figure 1A).

4.6. Self-administration data analysis

Active and inactive lever responding during self-administration and extinction sessions were analyzed in separate mixed factorial ANOVAs with the respective lever responding as the within subjects factor and environmental condition (EC, IC, SC) and treatment (CTX or saline) as the between subjects factors. During the reinstatement tests, we compared active lever responding during the last extinction session to active lever responding during the reinstatement session. Active lever responses during both sessions were the within subjects factor and treatment (CTX or saline) and environmental condition (EC, IC, SC) were the between subjects factors. For experiment 1, we also compared responding across treatment and environmental condition within the reinstatement session itself. For all ANOVAs, the alpha level was set at 0.05. Following the ANOVAs, Bonferroni-corrected planned simple effects comparisons were performed, and the resulting F values are reported. All datasets are available on request after publication. The raw data supporting the conclusions of this manuscript will be made available by the authors to any qualified researcher institution or organization after publication.

4.7. Experiment 2: Effect of daily amphetamine on GLT1 or xCT in the NAc and mPFC in standard housed rats (n = 24)

Cocaine induces downregulation of GLT1 and xCT in the NAc (Knackstedt et al., 2010), but it is not yet determined if experimenter-administered amphetamine injections induces the same downregulation. Given that our previous experiment did not result in a downregulation of GLT1 expression in the NAc, we designed this experiment to determine if controlled injections of amphetamine resulted in reduced expression of GLT1 or xCT in the NAc or the medial prefrontal cortex (mPFC). To test the effects of repeated amphetamine exposure on GLT1 and xCT expression, we used a separate cohort of 24 pair-housed male Sprague-Dawley rats and did not examine differential rearing. Rats arrived between 100-125g, and they were housed two rats per cage. After three weeks in our colony room, rats were randomly assigned to experimental or control groups: Amp-CTX, Amp-Sal, Sal-CTX, and Sal-Sal. Amphetamine (1.0 mg/kg, s.c.) or saline was administered every day for eight days. Locomotor activity in response to amphetamine or saline was measured on day 1 and day 8. Following the amphetamine exposure phase, rats were administered CTX (200 mg/kg, i.p.) or saline for 10 exposures. After the last CTX or saline exposure the mPFC and the NAc was removed (see below). Locomotor data was analyzed with a mixed factorial repeated measures ANOVA, drug (amphetamine or saline) x treatment (CTX or saline) x session, and all significance was set at <0.05.

4.8. Brain extraction and tissue dissection

Rats were briefly exposed to isoflurane gas until effect and rapidly decapitated. The whole brain was extracted and immediately frozen in powdered dry ice and stored at −80°C until processing. The brain was sliced into 1mm coronal sections using the brain matrix and the NAc and the mPFC were dissected using the 1mm biopsy punches. A rat brain stereotaxic atlas (Paxinos and Watson 1998) was used to identify regions of interest. The tissue punches from each brain region were homogenized separately in extraction buffer (150mM NaCl, 50mM Tris-Base pH 8.0)+EDTA+protease inhibitors (ThermoScientific Protease cocktail) or in experiment 2 tissue punches were placed in 1ml of sucrose harvest buffer (0.32M sucrose, 2mM EDTA, 50mM Tris-HCl pH 7.4)+protease inhibitor (ThermoScientific Protease cocktail) and homogenized. The homogenized samples were stored in the sucrose harvest buffer at −80°C until the subcellular fractionation was completed.

4.8.1. Subcellular fractionation and western blot

Homogenized samples were removed from the −80°C freezer and thawed prior to cellular fractionation. The samples were spun at 1,000g for 5 min to remove the nucleus and insoluble cellular materials at 4°C. The supernatant (S1) was transferred to a new aliquot and contained the crude membrane and cytosolic fractions. The S1 aliquot was returned to the refrigerated centrifuge and spun at 10,000g for 20 min at 4°C. The resulting pellet (P2) contained the crude membrane fraction and the supernatant was the cleared cytosol which was discarded. The P2 pellet was resuspended in 200μl of NP-40 lysis buffer+protease inhibitor and vortexed until the pellet was dissolved. The P2 fraction was used for all protein quantification and western blot procedures for both experiments (Knackstedt et al., 2010; Lacrosse et al., 2016). The total protein quantification from samples was interpolated using the Pierce BCA protein assay using standard bovine albumin. (ThermoScientific) and Nanodrop 8000. Each sample was measured twice and averaged to determine the total protein concentration.

Ten micrograms of total protein homogenate was loaded into each lane of a precast acrylamide gels in the gradient of 4-20% bis-acrylamide (1mm; Bio Rad). Laemmli loading buffer (Bio Rad) was added to the tissue samples (10μg) under reducing conditions (βME) to reach a standard loading volume 30μl. Proteins were separated through the acrylamide gel using 100v for 90 minutes. The gel was rinsed with deionized water and the separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane using a wet transfer method at 75-80v for 120 min. The visible protean standards (Precision Plus Protein Standard Dual Color, Bio Rad) were marked with an ink pen to prevent fading during blocking/rinsing and antibody application.

The PVDF membrane was blocked with a 3% non-fat dry milk+tris-buffered-saline+tween20 (TBST) solution for 30 min before primary antibody application. Primary antibodies were applied overnight at 4°C: Anti-GLT1 (AB41621, Abcam, 1:10,000), anti-xCT (AB175186, Abcam, 1:2,000) antibody concentration. All primary antibodies were mixed in a new 3% non-fat dry milk+0.1TBST solution. All secondary antibody (AB97051, Abcam1:20,000 or 1:30,000) were mixed in a new 5% non-fat dry milk+0.1TBST solution. The proteins of interest (GLT1 and xCT) were normalized against calnexin (AB22595, Abcam, 1:10,000).

After each primary and secondary antibody application the PVDF membrane was washed in 0.1% TBST for 60 min, exchanging the TBST with fresh TBST every 10 min. The horseradish peroxidase conjugate was probed using 1ml of chemiluminescent solution (Clarity ECL; Bio Rad) and imaged with a Kodak Image Station. Digital files (.TIF) were analyzed in ImageJ. The PVDF membranes were stripped of protein of interest antibodies and calnexin was probed. The signal of each protein of interest was normalized to calnexin. To make comparisons across experimental groups, values were normalized to the SC rats in the saline condition for experiment 1. For experiment 2, the values were normalized to the Sal-Sal rats to make comparisons across experimental groups. The normalized value for each protein and brain area was analyzed using a factorial ANOVA with drug condition (amphetamine or saline) and CTX treatment group (CTX or saline) as the between subjects factors.

Highlights.

  • Enrichment synergizes with ceftriaxone to suppress cue-induced reinstatement

  • Ceftriaxone suppresses amphetamine-induced reinstatement in standard and enriched housed rats

  • Amphetamine self-administration does not change GLT1 expression in the mPFC or NAc

  • Amphetamine behavioral sensitization does not change GLT1 or xCT expression in the mPFC or NAc

Acknowledgments

This work was supported by DA035435. DLA was supported by DA035435. EJG was supported by DA035435-S1. The authors do not have any conflicts of interests that would confound the interpretation of the research or data presented. The biochemical results were collected at the Molecular Biology Core supported by Kansas State University College of Veterinary Medicine.

Footnotes

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