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Continuous exposure to dizocilpine facilitates the acquisition and escalation of cocaine consumption in male Sprague-Dawley rats
Drug and Alcohol Dependence 147 (2015) 137–143
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Drug and Alcohol Dependence journal homepage: www.elsevier.com/locate/drugalcdep
Continuous exposure to dizocilpine facilitates the acquisition and escalation of cocaine consumption in male Sprague-Dawley rats Richard M. Allen ∗ , Bruce H. Mandt, Jillian Jaskunas, Amanda Hackley, Alyssa Shickedanz, David Bergkamp Department of Psychology, University of Colorado Denver, 1200 Larimer Street, Denver, CO 80204, United States
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Article history: Received 17 September 2014 Received in revised form 25 November 2014 Accepted 26 November 2014 Available online 9 December 2014 Keywords: Cocaine self-administration Escalation Acquisition N-methyl-d-aspartate (NMDA) receptor (NMDAR) Dizocilpine
a b s t r a c t Background: Blocking N-methyl-d-aspartate (NMDA) glutamate receptors (NMDARs) prevents cocaine locomotor sensitization, but facilitates escalation of cocaine self-administration and produces ambiguous effects on acquisition of cocaine self-administration. This study used a recently described model of acquisition and escalation to test the hypothesis that continuous NMDAR antagonism functionally increases the effects of a given dose of cocaine. Methods: We assessed acquisition of cocaine self-administration (0.6 mg/kg/infusion) in rats treated continuously with either vehicle or the NMDAR antagonist dizocilpine (0.4 mg/kg/day) for 14 consecutive 2 h fixed ratio 1 (FR1) sessions. In a separate experiment that assessed the effect of dizocilpine treatment on escalation of cocaine self-administration, rats acquired cocaine self-administration (0.6 mg/kg/infusion) prior to vehicle or dizocilpine treatment. Then, immediately post-acquisition, rats were treated continuously with either vehicle or dizocilpine and allowed to self-administer either 0.6 or 1.2 mg/kg/infusion cocaine for an additional seven consecutive 2 h FR1 sessions. Results: Relative to vehicle-treated rats, a significantly greater percentage of dizocilpine-treated rats acquired cocaine self-administration. During the escalation experiment, both vehicle- and dizocilpinetreated rats escalated intake of 1.2 mg/kg/infusion cocaine. Whereas vehicle-treated rats exhibited stable intake of 0.6 mg/kg/infusion cocaine, dizocilpine-treated rats escalated intake of this moderate cocaine dose to levels indistinguishable from intake levels produced by self-administration of the high cocaine dose (i.e., 1.2 mg/kg/infusion). Conclusions: These findings suggest that chronic NMDAR blockade potentiates, rather than attenuates, cocaine’s effects and argue for reconsideration of the role of NMDARs in cocaine “addiction-like” behavior. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction About 25 years ago, Karler et al. (1989) demonstrated that repeatedly co-administering the non-competitive N-methyl-daspartate (NMDA) receptor (NMDAR) antagonist dizocilpine with cocaine prevents the development of cocaine-induced locomotor sensitization. This finding has been replicated and extended in many ways since then. For example, chronic blockade of NMDARs prevents not only the behavioral sensitization induced by repeated experimenter-administered injections of cocaine, but also the neurobiological correlates of these changes, such as altered dopamine
∗ Corresponding author. Tel.: +1 303 556 6740; fax: +1 303 556 3520. E-mail address: [email protected] (R.M. Allen). http://dx.doi.org/10.1016/j.drugalcdep.2014.11.027 0376-8716/© 2014 Elsevier Ireland Ltd. All rights reserved.
receptor sensitivity (Li et al., 1999) and up-regulation of glutamate receptor subunits (Scheggi et al., 2002). Additionally, repeated experimenter-administered injections of cocaine and other psychostimulants prior to the start of cocaine self-administration sessions (i.e., pre-exposure) can facilitate subsequent acquisition of cocaine self-administration (Horger et al., 1990; Mandt et al., 2008; Zhang and Kosten, 2007) and motivation to respond for cocaine under a progressive ratio (PR) schedule of reinforcement (Suto et al., 2002, 2003); this has been interpreted as reflecting the development of sensitization to the reinforcing effectiveness of cocaine. As with locomotor sensitization, blockade of NMDARs during this “pre-exposure” can block both the facilitation of acquisition (Schenk et al., 1993b) as well as the increase in responding for cocaine under the PR procedure (Suto et al., 2003). In contrast, blocking NMDARs during cocaine selfadministration produces effects that are either somewhat
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equivocal or in frank opposition to the blockade of behavioral sensitization or simple forms of associative learning produced in the context of experimenter-administered cocaine. For example, rats injected with dizocilpine before the start of daily self-administration sessions fail to discriminate the active from inactive levers for cocaine (Schenk et al., 1993a). However, these rats consume more cocaine than rats in the vehicle control group. Similarly, blockade of NMDARs with either LY235959 (Allen et al., 2007a,b) or dizocilpine (Allen, 2014) facilitates (rather than prevents) the escalation of cocaine consumption that occurs when rats self-administer cocaine in long-access sessions. This effect is most clearly demonstrated when the NMDAR antagonist is administered continuously via osmotic minipump; rats given pre-session injections with LY235959 show marked disruptions in behavior (despite high rates of intake in some subjects; Allen et al., 2007a). In addition to the facilitation of escalation, continuous exposure to LY235959 leads to post-escalation “upward” shifts in a cocaine dose-response curve (Allen et al., 2007a) and increases in responding for cocaine under a PR schedule of reinforcement (Allen et al., 2007b). All together, these data suggest that cocaine produces larger effects on post-consumption measures (e.g., escalation, break point) when administered together with an NMDAR antagonist. But if continuous blockade of NMDARs produces effects functionally equivalent to increasing the dose of cocaine, then this treatment should facilitate acquisition of cocaine self-administration, rather than attenuate it (e.g., Schenk et al., 1993a). Further, administering an NMDAR antagonist via osmotic minipump should reduce the behavioral impairments that might interfere with lever discrimination during acquisition. Recently, we have developed a procedure to quantify acquisition of cocaine self-administration early in a rat’s self-administration history and subsequent escalation of cocaine consumption in these subjects with minimal exposure to cocaine (Mandt et al., 2012a,b). In the model, acquisition of cocaine is defined as selfadministration of at least 4 mg/kg cocaine per session in each of three consecutive 2 h sessions. Rats that meet this criterion clearly discriminate the active from inactive levers (Mandt et al., 2012a,b). Further, post-acquisition escalation of consumption of cocaine can be measured in 2 h sessions and is dose-dependent; escalation is observed when rats self-administer 1.2 mg/kg/infusion cocaine but not 0.6 mg/kg/infusion cocaine in daily 2 h self-administration sessions (Mandt et al., 2012b). The aim of the present study was to test the effects of continuous infusion of dizocilpine on (1) the acquisition of cocaine selfadministration and (2) the escalation of cocaine self-administration using this novel experimental procedure that allows manipulation at each phase (i.e., acquisition and post-acquisition escalation; Mandt et al., 2012b). Further, dizocilpine was administered via osmotic minipump to minimize the disruptions in behavior that can occur with bolus injections of the drug (e.g., Carter, 1994; Ginski and Witkin, 1994). Our results show that dizocilpine facilitates both the acquisition and escalation of cocaine self-administration.
2. Materials and methods 2.1. Animals Adult outbred male Sprague-Dawley rats (n = 66) weighing between 225 and 250 g (∼8 weeks of age) were purchased from Charles River Laboratories (Portage, MI) and used in the two different experiments of this study. Rats in the first experiment were used to assess the impact of dizocilpine on the acquisition of cocaine self-administration (0.6 mg/kg/infusion; n = 31) and rats in the second experiment were used to assess the impact of dizocilpine on
the escalation of cocaine self-administration induced by a switch in cocaine dose (e.g., Mandt et al., 2012b; 0.6 → 1.2 mg/kg group, n = 16; 0.6 → 0.6 mg/kg group, n = 19). All rats were housed individually with ad libitum access to food and water in an animal care facility at the University of Colorado Denver (CU Denver). Rats were housed on a 12 h light/dark cycle (lights on at 0700 h), and all testing was conducted during the light cycle. All animal care and use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the CU Denver Institutional Animal Care and Use Committee. 2.2. Catheter construction and placement Intravenous catheters were constructed in the laboratory and surgically implanted into the right jugular vein under ketamine (100 mg/kg, i.m.) and xylazine (10 mg/kg, i.m.) anesthesia using established procedures (Thomsen and Caine, 2005). Rats received acetaminophen as an analgesic in their drinking water (20 mg/ml) for 48 h pre- and post-surgery. Rats recovered from surgery for at least 1 week before self-administration training began. Catheters were flushed with 0.3 ml of bacteriostatic 0.9% sodium chloride containing heparin (30 Units/ml) both before and after each self-administration session. To verify catheter patency, sodium thiopental (20 mg/kg, i.v.) was administered at the conclusion of each experiment (i.e., acquisition or escalation) or when a problem was suspected. 2.3. Subcutaneous osmotic minipump placement To establish steady-state dizocilpine levels and avoid the behavioral impairments associated with repeated i.p. dizocilpine injections, rats were implanted with subcutaneous osmotic minipumps (Alzet, Cupertino, CA; 2ML2—acquisition experiment; 2ML1—escalation experiment). All rats were implanted with minipumps containing either vehicle (0.9% sodium chloride) or dizocilpine (0.4 mg/kg/day; e.g., Allen, 2014) at one of two different time-points. In the acquisition experiment, minipumps were implanted in rats 48 h prior to beginning cocaine selfadministration. In the escalation experiment, minipumps were implanted following acquisition of cocaine self-administration (i.e., following session x + 2; see Section 2.4). Rats in the escalation experiment were allowed to recover for 48 h prior to returning to self-administration testing. For both the acquisition and escalation experiments, rats were randomly assigned to the different treatment conditions (i.e., vehicle or dizocilpine). 2.4. Self-administration training Rats self-administered cocaine in one of 16 Plexiglas and metal operant conditioning chambers (29 × 24 × 21 cm; Med Associates, St. Albans, VT, USA) housed within sound-attenuating cabinets. The chambers had two retractable levers on the front wall with stimulus lights positioned 6 cm above each lever. A tone presentation speaker (Sonalert Tone Generator, 2900 Hz) and a white noise speaker (90 dB) were mounted 12 cm above the floor on the wall opposite the levers. A houselight (100 mA) was mounted 6 cm above the tone speaker, and a computer-controlled syringe pump delivered cocaine infusions. All behavioral events were monitored and controlled by a personal computer using MED-PC for Windows software (Med Associates). All self-administration sessions began with the extension of the retractable levers, white noise activation, and illumination of the stimulus light above the active lever. One s after session initiation, a single cocaine priming infusion was delivered. Priming infusions were always the same dose as the self-administered dose: either 0.6 or 1.2 mg/kg/infusion, depending on the experimental condition.
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During this priming infusion and all subsequent self-administered infusions, the stimulus light over the active lever was turned off, and a tone-houselight stimulus complex was activated for 15 s coinciding with a “time-out” period. All self-administration sessions were 2 h in duration. During acquisition, responses on the right lever were reinforced with a cocaine infusion (0.6 mg/kg delivered over 5–7 s based on the weight of the rat) according to a fixed ratio 1 (FR1) schedule of reinforcement. Responses emitted on the right lever during cocaine infusion and the 15 s stimulus complex were not reinforced and were recorded separately from reinforced responses. Responses on the left lever were recorded but had no programmed consequence. Acquisition was defined as the first of three consecutive sessions during which a rat consumed at least 4 mg/kg cocaine; this session was designated “x”, the day of acquisition (Mandt et al., 2012a,b). In our lab, rats that meet these criteria continue to reliably self-administer cocaine; and these criteria are similar to intakebased acquisition criteria used by other labs (e.g., Carroll and Lac, 1997; Mantsch et al., 2001). Rats in the acquisition experiment were tested for 15 consecutive days (coinciding with the expected functional life of the model 2ML2 minipump). Rats in the escalation experiment were implanted with minipumps immediately upon meeting acquisition criteria (i.e., after session x + 2) and allowed to recover for 48 h. These rats then self-administered either 0.6 mg/kg/infusion cocaine or 1.2 mg/kg/infusion cocaine under an FR1 schedule in 2 h sessions. During acquisition, rats in the escalation experiment were tested 5 days a week. However, following acquisition and minipump implantation, rats in the escalation experiment were tested for an additional 7 consecutive days (i.e., sessions x + 3 to x + 9; coinciding with the expected functional life of the model 2ML1 minipump). 2.5. Exclusions and exceptions In total, 17 of the 66 rats used in this study were excluded from final analysis. Three rats were removed from the acquisition experiment because they repeatedly removed their minipump incision site sutures. Six rats in the 0.6 → 1.2 mg/kg/infusion group and four rats in the 0.6 → 0.6 mg/kg/infusion group of the escalation experiment were excluded because they did not acquire self-administration. Finally, in the 0.6 → 0.6 mg/kg/infusion escalation group, two rats (one vehicle and one dizocilpine treated) unexpectedly died during the study and two rats (one vehicle and one dizocilpine treated) became detached from the cocaine delivery apparatus during their escalation sessions, resulting in lost data points and exclusion from statistical analysis.
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2.7. Drugs The National Institute on Drug Abuse generously provided the (−) cocaine hydrochloride used in these experiments. For i.v. infusions, cocaine was dissolved in sterile 0.9% sodium chloride. Dizocilpine was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in 0.9% sodium chloride. To check catheter patency, sodium thiopental (Sigma-Aldrich) was dissolved in 0.9% sodium chloride and administered i.v. (20 mg/kg). Ketamine was purchased from MWI (Boise, ID, USA) and xylazine was purchased from SigmaAldrich. Drug weights refer to the salt.
3. Results 3.1. Acquisition of cocaine self-administration Acquisition of cocaine self-administration (0.6 mg/kg/infusion) was measured in rats that received continuous subcutaneous infusion of dizocilpine (0.4 mg/kg/day) or vehicle through surgically implanted osmotic minipumps. A Kaplan–Meier survival analysis of the acquisition data revealed a between-group difference (log rank Chi-square = 6.95; p < 0.01). A significantly higher percentage of dizocilpine-treated rats learned to self-administer cocaine by session 15 than did vehicle-treated rats (12/15 or 80% vs. 4/13 or 31%, respectively; Fig. 1A). Dizocilpine- and vehicle-treated rats that acquired self-administration, however, did not differ in the average number of days to reach acquisition (5.3 ± 1.0 vs. 5.3 ± 0.9 days, respectively). Cocaine intake during the earliest pre- and post-acquisition sessions did not differ between dizocilpine- and vehicle-treated rats (Fig. 1B). A two-way RMANOVA of cocaine intake during those sessions (i.e., x − 1, x, x + 1, x + 2) revealed a main effect of session [F3, 24 = 14.65; p < 0.001], but not a main effect of treatment or a session by treatment interaction. Rats in both groups consumed significantly more cocaine during sessions x, x + 1 and x + 2 than before session x. Importantly, both groups discriminated the active and inactive levers after meeting acquisition criteria (sessions x, x + 1, and x + 2), but not before (sessions x − 2 and x − 1; Fig. 1C). Total active lever (reinforced responses plus active lever responses during the “time-out” period) and inactive lever responses also are presented on each of the 14 sessions for all vehicle- and dizocilpinetreated rats that acquired self-administration (Fig. 2). Aligning data according to session, rather than day of acquisition, also revealed that both vehicle- and dizocilpine-treated rats displayed clear lever discrimination by the end of the acquisition experiment.
3.2. Escalation of cocaine consumption 2.6. Data analysis All statistical analyses were conducted using PASW Statistics, version 21.0 (IBM Corp., Somers, NY, USA). For the acquisition experiment, the percentages of vehicle and dizocilpine treated rats that acquired self-administration on each of the 14 consecutive sessions were analyzed with Kaplan–Meier survival analysis. Cocaine intake during acquisition was analyzed on the session prior to acquisition (i.e., x − 1) and the three post-acquisition sessions (i.e., x to x + 2) either with two-way (acquisition experiment) or three-way (escalation experiment) repeated measures ANOVA (RMANOVA). Treatment (vehicle or dizocilpine), cocaine dose (0.6 or 1.2 mg/kg/infusion), and session (within-subjects variable) were treated as independent variables; intake was treated as the dependent variable. When main or interaction effects were revealed, one-way ANOVA or RMANOVA was used for post-hoc analysis.
The effect of dizocilpine treatment on escalation of cocaine consumption was measured in rats that acquired cocaine self-administration under control conditions. After learning to self-administer cocaine (0.6 mg/kg/infusion), osmotic minipumps containing dizocilpine (0.4 mg/kg/day) or vehicle were surgically implanted subcutaneously. After recovery, rats were permitted to self-administer either 0.6 or 1.2 mg/kg/infusion cocaine in daily 2 h sessions. There were no statistically significant between-group differences in cocaine intake during acquisition of cocaine self-administration (i.e., through session x + 2) prior to dizocilpine treatment (Fig. 2). As previously demonstrated (Mandt et al., 2012b), control (i.e., vehicle-treated) rats that selfadministered 1.2 mg/kg/infusion cocaine escalated intake over the next seven sessions but rats that continued to self-administer 0.6 mg/kg/infusion cocaine did not (Fig. 3). In contrast, dizocilpinetreated rats that self-administered either dose of cocaine (0.6
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Fig. 3. Effects of continuous dizocilpine treatment on escalation of cocaine consumption. Cocaine intake is shown for the acquisition phase (i.e., x to x + 2; 0.6 mg/kg/infusion cocaine) prior to minipump implantation, and for the seven postacquisition sessions (either 0.6 or 1.2 mg/kg/infusion cocaine) in rats subsequently treated with either vehicle (Veh) or dizocilpine (Diz; 0.4 mg/kg/day). X represents the day of acquisition. The dashed line indicates the 48-h break in testing immediately after acquisition to allow for minipump implantation and recovery. Data are mean values ± SEM and N values are given in parentheses. & p < 0.05: All other groups vs. the veh-treated 0.6 → 0.6 mg/kg/inf group; # p < 0.05: session vs. the first post-acquisition escalation session (i.e., x + 3).
Fig. 1. Effects of continuous dizocilpine treatment on acquisition of cocaine selfadministration. (A) Percentages of vehicle (Veh) and dizocilpine (Diz; 0.4 mg/kg/day) treated rats that acquired cocaine (0.6 mg/kg/infusion) self-administration on each of the 15 consecutive sessions. (B) Cocaine intake (0.6 mg/kg/infusion) is shown for the two sessions prior to and three acquisition sessions in Veh and Diz treated rats. X represents the day of acquisition. (C) Total active lever (reinforced responses plus active lever responses during the “time-out” period; (AL) and inactive lever (IL) responding for the same acquisition sessions as in (B). Data in (B) and (C) are mean values ± SEM, and N values are given in parentheses.
or 1.2 mg/kg/infusion) escalated their rate of self-administration (Fig. 3). A three-way RMANOVA of cocaine intake in the sessions after acquisition (x + 3 through x + 9) revealed main effects of session [F6, 102 = 14.61; p < 0.001] and cocaine dose [F1, 17 = 9.86; p = 0.006], and a session x treatment x cocaine dose interaction [F6, 102 = 4.81; p < 0.001]. Within-subjects one-way RMANOVAs of cocaine intake revealed increases across the seven self-administration sessions in the 1.2-vehicle group [F6, 24 = 10.02; p < 0.001], the 1.2-dizocilpine group [F6, 24 = 4.78; p = 0.002], and the 0.6-dizocilpine group [F6, 24 = 9.22; p < 0.001], but not the 0.6-vehicle group. Betweensubjects post-hoc analyses with one-way ANOVA revealed that cocaine intake did not differ between groups on the first or second sessions (i.e., x + 3 and x + 4), but did differ between groups on all subsequent sessions. Beginning with the third escalation session (i.e., x + 5), all other groups consumed more cocaine than the 0.6-vehicle group (p < 0.01 for all comparisons), but did not differ from each other. Total active lever (reinforced responses plus active lever responses during the “time-out” period) and inactive lever responses are shown for vehicle- and dizocilpine-treated rats on each of the seven escalation sessions (i.e., x + 3 to x + 9) in the 0.6 → 0.6 mg/kg/infusion and 0.6 → 1.2 mg/kg/infusion escalation groups (Fig. 4A and B, respectively). Regardless of treatment or escalation dose condition, every group maintained a clear lever preference throughout the escalation experiment. 4. Discussion
Fig. 2. Lever discrimination over the course of the 14-session acquisition experiment in vehicle (Veh)- and dizocilpine (Diz)-treated rats. Total active lever (reinforced responses plus active lever responses during the “time-out” period; (AL) and inactive lever (IL) responses are shown for the same Veh- and Diz-treated rats shown in Fig. 1. Data are mean values ± SEM. Veh, n = 4; Diz, n = 12.
In this study, we found that chronic blockade of NMDARs with dizocilpine facilitates both the acquisition of cocaine self-administration as well as post-acquisition escalation of consumption under high-dose short access conditions. Both findings are novel, and important extensions of our previous work (Allen et al., 2007a,b; Allen, 2014). All together, the findings demonstrate that chronic blockade of NMDARs during cocaine self-administration produces effects functionally equivalent to increasing the self-administered dose of cocaine. Interestingly, the dizocilpine-induced potentiation of cocaine’s effects reported here is consistent with some reports of the effects of NMDAR blockade in humans: relative to placebo, the uncompetitive NMDAR antagonist memantine increases the subjective responses to smoked
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Fig. 4. Lever discrimination over the course of the seven-session escalation experiment in vehicle (Veh)- and dizocilpine (Diz)-treated rats. Total active lever (reinforced responses plus active lever responses during the “time-out” period; (AL) and inactive lever (IL) responses are shown for the same Veh- and Diz-treated rats shown in Fig. 3. Panel A shows lever responses for rats in the 0.6 → 0.6 mg/kg/inf group, whereas panel B shows responses for rats in the 0.6 → 1.2 mg/kg/inf group. Data are mean values ± SEM. Veh-0.6 mg/kg, n = 6; Diz-0.6 mg/kg, n = 5; Veh1.2 mg/kg, n = 5; Diz-1.2 mg/kg, n = 5.
cocaine (Collins et al., 1998, 2007). Thus, our study further highlights the need for reconsideration of the role of NMDARs in cocaine “addiction-like” behavior. In the present study, a higher percentage of rats treated with 0.4 mg/kg/day dizocilpine learned to self-administer 0.6 mg/kg/infusion cocaine compared with rats treated continuously with vehicle under identical conditions (80% vs. 31%, respectively). This facilitation is consistent with our hypothesis that chronic blockade of NMDARs functionally increases the reinforcing effectiveness of a dose of cocaine. In our previous work with this acquisition procedure, we showed that acquisition of cocaine self-administration is a function of dose, where the percent of rats that acquire increases with increases in the self-administered dose of cocaine (Mandt et al., 2012a). Indeed, increasing the selfadministered dose of cocaine increases reinforcer effectiveness and/or motivation to self-administer cocaine across a number of procedures including (but not limited to) acquisition under continuous reinforcement schedules (Schenk and Partridge, 2000), break point under PR schedules of reinforcement (Roberts et al., 1989), and selection of cocaine in choice procedures (Negus, 2003). We chose the single cocaine dose in our current acquisition study (i.e., 0.6 mg/kg/infusion), in part, to enable determination of two possible effects of dizocilpine on acquisition: (1) facilitation (as revealed here) or (2) blockade (as concluded by Schenk et al., 1993a). Rats injected with dizocilpine subcutaneously 30 min prior to the start of daily sessions fail to discriminate active from inactive levers when learning to self-administer cocaine (Schenk
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et al., 1993a), a finding interpreted as a blockade of acquisition (in contrast, our subjects showed clear lever discrimination; Figs. 1C and 2). However, close inspection of that published data set reveals earlier and greater consumption of cocaine in rats injected with dizocilpine, compared with vehicle controls, despite the lack of lever discrimination. These data are strikingly similar to our present findings in which continuous NMDAR blockade facilitates both the acquisition and escalation of cocaine self-administration. In our own studies, bolus injections of the competitive NMDAR antagonist LY235959 30 min before the start of a long-access self-administration session produced erratic behavior that included very high rates of cocaine consumption: administering the drug via osmotic minipump revealed the facilitation of escalation without the erratic behavior (Allen et al., 2007a). Considered broadly and together with the present findings, it is difficult to argue that NMDAR antagonism blocks acquisition of cocaine self-administration. Although lever discrimination is clearly an important facet of learning to self-administer cocaine, the current data reveal that NMDAR blockade actually facilitates acquisition of cocaine self-administration. In our current acquisition study, we were surprised that the rate of acquisition in our control group was so low (4/13 or 31% of rats). In contrast, prior to mini-pump implantation, 25/35 (71%) rats in the escalation experiment acquired self-administration of this same dose of cocaine. This higher percentage also is consistent with our previous work with this cocaine dose (e.g., Mandt et al., 2012b). However, the current acquisition study was different from the escalation study, and our previous work, in two key ways. First, rats in the acquisition study were the only rats surgically implanted with an osmotic mini-pump before learning to self-administer cocaine. Second, rats in the current acquisition study were the only rats to self-administer cocaine during 15 continuous daily sessions: all other rats (including our previous studies) learned to self-administer cocaine in 5 consecutive weekday sessions, with no sessions on weekends. Thus, rats in all other conditions had intermittent 72 h withdrawal periods during their self-administration training. In light of studies demonstrating that the withdrawal from cocaine self-administration is necessary to reveal certain neurobiological and behavioral changes (e.g., glutamate receptor expression or the “incubation of cocaine craving”; Pickens et al., 2011), we believe the differences in withdrawal period in our study could partly explain the lower rates of acquisition. We have not yet empirically tested the effect of these different training conditions on rate of acquisition and as such, these explanations are speculative; however, our current study suggests these variables warrant further investigation. Despite the low rate of acquisition in the control group, we remain confident that we can conclude NMDAR blockade facilitated acquisition of cocaine self-administration. First, vehicle- and dizocilpine-treated rats were from the same cohort of animals, and data were collected from each condition at the same time. Second, the data presented here represent data collected from two cohorts of animals, each with equivalent numbers of vehicle- and dizocilpine-treated rats. In each instance, a higher percentage of dizocilpine-treated rats acquired cocaine self-administration compared with vehicle-treated rats (cohort 1: 8/8 dizocilpine-treated vs. 3/6 vehicle-treated; cohort 2: 4/8 dizocilpine-treated vs. 1/7 vehicle-treated; total: 12/15 dizocilpine-treated vs. 4/13 vehicletreated). Thus, in two separate cohorts, rats randomly assigned to the dizocilpine-treatment group were more likely to acquire cocaine self-administration than rats randomly assigned to the vehicle-treatment group. In the present study, continuous infusion of dizocilpine facilitated the escalation of cocaine self-administration, also, when administered to rats post-acquisition. This finding both replicates and extends our earlier work (Allen et al., 2007a,b; Allen, 2014)
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in which continuous infusion of competitive (i.e., LY235959) and non-competitive (i.e., dizocilpine) NMDAR antagonists facilitated escalation of consumption in rats that self-administered cocaine under long-access conditions (Ahmed and Koob, 1998). This concordance suggests that the mechanisms that underlie escalation in our model (present study; Mandt et al., 2012b) and a commonly used long-access procedure (Ahmed and Koob, 1998) are similar. To the extent that the escalation is mediated by similar mechanisms, our model, in which rats self-administer cocaine in 2 h sessions immediately after acquiring the operant, can assess these changes much more efficiently than can be done with long-access (i.e., 6 h) sessions. Dizocilpine facilitated escalation of consumption only in rats that self-administered the low dose of cocaine (0.6 mg/kg/infusion), a dose that does not engender any escalation under control conditions. The result was a rate and magnitude of escalation equivalent to that observed in rats that self-administered twice the dose of cocaine alone (1.2 mg/kg/infusion cocaine, vehiclefilled minipump), representing a 50% increase over seven days. Dizocilpine did not further increase this rate of escalation in rats that self-administered cocaine under the high-dose condition (1.2 mg/kg/infusion cocaine). As we have not observed greater rates of escalation previously (Mandt et al., 2012b), this may reflect a ceiling on the rate of escalation possible with our procedure. To note, rats treated continuously with dizocilpine increase their rate of cocaine consumption (1 mg/kg/infusion) by 38% over the first five long-access (i.e., 6 h) sessions, previously the highest rate of escalation we have observed in our studies (Allen, 2014). However, to fully evaluate the effects of dizocilpine on rate of escalation of cocaine consumption in our novel escalation procedure, a complete dose-response study would be required. It is possible that the single priming infusion at the start of each self-administration session produced sensitization to cocaine-induced effects (e.g., locomotor sensitization). Indeed, we have observed locomotor sensitization to cocaine following five repeated single daily experimenter-administered intravenous infusions of cocaine delivered in the self-administration context (i.e., single priming infusions; unpublished observation). There is a large body of literature (and our own unpublished observations) showing that chronic NMDAR antagonism blocks the development of sensitization to the locomotor stimulating effects of cocaine (e.g., Karler et al., 1989; Li et al., 1999; Scheggi et al., 2002). If sensitization induced by the priming infusion is associated with the acquisition of self-administration, then dizocilpine would be expected to attenuate, rather than facilitate, this process; previous research has shown that dizocilpine blocks the enhancement of acquisition produced by repeated non-contingent amphetamine pre-treatment (Schenk et al., 1993b). However, this was not the case in the present study: dizocilpine-treatment facilitated the acquisition of cocaine self-administration. Thus, these data further disambiguate the role of locomotor sensitization and its associated mechanisms from cocaine self-administration, and the role of NMDARs in these processes. It is challenging to link glutamatergic mechanisms described for other cocaine exposure conditions and behavioral procedures to the data we collected here. However, the facilitation of acquisition and escalation we observe with chronic NMDAR blockade in our studies is consistent with data that show hypoglutamatergic effects and decreased neuronal excitability produced by cocaine exposure. For example, cocaine exposure produces decreases in basal glutamate levels (Baker et al., 2003; Bell et al., 2000; Hotsenpiller et al., 2001; Moran et al., 2005), decreases in glutamate turnover rates (Smith et al., 2003), decreased neural responses to iontophoretic application of glutamate (White et al., 1995), and decreases in glutamate immunolabelling (Keys et al., 1998; Kozell and Meshul, 2001, 2003, 2004). Decreases in nucleus accumbens medium spiny
neuron excitability have been measured also following both repeated, experimenter-administered injections of cocaine (Kourrich et al., 2007; Kourrich and Thomas, 2009) and cocaine self-administration when assessed in early (i.e., 1 to 2 days) abstinence (Schramm-Sapyta et al., 2006; Ortinski et al., 2012). In the present escalation study, all cocaine self-administration sessions were conducted within 24 to 72 h from the prior selfadministration session, and thus were measured during brief withdrawal periods. This is important, because many of the changes that contribute to neuronal excitability and cocaine induced synaptic plasticity are withdrawal-time dependent, in some cases reversing after prolonged periods of abstinence (e.g., Kourrich et al., 2007; Conrad et al., 2008; Ortinski et al., 2012). Clearly, the next step in this line of research is to measure these neurobiological changes directly in this novel acquisition and escalation model. Author disclosures Role of funding source David Bergkamp’s effort on this project was supported by National Institutes of Health Grant R25 GM083333. The funding source did not have any other role in this study. Contributors Allen, Mandt, Jaskunas, Hackley, Schickedanz, and Bergkamp participated in research design. Allen, Mandt, Jaskunas, Hackley, Schickedanz, and Bergkamp conducted experiments. Allen and Mandt performed data analysis and wrote the manuscript. All authors have and read and approved the submission of this version of the manuscript. Conflict of interest statement No conflict declared. Acknowledgments We gratefully acknowledge Irma Spahic and Leland Copenhagen for technical support. References Ahmed, S.H., Koob, G.F., 1998. Transition from moderate to excessive drug intake: change in hedonic set point. Science 282, 298–300. Allen, R.M., Dykstra, L.A., Carelli, R.M., 2007a. Continuous exposure to the competitive N-methyl-d-aspartate receptor antagonist, LY235959, facilitates escalation of cocaine consumption in Sprague-Dawley rats. Psychopharmacology (Berl.) 191, 341–351. Allen, R.M., Uban, K.A., Atwood, E.M., Albeck, D.S., Yamamoto, D.J., 2007b. Continuous intracerebroventricular infusion of the competitive NMDA receptor antagonist, LY235959, facilitates escalation of cocaine self-administration and increases break point for cocaine in Sprague-Dawley rats. Pharmacol. Biochem. Behav. 88, 82–88. Allen, R.M., 2014. Continuous exposure to dizocilpine facilitates escalation of cocaine consumption in male Sprague-Dawley rats. Drug Alcohol Depend. 134, 38–43, http://dx.doi.org/10.1016/j.drugalcdep.2013.09.005. Baker, D.A., McFarland, K., Lake, R.W., Shen, H., Tang, X.C., Toda, S., Kalivas, P.W., 2003. Neuroadaptations in cystine–glutamate exchange underlie cocaine relapse. Nat. Neurosci. 6, 743–749. Bell, K., Duffy, G.A., Kalivas, P.W., 2000. Context-specific enhancement of glutamate transmission by cocaine. Neuropsychopharmacology 23, 335–344. Carter, A.J., 1994. Many agents that antagonize the NMDA receptor-channel complex in vivo also cause disturbances of motor coordination. J. Pharmacol. Exp.Ther. 269, 573–580. Carroll, M.E., Lac, S.T., 1997. Acquisition of i.v. amphetamine and cocaine selfadministration in rats as a function of dose. Psychopharmacology (Berl.) 129, 206–214.
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Contents lists available at ScienceDirect
Drug and Alcohol Dependence journal homepage: www.elsevier.com/locate/drugalcdep
Continuous exposure to dizocilpine facilitates the acquisition and escalation of cocaine consumption in male Sprague-Dawley rats Richard M. Allen ∗ , Bruce H. Mandt, Jillian Jaskunas, Amanda Hackley, Alyssa Shickedanz, David Bergkamp Department of Psychology, University of Colorado Denver, 1200 Larimer Street, Denver, CO 80204, United States
a r t i c l e
i n f o
Article history: Received 17 September 2014 Received in revised form 25 November 2014 Accepted 26 November 2014 Available online 9 December 2014 Keywords: Cocaine self-administration Escalation Acquisition N-methyl-d-aspartate (NMDA) receptor (NMDAR) Dizocilpine
a b s t r a c t Background: Blocking N-methyl-d-aspartate (NMDA) glutamate receptors (NMDARs) prevents cocaine locomotor sensitization, but facilitates escalation of cocaine self-administration and produces ambiguous effects on acquisition of cocaine self-administration. This study used a recently described model of acquisition and escalation to test the hypothesis that continuous NMDAR antagonism functionally increases the effects of a given dose of cocaine. Methods: We assessed acquisition of cocaine self-administration (0.6 mg/kg/infusion) in rats treated continuously with either vehicle or the NMDAR antagonist dizocilpine (0.4 mg/kg/day) for 14 consecutive 2 h fixed ratio 1 (FR1) sessions. In a separate experiment that assessed the effect of dizocilpine treatment on escalation of cocaine self-administration, rats acquired cocaine self-administration (0.6 mg/kg/infusion) prior to vehicle or dizocilpine treatment. Then, immediately post-acquisition, rats were treated continuously with either vehicle or dizocilpine and allowed to self-administer either 0.6 or 1.2 mg/kg/infusion cocaine for an additional seven consecutive 2 h FR1 sessions. Results: Relative to vehicle-treated rats, a significantly greater percentage of dizocilpine-treated rats acquired cocaine self-administration. During the escalation experiment, both vehicle- and dizocilpinetreated rats escalated intake of 1.2 mg/kg/infusion cocaine. Whereas vehicle-treated rats exhibited stable intake of 0.6 mg/kg/infusion cocaine, dizocilpine-treated rats escalated intake of this moderate cocaine dose to levels indistinguishable from intake levels produced by self-administration of the high cocaine dose (i.e., 1.2 mg/kg/infusion). Conclusions: These findings suggest that chronic NMDAR blockade potentiates, rather than attenuates, cocaine’s effects and argue for reconsideration of the role of NMDARs in cocaine “addiction-like” behavior. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction About 25 years ago, Karler et al. (1989) demonstrated that repeatedly co-administering the non-competitive N-methyl-daspartate (NMDA) receptor (NMDAR) antagonist dizocilpine with cocaine prevents the development of cocaine-induced locomotor sensitization. This finding has been replicated and extended in many ways since then. For example, chronic blockade of NMDARs prevents not only the behavioral sensitization induced by repeated experimenter-administered injections of cocaine, but also the neurobiological correlates of these changes, such as altered dopamine
∗ Corresponding author. Tel.: +1 303 556 6740; fax: +1 303 556 3520. E-mail address: [email protected] (R.M. Allen). http://dx.doi.org/10.1016/j.drugalcdep.2014.11.027 0376-8716/© 2014 Elsevier Ireland Ltd. All rights reserved.
receptor sensitivity (Li et al., 1999) and up-regulation of glutamate receptor subunits (Scheggi et al., 2002). Additionally, repeated experimenter-administered injections of cocaine and other psychostimulants prior to the start of cocaine self-administration sessions (i.e., pre-exposure) can facilitate subsequent acquisition of cocaine self-administration (Horger et al., 1990; Mandt et al., 2008; Zhang and Kosten, 2007) and motivation to respond for cocaine under a progressive ratio (PR) schedule of reinforcement (Suto et al., 2002, 2003); this has been interpreted as reflecting the development of sensitization to the reinforcing effectiveness of cocaine. As with locomotor sensitization, blockade of NMDARs during this “pre-exposure” can block both the facilitation of acquisition (Schenk et al., 1993b) as well as the increase in responding for cocaine under the PR procedure (Suto et al., 2003). In contrast, blocking NMDARs during cocaine selfadministration produces effects that are either somewhat
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equivocal or in frank opposition to the blockade of behavioral sensitization or simple forms of associative learning produced in the context of experimenter-administered cocaine. For example, rats injected with dizocilpine before the start of daily self-administration sessions fail to discriminate the active from inactive levers for cocaine (Schenk et al., 1993a). However, these rats consume more cocaine than rats in the vehicle control group. Similarly, blockade of NMDARs with either LY235959 (Allen et al., 2007a,b) or dizocilpine (Allen, 2014) facilitates (rather than prevents) the escalation of cocaine consumption that occurs when rats self-administer cocaine in long-access sessions. This effect is most clearly demonstrated when the NMDAR antagonist is administered continuously via osmotic minipump; rats given pre-session injections with LY235959 show marked disruptions in behavior (despite high rates of intake in some subjects; Allen et al., 2007a). In addition to the facilitation of escalation, continuous exposure to LY235959 leads to post-escalation “upward” shifts in a cocaine dose-response curve (Allen et al., 2007a) and increases in responding for cocaine under a PR schedule of reinforcement (Allen et al., 2007b). All together, these data suggest that cocaine produces larger effects on post-consumption measures (e.g., escalation, break point) when administered together with an NMDAR antagonist. But if continuous blockade of NMDARs produces effects functionally equivalent to increasing the dose of cocaine, then this treatment should facilitate acquisition of cocaine self-administration, rather than attenuate it (e.g., Schenk et al., 1993a). Further, administering an NMDAR antagonist via osmotic minipump should reduce the behavioral impairments that might interfere with lever discrimination during acquisition. Recently, we have developed a procedure to quantify acquisition of cocaine self-administration early in a rat’s self-administration history and subsequent escalation of cocaine consumption in these subjects with minimal exposure to cocaine (Mandt et al., 2012a,b). In the model, acquisition of cocaine is defined as selfadministration of at least 4 mg/kg cocaine per session in each of three consecutive 2 h sessions. Rats that meet this criterion clearly discriminate the active from inactive levers (Mandt et al., 2012a,b). Further, post-acquisition escalation of consumption of cocaine can be measured in 2 h sessions and is dose-dependent; escalation is observed when rats self-administer 1.2 mg/kg/infusion cocaine but not 0.6 mg/kg/infusion cocaine in daily 2 h self-administration sessions (Mandt et al., 2012b). The aim of the present study was to test the effects of continuous infusion of dizocilpine on (1) the acquisition of cocaine selfadministration and (2) the escalation of cocaine self-administration using this novel experimental procedure that allows manipulation at each phase (i.e., acquisition and post-acquisition escalation; Mandt et al., 2012b). Further, dizocilpine was administered via osmotic minipump to minimize the disruptions in behavior that can occur with bolus injections of the drug (e.g., Carter, 1994; Ginski and Witkin, 1994). Our results show that dizocilpine facilitates both the acquisition and escalation of cocaine self-administration.
2. Materials and methods 2.1. Animals Adult outbred male Sprague-Dawley rats (n = 66) weighing between 225 and 250 g (∼8 weeks of age) were purchased from Charles River Laboratories (Portage, MI) and used in the two different experiments of this study. Rats in the first experiment were used to assess the impact of dizocilpine on the acquisition of cocaine self-administration (0.6 mg/kg/infusion; n = 31) and rats in the second experiment were used to assess the impact of dizocilpine on
the escalation of cocaine self-administration induced by a switch in cocaine dose (e.g., Mandt et al., 2012b; 0.6 → 1.2 mg/kg group, n = 16; 0.6 → 0.6 mg/kg group, n = 19). All rats were housed individually with ad libitum access to food and water in an animal care facility at the University of Colorado Denver (CU Denver). Rats were housed on a 12 h light/dark cycle (lights on at 0700 h), and all testing was conducted during the light cycle. All animal care and use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the CU Denver Institutional Animal Care and Use Committee. 2.2. Catheter construction and placement Intravenous catheters were constructed in the laboratory and surgically implanted into the right jugular vein under ketamine (100 mg/kg, i.m.) and xylazine (10 mg/kg, i.m.) anesthesia using established procedures (Thomsen and Caine, 2005). Rats received acetaminophen as an analgesic in their drinking water (20 mg/ml) for 48 h pre- and post-surgery. Rats recovered from surgery for at least 1 week before self-administration training began. Catheters were flushed with 0.3 ml of bacteriostatic 0.9% sodium chloride containing heparin (30 Units/ml) both before and after each self-administration session. To verify catheter patency, sodium thiopental (20 mg/kg, i.v.) was administered at the conclusion of each experiment (i.e., acquisition or escalation) or when a problem was suspected. 2.3. Subcutaneous osmotic minipump placement To establish steady-state dizocilpine levels and avoid the behavioral impairments associated with repeated i.p. dizocilpine injections, rats were implanted with subcutaneous osmotic minipumps (Alzet, Cupertino, CA; 2ML2—acquisition experiment; 2ML1—escalation experiment). All rats were implanted with minipumps containing either vehicle (0.9% sodium chloride) or dizocilpine (0.4 mg/kg/day; e.g., Allen, 2014) at one of two different time-points. In the acquisition experiment, minipumps were implanted in rats 48 h prior to beginning cocaine selfadministration. In the escalation experiment, minipumps were implanted following acquisition of cocaine self-administration (i.e., following session x + 2; see Section 2.4). Rats in the escalation experiment were allowed to recover for 48 h prior to returning to self-administration testing. For both the acquisition and escalation experiments, rats were randomly assigned to the different treatment conditions (i.e., vehicle or dizocilpine). 2.4. Self-administration training Rats self-administered cocaine in one of 16 Plexiglas and metal operant conditioning chambers (29 × 24 × 21 cm; Med Associates, St. Albans, VT, USA) housed within sound-attenuating cabinets. The chambers had two retractable levers on the front wall with stimulus lights positioned 6 cm above each lever. A tone presentation speaker (Sonalert Tone Generator, 2900 Hz) and a white noise speaker (90 dB) were mounted 12 cm above the floor on the wall opposite the levers. A houselight (100 mA) was mounted 6 cm above the tone speaker, and a computer-controlled syringe pump delivered cocaine infusions. All behavioral events were monitored and controlled by a personal computer using MED-PC for Windows software (Med Associates). All self-administration sessions began with the extension of the retractable levers, white noise activation, and illumination of the stimulus light above the active lever. One s after session initiation, a single cocaine priming infusion was delivered. Priming infusions were always the same dose as the self-administered dose: either 0.6 or 1.2 mg/kg/infusion, depending on the experimental condition.
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During this priming infusion and all subsequent self-administered infusions, the stimulus light over the active lever was turned off, and a tone-houselight stimulus complex was activated for 15 s coinciding with a “time-out” period. All self-administration sessions were 2 h in duration. During acquisition, responses on the right lever were reinforced with a cocaine infusion (0.6 mg/kg delivered over 5–7 s based on the weight of the rat) according to a fixed ratio 1 (FR1) schedule of reinforcement. Responses emitted on the right lever during cocaine infusion and the 15 s stimulus complex were not reinforced and were recorded separately from reinforced responses. Responses on the left lever were recorded but had no programmed consequence. Acquisition was defined as the first of three consecutive sessions during which a rat consumed at least 4 mg/kg cocaine; this session was designated “x”, the day of acquisition (Mandt et al., 2012a,b). In our lab, rats that meet these criteria continue to reliably self-administer cocaine; and these criteria are similar to intakebased acquisition criteria used by other labs (e.g., Carroll and Lac, 1997; Mantsch et al., 2001). Rats in the acquisition experiment were tested for 15 consecutive days (coinciding with the expected functional life of the model 2ML2 minipump). Rats in the escalation experiment were implanted with minipumps immediately upon meeting acquisition criteria (i.e., after session x + 2) and allowed to recover for 48 h. These rats then self-administered either 0.6 mg/kg/infusion cocaine or 1.2 mg/kg/infusion cocaine under an FR1 schedule in 2 h sessions. During acquisition, rats in the escalation experiment were tested 5 days a week. However, following acquisition and minipump implantation, rats in the escalation experiment were tested for an additional 7 consecutive days (i.e., sessions x + 3 to x + 9; coinciding with the expected functional life of the model 2ML1 minipump). 2.5. Exclusions and exceptions In total, 17 of the 66 rats used in this study were excluded from final analysis. Three rats were removed from the acquisition experiment because they repeatedly removed their minipump incision site sutures. Six rats in the 0.6 → 1.2 mg/kg/infusion group and four rats in the 0.6 → 0.6 mg/kg/infusion group of the escalation experiment were excluded because they did not acquire self-administration. Finally, in the 0.6 → 0.6 mg/kg/infusion escalation group, two rats (one vehicle and one dizocilpine treated) unexpectedly died during the study and two rats (one vehicle and one dizocilpine treated) became detached from the cocaine delivery apparatus during their escalation sessions, resulting in lost data points and exclusion from statistical analysis.
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2.7. Drugs The National Institute on Drug Abuse generously provided the (−) cocaine hydrochloride used in these experiments. For i.v. infusions, cocaine was dissolved in sterile 0.9% sodium chloride. Dizocilpine was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in 0.9% sodium chloride. To check catheter patency, sodium thiopental (Sigma-Aldrich) was dissolved in 0.9% sodium chloride and administered i.v. (20 mg/kg). Ketamine was purchased from MWI (Boise, ID, USA) and xylazine was purchased from SigmaAldrich. Drug weights refer to the salt.
3. Results 3.1. Acquisition of cocaine self-administration Acquisition of cocaine self-administration (0.6 mg/kg/infusion) was measured in rats that received continuous subcutaneous infusion of dizocilpine (0.4 mg/kg/day) or vehicle through surgically implanted osmotic minipumps. A Kaplan–Meier survival analysis of the acquisition data revealed a between-group difference (log rank Chi-square = 6.95; p < 0.01). A significantly higher percentage of dizocilpine-treated rats learned to self-administer cocaine by session 15 than did vehicle-treated rats (12/15 or 80% vs. 4/13 or 31%, respectively; Fig. 1A). Dizocilpine- and vehicle-treated rats that acquired self-administration, however, did not differ in the average number of days to reach acquisition (5.3 ± 1.0 vs. 5.3 ± 0.9 days, respectively). Cocaine intake during the earliest pre- and post-acquisition sessions did not differ between dizocilpine- and vehicle-treated rats (Fig. 1B). A two-way RMANOVA of cocaine intake during those sessions (i.e., x − 1, x, x + 1, x + 2) revealed a main effect of session [F3, 24 = 14.65; p < 0.001], but not a main effect of treatment or a session by treatment interaction. Rats in both groups consumed significantly more cocaine during sessions x, x + 1 and x + 2 than before session x. Importantly, both groups discriminated the active and inactive levers after meeting acquisition criteria (sessions x, x + 1, and x + 2), but not before (sessions x − 2 and x − 1; Fig. 1C). Total active lever (reinforced responses plus active lever responses during the “time-out” period) and inactive lever responses also are presented on each of the 14 sessions for all vehicle- and dizocilpinetreated rats that acquired self-administration (Fig. 2). Aligning data according to session, rather than day of acquisition, also revealed that both vehicle- and dizocilpine-treated rats displayed clear lever discrimination by the end of the acquisition experiment.
3.2. Escalation of cocaine consumption 2.6. Data analysis All statistical analyses were conducted using PASW Statistics, version 21.0 (IBM Corp., Somers, NY, USA). For the acquisition experiment, the percentages of vehicle and dizocilpine treated rats that acquired self-administration on each of the 14 consecutive sessions were analyzed with Kaplan–Meier survival analysis. Cocaine intake during acquisition was analyzed on the session prior to acquisition (i.e., x − 1) and the three post-acquisition sessions (i.e., x to x + 2) either with two-way (acquisition experiment) or three-way (escalation experiment) repeated measures ANOVA (RMANOVA). Treatment (vehicle or dizocilpine), cocaine dose (0.6 or 1.2 mg/kg/infusion), and session (within-subjects variable) were treated as independent variables; intake was treated as the dependent variable. When main or interaction effects were revealed, one-way ANOVA or RMANOVA was used for post-hoc analysis.
The effect of dizocilpine treatment on escalation of cocaine consumption was measured in rats that acquired cocaine self-administration under control conditions. After learning to self-administer cocaine (0.6 mg/kg/infusion), osmotic minipumps containing dizocilpine (0.4 mg/kg/day) or vehicle were surgically implanted subcutaneously. After recovery, rats were permitted to self-administer either 0.6 or 1.2 mg/kg/infusion cocaine in daily 2 h sessions. There were no statistically significant between-group differences in cocaine intake during acquisition of cocaine self-administration (i.e., through session x + 2) prior to dizocilpine treatment (Fig. 2). As previously demonstrated (Mandt et al., 2012b), control (i.e., vehicle-treated) rats that selfadministered 1.2 mg/kg/infusion cocaine escalated intake over the next seven sessions but rats that continued to self-administer 0.6 mg/kg/infusion cocaine did not (Fig. 3). In contrast, dizocilpinetreated rats that self-administered either dose of cocaine (0.6
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Fig. 3. Effects of continuous dizocilpine treatment on escalation of cocaine consumption. Cocaine intake is shown for the acquisition phase (i.e., x to x + 2; 0.6 mg/kg/infusion cocaine) prior to minipump implantation, and for the seven postacquisition sessions (either 0.6 or 1.2 mg/kg/infusion cocaine) in rats subsequently treated with either vehicle (Veh) or dizocilpine (Diz; 0.4 mg/kg/day). X represents the day of acquisition. The dashed line indicates the 48-h break in testing immediately after acquisition to allow for minipump implantation and recovery. Data are mean values ± SEM and N values are given in parentheses. & p < 0.05: All other groups vs. the veh-treated 0.6 → 0.6 mg/kg/inf group; # p < 0.05: session vs. the first post-acquisition escalation session (i.e., x + 3).
Fig. 1. Effects of continuous dizocilpine treatment on acquisition of cocaine selfadministration. (A) Percentages of vehicle (Veh) and dizocilpine (Diz; 0.4 mg/kg/day) treated rats that acquired cocaine (0.6 mg/kg/infusion) self-administration on each of the 15 consecutive sessions. (B) Cocaine intake (0.6 mg/kg/infusion) is shown for the two sessions prior to and three acquisition sessions in Veh and Diz treated rats. X represents the day of acquisition. (C) Total active lever (reinforced responses plus active lever responses during the “time-out” period; (AL) and inactive lever (IL) responding for the same acquisition sessions as in (B). Data in (B) and (C) are mean values ± SEM, and N values are given in parentheses.
or 1.2 mg/kg/infusion) escalated their rate of self-administration (Fig. 3). A three-way RMANOVA of cocaine intake in the sessions after acquisition (x + 3 through x + 9) revealed main effects of session [F6, 102 = 14.61; p < 0.001] and cocaine dose [F1, 17 = 9.86; p = 0.006], and a session x treatment x cocaine dose interaction [F6, 102 = 4.81; p < 0.001]. Within-subjects one-way RMANOVAs of cocaine intake revealed increases across the seven self-administration sessions in the 1.2-vehicle group [F6, 24 = 10.02; p < 0.001], the 1.2-dizocilpine group [F6, 24 = 4.78; p = 0.002], and the 0.6-dizocilpine group [F6, 24 = 9.22; p < 0.001], but not the 0.6-vehicle group. Betweensubjects post-hoc analyses with one-way ANOVA revealed that cocaine intake did not differ between groups on the first or second sessions (i.e., x + 3 and x + 4), but did differ between groups on all subsequent sessions. Beginning with the third escalation session (i.e., x + 5), all other groups consumed more cocaine than the 0.6-vehicle group (p < 0.01 for all comparisons), but did not differ from each other. Total active lever (reinforced responses plus active lever responses during the “time-out” period) and inactive lever responses are shown for vehicle- and dizocilpine-treated rats on each of the seven escalation sessions (i.e., x + 3 to x + 9) in the 0.6 → 0.6 mg/kg/infusion and 0.6 → 1.2 mg/kg/infusion escalation groups (Fig. 4A and B, respectively). Regardless of treatment or escalation dose condition, every group maintained a clear lever preference throughout the escalation experiment. 4. Discussion
Fig. 2. Lever discrimination over the course of the 14-session acquisition experiment in vehicle (Veh)- and dizocilpine (Diz)-treated rats. Total active lever (reinforced responses plus active lever responses during the “time-out” period; (AL) and inactive lever (IL) responses are shown for the same Veh- and Diz-treated rats shown in Fig. 1. Data are mean values ± SEM. Veh, n = 4; Diz, n = 12.
In this study, we found that chronic blockade of NMDARs with dizocilpine facilitates both the acquisition of cocaine self-administration as well as post-acquisition escalation of consumption under high-dose short access conditions. Both findings are novel, and important extensions of our previous work (Allen et al., 2007a,b; Allen, 2014). All together, the findings demonstrate that chronic blockade of NMDARs during cocaine self-administration produces effects functionally equivalent to increasing the self-administered dose of cocaine. Interestingly, the dizocilpine-induced potentiation of cocaine’s effects reported here is consistent with some reports of the effects of NMDAR blockade in humans: relative to placebo, the uncompetitive NMDAR antagonist memantine increases the subjective responses to smoked
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Fig. 4. Lever discrimination over the course of the seven-session escalation experiment in vehicle (Veh)- and dizocilpine (Diz)-treated rats. Total active lever (reinforced responses plus active lever responses during the “time-out” period; (AL) and inactive lever (IL) responses are shown for the same Veh- and Diz-treated rats shown in Fig. 3. Panel A shows lever responses for rats in the 0.6 → 0.6 mg/kg/inf group, whereas panel B shows responses for rats in the 0.6 → 1.2 mg/kg/inf group. Data are mean values ± SEM. Veh-0.6 mg/kg, n = 6; Diz-0.6 mg/kg, n = 5; Veh1.2 mg/kg, n = 5; Diz-1.2 mg/kg, n = 5.
cocaine (Collins et al., 1998, 2007). Thus, our study further highlights the need for reconsideration of the role of NMDARs in cocaine “addiction-like” behavior. In the present study, a higher percentage of rats treated with 0.4 mg/kg/day dizocilpine learned to self-administer 0.6 mg/kg/infusion cocaine compared with rats treated continuously with vehicle under identical conditions (80% vs. 31%, respectively). This facilitation is consistent with our hypothesis that chronic blockade of NMDARs functionally increases the reinforcing effectiveness of a dose of cocaine. In our previous work with this acquisition procedure, we showed that acquisition of cocaine self-administration is a function of dose, where the percent of rats that acquire increases with increases in the self-administered dose of cocaine (Mandt et al., 2012a). Indeed, increasing the selfadministered dose of cocaine increases reinforcer effectiveness and/or motivation to self-administer cocaine across a number of procedures including (but not limited to) acquisition under continuous reinforcement schedules (Schenk and Partridge, 2000), break point under PR schedules of reinforcement (Roberts et al., 1989), and selection of cocaine in choice procedures (Negus, 2003). We chose the single cocaine dose in our current acquisition study (i.e., 0.6 mg/kg/infusion), in part, to enable determination of two possible effects of dizocilpine on acquisition: (1) facilitation (as revealed here) or (2) blockade (as concluded by Schenk et al., 1993a). Rats injected with dizocilpine subcutaneously 30 min prior to the start of daily sessions fail to discriminate active from inactive levers when learning to self-administer cocaine (Schenk
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et al., 1993a), a finding interpreted as a blockade of acquisition (in contrast, our subjects showed clear lever discrimination; Figs. 1C and 2). However, close inspection of that published data set reveals earlier and greater consumption of cocaine in rats injected with dizocilpine, compared with vehicle controls, despite the lack of lever discrimination. These data are strikingly similar to our present findings in which continuous NMDAR blockade facilitates both the acquisition and escalation of cocaine self-administration. In our own studies, bolus injections of the competitive NMDAR antagonist LY235959 30 min before the start of a long-access self-administration session produced erratic behavior that included very high rates of cocaine consumption: administering the drug via osmotic minipump revealed the facilitation of escalation without the erratic behavior (Allen et al., 2007a). Considered broadly and together with the present findings, it is difficult to argue that NMDAR antagonism blocks acquisition of cocaine self-administration. Although lever discrimination is clearly an important facet of learning to self-administer cocaine, the current data reveal that NMDAR blockade actually facilitates acquisition of cocaine self-administration. In our current acquisition study, we were surprised that the rate of acquisition in our control group was so low (4/13 or 31% of rats). In contrast, prior to mini-pump implantation, 25/35 (71%) rats in the escalation experiment acquired self-administration of this same dose of cocaine. This higher percentage also is consistent with our previous work with this cocaine dose (e.g., Mandt et al., 2012b). However, the current acquisition study was different from the escalation study, and our previous work, in two key ways. First, rats in the acquisition study were the only rats surgically implanted with an osmotic mini-pump before learning to self-administer cocaine. Second, rats in the current acquisition study were the only rats to self-administer cocaine during 15 continuous daily sessions: all other rats (including our previous studies) learned to self-administer cocaine in 5 consecutive weekday sessions, with no sessions on weekends. Thus, rats in all other conditions had intermittent 72 h withdrawal periods during their self-administration training. In light of studies demonstrating that the withdrawal from cocaine self-administration is necessary to reveal certain neurobiological and behavioral changes (e.g., glutamate receptor expression or the “incubation of cocaine craving”; Pickens et al., 2011), we believe the differences in withdrawal period in our study could partly explain the lower rates of acquisition. We have not yet empirically tested the effect of these different training conditions on rate of acquisition and as such, these explanations are speculative; however, our current study suggests these variables warrant further investigation. Despite the low rate of acquisition in the control group, we remain confident that we can conclude NMDAR blockade facilitated acquisition of cocaine self-administration. First, vehicle- and dizocilpine-treated rats were from the same cohort of animals, and data were collected from each condition at the same time. Second, the data presented here represent data collected from two cohorts of animals, each with equivalent numbers of vehicle- and dizocilpine-treated rats. In each instance, a higher percentage of dizocilpine-treated rats acquired cocaine self-administration compared with vehicle-treated rats (cohort 1: 8/8 dizocilpine-treated vs. 3/6 vehicle-treated; cohort 2: 4/8 dizocilpine-treated vs. 1/7 vehicle-treated; total: 12/15 dizocilpine-treated vs. 4/13 vehicletreated). Thus, in two separate cohorts, rats randomly assigned to the dizocilpine-treatment group were more likely to acquire cocaine self-administration than rats randomly assigned to the vehicle-treatment group. In the present study, continuous infusion of dizocilpine facilitated the escalation of cocaine self-administration, also, when administered to rats post-acquisition. This finding both replicates and extends our earlier work (Allen et al., 2007a,b; Allen, 2014)
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in which continuous infusion of competitive (i.e., LY235959) and non-competitive (i.e., dizocilpine) NMDAR antagonists facilitated escalation of consumption in rats that self-administered cocaine under long-access conditions (Ahmed and Koob, 1998). This concordance suggests that the mechanisms that underlie escalation in our model (present study; Mandt et al., 2012b) and a commonly used long-access procedure (Ahmed and Koob, 1998) are similar. To the extent that the escalation is mediated by similar mechanisms, our model, in which rats self-administer cocaine in 2 h sessions immediately after acquiring the operant, can assess these changes much more efficiently than can be done with long-access (i.e., 6 h) sessions. Dizocilpine facilitated escalation of consumption only in rats that self-administered the low dose of cocaine (0.6 mg/kg/infusion), a dose that does not engender any escalation under control conditions. The result was a rate and magnitude of escalation equivalent to that observed in rats that self-administered twice the dose of cocaine alone (1.2 mg/kg/infusion cocaine, vehiclefilled minipump), representing a 50% increase over seven days. Dizocilpine did not further increase this rate of escalation in rats that self-administered cocaine under the high-dose condition (1.2 mg/kg/infusion cocaine). As we have not observed greater rates of escalation previously (Mandt et al., 2012b), this may reflect a ceiling on the rate of escalation possible with our procedure. To note, rats treated continuously with dizocilpine increase their rate of cocaine consumption (1 mg/kg/infusion) by 38% over the first five long-access (i.e., 6 h) sessions, previously the highest rate of escalation we have observed in our studies (Allen, 2014). However, to fully evaluate the effects of dizocilpine on rate of escalation of cocaine consumption in our novel escalation procedure, a complete dose-response study would be required. It is possible that the single priming infusion at the start of each self-administration session produced sensitization to cocaine-induced effects (e.g., locomotor sensitization). Indeed, we have observed locomotor sensitization to cocaine following five repeated single daily experimenter-administered intravenous infusions of cocaine delivered in the self-administration context (i.e., single priming infusions; unpublished observation). There is a large body of literature (and our own unpublished observations) showing that chronic NMDAR antagonism blocks the development of sensitization to the locomotor stimulating effects of cocaine (e.g., Karler et al., 1989; Li et al., 1999; Scheggi et al., 2002). If sensitization induced by the priming infusion is associated with the acquisition of self-administration, then dizocilpine would be expected to attenuate, rather than facilitate, this process; previous research has shown that dizocilpine blocks the enhancement of acquisition produced by repeated non-contingent amphetamine pre-treatment (Schenk et al., 1993b). However, this was not the case in the present study: dizocilpine-treatment facilitated the acquisition of cocaine self-administration. Thus, these data further disambiguate the role of locomotor sensitization and its associated mechanisms from cocaine self-administration, and the role of NMDARs in these processes. It is challenging to link glutamatergic mechanisms described for other cocaine exposure conditions and behavioral procedures to the data we collected here. However, the facilitation of acquisition and escalation we observe with chronic NMDAR blockade in our studies is consistent with data that show hypoglutamatergic effects and decreased neuronal excitability produced by cocaine exposure. For example, cocaine exposure produces decreases in basal glutamate levels (Baker et al., 2003; Bell et al., 2000; Hotsenpiller et al., 2001; Moran et al., 2005), decreases in glutamate turnover rates (Smith et al., 2003), decreased neural responses to iontophoretic application of glutamate (White et al., 1995), and decreases in glutamate immunolabelling (Keys et al., 1998; Kozell and Meshul, 2001, 2003, 2004). Decreases in nucleus accumbens medium spiny
neuron excitability have been measured also following both repeated, experimenter-administered injections of cocaine (Kourrich et al., 2007; Kourrich and Thomas, 2009) and cocaine self-administration when assessed in early (i.e., 1 to 2 days) abstinence (Schramm-Sapyta et al., 2006; Ortinski et al., 2012). In the present escalation study, all cocaine self-administration sessions were conducted within 24 to 72 h from the prior selfadministration session, and thus were measured during brief withdrawal periods. This is important, because many of the changes that contribute to neuronal excitability and cocaine induced synaptic plasticity are withdrawal-time dependent, in some cases reversing after prolonged periods of abstinence (e.g., Kourrich et al., 2007; Conrad et al., 2008; Ortinski et al., 2012). Clearly, the next step in this line of research is to measure these neurobiological changes directly in this novel acquisition and escalation model. Author disclosures Role of funding source David Bergkamp’s effort on this project was supported by National Institutes of Health Grant R25 GM083333. The funding source did not have any other role in this study. Contributors Allen, Mandt, Jaskunas, Hackley, Schickedanz, and Bergkamp participated in research design. Allen, Mandt, Jaskunas, Hackley, Schickedanz, and Bergkamp conducted experiments. Allen and Mandt performed data analysis and wrote the manuscript. All authors have and read and approved the submission of this version of the manuscript. Conflict of interest statement No conflict declared. Acknowledgments We gratefully acknowledge Irma Spahic and Leland Copenhagen for technical support. References Ahmed, S.H., Koob, G.F., 1998. Transition from moderate to excessive drug intake: change in hedonic set point. Science 282, 298–300. Allen, R.M., Dykstra, L.A., Carelli, R.M., 2007a. Continuous exposure to the competitive N-methyl-d-aspartate receptor antagonist, LY235959, facilitates escalation of cocaine consumption in Sprague-Dawley rats. Psychopharmacology (Berl.) 191, 341–351. Allen, R.M., Uban, K.A., Atwood, E.M., Albeck, D.S., Yamamoto, D.J., 2007b. Continuous intracerebroventricular infusion of the competitive NMDA receptor antagonist, LY235959, facilitates escalation of cocaine self-administration and increases break point for cocaine in Sprague-Dawley rats. Pharmacol. Biochem. Behav. 88, 82–88. Allen, R.M., 2014. Continuous exposure to dizocilpine facilitates escalation of cocaine consumption in male Sprague-Dawley rats. Drug Alcohol Depend. 134, 38–43, http://dx.doi.org/10.1016/j.drugalcdep.2013.09.005. Baker, D.A., McFarland, K., Lake, R.W., Shen, H., Tang, X.C., Toda, S., Kalivas, P.W., 2003. Neuroadaptations in cystine–glutamate exchange underlie cocaine relapse. Nat. Neurosci. 6, 743–749. Bell, K., Duffy, G.A., Kalivas, P.W., 2000. Context-specific enhancement of glutamate transmission by cocaine. Neuropsychopharmacology 23, 335–344. Carter, A.J., 1994. Many agents that antagonize the NMDA receptor-channel complex in vivo also cause disturbances of motor coordination. J. Pharmacol. Exp.Ther. 269, 573–580. Carroll, M.E., Lac, S.T., 1997. Acquisition of i.v. amphetamine and cocaine selfadministration in rats as a function of dose. Psychopharmacology (Berl.) 129, 206–214.
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