Hippocampal memory system function and the regulation of cocaine self-administration behavior in rats

Behavioural Brain Research 151 (2004) 225–238

Research report

Hippocampal memory system function and the regulation of cocaine self-administration behavior in rats Yolanda D. Black a , Kristen Green-Jordan a , Howard B. Eichenbaum b , Kathleen M. Kantak a,∗ a

Laboratory of Behavioral Neuroscience, Department of Psychology and Program in Neuroscience, Boston University, 64 Cummington Street, Boston, MA 02215, USA b Laboratory of Cognitive Neurobiology, Department of Psychology and Program in Neuroscience, Boston University, 64 Cummington Street, Boston, MA 02215, USA Received 8 April 2003; received in revised form 22 August 2003; accepted 30 August 2003

Abstract There is considerable interest in elucidating neurocognitive mechanisms of cocaine addiction. This report focuses on the hippocampal memory system. Using food reward, two cognitive tasks were examined after lidocaine inactivation of the dorsal (dSUB) or ventral (vSUB) subiculum, the primary hippocampal output regions in rats. These tasks were conducted to first identify functionally relevant stereotaxic coordinates within the hippocampal memory system, in order to then examine its role in regulating drug-seeking and drug-taking behavior studied under a contextually discriminable FI 5-min(FR5:S) second-order schedule of cocaine and brief stimulus delivery. Inactivation of the dSUB and vSUB with 10 ␮g lidocaine impaired hippocampal-dependent win-shift performance. Amygdalar-dependant conditioned cue preference, used as a test for behavioral specificity of lidocaine, was not affected following inactivation of either site. Inactivation of the dSUB with 100 ␮g lidocaine significantly reduced drug-seeking and drug-taking behavior studied during the cocaine self-administration maintenance phase. Following extinction, inactivation of neither the dSUB nor vSUB influenced reinstatement of drug-seeking behavior induced by drug-paired cues presented alone or with a cocaine priming injection. The impairments in win-shift performance are consistent with the spatial processing functions of the dSUB and vSUB, and the reduction in drug-taking behavior is consistent with the non-spatial temporal processing functions of the dSUB. The lack of an effect of dSUB and vSUB inactivation on reinstatement of drug-seeking behavior may relate to the fact that the contextual associations with cocaine were well-practiced at the time of cue reinstatement testing, and therefore, drug-seeking behavior was likely regulated by nonhippocampal-dependent mechanisms. © 2003 Elsevier B.V. All rights reserved. Keywords: Conditioned cue preference; Cocaine; Drug-seeking behavior; Drug-taking behavior; Lidocaine; Self-administration; Subiculum; Win-shift

1. Introduction Research suggesting a link between limbic and cortical structures in mediating drug use and craving underscores the possible importance of neurocognitive processes for regulating some aspects of drug addiction. Studies using brain-imaging techniques demonstrate that craving induced by cocaine-associated cues or a priming injection of cocaine produces specific changes in the pattern of activation in the amygdala, anterior cingulate, hippocampus, basal ganglia, dorsolateral prefrontal cortex and cerebellum [2,4,17,26,48]. Animal studies examining neurocognitive

∗

Corresponding author. Tel.: +1-617-353-9201; fax: +1-617-353-2894. E-mail address: [email protected] (K.M. Kantak).

0166-4328/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2003.08.020

aspects of addiction-related behavior are also emerging, especially in reference to the basolateral amygdala (e.g. [21,33,49]) and prefrontal cortex (e.g. [15,38,50]). The research goal of this laboratory is to characterize how several different memory systems play a role in regulating behavior related to cocaine addiction. Using a neural systems/brain inactivation approach in rats, we first confirm that inactivation of discrete brain sites within the different memory systems could each produce specific deficits in cognitive task performance in the absence of the drug or drug-paired environment [20]. Then, following the completion of these studies, the function of each specific site during different phases of the addiction process is examined [21,22]. In the learning and memory literature, it has been well documented that there are at least four distinct memory

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systems. These systems aid in the acquisition and performance of various behavioral tasks. For instance, a series of experiments by McDonald and White [31] and Floresco et al. [13] delineated specific eight-arm radial maze tasks that could dissociate the involvement of the amygdalar, hippocampal, dorsal striatal, and prefrontal cortical memory systems in regulating associative functions such as conditioned-stimulus reward learning, stimulus–stimulus learning, stimulus–response learning, and working memory examined over a delay, respectively. Although many studies have traditionally used electrolytic or neurotoxic lesions to delineate the roles of these memory systems in behavior regulation during cognitive tasks (e.g. [31,36,37]), our laboratory uses lidocaine inactivation to address such an issue [20]. The use of lidocaine to reversibly inactivate discrete brain sites facilitates a within-subject design across tasks, and allows temporally specific manipulations. In order to continue our investigation of the role that different memory systems play in regulating behavior related to cocaine addiction, the present study examined the hippocampal memory system, which is composed of the hippocampal formation and parahippocampal region [11]. The hippocampus and subicular regions, considered together, make up the area defined as the hippocampal formation [11]. The hippocampus is thought to be critical for the formation of episodic memory representations. It processes information about spatial as well as non-spatial temporal and contextual details in order to create sequential representations of the relationships among the various stimuli present in an episode (see [10], for review). The dorsal subiculum (dSUB) and ventral subiculum (vSUB), which are the focus of the present investigation, are the primary output regions of the hippocampus and provide a major source of hippocampal innervation to the nucleus accumbens [52]. Some researchers acknowledge a possible function for the subicular regions in the associative-learning processes involved with reward in general, however, few comprehensive studies have specifically examined drug reward and relapse. For example, Caine and colleagues [3] demonstrated that lesions of the vSUB and dSUB produced moderate deficits in the acquisition of cocaine self-administration, but do not impact the cocaine dose–response functions later in training. In addition, Vorel et al. [47] provided evidence that theta burst stimulation of the vSUB can reinstate drug-seeking behavior following a period of extinction. In a comprehensive design similar to two previous reports from this laboratory [21,22], the study described herein first investigated basic cognitive functions of the dSUB and vSUB in one set of rats, and then evaluated the role of each functionally identified site in regulating behavior during the maintenance and reinstatement phases of cocaine self-administration in another set of rats. A lidocaine inactivation technique was used in both studies. This dual approach was used to make inferences regarding a role for neurocognitively functional sites of the hippocampal memory system in the regulation of cocaine-seeking and cocaine-taking behavior.

2. Materials and methods 2.1. Subjects Male Crl(WI)BR rats (Wistar strain, Charles River Breeding Labs, Portage, MI), weighing approximately 250 g upon arrival, were maintained at 85% of an adjusted ad libitum body weight throughout the duration of each study by restricting food to approximately 16 g per day. Between experimental sessions, the rats had access to water in their home cages. Rats were housed in individual clear plastic cages (24 cm × 22 cm × 20 cm) in a temperature- (21–23 ◦ C) and light- (08:00 h on, 20:00 h off) controlled vivarium. The policies and procedures set forth in the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health (vol. 25, no. 28, revised 1996) were followed. 2.2. Apparatus Cognitive task experiments were conducted in a modular 8-arm radial maze (Model ENV-538; Med Associates, Georgia, VT). The maze and the complete experimental environment have been previously described in detail [20]. A curtain surrounding the maze was used to control exposure to extramaze visual cues (see below). Activity in the maze was monitored remotely on a video screen connected to a ceiling-mounted video camera (ProVideo, Model CVC-100L, Amityville, NY). An interface-coupled switch box was used for manual input of arm entries and exits. For self-administration studies, experimental chambers (Model ENV-008CT; Med Associates, East Fairfield, VT) were each equipped with two response levers positioned 8 cm to the left and right of a center-mounted food receptacle and 7 cm from the grid floor. Connected to the food receptacle was a pellet dispenser that delivered 45-mg food pellets (Traditional Formula; Noyes, Lancaster, NH). A white stimulus light was located 7 cm above either the right or left lever. In addition, a houselight located on the opposite wall from the levers provided general illumination of the chamber. Each chamber contained a single channel fluid swivel and spring leash assembly connected to a counterbalanced arm assembly (Med Associates). A sound-attenuating cubicle enclosed each experimental chamber and was equipped with a fan to provide ventilation and an 8- speaker that provided auditory stimuli. Motor driven syringe pumps (Model PHM-100, Med Associates) located outside of each sound-attenuating cubicle were used for drug delivery. The same model syringe pump was used for intracerebral infusions. A standard personal computer programmed in Medstate Notation and connected to an interface (Med Associates), controlled experimental events. 2.3. Drugs The drugs used were cocaine hydrochloride (gift from NIDA, Bethesda, MD) and lidocaine hydrochloride (Sigma

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Chemical Co., St. Louis, MO). Cocaine was dissolved in sterile 0.9% saline solution containing 3 IU heparin/ml. For all self-administration experiments, a 1.0 mg/kg unit infusion dose of cocaine was used and delivered intravenously (i.v.) at a rate of 1.8 ml/min. To attain a dose of 1.0 mg/kg, infusion volume was adjusted for body weight, resulting in drug delivery times of approximately 4–5 s in individual rats. During saline self-administration sessions, heparinized saline solution was substituted for cocaine. Lidocaine, which was infused intracerebrally, was dissolved in sterile 0.9% saline to make 2% (20 mg/ml), 6% (60 mg/ml), 11% (112 mg/ml) or 20% (200 mg/ml) solutions. The pH of all solutions, including saline, was 5.0. A total volume of 0.5 ␮l, resulting in lidocaine doses of 10, 30, 56, 100 ␮g, respectively, was infused bilaterally at a rate of 0.59 ␮l/min. The 28-gauge stainless steel infusion cannula extended 1 mm beyond the tip of the guide cannula and was left in place for 1 min following the infusion. Sterile 0.9% saline infusions were used as the control for lidocaine infusions. 2.4. Surgery and histology Rats were anesthetized with an intraperitoneal (i.p.) injection of 90 mg/kg ketamine plus 10 mg/kg xylazine. Bilateral 22-gauge stainless steel guide cannulae (Plastics One, Roanoke, VA) were stereotaxically implanted into either the dSUB of the hippocampus (AP: −5.7 mm; L: ±2.5 mm; DV: −2.3 mm) or vSUB of the hippocampus (AP: −5.7 mm; L: ±4.5 mm; DV: −7.8 mm). Guide cannulae were positioned 1 mm above the intended sites and placements were based on the bregma coordinate system provided by Swanson [43]. The guide cannulae were attached to the skull with three stainless steel jeweler screws and dental cement. Two 28-gauge stainless steel obturators (Plastics One) were used to occlude the guide cannulae between infusions. For animals participating in self-administration studies, a catheter made of silicon tubing (i.d. = 0.51 mm, o.d. = 0.94 mm) was implanted into the right jugular vein, as previously described [19], prior to guide cannulae implantation. Wounds were treated daily with nitrofurazone powder until healed. Catheters were maintained by flushing them daily with 0.1 ml of a 0.9% saline solution containing 0.3 IU heparin (LymphoMed, Rosemont, IL) and 6.7 mg timentin (SmithKlineBeecham Pharmaceuticals, Philadelphia, PA). Additionally, catheters were checked for function weekly by infusing a 0.1 ml solution containing 1 mg methohexital sodium (Brevital; Lilly, Indianapolis, IN) and noting the presence or absence of sedation. A new catheter was implanted into either the left jugular vein or right femoral vein to replace leaky or non-functional catheters. All rats were allowed to recover from surgery for 1 week before initiation of the experiments. Upon completion of the experiments, rats were given an overdose of sodium pentobarbital and then intracardially perfused with saline and a 10% formalin solution. Brains were extracted and stored in 10% formalin. Twenty-four

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hours prior to sectioning, brains were transferred to a 20% glycerin solution and stored at 4 ◦ C. Coronal brain sections (50 ␮m) were cut and stained with thionin to verify cannulae placements. 2.5. Cognitive task procedures 2.5.1. Conditioned cue preference The procedures used for conditioned cue preference were similar to those described by Kantak et al. [20]. Briefly, two non-adjacent arms of the 8-arm radial maze were randomly assigned to each food-restricted rat. One was designated as the “dark” arm, and the other as the “lit” arm, as the stimulus lights over the entrance and inside the runway were illuminated in the latter arm. Access to all other arms was blocked. During the first and second sessions, the rats were free to move around the center hub and the two arms for 10 min. The time spent in each arm and the frequency of entries into each arm was recorded during the second session as a measure of preconditioning arm preference. Next, eight conditioning trials were conducted where individual rats were confined to either the lit or the dark arm of the test chamber on alternating days. The non-preferred arm was paired with food (S+ ; 70 “Froot Loops” cereal pieces) and the preferred arm was without food (S− ) for 30 min. The day after the last conditioning session, a preference test was conducted to reassess arm preference. During this test, no food was available in the apparatus and rats could move freely between the lit and dark arms. The time spent in each arm and the frequency of entries into each arm were recorded for 10 min. A preference ratio [20] was calculated that took into consideration the performance of rats in both the S+ and S− arms prior to and following conditioning. The preference ratio was calculated in the following manner: the proportion of time (in s) spent in the S+ (food-paired) arm after conditioning relative to before conditioning (S+ post/S+ pre) divided by the proportion of time spent in the S− arm after conditioning relative to before conditioning (S− post/S− pre). 2.5.2. Spatial win-shift The methods for the win-shift task were similar to those described by McDonald and White [31]. Specifically, four unique and visually distinct extra-maze cues were placed at 90◦ intervals on the curtain surrounding the maze. Cues were made from black and white felt material and measured 21 in. × 22 in. in total size. Cues were attached to the curtain with Velcro strips. During training, all arms of the 8-arm radial maze were accessible and a single piece of Froot Loops cereal was placed in the food well at the distal end of each arm. The rat was placed in the center of the apparatus with all doors initially open. After a choice was made, doors located at the proximal ends of non-selected arms were closed. Upon the rat’s return to the center platform, the previously selected door closed, and a 10-s waiting period was initiated. After the waiting period, all doors were again opened until the next choice was made. The session terminated after ei-

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ther 10 min had elapsed or all eight Froot Loops reinforcers were retrieved. The number of errors in a test session, an indication of performance on the task, was determined by tabulating the number of revisits to previously selected arms within the first eight choices. Criterion for acquisition of the task was reached when one or fewer errors were made within a session for two consecutive sessions in individual rats [31]. 2.6. Self-administration procedures 2.6.1. Initial training Before surgery, rats were trained to respond on a lever for food reinforcement under a fixed-ratio 1 (FR 1) schedule of food pellet delivery. After food training was complete, right jugular vein catheters and guide cannulae were implanted. One week later, self-administration training began. Initial sessions were 2 h in duration and rats were incrementally trained to self-administer cocaine under a terminal fixed-interval (FI)-based second-order schedule of drug delivery that incorporated contextual sound and conditioned light stimuli, as previously described in Kantak et al. [21,22]. For half of the rats, the right lever was designated as the active lever, and for the remaining rats, the left lever was designated as active. Delivery of cocaine was contingent on the completion of five responses on the active lever after the 5-min FI elapsed. The light stimulus over the active lever remained illuminated for the duration of the infusion and during a 20-s timeout (TO) period that followed each infusion. The TO period was signaled by extinguishing the house light. During the FI, a 2-s brief stimulus light was presented after every fifth response on the active lever. This schedule is termed an FI 5-min(FR5:S) second-order schedule of drug and brief stimulus delivery. Training sessions were conducted 5 days a week during the light phase and continued until cocaine intake was stable (number of infusions did not deviate by more than 20%) and the number of responses on the inactive lever was no greater than 25 per session for a 5-day period. 2.6.2. Discrimination baseline After initial training was complete, the discrimination training method of Weiss et al. [49] was adapted for use with the FI 5-min(FR5:S) second-order schedule in order to provide training with unique drug-associated (S+ ) and saline-associated (S− ) sound and light cues. This method was used because it results in reliable reinstatement of drug-seeking behavior over multiple sessions without providing access to cocaine. For each day of discrimination training, rats were given two 1-h sessions, separated by 1 h. For all rats, cocaine was available under a FI 5-min(FR5:S) schedule of drug delivery during one session and saline was available under a FI 5-min(FR5:S) schedule of saline delivery for the remaining session. Determinations of which session was first and which was second were made randomly everyday. During cocaine and saline sessions, a unique discriminative sound stimulus (intermittent tone versus

white noise) was presented and counterbalanced for each condition. These discriminative sound cues were presented throughout the session. In addition, to provide a unique conditioned light stimulus for saline sessions, the stimulus light present over the active lever flashed, instead of remaining constant as in cocaine sessions, during presentation of the brief stimulus, drug infusion, and TO period. Discrimination training took place 5 days a week. After 10 discrimination sessions, rats were tested for the effects of lidocaine inactivation on drug-seeking and drug-taking behavior maintained by an FI 5-min(FR5:S) schedule of cocaine delivery that utilized the S+ sound and light cues (see below). 2.6.3. Extinction and abstinence Rats underwent extinction training followed by a home-cage abstinence period prior to reinstatement tests (see below). During extinction, in order to specifically reduce the lever press response, rats were placed in the operant box in the absence of cocaine or saline. S+ and S− paired conditioned light and discriminative sound cues were also omitted, as a reduction in the salience of these cues was not desired. Extinction sessions were 2 h in duration and continued until each individual rat responded less than secen times on the active lever for three consecutive sessions. A 1-week abstinence period followed extinction in order to further dissociate rats from the cues associated with self-administration. During this period, rats remained in their home cages. Reinstatement testing commenced immediately following abstinence. 2.7. Experimental design 2.7.1. Experiment 1: cognitive task performance following lidocaine inactivation of the dSUB and vSUB The purpose of completing Experiment 1 was to determine if the stereotaxic coordinates chosen would selectively mediate an associative learning function ascribed to the hippocampus. Thus, we examined the effects of lidocaine inactivation of the dSUB and vSUB on the hippocampal-dependent win-shift task [31], and a control task, the amygdala-dependent conditioned cue preference task [20]. If the selected subicular sites selectively disrupted hippocampal-dependent associative learning, then these same stereotaxic coordinates would be used in new rats to determine if sites important for regulating hippocampal-dependent associative learning are involved in regulating drug-seeking and drug-taking behavior as studied in our maintenance/reinstatement model [21,22]. Rats were first exposed to the conditioned cue preference task. Two groups of rats were implanted with cannulae aimed at the dSUB (n = 8) or vSUB (n = 8). Bilateral infusions were made 5-min before the start of the post-conditioning preference test. Half of the rats from each group were infused with sterile 0.9% saline and the other half were infused with a 2% lidocaine solution (10 ␮g) in order to measure the expression of conditioned preference 1-day after the last

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conditioning session. A total volume of 0.5 ␮l was infused into each cannula at a rate of 0.59 ␮l/min by means of a motor driven syringe pump (Model PHM-100; Med Associates, Georgia, VT). A piece of PE-20 tubing was used to connect a 5-␮l Hamilton syringe to a 28-gauge stainless steel infusion cannula that extended 1 mm beyond the tip of the guide cannula. The infusion cannula was left in place for 1 min following the infusion. One week after completion of the conditioned cue preference task, training for the win-shift task was initiated. No experimental manipulations took place during the acquisition of the task. To study the effects of lidocaine inactivation on win-shift performance, infusions began after an individual rat reached criterion performance of this task. Half of the rats in each group received bilateral infusions of sterile 0.9% saline 5 min before the first performance test and 10 ␮g lidocaine 5 min before the second performance test. The remaining rats received these two treatments in the reverse order. 2.7.2. Experiment 2: drug-seeking and drug-taking behavior after lidocaine inactivation of the dSUB and vSUB under cocaine maintenance conditions After initial training and the completion of 10 discrimination sessions in 11 new rats (dSUB, n = 6; vSUB, n = 5), bilateral 0.5 ␮l infusions containing either saline, 30, 56, or 100 ␮g lidocaine were made in a counterbalanced order 5-min prior to a 1-h cocaine self-administration session. Several doses of lidocaine were examined in this experiment and in the cocaine prime reinstatement tests (see Experiment 4) because previous research in this laboratory has indicated that under testing conditions where cocaine is present, changes in behavior would only be expected after infusion of 5- to 10-fold higher doses than the typical 10 ␮g dose of lidocaine (see [21,22] for further discussion of this issue). Maintenance phase tests were administered 3 days apart with discrimination sessions on intervening days. 2.7.3. Experiment 3: drug-seeking behavior after lidocaine inactivation of the dSUB and vSUB under cocaine-associated cue reinstatement conditions Following maintenance testing, extinction, and abstinence from cocaine self-administration, cued reinstatement phase testing began in the same 11 rats. Tests took place every 3 days and animals remained in their homes cages on intervening days. During reinstatement tests, no saline or cocaine was available for self-administration. In order to assess discriminative control over responding, the first test session consisted of the presentation of the S− stimulus cues. The S− discriminative sound stimulus was presented for the duration of the session, while the flashing S− conditioned light cue was presented according to a FI 5-min(FR5:S) schedule, i.e. for 2-s following the completion of every fifth response during the FI 5-min, for the length of time corresponding to the infusion after the FI had elapsed and five responses were emitted, and for the 20-s TO period. Under

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S+ conditions, either 10 ␮g lidocaine or saline was bilaterally infused into the dSUB or vSUB 5-min prior to the second and third 1-h test sessions. The order of infusions was counterbalanced across subjects. Similar to the S− sessions, cocaine-associated (S+ ) discriminative sound stimuli were presented throughout the session. S+ conditioned light cues were presented according to an FI 5-min(FR5:S) schedule, as above. Doses of lidocaine higher than 10 ␮g were not evaluated as no cocaine was ever presented to the rats under these test conditions. Using only a 10 ␮g dose of lidocaine is justified because if this dose were ineffective in altering behavior measured in the absence of cocaine, but higher doses of lidocaine were effective, then these latter results would not be interpretable or would represent non-specific effects since 10 ␮g lidocaine is sufficient to inactivate over 90% of neurons in the absence of cocaine [41]. 2.7.4. Experiment 4: drug-seeking behavior after lidocaine inactivation of the dSUB and vSUB under cocaine prime + cue reinstatement conditions Following the completion of cue reinstatement testing, test sessions were conducted in the same 11 rats to examine the effects of lidocaine inactivation of the dSUB and vSUB on drug-seeking behavior induced by cocaine-associated cues presented in conjunction with a single cocaine priming injection. Cocaine-associated conditioned light and discriminative sound cues were presented in an identical manner to the cue reinstatement tests mentioned above. In addition, a single injection of 20 mg/kg cocaine (i.p.), shown to produce maximal reinstatement of drug-seeking behavior in Wistar strain rats [27], was given 30-min prior to the 1-h test session. The 30-min post-injection interval was chosen to avoid decreases in responding seen for the first 30 min of a cocaine self-administration session that follows a non-contingent injection of cocaine [30]. Saline and the 30, 56 or 100 ␮g lidocaine doses were bilaterally infused in a counterbalanced order 5-min prior to the sessions. Tests were spaced 3 days apart and animals remained in their home cages on intervening days.

2.8. Data analysis In the design of Experiment 1, the conditioned cue preference task always occurred first. Therefore, the Pearson correlation statistic was calculated to determine if there were any carryover effects of cue preference conditioning, as measured by the preference ratio, which could influence the rate of win-shift acquisition. Preference ratios were not correlated with the number of sessions to achieve acquisition criterion in the win-shift task (r 2 = 0.19, P ≤ 0.54). Thus, the tasks were considered to be independent tests of cognition for the data analyses described below. For the conditioned cue preference task, a preference ratio that is significantly greater than 1.0 indicates a conditioned

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preference for the food-paired arm, while a ratio significantly less than 1.0 indicates a conditioned aversion to the food-paired arm. Preference ratios equal to 1.0 are indicative of a blockade of the effects of conditioning. The 95% confidence limits of the treatment means were used to determine significance. Further, in order to examine differences in the magnitude of conditioning observed, a two-factor ANOVA (brain site × infusion treatment) with repeated measures was used to compare the preference ratios of saline- and lidocaine-treated dSUB- and vSUB-implanted rats. The frequency of entries into the S+ and S− arms was tabulated to monitor general activity level during each preference test. Frequency of arm entries was compared by means of a three-factor (infusion treatment × preference test number × food stimulus condition) repeated-measures ANOVA for each brain site. The number of Froot Loops consumed during S+ conditioning sessions for each brain site was analyzed by means of Student t-tests for independent samples. During win-shift acquisition, accuracy prior to any treatment initiation was first compared by means of a two-factor (brain site × session number) repeated-measures ANOVA. During performance tests following treatment initiation, the number of errors made after saline and lidocaine infusion was compared by means of Student t-tests for dependent samples in each brain site. For self-administration studies, three dependent measures were calculated: (1) the number of active lever responses made (drug-seeking behavior); (2) the number of inactive lever responses made (non-specific behavior); and (3) the number of infusions earned (drug-taking behavior), or, as is the case with all reinstatement tests, the number of infusion-paired light deliveries earned during the test session. As drug-seeking behavior is traditionally defined as responding maintained by drug-associated cues at times when drug is not immediately available under a second-order schedule of drug and brief stimulus light delivery [16], and more recently defined as responding maintained by drug-associated cues and/or a priming injection following extinction [42], drug-seeking behavior is measured under our three experimental phases. Data over the last three cocaine and saline discrimination sessions and the last three extinction sessions were averaged from individual rats and used as a measure of baseline behavior. A series of single factor ANOVAs with repeated measures was used, with each ANOVA comparing baseline measures, maintenance test measures, prime reinstatement test measures or cue reinstatement test measures. The Dunnett’s t-test was used for all post-hoc comparisons. The control group for each post-hoc test was the cocaine condition for the baseline measures and the respective 0 ␮g lidocaine dose for the maintenance, prime reinstatement and cue reinstatement test measures. A paired t-test for dependent samples was used to compare the number of infusions earned during baseline sessions.

3. Results 3.1. Functional spread of lidocaine and histology The volume of lidocaine required to inactivate over 90% of neurons within a particular radius from the infusion site is governed by the spherical volume equation, V = 4/3πr 3 [44]. Based on this equation, the functional spread of 0.5 ␮l of lidocaine, which depends on the volume of lidocaine, not the concentration [40,41], was estimated to have been 0.49 mm from the infusion site in the present study. Histological verification of placements and functional spread of lidocaine or saline for animals with cannulae aimed at the dSUB and vSUB of the hippocampus are depicted in Fig. 1 (top row and bottom row, respectively). For every animal, cannulae were within 0.5 mm of the intended placement in the anterior–posterior position. For animals with cannulae implanted in the dSUB, bilateral placement was confirmed for six of eight rats performing the cognitive tasks and for six of six animals involved in self-administration studies. Since two rats used in the cognitive behavioral studies had bilateral placements outside the dSUB, their data were excluded from the analyses. The only brain structure within the functional spread zone that was common to the 12 rats whose data were included in the analyses of Experiments 1–4 was the dSUB. In several rats, the functional spread zone overlapped with the dorsal hippocampal commissure, and in one rat with the striatum radium. For animals with cannulae aimed at the vSUB, bilateral placement was confirmed for seven of eight rats performing cognitive tasks and for five of five rats involved in self-administration studies. Since one rat used in the cognitive behavioral studies had only a unilateral cannula placement in the vSUB, its data were excluded from the analyses. The only brain structure within the functional spread zone that was common to the 12 rats whose data were included in the analyses of Experiments 1–4 was the vSUB. In several rats, the functional spread zone overlapped with the dentate gyrus. Photomicrographs of a dSUB and a vSUB guide cannula placement are shown in Fig. 1 in the left column. The above analyses indicate that there was a sufficient separation of placements from the dSUB and vSUB to delineate two discrete sites of interest. Furthermore, for all rats included in the data analyses, cannulae placements verify that lidocaine inactivation predominantly encompassed the site of interest. One caveat, however, is that lidocaine may have spread “up” the cannula tract to inactivate areas more dorsal to the selected sites. If this had occurred after vSUB infusion, the dentate gyrus, a subregion of the hippocampus itself, would have been affected. Spread of lidocaine “up” from the dSUB would have impacted regions outside the hippocampus. However, several studies have shown that lesions of the neocortical regions located dorsal to the dorsal hippocampus have no significant impact on cognitive functions defined to be hippocampal, such

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Fig. 1. Cannulae placements for the dSUB (top) and vSUB (bottom) were verified using Nissl staining techniques (left column). These schematics represent coronal sections of rat brain encompassing the midpoint of placements in the anterior–posterior plane (left). All drawings are based upon the atlas of Swanson [43] with the anterior–posterior reference measured from bregma. Circles, drawn to scale, indicate the location and approximate diffusion plane of lidocaine or saline, and are based on the spherical volume equation [43]. Photomicrographs of guide cannulae placements, which end 1 mm above the intended site, are shown on the right.

as contextual fear conditioning, paired-associate learning, and processing the temporal order of a sequence of odors [5,24,32].

3.2.2. Win-shift performance Acquisition rates in the win-shift task prior to any treatment initiation were significantly faster in rats with cannulae aimed at the dSUB than the vSUB (F(1, 12) = 8.80;

3.2. Experiment 1: cognitive task performance following lidocaine inactivation of the dSUB and vSUB

Preference Ratio

3.2.1. Conditioned cue preference Lidocaine-induced inactivation of the dSUB and vSUB did not impair expression of conditioned cue preference (Fig. 2). Both the saline control rats and the lidocaine-treated rats showed a conditioned preference for the S+ (food-paired) arm in that preference ratios were significantly greater than 1.0 (P ≤ 0.05). There were no differences in the magnitude of conditioning between saline- and lidocaine-treated dSUB- and vSUB-implanted rats. Regarding arm entries, there were no significant treatment differences between the number of entries into the food-paired and nonfood-paired arms prior to and following conditioning, nor were there differences in the number of Froot Loops consumed between saline and lidocaine-treated rats from each site that could influence the strength of conditioning (Table 1).

9 8 Saline Lidocaine

7 6 5 4 3 2 1 0

vSUB

dSUB

Infusion Site Fig. 2. Conditioned cue preference expressed as the mean (±S.E.M.) preference ratios in saline- and lidocaine-treated rats with cannulae aimed at either the vSUB or dSUB. The dashed line refers to a ratio of 1.0. (*): Significantly different (P ≤ 0.05) from a value of 1.0.

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Table 1 Mean (±S.E.M.) number of Froot Loops (#FL) consumed during conditioning sessions and frequency of entries into the food-paired (S+ ) arm and nonfood-paired (S− ) arm during the pre- and post-conditioning preference tests in saline- and lidocaine-treated rats for each brain site Site

Arm

Pre

Post

#FL

Pre

Post

#FL

dSUB

S+

S−

7±1 14 ± 2

10 ± 2 10 ± 1

26 ± 4

8±2 10 ± 1

11 ± 1 9±1

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P ≤ 0.013). Rats from the dSUB group acquired the task to criterion levels after an average of 13 ± 1.6 sessions, while it took rats with cannulae aimed at the vSUB an average of 23 ± 3.0 sessions to acquire the task to the same criterion level as animals with cannulae aimed at the dSUB (data not shown). However, after acquisition criterion levels were achieved, infusion of saline resulted in near perfect win-shift performance in both groups of rats (Fig. 3). Lidocaine inactivation of the dSUB (t(5) = 2.08; P ≤ 0.046) and vSUB (t(6) = 3.24; P ≤ 0.01) significantly disrupted win-shift performance compared to saline control (Fig. 3). After lidocaine inactivation, the average number of errors was approximately 10-fold greater than after saline for both sites. 3.3. Experiment 2: drug-seeking and drug-taking behavior after lidocaine inactivation of the dSUB and vSUB under cocaine maintenance conditions During discrimination baseline sessions, cocaine maintained high levels of active lever responding which were significantly greater (P ≤ 0.05) than the levels of active lever responding observed during saline baseline and extinction conditions for animals with cannulae aimed at the dSUB

(F(2, 10) = 18.07; P ≤ 0.001) (Fig. 4, top panel A) and the vSUB (F(2, 8) = 33.62; P ≤ 0.001) (Fig. 6, top panel A). Inactive lever responding for dSUB-implanted rats averaged fewer than 14 responses during baseline, maintenance, and across all reinstatement test conditions (Fig. 4, bottom). An average of 17 or fewer responses on the inactive lever was seen for vSUB-implanted rats on the same measure (Fig. 6, bottom). The number of infusions earned during cocaine sessions was significantly higher than the number earned during saline sessions for both dSUB-implanted (t(5) = 8.89; P ≤ 0.001) (Fig. 5, panel A) and vSUB-implanted (t(4) = 6.67; P ≤ 0.005) (Fig. 7, panel A) rats. Inactivation of the dSUB with lidocaine reduced drug-seeking behavior (F(3, 15) = 3.25; P ≤ 0.05), with modest, but significant reductions (P ≤ 0.05) seen after 100 ␮g lidocaine as compared to 0 ␮g lidocaine (Fig. 4, top panel B). Drug-taking behavior (F(3, 15) = 3.84; P ≤ 0.05), reflected by the number of infusions earned, was also modestly, but significantly reduced (P ≤ 0.05) in these animals after 100 ␮g lidocaine as compared to 0 ␮g lidocaine (Fig. 5, panel B). Inactivation of the vSUB, however, had no significant effect on drug-seeking or drug-taking behavior after 30–100 ␮g lidocaine as compared to 0 ␮g lidocaine.

1.6 Saline Lidocaine

Number of Errors

1.4 1.2 1 0.8 0.6 0.4 0.2 0

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Infusion Site Fig. 3. Mean (±S.E.M.) number of errors made during win-shift performance tests following saline and lidocaine infusions into either the vSUB or dSUB. (*): Significantly different from saline control (P ≤ 0.05).

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Fig. 4. Drug-seeking behavior after lidocaine inactivation of the dSUB during 1-h test sessions. Responses on the active lever are shown on the top panel and responses on the inactive lever are shown on the bottom panel for each phase of the experiment. (A) Cocaine, saline, and extinction baselines, (B) cocaine maintenance tests, (C) cocaine-associated cue reinstatement tests, (D) cocaine prime + cue reinstatement tests. Values represent the mean ± S.E.M. S+ refers to presentation of drug-associated cues and S− refers to presentation of saline-associated cues. (*): Significantly different (P ≤ 0.05) from the appropriate control value (see Section 2 for details).

3.4. Experiment 3: drug-seeking behavior after lidocaine inactivation of the dSUB and vSUB under cocaine-associated cue reinstatement conditions The presentation of S− (saline-associated) cues after extinction resulted in levels of performance similar to saline baseline in both groups of rats (Figs. 4 and 6, top panel C). Based on Dunnett’s t-test analyses, the presentation of S+ (cocaine-associated) cues after extinction reinstated drug-seeking behavior in that there were significantly more responses under S+ compared to S− conditions for both dSUB-implanted (P ≤ 0.05) and vSUB-implanted (P ≤ 0.05) rats, although the overall ANOVAs for either site did not reach the 0.05 level of significance. The overall ANOVAs were significant for the number of infusion-paired light deliveries earned after extinction in both dSUB-implanted (F(2, 8) = 4.63; P ≤ 0.05) and vSUB-implanted (F(2, 8) = 7.04; P ≤ 0.05) rats. There were significantly more infusion-paired light deliveries earned under the S+ compared to the S− condition for both the dSUB (Fig. 5, panel C) and vSUB

(Fig. 7, panel C) sites (P ≤ 0.05). In both sites, the reinstatement of drug-seeking behavior and the number of infusion-paired light deliveries earned after extinction did not significantly change after administration of 10 ␮g lidocaine as compared to the S+ 0 ␮g lidocaine control. 3.5. Experiment 4: drug-seeking behavior after lidocaine inactivation of the dSUB and vSUB under cocaine prime + cue reinstatement conditions After a saline infusion, the administration of a single 20 mg/kg priming injection of cocaine prior to the presentation of S+ cocaine-associated cues reinstated drug-seeking behavior for animals with cannulae aimed at the dSUB (Fig. 4, top panel D) and vSUB (Fig. 6, top panel D). Lidocaine inactivation (30–100 ␮g) of neither the dSUB nor the vSUB significantly altered either the reinstatement of drug-seeking behavior or the number of infusion-paired light deliveries earned after the cocaine priming injection (Figs. 5 and 7, panel D).

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Fig. 5. Number of infusions earned or number of infusion-paired light deliveries earned in 1-h test sessions after lidocaine inactivation of the dSUB for each phase of the experiment. (A) Cocaine, saline, and extinction baselines, (B) cocaine maintenance tests, (C) cocaine-associated cue reinstatement tests, (D) cocaine prime + cue reinstatement phase tests. Values represent the mean ± S.E.M. S+ refers to presentation of the cocaine-associated cues and S− refers to presentation of saline-associated cues. (*): Significantly different (P ≤ 0.05) from the appropriate control value (see Section 2 for details).

4. Discussion The purpose of this study was to verify the effects of temporary neuronal inactivation of the dSUB and vSUB on a hippocampal-dependent associative learning task, and then determine if these same sites were important for regulating addiction-related behavior. The hippocampal-dependent win-shift task [31] was used as a simple tool for identifying functionally relevant stereotaxic coordinates within the hippocampal formation. Besides serving this purpose, the results of the win-shift task revealed novel findings regarding the impact of lidocaine inactivation of the dSUB on spatial working memory, and replicated previous finding regarding vSUB function and lidocaine inactivation during the win-shift task [12,13]. Previous investigations using permanent lesioning techniques have shown that both the dorsal and ventral hippocampi are important for spatial processing [12,34,46]. In addition, the neuronal connections from the hippocampus via the vSUB to the nucleus accumbens have been shown to be critical for the successful processing of spatial information [12]. Therefore, in the present study, inactivation of the dSUB and vSUB appears to have sufficiently blocked the output of the dorsal and ventral hippocampi to impair performance on a hippocampal-dependent spatial learning task. It is important to note that the impaired performance observed following lidocaine inactivation of the dSUB and vSUB is not likely due to non-specific factors. First, each rat received a total of three intracerebral infusions. It is unlikely that this number of repeated infusions caused tissue damage at the cannula tip that interfered with the spread of lido-

caine or non-specifically disrupted behavioral performance. Previous research has indicated that 20 daily infusions of lidocaine do not alter its effectiveness for disrupting behavioral performance and that 20 daily infusions of saline do not cause deficits in behavioral performance [20]. Further, the effects of lidocaine inactivation of the dSUB and vSUB appear to be specific for disrupting a form of associative learning dependent on the hippocampal formation, because in the conditioned cue preference task, previously shown to depend on the function of the basolateral amygdala [20], neither dSUB nor vSUB inactivation influenced conditioned stimulus-reward learning. The impairments in spatial processing explain why win-shift task accuracy was significantly impaired after lidocaine inactivation of either site, but cannot explain the differential effects of dSUB and vSUB inactivation during the maintenance phase or the lack of an effect of inactivation of either site on the reinstatement of drug-seeking behavior. The cognitive demands of the win-shift task (spatial) are not the same as the cognitive demands of the cocaine self-administration task (contextual and temporal). Spatial ability is most likely not critical for performance during our cocaine self-administration maintenance/reinstatement testing procedure. The hippocampus [14,24], more specifically the dorsal aspect, is also essential for processing the temporal elements of an event or episode [7,18,25]. A role for the ventral hippocampus or vSUB in processing temporal information has not yet been identified. As the dSUB itself has been shown to be involved in creating a temporal link between events encoded in the hippocampus [18], a loss of dSUB function during maintenance may have interfered

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Fig. 6. Drug-seeking behavior after lidocaine inactivation of the vSUB during 1-h test sessions. Responses on the active lever are shown on the top panel and responses on the inactive lever are shown on the bottom panel for each phase of the experiment. (A) Cocaine, saline, and extinction baselines, (B) cocaine maintenance tests, (C) cocaine-associated cue reinstatement tests, (D) cocaine prime + cue reinstatement tests. Values represent the mean ± S.E.M. S+ refers to presentation of drug-associated cues and S− refers to presentation of saline-associated cues. (*): Significantly different (P ≤ 0.05) from the appropriate control value (see Section 2 for details).

with the temporal pattern of drug-taking behavior. The dSUB, by maintaining a record of time between infusion events during the fixed-interval portion of the second-order schedule, may have influenced the efficiency by which infusions are earned. Using a DRL (differential reinforcement of low rates of responding) schedule, others have shown impairments in reward efficiency after transection of fibers projecting from the subiculum to the nucleus accumbens [45]. As unit activity in the dorsal hippocampus has been shown to increase at the beginning of the DRL interval, followed by a gradual decrease throughout the interval [53], this region of the hippocampus may be part of a critical timing circuit for mediating reward efficiency under a variety of conditions. Importantly, the reductions in drug-seeking and drugtaking behavior seen after 100 ␮g lidocaine are not likely due to an impaired ability to respond on the lever, because the same dose did not impact behavior during the prime + cue reinstatement tests. Furthermore, inactivation of the vSUB with 100 ␮g lidocaine had no significant ef-

fects on behavior, again arguing against non-specific effects of this dose of lidocaine. During the reinstatement phases, inactivation of the dSUB and vSUB with lidocaine did not affect the reinstatement of drug-seeking behavior. A close examination of contextual processing functions of the hippocampus [29,39] and subiculum [28] during the learning and memory process may explain these results. Investigations have shown that the hippocampus plays an essential role in episodic learning and memory, specifically the encoding and consolidation of personal experiences and events [9]. Further, detailed evaluations suggest that novelty and training history are critical determinants for whether or not the hippocampus has a role in regulating behavior [1,23]. Taken together, this literature supports the hypothesis that the hippocampal formation may be more important for encoding and consolidating the contextual details of an episode, and less important for the long term processing or recall of these contextual details [6,54]. If this were the case, then inactivation of the hippocampal output regions should not influence the rein-

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Fig. 7. Number of infusions earned or number of infusion-paired light deliveries earned in 1-h test sessions after lidocaine inactivation of the vSUB for each phase of the experiment. (A) Cocaine, saline, and extinction baselines, (B) cocaine maintenance tests, (C) cocaine-associated cue reinstatement tests, (D) cocaine prime + cue reinstatement phase tests. Values represent the mean ± S.E.M. S+ refers to presentation of the cocaine-associated cues and S− refers to presentation of saline-associated cues. (*): Significantly different (P ≤ 0.05) from the appropriate control value (see Section 2 for details).

statement of drug-seeking behavior in animals with a long and extensive training history with contextual stimuli, as processing of the discriminable contextual elements of the self-administration environment would be done early during the acquisition phases of training, as well as following early exposure to the S+ and S− contextual cues of the discrimination sessions. In support of this idea, Neisewander and colleagues [35] showed no changes in Fos protein expression in the subiculum following cued and prime reinstatement tests in well-trained animals. Also in well-trained animals, Ciccocioppo and colleagues [8] demonstrated that the reinstatement of drug-seeking behavior by discriminable contextual cues was associated with increases in Fos protein expression only in the basolateral amygdala and medial prefrontal cortex. These results, along with those found in the present study, are consistent with the neurocognitive view that once contextual associations are consolidated, further processing of this information takes place outside the hippocampal memory system [1]. Human imaging studies have identified links between cortical/subcortical memory systems and cocaine use/craving (e.g. [2,4]), but the functional significance of these associations is not clear. Combining studies addressing the neural mechanisms of addictive drugs and their effects on behavior with studies investigating the neurobiology of learning and memory may be the best method to definitively ascertain the function of each memory system during the various stages of the addiction process. Although this study has elucidated a potential function of the dSUB for regulating temporal aspects of drug-taking behavior, the design of experiments in future investigations will need to more directly examine

other functions already ascribed to the hippocampal memory system, such as contextual encoding and spatial processing. For example, during the acquisition phase of cocaine self-administration, it is possible that the dSUB and, even in this case, the vSUB are essential for encoding the relationship between the external context, known to be processed by the hippocampal formation, and the internal affective state that results after intake of cocaine [51]. Once acquired, sites outside of the hippocampal memory system may process the consolidated contextual information, as suggested by the findings of the present reinstatement tests and results of others [3,8,35]. Though this conclusion would most likely predict minimal subicular involvement during reinstatement, Vorel et al. [47] found that stimulation of the vSUB did reinstate drug-seeking behavior. Interestingly, the rats in that study had a relatively short cocaine self-administration training history (1 week) followed by 7–20 days of extinction. With this in mind, training history may be a significant factor for showing hippocampal memory system involvement in regulating the reinstatement of drug-seeking behavior. In conclusion, focused investigations, such as those described herein, will allow successful elucidation of how neurocognitive mechanisms may regulate addiction and relapse, not only in hippocampal, but in non-hippocampal memory systems as well.

Acknowledgements This research was supported by the National Institute on Drug Abuse Grant DA11716. We thank Francisco Ugalde

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and Drew Kresman for their technical assistance with the self-administration and radial arm maze experiments.

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