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. Author manuscript; available in PMC: 2014 Jan 1.
Published in final edited form as: Int J Neuropsychopharmacol. 2013 Jan 29;16(6):1295–1307. doi: 10.1017/S146114571200154X

Evaluation of Animal Models of Obsessive-Compulsive Disorder: Correlation with Phasic Dopamine Neuron Activity

Thibaut Sesia 1, Brandon Bizup 2, Anthony A Grace 2
PMCID: PMC3674214  NIHMSID: NIHMS455680  PMID: 23360787

Abstract

Obsessive compulsive disorder (OCD) is a psychiatric condition defined by intrusive thoughts (obsessions) associated with compensatory and repetitive behavior (compulsions). However, advancement in our understanding of this disorder has been hampered by the absence of effective animal models, and correspondingly analysis of the physiological changes that may be present in these models To address this, we have evaluated two current rodent models of OCD; repeated injection of dopamine D2 agonist quinpirole and repeated adolescent injection of the tricyclic agent clomipramine in combination with a behavioral paradigm designed to produce compulsive lever pressing. These results were then compared with their relative impact on the state of activity of the mesolimbic dopaminergic system using extracellular recoding of spontaneously active dopamine (DA) neurons in the ventral tegmental area (VTA). The clomipramine model failed to exacerbate compulsive lever pressing, and VTA DA neurons in clomipramine-treated rats had mildly diminished bursting activity. In contrast, quinpirole treated animals showed significant increases in compulsive lever pressing, which was concurrent with a substantial diminution of bursting activity of VTA DA neurons. Therefore, VTA DA activity correlated with the behavioral response in these models. Taken together, these data support the view that compulsive behaviors likely reflect, at least in part, a disruption of the dopaminergic system, more specifically by a decrease in baseline phasic dopamine signaling mediated by burst firing of DA neurons.

Keywords: Obsessive compulsive disorder, phasic dopamine, animal models, compulsive lever pressing

Introduction

Obsessive compulsive disorder (OCD) is characterized by obsessions (intrusive recurrent thoughts) and compulsions (repetitive aberrant behavior). Current effective therapeutic intervention in patients suggests that dopaminergic and serotonergic systems are involved in controlling compulsive behavior (Denys et al., 2004b; Koo et al., 2010). Thus, dopamine (DA) antagonists are often substituted or added to serotonin reuptake inhibitors to enhance therapeutic efficacy (Koo et al., 2010). Furthermore, imaging studies suggest the presence of a complex imbalance in the DA system in OCD patients (Denys et al., 2004a; Hesse et al., 2005; Kim et al., 2003; Moresco et al., 2007; Olver et al., 2009; Perani et al., 2008; van der Wee et al., 2004). Albeit debatable, the data suggest that OCD patients exhibit lower DA receptor availability primarily in the striatum secondary to increased DA drive.

Obsessions cannot be effectively approximated in animal models. Thus, research has focused on developing models for compulsivity (for review see Albelda and Joel, 2011). Szechtman and colleagues (1998) proposed a pharmacological model produced by repeated injection of the DA D2 agonist quinpirole that induces compulsive checking. This model is responsive to treatments used therapeutically for OCD such as clomipramine (Szechtman et al., 1998) and deep brain stimulation (Klavir et al., 2009; Mundt et al., 2009; Winter et al., 2008). Recently, Andersen and colleagues (2010) introduced an OCD model based on repeated injection of a drug during a sensitive developmental period. Adult rats showed increased anxiety level, perseveration, and working memory deficits which is occurred in concert with increased levels of mRNA for serotonin 5HT2c receptors and DA D2 receptors in the orbitofrontal cortex and striatum.

In this manuscript, we challenge these two models in combination with post-training signal attenuation (PTSA) procedure, a behavioral model developed to assess compulsive lever pressing (Joel and Avisar, 2001). Additionally we examined elevated plus maze and spontaneous alternation behavior to compare with previously published data on these models. Finally, in order to further characterize the role of the DA system in compulsive behavior, we evaluated the activity state of mesolimbic DA neurons to parallel the behavioral testing.

Materials and Methods

Subjects

Male Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN) were housed pairwise on a 12-hour light/dark cycle. Litters for clomipramine treatment were acquired at postnatal day 4 (PD 4) and were initially housed with the dam in a normal light cycle. Weaning occurred at PD24, and the mother and all the females were euthanized. An average litter provided 5–6 males, which were moved to a reverse cycle room at PD 75; 10 days prior any behavioral experiments. The quinpirole treatment rats arrived weighting ~300g and were placed into reverse light cycle housing. Animals that underwent electrophysiological recording remained under normal light cycle. All protocols were consistent with the guidelines outlined in the USPHS Guide for the Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh.

Procedures

Spontaneous Alternation Behavior

Spontaneous alternation behavior (SAB) is T-maze based. Subjects are expected to randomly alternate between right and left arm choices to optimize the area investigated during exploration. Arm choice was recorded for a total of nine trials. Failure to choose within 90 seconds resulted in trial cancellation. The maze was cleaned with 70% ethanol solution between each attempted trial; the subject waited in a holding cage.

Elevated Plus Maze

The elevated plus maze (EPM) consisted of two open and two closed arms attached to a central platform (hub) and raised 50 cm above the floor. Each rat’s exploratory behavior was examined over a 5-minute period and included measures of time in each compartment (open arms, closed arm and hub) and total entries into the open and closed arms.

Signal attenuation and regular extinction

The signal attenuation model was developed (Joel and Avisar, 2001) based on the concept that compulsive behaviors result from a deficit in feedback associated with performance of normal goal-directed responses.

Three days before experiments, rats were subjected to food deprivation that maintained ≥ 85% of free feeding body weight, monitored daily prior to testing.

During stage 1, day 1–3, rats were trained to collect single food pellets paired with a compound stimulus (CS: magazine light and tone). The CS was turned off after the rat visited the food magazine or after 15 s, followed 30 s intertrial intervals. Daily training continued until 30 collected trials (e.g. when the rat visited the food magazine during CS), or until 40 total trials were reached.

During stage 2, day 4–6, rats were presented with two levers into the chamber. Responding on the reinforced lever (RL) resulted in the delivery of a single food pellet into the magazine contingent to CS presentation. The lever designated as RL was counter-balanced among subjects, but was constant for each rat across the procedure. Further lever-presses on the RL or responses on the non-reinforced lever (NRL) had no programmed outcome, but were recorded. The levers were retracted and the CS was turned off following entry into the magazine panel or 15 s following the initial press on the RL. Each trial was followed by a 30 s intertrial interval. A completed trial (CT) means that a RL press is followed by magazine entry during CS presentation. On Day 4, the training stopped after 24 CTs or 60 total trials. Rats that failed to reach 20 CTs were returned to the test chamber at the end of the day for an additional session; rats that failed for the second time to reach 20 CT were excluded from the study. On Days 5 and 6, training ended following 40 CT or 60 total trials.

We monitored the number of CT, the number of unpressed trials (UPT: no RL press) and uncompleted trials (UCT: rat pressed RL but did not visit magazine). In addition, the general behavioral output was assessed in terms of NRL press (NRLP) and extra lever presses after the first RL press (ELP). For the latter measure we also monitored whether ELPs occurred during uncompleted trials (ELP-U) or during completed trials (ELP-C).

In stage 3, days 3–9, rats were subjected to regular extinction (RE; 3 days in home cage) or to signal attenuation post-training (PTSA). PTSA consisted of presentation of the CS without food delivery.. No levers were presented. Daily Sessions lasted 30 trials. The number of first visits to the magazine following CS (collected trial) was monitored.

On stage 4, day 10, rats were tested as in stage 2, but pressing the lever resulted in the presentation of the CS without food delivery. The session lasted for 50 trials. The behavioral measures monitored were identical to stage 2.

A pro-compulsive effect is evidenced by an increase in extra lever presses that are not followed by magazine entry in rats that underwent signal attenuation but not regular extinction (see Joel, 2006 for review). A correct extinction response is defined as a decrease in CT and increase in UPT. General behavioral output is estimated with ELPs and NRLP. A contingent change in UCT and ELP-U was interpreted as an increase (or decrease) in compulsive lever pressing.

Extracellular recordings from VTA dopamine neurons

Male Sprague-Dawley rats (300–450 g) were anaesthetized with chloral hydrate (400 mg/kg i.p.) and placed in a stereotaxic apparatus. Chloral hydrate was utilized for all recordings as under this anesthetic, DA neuron activity states more closely resemble that observed in freely moving rats (Hyland et al., 2002). Anesthesia was maintained by supplemental administration of chloral hydrate i.p. as required to maintain suppression of the hindlimb compression withdrawal reflex. VTA recording was performed as previously described (Lodge and Grace, 2006b). DA neuron activity was determined by counting the number of spontaneously active DA neurons encountered while making 6–9 vertical electrode passes, separated by 200 μm, in a predetermined pattern to sample equivalent regions of the VTA (cells per track, CPT); spontaneous firing rate and pattern was recorded for each neuron encountered. DA neurons were identified using previously described criteria and open filter settings (low cutoff: 3Hz, high cutoff: 30kHz) (Grace and Bunney, 1983; Ungless and Grace, 2012). Each neuron was recorded for a minimum of 6 min and analysis was performed on blocks of 3 min. DA neurons were subdivided into bursting (at least two 3-spike bursts per 500 spike epoch) or non-bursting (Grace and Bunney, 1984). A burst was defined as at least two spikes with an interspike interval ≤ 80 ms, with burst termination defined as a subsequent interspike interval < 160 ms (Grace and Bunney, 1984). Bursting was expressed as the proportion of spikes occurring in bursts (%SIB). The bursting neuron population was calculated and the bursting neuron ratio (BNR) is defined as the number of bursting DA neurons divided by the total number of active DA neurons recorded. Bursting neurons were further analyzed to determine the burst frequency (BF), the average number of spikes per burst (SPB) and the interburst interval (iBi).

Drug Pretreatment and procedures

Clomipramine-treated rats

Injection schedule and drug administration

The injection schedule was based on the published OCD model (Andersen et al., 2010). Between PD9 and PD16, rats were weighed daily at 9:00 AM and given an intraperitoneal injection of physiological saline vehicle or 15 mg/kg clomipramine hydrochloride dissolved in saline (0.9 mg/ml; Sigma-Aldricht, St Louis, MO), followed 7 hours later by a second injection. Pups and matched littermates received a total of 16 injections. After one week of handling the testing periods started at PD 85. As a general observation, during injection schedule rats exposed to clomipramine tended to weigh less than controls (10–20%, data not shown), but was no longer present at testing.

Single exposure behavioral battery

For this behavioral series, four litters were divided into clomipramine exposure (CLO) and saline (control) groups. Rats were tested during the 2 weeks following PD85 (10 CLO and 12 controls). The first week rats were subjected to SAB and the following week to EPM; testing occurred during the dark phase. Three days prior to each experiment the rats were acclimated to the transportation and the testing environment for one hour per day. During EPM testing, 2 CLO and 1 control were removed from the analysis due to falling off the open arms.

Post Training Signal Attenuation procedure

Six litters of rats that were pretreated with either clomipramine or vehicle (saline) were subjected to PTSA procedure. Training started at PD85. Two rats (one from each group) were excluded because they failed stage 1, leaving 16 rats in the CLO group (post-training procedure: 10 signal attenuation and 6 regular extinction, respectively CLO-SA and CLO-RE) and 16 rats in the control group (post-training procedure: 10 signal attenuation and 6 regular extinction, respectively SAL-SA and SAL-RE).

VTA activity recording

Recordings were performed on four litters, evenly divided between saline and clomipramine injection. One rat died during the surgery (CLO) and two rats were excluded due to incorrect electrode placements. The final analysis of the collected data is based on 10 rats subjected to clomipramine and 10 rats subjected to saline, yielding recordings from 70 and 62 DA neurons, respectively.

Quinpirole-treated rats

Injection schedule and drug administration

Quinpirole hydrochloride (Sigma-Aldrich, St Louis, MO) was dissolved in physiological saline (0.5 mg/ml) and injected subcutaneously under the nape of the neck (QUIN; 0.5 mg/kg) twice per week at 3–4 days intervals for 7 weeks, for a total of 14 injections (Szechtman et al., 1994). Vehicle animals were injected in parallel with equal volume of vehicle (saline).

Single exposure behavioral battery

Twenty-four animals were subjected to same order of testing as described for the clomipramine group. Additionally, the rats were tested for SAB and EPM 30–60 min following respectively the 12th and 14th injection of quinpirole or saline. Each group initially consisted of 12 rats; 2 QUIN animals did not reach a total of 9 choices after 50 attempted trials during the SAB test and were excluded from the SAB analysis. Additionally, 5 rats fell of the maze and were excluded from the EPM analysis, leaving 9 QUIN rats and 8 control rats.

Post Training Signal Attenuation procedure

Forty rats were injected with quinpirole (QUIN) or saline (control) for 5 weeks prior to starting the PTSA procedure. Day 1 of training occurred following the 11th injection while day 10 occurred at the last injection of the schedule (i.e. 14th).

One control rat failed stage 1 and three failed at stage 2 and were excluded from analysis. Therefore, 20 rats in QUIN group (10 signal attenuation and 10 regular extinction; respectively QUIN-SA and QUIN-RE) and 19 rats in saline group (10 signal attenuation and 9 regular extinction; respectively SAL-SA and SAL-RE) were analyzed.

VTA activity recording

VTA DA neurons were recorded in three different treatment groups and their respective saline controls. Recordings were initiated 72 hours after the last of 14 injections, defined as quinpirole chronic withdrawn group (QUIN-WTH); these rats had been exposed to PTSA training previously based on Joel and Avisar (2001). A second group was treated identically but recordings were initiated 30 min following the last injection, defined as the quinpirole chronic (QUIN-CHC) group, in which the paradigm corresponded to that of the behavioral data collected here. Recordings were also performed on animals exposed only once to quinpirole (30 min prior recording), defined as the quinpirole acute (QUIN-ACU) group, to compare acute drug effects with the repeated treatment QUIN-CHC group.

A total of 40 rats were divided into 6 groups. Two rats died during recordings, and histology of two other rats showed electrode misplacement. The statistical analysis of the recorded data is based on 7 rats in QUIN-WTH and 7 control rats (yielding respectively 64 and 47 DA neurons), 6 QUIN-CHC and 4 controls (providing respectively 23 and 31 DA neurons), and 6 QUIN-ACU and 6 controls (yielding 21 and 41 DA neurons, respectively)

Apparatus

A plus maze with one arm closed was used for the SAB test. The structure was made locally out of dark grey plastic (0.5 cm thick). The apparatus consisted of three arms (40 cm × 12.7 cm × 25 cm) connected via a hub (14 cm × 14 cm × 25 cm). EPM test was performed on a near infrared backlit maze from Med Associates (175.3 cm × 142.2 cm × 114.3 cm); data were collected using the standard plus maze software (SOF-700RA-3).

Four operant chamber units from Med Associates connected to an 8 channel interface (MED-SYST-8) were used for PTSA and run using software provided by the manufacturer (MED-PC IV). Each unit is composed of an operant chamber housed in a sound-attenuated box. A standard modular test chamber (ENV-008) was used, presenting on one wall a 5 cm wide magazine (2 cm from the grilled floor) flanked on both sides by two 5 cm wide retractable levers (6 cm from the grilled floor and 3 cm apart from the magazine corner to corner). The magazine was equipped with photoreceptors to detect entries into the aperture and a light to illuminate the magazine. The sound generator was located above the magazine (25.5 cm from the grilled floor; 65dB, 2900Hz). The house light was located at the center of the opposite wall (25.5 cm from the grilled floor).

Histology

Following each electrophysiological experiment, the recording site was marked using electrophoretic ejection of Chicago Sky Blue dye (−20 μA constant current: 20–30 minutes). Rats were euthanized with an overdose of chloral hydrate (400mg/kg i.v.), decapitated, and their brains removed. Brains were submerged in 8% paraformaldehyde (in PBS) for fixation for a period of at least 48 hours, and then transferred to a 25% sucrose solution (in PBS) for cryoprotection before being frozen and sectioned on a cryostat in the coronal plane (thickness: 60μm). Sections were placed on gelatin-chromalum-coated slides and stained using cresyl violet for histochemical verification of electrode placement with reference to a stereotaxic atlas (Paxinos and Watson, 1998).

Statistics

For electrophysiological recordings, CLO data were analyzed using the Student’s t test for all parameters. The three control QUIN treatments were compared using one way Analysis of Variance (ANOVA). Because no differences were observed (all F’s<0.36; n.s), the control data were combined. The three treatment groups and the controls were compared using one way ANOVA for all parameters, and if significant, the Dunnett t (2-sided) post-hoc test was performed (using control group as reference).

The behavioral data for stage 2 and 3 was compared between treated animal (CLO and QUIN) and their respective control groups using Student’s t test for all parameters. Stage 2 and stage 4 were compared using a 2 way ANOVA for repeated measures with post-training period (day 6 and day 10) and treatment (treated vs. controls) as independent factors. This analysis was done separately if rats underwent RE or PTSA. Stage 2 has 40 trials while stage 4 has 50 trials in total. We computed a ratio of type of trials (CT, UPT and UCT) to allow for a comparison. At day 6 none of the animals expressed UCT so no ELP-U could be measured.

Finally, the data collected at stage 4 for RE and PTSA groups were compared using a 2 way ANOVA with extinction type (RE vs. SA) and treatment (treated vs. controls). The least significant difference (LSD) method was used for post-hoc analysis.

For all tests, a two-tailed p < 0.05 was considered to be significantly different between the tested groups.

Results

Clomipramine model

All CLO-series behavioral measures are summarized in Table 1 (EPM and SAB) and 2 (PTSA).

Table 1.

Summary of the behavioral measurements collected during SAB and EPM tests of the animals exposed to clomipramine or quinpirole chronically and their respective controls. Results are reported as mean ± SEM.

Behavioral Measures Controls Clomipramine
Treated with Clomipramine
Spontaneous Altermation Behavior
 Number of alternations 4.8 ± 0.4 4.3 ± 0.4
Elevated Plus Maze
 % of time Spent in Open Arms 0.4 ± 0.1 0.4 ± 0.1
 % of time Spent in Closed Arms 0.5 ± 0.0 0.5 ± 0.1
Treated with Quinpirole
Spontaneous Altermation Behavior
 Number of alternations 4.3 ± 0.4 2.7 ± 0.6 *
Elevated Plus Maze
 % of time Spent in Open Arms 0.4 ± 0.1 0.5 ± 0.1
 % of time Spent in Closed Arms 0.5 ± 0.1 0.4 ± 0.1
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*

indicates significant effect compare to respective controls (p<0.05).

Spontaneous Alternation, Elevated Plus Maze, and Post Training Signal Attenuation Behavior

There was no difference between the clomipramine-treated rats and the saline controls in alternations at PD85 (t20=0.90, n.s). Furthermore, No difference between groups were found during EPM test (time in closed arms: t17=0.25, n.s.; time in open arm: t17=0.32, n.s.) or in CT at stage 2 (day 6) or in general behavioral output and type of trials composition at stage 3 (day 9) (all t’s <1, n.s.).

Effect of juvenile exposure to clomipramine on regular extinction

Both saline and clomipramine rats at stage 4 were affected by the regular extinction (RE) procedure (all F1,10>18.72; p’s<0.001). Post-hoc analysis showed that following RE the ratio of CT decreased, while the UPT and UCT increased. This is indicative of an extinction process in reaction to the absence of expected reward.

Both behavioral output measures increased after RE, but only NRLP was sensitive to clomipramine treatment. Rats produce more ELP-C following RE (post-training period: F1,10 = 42.82; p < 0.001), without treatment effect or interaction (all F’s< 1.32; p>0.05). However, the number of NRLP was affected by both clomipramine exposure (treatment: F1,10=5.17; p= 0.046) and RE (post-training period: F1,10=25.04; p=0.001) and their interaction (F1,10= 5.16; p=0.046). Animals exposed to clomipramine showed a higher increase of NRLP than controls after RE. The increase of lever presses following RE matches previous observations and was referred to as extinction burst (Joel and Avisar, 2001).

Effect of juvenile exposure to clomipramine on post-training signal attenuation

The ratio of CT decreased in saline and clomipramine rats at day 10, while UPT and UCT increased (post-training period: F’s>73.21; p’s<0.001) indicating an extinction process.

NRLP were sensitive to SA (post-training period: F1,19= 42.82; p < 0.001), but not to clomipramine treatment or their interaction (F’s<1.83; n.s.); NRLP were greater at day 10. ELP-C remained unaffected by clomipramine (F1,19=2.14; n.s.) or SA (F1,19=3.51; n.s.). Overall, clomipramine treatment did not affect the general behavioral output following SA.

Comparison between regular extinction and signal attenuation at day 10

The number of UCT was higher in rats that underwent SA than RE (figure 1B; extinction type: F1,29=9.83, p=0.004). No treatment effect or interaction was found (F’s<2.5, n.s.). Similarly, rats produced more ELP-U following SA (extinction type: F1,29=6.46, p=0.017) without treatment or interaction effects (F’s< 2.2, n.s.; Fig. 1D). Clomipramine exposure did not potentiate compulsive behavior induced by PTSA.

Figure 1.

Figure 1

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Juvenile exposure to clomipramine does not further affect compulsive lever pressing following signal attenuation post training or performance after regular extinction. The upper panel shows the mean (± SEM) number of (A) completed, CT, and (B) uncompleted trials, UCT, in rats exposed to clomipramine (black bars) and their controls (white bars). The mean number of CT decreases when exposed to signal attenuation in both groups and the number of UCT increased in both group as well. The lower panels shows the number (mean ± SEM ) of extra lever presses that were followed by an attempt to collect a reward (C), ELP-C, and that were not followed by an attempt to collect a reward (D), ELP-U. The mean number of ELP-C decreases when trained with signal attenuation while the number of ELP-U increases in both controls and treated animals. * indicates significant effect (p<0.005).

Rats that underwent RE exhibited more completed trials than the groups trained with SA (extinction type: F1,29=24.55, p<0.001 Figure 1A). Conversely, rats exposed to SA produced more UPT than rats exposed to RE (extinction type: F1,29=14.85; p=0.001). No treatment effect or interaction was found (all F’s<2.6, n.s.). SA increased the sensitivity to reward extinction.

Animals exposed to RE produced more ELP-C (extinction type: F1,29=8,71, p= 0.006). No treatment or interaction effects were observed (F’s<2.3, n.s; figure 1C). Despite no extinction type or treatment effects (F’s<0.67, n.s.), NRLP analysis showed an interaction effect (F1,29=6.60, p=0.016): CLO-RE animals expressed more NRLP than any other group. Clomipramine exposure did not alter the general behavioral output in this paradigm.

VTA Recordings

No differences were found between controls and CLO group in number of active DA neurons (firing t18=0.09, n.s.; Fig. 2A, Table 3), their average firing rate (t130=1.62, n.s.; Fig. 2B) or the ratio of bursting DA neurons (t18=1.50, n.s.; Fig.2C). Furthermore, there were no differences in activity across the anterior-posterior axis of the VTA. However, DA neurons in clomipramine-treated animals exhibited fewer spikes occurring in bursts (t130=2.97, p = 0.04; Fig. 2D) and burst frequency (t84=3.51, p= 0.001), plus a higher interburst interval (t84=3.35, p= 0.001). No effect was found in the average number of spikes per burst (t84=1.699, n.s.). Therefore, clomipramine exposure led to a decrease in bursting activity in the VTA by altering burst frequency but not burst structure.

Figure 2.

Figure 2

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Juvenile exposure to clomipramine decreases the bursting activity of dopamine neurons in the VTA at the same age as that of behavioral testing. Measures are presented as mean ± SEM, controls are showed in white bars and clomipramine-exposed animals (CLO) in black bars. The upper panels show the average number of active dopamine neurons per electrode track (A) and their average firing rate (B). The lower panels present the proportion of bursting neurons in the recorded population (C) and the percentage of spikes that occurred in bursts for the spontaneously active DA neurons (D). Only the percentage of spikes in bursts was affected by the clomipramine pre-treatment and showed a 41% decrease compare to controls. * indicates significant effect (p<0.005).

Table 3.

Summary of the extracellular recording data from DA neurons in the VTA of the animals exposed to clomipramine, quinpirole chronically and then withdrawn (QUIN-WTH), chronically (QUIN-CHC), acutely (QUIN-ACU), and their respective controls. Results are reported as mean ± SEM.

VTA Recording measures Controls QUIN WTH QUIN CHC QUIN ACU Statistical significance (p)
Bursting Neuron Ratio 0.66 ± 0.05 0.63 ± 0.05 0.27 ± 0.10* 0.43 ± 0.15 0.009
Cells per track 1.01 ± 0.05 1.49 ± 0.09* 0.60 ± 0.09* 0.43 ± 0.08* <0.001
Firing Rate (Hz) 3.41 ± 0.18 4.07 ± 0.25 2.48 ± 0.33 2.24 ± 0.35* <0.001
Spikes in Bursts (%) 28.03 ± 2.33 27.35 ± 3.38 8.75 ± 3.42* 13.48 ± 4.48* 0.001
Burst Frequency (Hz) 0.52 ± 0.04 0.66 ± 0.06 0.25 ± 0.07 0.37 ± 0.10 n.s.
Spikes per Burst 3.29 ± 0.19 3.21 ± 0.16 3.35 ± 0.45 2.84 ± 0.15 n.s.
Interburst Interval (sec) 3.35 ± 0.38 2.54 ± 0.42 5.15 ± 1.67 5.33 ± 2.05 n.s.
VTA Recording measures Control Clomipramine Statistical significance (p)
Bursting Neuron Ratio 0.72 ± 0.07 0.56 ± 0.08 n.s.
Cells per track 0.99 ± 0.06 0.98 ± 0.10 n.s.
Firing Rate (Hz) 3.69 ± 0.21 3.24 ± 0.18 n.s.
Spikes in Bursts (%) 24.83 ± 2.93 14.68 ± 1.89 0.004
Burst Frequency (Hz) 0.53 ± 0.06 0.29 ± 0.04 0.001
Spikes per Burst 2.85 ± 0.11 2.62 ± 0.08 n.s.
Interburst Interval (sec) 2.92 ± 0.42 5.94 ± 0.80 0.001
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*

indicates significant difference from controls (p<0.005).

Quinpirole model

All behavioral measures are summarized in Table 1 (EPM and SAB) and 2 (PTSA).

Spontaneous Alternation, Elevated Plus Maze, and Post Training Signal Attenuation Behavior

Animals exposed to quinpirole performed significantly less alternations than controls (t20= 2.225, p = 0.038). There was no difference between the quinpirole-treated rats and controls when exposed to the EPM (time in closed arms: t15= 0.33, n.s.; time in the open arms: t15= 0.68, n.s.) or in CT at stage 2 (day 6) or in general behavioral output and type of trials composition at stage 3 (day 9) (all t values <1.1; n.s.).

Effect of chronic exposure to quinpirole on regular extinction

At day 10, CT and UPT occurrences were affected by RE (post-training period: CT: F1,17=92,73, p<0.001 and UPT: F1,17= 72.37, p<0.001), quinpirole exposure (treatment: CT: F1,17=9.07, p=0.008 and UPT: F1,17= 9.60, p= 0.007) and their interaction (CT: F1,17=10.55, p=0.005 and UPT: F1,17= 11.80, p=0.003). Quinpirole rats expressed more CT and less UPT than controls (SAL-RE) after regular extinction, which could indicate an impaired extinction process.

The ratio of UCT was sensitive to RE (post-training period: F1,17=41.40, p<0.001) with no effect of quinpirole injections and no interaction (F’s<0.20, n.s.). UCT were nonexistent at stage 2 (day 6) but represented 11% of the trials at stage 4 (day 10).

ELP-C were unaffected by RE or quinpirole injections (F’s< 1.2, n.s.). RE procedure promoted NRLP (post-training period: F1,17= 5.38, p=0.033), but only in controls (interaction: F1,17=12.54, p= 0.003). Quinpirole-treated rats expressed less NRLP than controls (treatment: F1,17= 5.28, p= 0.035). Unlike clomipramine rats, quinpirole-treated animals did not express “extinction burst”.

Effect of chronic exposure to quinpirole on post-training signal attenuation at day 10

The ratio of UCT was affected by SA (post-training period: F1,18=78.07, p<0.001), quinpirole injections (treatment: F1,18=5.94, p=0.025) and their interaction (F1,18=5.94, p=0.025). This indicates that the signal attenuation post-training triggered more non goal-directed behavior in quinpirole-exposed animals (QUIN-SA) than in controls (SAL-SA).

Signal attenuation affected both the ratio of CT (post-training period: F1,18=327,57, p<0.001) and UPT (post-training period: F1,18=99.65, p<0.001), respectively decreasing and increasing these measures following SA. Only UPT was also sensitive to quinpirole exposure (treatment: F1,18=9.77, p=0.006) and their interaction (F1,18=9.79, p=0.006). SAL-SA group expressed more UPT than QUIN-SA. This effect could result from the great increase in UCT which would have had to occur at the expense of another type of response and did not necessarily preclude any extinction process deficiency.

Similar to that in regular extinction groups, no differences were found with extra lever presses during completed trials (ELP-C: F’s<2.80, n.s.). In addition, NRLP was sensitive to SA (post-training period: F1,18=13.92, p=0.002), quinpirole exposure (treatment: F1,18=9.20, p=0.007) and their interaction (F1,18=10.41, p=0.005). The number of NRLP increased following stage 3 only in controls, which together shows that the general behavioral output is lower in quinpirole-treated animals.

Comparison between regular extinction and signal attenuation post-training

The number of UCT was higher when animals underwent signal attenuation (extinction type: F1,35=13.86, p= 0.001; Fig. 3B). There was an interaction between treatment and extinction type factors (F1,35= 7.88, p= 0.008), but no treatment effect (F1,35= 1.44, n.s.). QUIN-SA UCT occurrences were significantly higher than in controls. Moreover, the number of ELP-U was increased by SA (extinction type: F1,35=5.37, p= 0.026; Fig. 3D). No other effect was found (F’s<2.89, n.s.). The compulsive behavior (UCT and ELP-U) was consistent with previously published work on signal attenuation post-training schedule (Joel and Doljansky, 2003); furthermore, chronic exposure to quinpirole increased the number of non-goal-directed responses following signal attenuation, and ELP-U apparently was not affected by the general decrease in behavioral output. Overall these data suggest a potentiation of the effect of the procedure on compulsive behavior following quinpirole injections.

Figure 3.

Figure 3

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Chronic exposure to quinpirole potentiated the compulsive behavior induced by signal attenuation. The upper panel shows the mean (± SEM) number of completed (A), CT, and uncompleted trials (B), UCT in rats exposed to quinpirole (black bar) and their controls (white bars). The mean number of CT decreased when exposed to signal attenuation in both groups and the number of UCT increased in both group as well; rats exposed to quinpirole produced more CT than controls. Rat exposed to quinpirole and signal attenuation performed more UCT than any other group. The lower panels show the number (mean ± SEM ) of extra lever presses that were followed by an attempt to collect a reward (C), ELP-C, and that were not followed by an attempt to collect a reward (D), ELP-U. The mean number of ELP-C was lower in animals treated with quinpirole in both conditions. The number of ELP-U increased in both controls and treated animal following signal attenuation. * indicates significant effect (p<0.005).

The number of CT was higher following regular extinction (Fig. 3A), while the number of UPT was higher in groups that underwent signal attenuation post-training (extinction type; CT: F1,35= 29.42, p<0.001; UPT: F1,35= 8.95, p= 0.005). Moreover, quinpirole treated animals performed more CT and less UPT than their respective controls, suggesting an alteration of extinction processes induced by the drug (treatment; CT: F1,35= 9.66, p=0.004; UPT: F1,35= 19.95, p<0.001). No interaction was found (F’s< 2.71; n.s.). SA again promoted the extinction process, while quinpirole injections tended to act against it.

The general behavioral output (ELP-C and NRLP) was affected in the same way by chronic quinpirole injection (treatment: respectively, F1,35= 4.37, p= 0.044; F1,35 = 26.08, p<0.001; ELP-C: Fig. 3C). Both types of lever presses were decreased in quinpirole-treated animals regardless of the type of post-training to which they were exposed (post-training and interactions: all F’s<2.98, n.s.).

VTA Recordings

All control groups were merged and compared to rats: withdrawn after chronic exposure (QUIN-WTH), chronically exposed (QUIN-CHC), and acutely exposed (QUIN-ACU) to quinpirole. All measurements are reported in Table 3.

The number of spontaneously active DA neurons was altered by the various quinpirole injections regimen (F3,32=34.18, p<0.001): QUIN-CHC and QUIN-ACU rats had significantly fewer active DA neurons per track and QUIN-WTH had more active DA neurons, compared to controls (Fig. 4A). Furthermore, there were no differences in activity across the anterior-posterior axis of the VTA. QUIN-CHC animals had fewer bursting DA neuron than controls (F3,32=4.52, p= 0.009; Fig. 4C). This change was associated with a lower percentage of spikes in bursts (%SIB: F3,223=5.55, p=0.001; Fig. 4D). Acute exposure to quinpirole (QUIN-ACU) resulted in a lower average firing rate compared to controls (F3,223=7.00, p<0.001; figure 4B), and this was associated with a lower %SIB (F3,223=5.57, p=0.001).

Figure 4.

Figure 4

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Exposure to quinpirole changes the activity of dopamine (DA) neurons in the ventral tegmental depending on the degree of chronicity. Measures are presented as mean ± SEM, controls are showed in white bars, chronically and then withdrawn from quinpirole (QUIN-WTH) group in black bars, chronic group (QUIN-CHC) in grey bars and acute group (QUIN-ACU) in dashed bars. The upper panels show the average number of active dopamine neuron per track recorded (A) and their firing rate (B). Withdrawal from quinpirole resulted in a higher number of active neurons per track, while chronic or acute exposure decreased their numbers. Acute exposure also decreased the average firing rate of the recoded neurons. The lower panels present the ration of bursting dopamine neurons in the recorded population (C) and the percentage of spikes that occur in bursts of the spontaneously active DA neurons (D). Both acute and chronic exposure to quinpirole decreased the bursting activity of recorded DA neurons, but only chronic exposure decreased the number bursting neurons in the population of DA neurons recorded. * indicates significant effect (p<0.005).

The burst structure was unaltered when comparing between groups. ANOVAs revealed a significant effect on burst frequency (F3,223=3.18, p=0.026), but post-hoc tests did not reveal any differences between treated and control groups. The other parameters were not affected (F’s<2.20, n.s.). Therefore, acute and chronic exposure to quinpirole decreased VTA activity both in terms of active cells and bursting activity; withdrawal in contrast increased overall VTA output.

Discussion

In comparing two models of OCD, we found that the clomipramine model, in our hands, did not robustly replicate the behavioral features of OCD adequately to use as a screen. The clomipramine model did not further affect indices of behavior using EPM, SAB or PTSA paradigms. In contrast, the quinpirole model showed several alterations consistent with an augmentation of compulsive behavior but without affecting anxiety level. This included the highest number of uncompleted trials and a 300 % increase in extra-lever pressing during these trials compared to the performance prior to the signal attenuation stage. We also found that DA neuron activity corresponded to behavioral outcomes. Exposure to clomipramine reduced VTA DA neuron bursting activity, whereas repeated injection of quinpirole reduced both the number of bursting DA neurons and the percentage of spikes that occurred in bursts. Given that this was a decrease in baseline burst firing, stimulus-evoked bursting should therefore have a greater relative impact in the quinpirole-treated rats.

Compulsive behavioral model

Rats treated with clomipramine did not show differences in EPM or SAB performance, which differs from a previous report (Andersen et al., 2010). Treated animals also did not exhibit potentiation in the PTSA paradigm. Behavioral outcomes for both regular extinction and signal attenuation groups were comparable to controls. The CLO RE group expressed a specific increase in NRLP. No other lever presses were affected; therefore, an increase in general behavioral output resulting from regular extinction is unlikely. Alternatively, it can indicate an enhanced sensitivity to reward extinction which translates to a greater extinction burst specific to the unrewarded lever.

Rats treated with quinpirole showed increased preservative behavior (decreased alternation in SAB) with no effect on the level of anxiety (EPM). A decrease in alternation has been reported previously for the quinpirole model (Einat and Szechtman, 1995). To our knowledge no data was reported previously on the effects of repeated injection of quinpirole on EPM exploration. However, it was reported that local infusion of high doses of quinpirole into the basal lateral amygdala resulted in less time spent in open arms without altering the time spent in closed arms (Bananej et al., 2011). Single local high dose may not compare to chronic systemic injection. Repeated injection of quinpirole potentiated the compulsive lever pressing (combined effect on UCT and ELP-U). The increase in UCT is substantial, while the effect on ELP-U is dampened by the general decrease in behavioral output. The increase in ELP-U following signal attenuation is specific because all other lever pressing parameters were decreased in QUIN groups regardless of the post-training (i.e. NRLP and ELP-C). Additionally, rats treated with quinpirole showed impaired extinction: the number of completed trials in the non-rewarded condition increased in all groups exposed to quinpirole injections at the expense of the unpressed trials (correct extinguished response) similar to that reported previously (Dubrovina and Zinov’eva, 2010; Kurylo, 2004; Kurylo and Tanguay, 2003). The effect of the treatment on uncompleted trials is specific to QUIN-SA (interaction effect between treatment and procedure factors). The fact that the QUIN-SA group expresses more UCT than any other group can explain the weaker drug effect on its CT numbers.

Mesolimbic dopaminergic activity and compulsive lever pressing

Clomipramine-exposed rats exhibited a decrease in VTA dopamine neurons without exhibiting behavioral alterations.

Acute injection of quinpirole decreased the number of active DA neurons in the VTA, and decreased the activity of the spontaneously firing DA neurons (lower firing rate and fewer spikes/burst). To our knowledge, no behavioral data are available for compulsive behavior at that stage.

Repeated injection of quinpirole is known to induce long-term changes in dopaminergic receptors by downregulating both the number of receptors available and their mRNA levels (Chen et al., 1993). If D2 receptors are downregulated, we expected the VTA activity in the QUIN-CHC group to be less affected by the last exposure prior to recording. However, chronic exposure to quinpirole induced stronger changes in VTA activity: it reduced the number of active neurons as well as produced a marked shift in DA neuron activity from bursting to non-bursting, with the remaining bursting neurons showing less spikes in bursts and less frequent burst episodes. Therefore, at baseline the amount of bursts reaching the VTA projection sites would be significantly diminished. Coincidentally, we observed the strongest effect on compulsive behavior following chronic treatment. The compulsive lever pressing observed in the PTSA schedule was hypothesized to reflect a decrease in phasic dopamine mediated by dopamine D1 receptors in the striatum (Joel and Doljansky, 2003). Both low-affinity D1 receptors (Dreyer et al.; Gonon, 1997; Lodge and Grace, 2006a) and intrasynaptic D2 receptors (Floresco et al., 2003) can be stimulated by phasic DA release secondary to burst firing of DA neurons (Floresco et al., 2003; Goto and Grace, 2005). Therefore, phasic burst firing and the number of DA neurons that can be recruited to burst fire (Lodge and Grace 2006a) could provide an effective correlate of DA disruption in OCD. Given that the baseline, non-stimulated burst firing is less following quinpirole, this would suggest that stimulus-driven bursting should have a greater impact on behavior, which is consistent with increased DA responsivity believed to contribute to this pathology. It has been reported that repeated daily injection of quinpirole induced more compulsive responses in the withdrawn state (Joel et al., 2001). Our results suggest that a withdrawn state induces an overactive VTA by increasing the number of active neurons. In this early work on the PTSA model, the effect on compulsive lever pressing was due to a general increase of extra lever presses (regardless of the type of trials during which it was produced), in which the majority of them were generated during completed trials. The number of UCT previously reported in the quinpirole-treated rats (Joel et al., 2001) was on average less than 6. Animals tested in our experiment generated on average 19.2±3.2 uncompleted trials.

Conclusion

In our hands neonatal injection of clomipramine failed to produce any sort of compulsive behavior. This model has also been used as an animal model for endogenous depression (Andersen et al., 2002; Bhagya et al., 2008; Cassano et al., 2006; Vogel et al., 1988; Vogel et al., 1990a; Vogel et al., 1990b; Vogel et al., 1990c; Vogel et al., 1990d). More investigation is needed to clarify which model juvenile exposure to clomipramine better approximates. In contrast, quinpirole-treated animals demonstrated clear increases in compulsive behavior in the PTSA test and a reliable perseveration in SAB test, which further strengthens the face validity of this model. Furthermore we found that the degree of alteration in bursting activity of the VTA is predictive of the amount of uncompleted type of trials observed following signal attenuation and the associated amount of repetitive lever pressing.

Table 2.

Summary of the behavioral measurements collected during PTSA procedure of the animals exposed to clomipramine or quinpirole chronically and their respective controls. Results are reported as mean ± SEM.

Behavioral Measures Controls (RE) Clomipramine (RE) Controls (SA) Clomipramine (SA)
Lever-Press Training
 Completed Trials 40.0 ± 0.0 40.0 ± 0.0 40.0 ± 0.0 40.0 ± 0.0
 Unpressed Trials 0.3 ± 0.2 0.0 ± 0.0 0.6 ± 0.2 1.0 ± 0.3
 Uncompleted Trial 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
 Extra-lever press - C 10.7 ± 1.4 10.8 ± 2.6 19.3 ± 6.3 12.7 ± 3.2
 Non Rewarded-lever Press 0.7 ± 0.5 1.0 ± 0.3 1.1 ± 0.5 5.8 ± 2.3
Signal Attenuation
 Completed Trials 10.0 ± 1.0 8.6 ± 1.1
 Uncompleted Trial 20.0 ± 1.0 21.4 ± 1.1
Test
 Completed Trials 24.2 ± 4.0 27.0 ± 2.3 12.4 ± 1.7a 13.0 ± 2.4a
 Unpressed Trials 20.7 ± 3.1 20.0 ± 2.1 27.2 ± 1.4a 29.4 ± 1.8a
 Uncompleted Trial 5.2 ± 1.8 3.0 ± 0.4 10.5 ± 1.7a 7.6 ± 1.2a
 Extra-lever press - C 34.5 ± 6.8 42.2 ± 4.4 27.7 ± 3.2a 21.7 ± 4.3a
 Extra-lever press - U 29.2 ± 10.1 22.5 ± 5.2 61.8 ± 10.1a 43.4 ± 10.3a
 Non Rewarded-lever Press 7.7 ± 1.6 19.7 ± 5.0c 18.2 ± 3.5 12.0 ± 2.6
Behavioral Measures Controls (RE) Quinpirole (RE) Controls (SA) Quinpirole (SA)
Lever-Press Training
 Completed Trials 40.0 ± 0.0 40.0 ± 0.0 40.0 ± 0.0 40.0 ± 0.0
 Unpressed Trials 0.7 ± 0.3 0.6 ± 0.4 0.1 ± 0.1 0.3 ± 0.2
 Uncompleted Trial 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
 Extra-lever press - C 23.6 ± 7.2 20.4 ± 4.7 17.0 ± 2.3 23.7 ± 6.6
 Non Rewarded-lever Press 3.7 ± 2.0 3.7 ± 1.6 3.5 ± 1.9 2.0 ± 0.9
Signal Attenuation
 Completed Trials 7.7 ± 1.4 11.2 ± 2.0
 Uncompleted Trial 22.3 ± 1.4 18.8 ± 2.0
Test
 Completed Trials 23.1 ± 2.5 36.4 ± 3.5b 12.5 ± 1.9a 16.6 ± 3.2a,b
 Unpressed Trials 17.9 ± 1.7 7.9 ± 2.6b 26.6 ± 1.9a 14.2 ± 3.5a,b
 Uncompleted Trial 9.0 ± 1.5 5.7 ± 1.7b 10.9 ± 1.2a 19.2 ± 3.2a,c
 Extra-lever press - C 36.5 ± 8.4 20.9 ± 5.0b 23.2 ± 6.1 12.4 ± 4.7b
 Extra-lever press - U 37.7 ± 4.3 9.0 ± 3.7 45.6 ± 4.7a 43.3 ± 16.2a
 Non Rewarded-lever Press 11.7 ± 2.2 2.0 ± 0.8b 15.9 ± 3.5 2.9 ± 0.9b
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a, b and c indicates significant effect respectively for extinction type, drug treatment and interaction of both (p<0.05).

Acknowledgments

The authors thank Niki MacMurdo for technical support and Sarah Schreiber for participation in this research. This research was founded by NIH (MH086400)

Footnotes

Statement of Interest: During the past years, Anthony A. Grace has received consulting honoraria from Johnson & Johnson, Lundbeck, Pfizer, GSK, Puretech Ventures, Merck, Takeda, Dainippon Sumitomo, Otsuka, Eli Lilly, and Roche.

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