Limited versus extended cocaine intravenous self‐administration: Behavioral effects and electrophysiological changes in insular cortex

Abstract Aims Limited vs extended drug exposure has been proposed as one of the key factors in determining the risk of relapse, which is the primary characteristic of addiction behaviors. The current studies were designed to explore the related behavioral effects and neuronal alterations in the insular cortex (IC), an important brain region involved in addiction. Methods Experiments started with rats at the age of 35 days, a typical adolescent stage when initial drug exposure occurs often in humans. The drug‐seeking/taking behaviors, and membrane properties and intrinsic excitability of IC pyramidal neurons were measured on withdrawal day (WD) 1 and WD 45‐48 after limited vs extended cocaine intravenous self‐administration (IVSA). Results We found higher cocaine‐taking behaviors at the late withdrawal period after limited vs extended cocaine IVSA. We also found minor but significant effects of limited but not extended cocaine exposure on the kinetics and amplitude of action potentials on WD 45, in IC pyramidal neurons. Conclusion Our results indicate potential high risks of relapse in young rats with limited but not extended drug exposure, although the adaptations detected in the IC may not be sufficient to explain the neural changes of higher drug‐taking behaviors induced by limited cocaine IVSA.

that IC lesions interrupt addictive behaviors, suggesting that the IC or the IC-mediated interoceptive system is sensitized in addicted subjects as a consequence of addiction history and a cause of relapse. However, a different picture was suggested by neuroimaging studies, in which reduced activity 9,10 and shrunken gray matter 5,11 in the IC were detected in individuals with substance abuse history. These apparently contradictory lines of evidence may indicate that the IC can both promote drug use (eg, via increased perception of craving for drugs) and weaken the processes that prevent drug use (such as decision-making and the evaluation of negative consequences). 12 The IC consists of 3 subregions, that is, posterior granular insular, anterior agranular insula (AAI), and the intermediate dysgranular insula). [13][14][15] Particular attention in addiction research has been payed to the AAI due to its rich chemoarchitecture, including the presence of dopamine and opioid receptors. 6,[16][17][18][19][20] The present study used a rat model of cocaine intravenous self-administration (IVSA) to explore the behavioral and electrophysiological consequences of limited vs extended IVSA after short (1 day) or prolonged (45 days) withdrawal periods (designated as WD1 and WD45, respectively). Experiments started with rats at the age of 35 days, a typical adolescent stage when initial drug exposure occurs often in humans. 21,22 2 | MATERIAL S AND ME THODS

| Experimental subjects
All procedures were performed in accordance with the United States Public Health Service Guide for Care and Use of Laboratory Animals and were approved by the institutional Animal Care and Use committee at the State University of New York, Binghamton and Indiana University School of Medicine. Experiments were conducted on male Sprague Dawley rats, bred in-house using breeders originally derived from Envigo (USA). With the day of birth being deemed as postnatal day (P) 0, rats were allowed to develop normally until P21-23 when animals were weaned and pair-housed in standard Plexiglas bins unless receiving cannula implantation procedure. Rats were maintained on a 7 am/7 pm light/ dark schedule with ad libitum access to food and water. Rats were allowed 5-7 days to acclimate to colony conditions and handled to habituate them to human contact prior to experimentation. Similar to our previous studies, rats received catheter implantation on postnatal ~35 days. Seven days later, rats received one overnight session, followed by 5 of 1-hour daily sessions (considered as limited training procedure) or 21 of 6-hour daily sessions (considered as extended training procedure) of cocaine IVSA training. 23,24 In total, 75 rats were used in this study, among which 8 rats were excluded due to patency failure of the catheter (n = 6) and behavioral outliers (n = 2). From the 67 remaining subjects, 21 were trained by limited (n = 11) or extended (n = 10) cocaine IVSA procedures, followed by cocaine-seeking test on WD1 and WD45, and cocaine-taking test on WD46-48 ( Figure 1A). The other 46 rats were trained by limited (n = 9 for saline and n = 14 for cocaine) or extended (n = 10 for saline and n = 13 for cocaine) IVSA procedures and whole-cell patch-clamp recordings were obtained either 1 or 45 days after the last IVSA session.

| Catheter implantation
As described previously, 23-27 a silastic catheter was inserted into the right jugular vein, and the distal end was led subcutaneously to the back between the scapulae. Catheters were constructed from silastic tubing (~8 cm; inner diameter 0.020 in, outer diameter 0.037 in) connected to a One Channel Rat Button (Instech Labs). Rats were allowed to recover for ~7 days. During recovery, the catheter was flushed daily with 1 mL/kg body weight of heparin (10 U/mL) and gentamicin antibiotic (5 mg/mL) in sterile saline to help protect against infection and catheter occlusion.

| Self-administration apparatus
Experiments were conducted in operant-conditioning chambers enclosed within sound-attenuating cabinets (Med Associates).
Each chamber contained two levers randomly assigned as active vs inactive levers, a food dispenser, and the conditioned stimulus (CS) light 9 cm above each lever. No food or water was provided in the chambers during the training or testing sessions.

| Intravenous cocaine selfadministration training
7 days after catheter implantation, cocaine self-administration training began with an overnight session at 7 pm to 7 am on the following day. The daily training session, either 1 hour per day for 5 days as limited cocaine access or 6 hours per day for 21 days as extended cocaine access, started on the day after. The same training protocol was used in overnight and daily sessions. Rats were placed in the self-administration chamber on a fixed ratio (FR) 1 reinforcement schedule with the house light on. Active lever press resulted in a cocaine infusion (0.75 mg/kg over 2-4 seconds) and illumination of a CS light above the active lever for 20 seconds with the house light off. In contrast, the inactive lever press led to no outcome but was also recorded. Rats that received at least 60 cocaine rewards in the overnight session were allowed to move to daily selfadministration of cocaine ~24 hr after the overnight training on an FR1 reinforcement schedule. Animals that did not meet this standard (n = 1) were removed from subsequent self-administration training.

| Cocaine-seeking test
Rats previously assigned for behavioral test were tested on both WD1 and WD45 for their cocaine-seeking behaviors. The cocaine-seeking test lasted for 1 hour, during which rats were reexposed to the operant chamber with the exact same conditions as during cocaine IVSA training except no cocaine IV delivery was programmed after active lever press. The active lever press during 1 hour seeking test was analyzed as an index of cocaine-seeking level ( Figure 1D).

| Cocaine-taking test
Multiple doses of cocaine were evaluated in the cocaine-taking test as described before. 28 Briefly, a 2-hour period was divided into four 30-minute segments, which allowed the assessment of four pseudorandomized doses of cocaine (0.1, 0.3, 1.0, and 3.0 mg/kg per injection) in a 2-hour session. Two adjacent 30-minute sessions were spaced by a ~5-minute interval. During each 30-minute session, the exact same conditions, although the dose of cocaine per injection was different, were applied to rats, which means rats received F I G U R E 1 Effects limited vs extended cocaine IVSA on cocaine-seeking and cocaine-taking behaviors during the withdrawal period. A, Timeline of cocaine-seeking and cocaine-taking test with specified age of days at each stage. Rats were trained by limited (upper line) or extended (lower line) cocaine IVSA procedures. P, postnatal days. B, The daily # of cocaine IV infusion and daily # of active lever press by rats exposed to limited cocaine IVSA procedure. C, The daily # of cocaine IV infusion and daily # of active lever press by rats exposed to extended cocaine IVSA procedure. D, Cocaine-taking behaviors on WD1 and WD45. E, Cocaine-taking behaviors tested by multiple dose of cocaine (ie, 0.1, 0.3, 1.0, 3.0 mg/kg per infusion) on WD46-48. F, cocaine (0.1 mg/kg per infusion)-taking behaviors of individual rat are represented by the circles in the columns. Data were analyzed by one-way ANOVA (each curve in B, C) and two-way ANOVA with repeated measures (D,E), followed by Bonferroni post-hoc tests or Student's t test (F). n = 11 in limited cocaine IVSA group; n = 10 in extended cocaine IVSA group. *P < .05; **P < .01, comparations between day 1 vs any of other days during the IVSA training (C), WD1 vs WD45 (D), or limited vs extended IVSA groups (E,F) cocaine IV delivery after active lever press, followed by a 20-second timeout during which the cue light signals were presented and no cocaine delivery was allowed. This 2-hour daily session was done once per day for 3 consecutive days (ie, WD46-48). The average number of cocaine infusions from 3 30-minute sessions with a specific dose of cocaine was analyzed as an index of cocaine-taking level.
The rat was decapitated, and then the brain was removed and glued to a block before slicing using a Leica VT1200s vibratome in 4°C cutting solution. Coronal slices of 250-µm thickness were cut such that the preparation contained the signature anatomical landmarks (eg, the rhinal fissure, the anterior commissure, and the corpus callosum) that clearly delineate the AAI area. After allowing at least 1 hour for recovery, slices were transferred from a holding chamber to a submerged recording chamber where it was continuously perfused with oxygenated ACSF maintained at Standard whole-cell current-or voltage-clamp recordings were obtained with a MultiClamp 700B amplifier (Molecular Devices), filtered at 3 kHz, amplified 5 times, and then digitized at 20 kHz with a Digidata 1550B analog-to-digital converter (Molecular Devices). The intrinsic excitability was examined using a series of depolarizing current pulses and by constructing input-output (I-O) functions.

| Data acquisition and analysis
Data were collected either 2 days or 21 days after the last IVSA session. All results are shown as mean ± SEM. Each experiment was replicated in at least 10 rats for in vivo behavioral tests and at

| Effects of limited vs extended cocaine IVSA on cocaine-seeking and cocaine-taking behaviors during the withdrawal period
Adolescent rats (n = 21), implanted with catheter on postnatal day 35 and recovered for ~1 week, were trained by limited (ie, 1 hour per day for 5 days) or extended (ie, 6 hours per day for 21 days) cocaine IVSA procedure, followed by a period of forced abstinence during which the rats were just housed in the home cage, except on WD1 and WD45 when they underwent the cocaine-seeking test, and on WD46-48 when they received the cocaine-taking test in the operant chambers as shown in Figure 1A.  Figure 1F). Drug-seeking and drugtaking behaviors after a prolonged withdrawal period have been well accepted as reliable indices of relapse. [29][30][31] Thus, we conclude that the extended cocaine IVSA procedure used in the current study did not increase the risk for relapse. Interestingly, the limited cocaine IVSA procedure may increase the relapse risk due to significantly increased drug-taking behaviors on WD45.

| Effects of limited vs extended cocaine IVSA on the kinetics and amplitude of evoked action potentials in insula pyramidal neurons
Whole-cell current clamp recordings in IC-containing coronal slices were performed in rats 1 day or 45 days after IVSA of saline or cocaine. The kinetics and amplitude of the action potential were analyzed by measuring the 2nd action potential induced by 300 pA current injection (Figure 2A). The insula pyramidal neurons from rats with a history of limited IVSA procedure, that is, 1 hour per day

| Effects of limited vs extended cocaine IVSA on intrinsic excitability of insula pyramidal neurons
The number of spikes evoked by depolarizing current injections ranging from 50 to 400 pA were also analyzed. Our data showed that, relative to saline controls, cocaine IVSA procedure did not Further data analyses showed no significant difference of RMPs in rats with a history with limited ( Figure 4A, saline/cocaine × WD1/WD45 interaction F 1,70 = 2.2, P = .14, saline/cocaine

| D ISCUSS I ON
Our data demonstrated no differences of drug-seeking behaviors after limited vs extended cocaine IVSA, although both limited and extended IVSA rats showed higher drug-seeking behaviors on WD45, compared with WD1, which indicates the duration of withdrawal period can significantly increase the drug-seeking behaviors as evidenced before. [32][33][34][35] Importantly, limited cocaine IVSA significantly increased cocaine-taking behaviors compared with the extended cocaine IVSA group as supported by the upward shift of the dose-response curve of cocaine taking at the later withdrawal stage. Thus, limited, compared with extended, cocaine history, may lead to a potential high risk of relapse to cocaine.
Notably, this behavioral outcome dispels the current belief that extended drug exposure has more robust effects on drug-seeking and drug-taking behaviors. For example, extended access to drugs significantly increased drug-taking behaviors in the withdrawal period [36][37][38] and compulsive drug-seeking behaviors could only be established in rats by extended but not limited cocaine IVSA procedure 39 . It is critical to point out the developmental stages when drug exposure and drug withdrawal occur. Relative to adults, adolescent rats took cocaine more readily, were more sensitive to lower doses, and showed greater escalation of cocaine intake. 40 Thus, our IVSA training was done at the adolescence stage. On another hand, it has been fashionable in addiction research to run drug exposure with extended daily sessions (≥6 hours) during a long period (eg, at least 10 days). [41][42][43][44] However, studies on limited vs extended drug exposure in adolescent animals were still lacking.
Most, if not all, of the previous studies comparing the prolonged effects of limited vs extended drug exposure on drug-seeking/taking behaviors used adult animals, whereas rats were adolescent when the current experiments started. We found high risk of relapse in rats with limited but not extended drugs at the adolescent stage. Thus, it is assumed the developmental stage of drug experience matters more than the amount of drug in the young brain. This is based on several observations: First, at the initial drug-taking stage, young brains may be more sensitive to the effects of drugs. This vulnerability of adolescents may be fully masked by chronic drug administration. 21,22 According to clinical observations, the onset of drug use during adolescence appears to lead to a prolonged sensitivity to drug relapse and dependence in humans. 45,46 Second, withdrawal experience of the young brain may trigger more intensive neuronal alterations that facilitate the drug-seeking and drug-taking behaviors even after a prolonged withdrawal period. Our previous works demonstrated active neuronal adaptations by the passage of the withdrawal period. 23,24,47 The current limited cocaine IVSA procedure involves at least two factors, which significantly increase the risk of drug-taking behaviors at the late withdrawal time. One is the limited exposure of cocaine, which is enough for young brains to be reshaped with the abusive potential of drugs. The other is the withdrawal period starting from the adolescent stage when the brain is highly malleable. Our current conclusion, that is, high risk of relapse associated with limited drug exposure at the adolescent stage, followed by a withdrawal period, is not contradictory but an essential complement to the established addiction theory.
Low drug taking observed in rats with a history of extended cocaine IVSA might be attributed to their poor learning and memory, which resulted in a poor retrieval of the previously learned association between the action (ie, lever press) and output (cocaine IV delivery and cue light) in the seeking/taking test during the withdrawal period. This can be excluded by the comparable drug-seeking behaviors between rats pre-treated by limited vs extended cocaine IVSA on either WD1 or WD45, and comparable drug-taking behaviors at 10 mg/kg cocaine per injection on WD46-48, indicating the retrieval of previous acquired operant behaviors was intact. Also, the poor locomotion could be a consequence of extended cocaine IVSA, which can be excluded by the comparable number of inactive lever presses in rats treated by limited vs extended cocaine IVSA (eg, active lever presses in 1-hour drug-seeking test on WD45: limited, 10.1 ± 3.4; extended, 8.7 ± 2.7, P = .34).
Our data showed escalated drug intake in rats with extended IVSA procedure, consistent with previous observations. 48 However, there are some exceptions; data from rhesus monkeys showed no escalated intake over time after extended access to cocaine IVSA. 49 We found that rats with escalated drug-taking behaviors during the extended cocaine IVSA procedure did not show high risk of relapse.
Together with the previous report that the presence of escalation did not affect reinstatement, 48 we assume that the presence of escalation has limited predictability on the risks of relapse. Escalation of cocaine taking was produced by continuous cocaine IVSA. 50 Although the critical involvement of the IC in addiction-related behaviors has been widely accepted, little was known about the intrinsic excitability of IC pyramidal neurons in rats which experienced limited vs extended IVSA. We found some minor but significant effects of limited but not extended cocaine exposure on the kinetics and amplitude of action potentials on WD 45 after limited cocaine IVSA, in pyramidal neurons from the AAI. Although our original hypothesis was that, compared with the saline controls, extended, but not limited, cocaine exposure may lead to significant changes (eg, modified intrinsic excitability) in pyramidal neurons in the AAI, we did not see any effects of extended cocaine IVSA on this electrophysiological readout in insular pyramidal neurons. These findings indicate that limited cocaine exposure has some, probably priming, effects on the capacity of IC neurons to generate APs and these effects are compensated after extended cocaine exposure. It is worth to note that the slower kinetics and smaller amplitude of action potentials observed on WD45 after limited cocaine IVSA could be also observed in unhealthy cells. Our cells in different groups were similar in terms of access resistance and holding current evaluated by Membrane Test in voltage clamp, indicating comparable quality of cells among groups. Thus, the observed changes in kinetics and amplitude of action potentials might be attributable to the adaptations of ion channels (eg, Na + and K + ).
The involvement of the IC in cocaine-related effects is well supported by previous literature. First, molecular alterations in the IC were detected 21 days but not 1 day after 5-day cocaine exposure. Specifically, the cocaine challenge produced a decrease in zif 268 expression after the 21-day, but not 1-day, withdrawal period. 51 Second, by using an extended procedure of cocaine access, the anterior IC was found to mediate the maintenance of escalated cocaine-taking behaviors but not the initial development of cocaine-taking behaviors. 52 Third, different subregions of IC may be involved in cocaine-seeking behaviors triggered by different factors. Agranular IC was involved in drug context-induced reinstatement of cocaine-seeking behavior without altering locomotor activity. 53 Anterior dorsal agranular IC participated in cue-associated cocaine-seeking behaviors, 54 whereas rostral agranular IC was reported to be related to cocaine-seeking behaviors intensified by negative affect. 55 Last but not least, human studies also demonstrated changes in the IC, as it was reported by human imaging studies that cocaine dependence is related to altered functional interactions of the IC with prefrontal networks. 56

ACK N OWLED G M ENTS
This work was supported by NIH grants (R01AA025784) and Brain & Behavior Research Foundation grant #24989.

CO N FLI C T O F I NTE R E S T
The authors report no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The authors confirm that the data supporting the findings of this study are available (1) within this article and (2) from the corresponding author upon reasonable request.