High frequency stimulation and temporary inactivation of the subthalamic nucleus reduce quinpirole-induced compulsive checking behavior in rats
Introduction
Obsessive-compulsive disorder (OCD) represents a highly impairing psychiatric disorder with a lifetime prevalence of 1-3% (Rasmussen and Eisen, 1992, Sasson et al., 1997). Effective treatment options comprise pharmacological interventions, preferentially with selective serotonin reuptake inhibitors (Masand and Gupta, 1999, Piccinelli et al., 1995, Pigott and Seay, 1999, Stein et al., 1995, Zohar et al., 1992) and behavioral therapy (Simpson et al., 2004). In patients refractory to pharmaco- and behavioral therapy, ablative lesions of structures and pathways within the basal ganglia-thalamo-cortical circuits have been shown to reverse clinical symptoms (Lopes et al., 2004). In the treatment of basal ganglia-related neurological disorders, such as Parkinson's disease, ablative lesions have widely been replaced by deep brain stimulation (DBS) at high frequencies (high frequency stimulation, HFS), a reversible and customizable procedure leading to a similar clinical outcome (Breit et al., 2004, Deuschl et al., 2006, Krack et al., 2003, Temel and Visser-Vandewalle, 2004).
In recent years there has been an attempt to establish HFS of structures within the basal ganglia-thalamo-cortical circuits also for the treatment of OCD. Several case reports have assessed the effects of HFS of the anterior limb of the internal capsule (Abelson et al., 2005, Gabriels et al., 2003), the ventral caudate nucleus (Aouizerate et al., 2004, Aouizerate et al., 2005) and the nucleus accumbens and ventral capsule/ventral striatum (Greenberg et al., 2006, Rauch et al., 2006, Sturm et al., 2003) in OCD patients, and there are also reports on the effects of HFS of the subthalamic nucleus (STN) in patients with comorbid Parkinson's disease and OCD (Fontaine et al., 2004, Mallet et al., 2002). The results of these studies are encouraging in showing that HFS may be effective in the treatment of OCD, and that HFS of the ventral striatum region may be particularly effective in alleviating symptoms in OCD. Yet, the inconsistency in the demonstration of beneficial effects and the variability in the time needed to obtain a therapeutic effect, highlight the need for identifying additional brain regions whose stimulation may produce beneficial effects in OCD patients. This goal may be advanced by the assessment of the effects of HFS in appropriate animal models of OCD. To date, experimental data in animal models are limited to only one such report: van Kuyck et al. (2003) found in rats an increase, rather than a decrease, of compulsive-like behavior after electrical stimulation or ablative lesion of the nucleus accumbens. Congruently, the authors cautiously interpreted their results by concluding that either electrical stimulation of the nucleus accumbens may not represent a potential target in the treatment of OCD, or that the model itself did not adequately reflect compulsive-like behavior. Another plausible reason for van Kuyck et al. 's finding is that they have used low frequency stimulation, whereas HFS is typically used in the clinical situation, and the frequency of stimulation has been shown to be a critical factor in determining the behavioral effect of stimulation (for review: Perlmutter and Mink, 2006).
In the present study we have used quinpirole- (QNP) induced compulsive checking behavior in rats as a model of OCD ((Szechtman et al., 1998); for recent reviews of this model and a comparison to other models of OCD: (Eilam and Szechtman, 2005, Joel, 2006, Man et al., 2004)). Rats treated chronically with the dopamine D2/D3 receptor agonist QNP develop compulsive-like behaviors that resemble compulsive checking behavior of OCD patients (Szechtman et al., 1998, Szechtman et al., 2001). The present study was designed to test the effects of HFS and of pharmacological inactivation (by intracerebral muscimol microinjections) of the STN on compulsive checking in QNP-treated rats. This experimental set up enables the assessment of the STN as a potential neurosurgical target in the treatment of OCD and the elucidation of the mechanism(s) underlying the influence of STN-HFS on compulsive-like behavior.
Section snippets
Animals
The present study was carried out in accordance with the European Communities Council Directive of November 24th, 1986 (86/609/EEC) for care of laboratory animals and after approval of the local ethic committee (senate of Berlin). All efforts were made to minimize animal suffering and to reduce the number of animals. Forty-seven naive male Wistar rats (Harlan-Winkelmann, Borchen, Germany, 220-450 g during the experiment) were housed in a temperature- and humidity-controlled vivarium with a
Apparatus and behavioral procedure
Prior to experiments, rats were handled for about 2 min daily for 5 days. With the start of the experiment, rats were injected subcutaneously twice weekly for a total of 13 or 15 injections with either saline (control group) or QNP (QNP group). Fifteen minutes after each injection, animals were placed in an open field and their behavior was videotaped continuously throughout a 30 min session. The open field consisted of a glass table (140 × 140 and 20 cm high) subdivided into 25 rectangles
Design
The experiment consisted of two phases. In phase I, rats received 10 injections (two injections per week with a 3–4 days test-free period) of either 0.5 mg/kg QNP (n = 26) or saline (controls, n = 21), followed by behavioral testing in the open field. Previous work has shown that the effects of chronic treatment with QNP reaches a plateau after 8 to 10 drug injections (Einat and Szechtman, 1993, Szechtman et al., 1994a, Szechtman et al., 1994b, Szumlinski et al., 1997) as well as reliable checking
Surgery
Stereotaxic operations were performed after the 10th session and were carried out under sodium pentobarbital anesthesia (60 mg/kg i.p.). For each operation, the incisor bar was set at 3.3 mm below the interaural line. Electrode implantation: Two electrodes (Concentric bipolar SNEX 100 with connector, RMI Woodland Hills, CA, USA) were implanted bilaterally into the STN -3.8 mm posterior and 2.5 mm lateral from bregma as well as - 7.6 mm ventral from dura (Paxinos and Watson, 1997). Cannula
Systemic and intracerebral drug administration
QNP hydrochloride (Sigma® Aldrich) was dissolved in 0.9% NaCl to a concentration of 0.5 mg/ml and injected subcutaneously under the nape of the neck at a dose of 0.5 mg/kg body weight. Control subjects received the same volume of saline.
Muscimol (Sigma® Aldrich) was dissolved in 0.9% NaCl to a dose of 0.01, 0.005, 0.001 μg per 0.5 μl. These doses have previously been shown to affect rats' performance following administration to the STN (Baunez et al., 2005, Baunez and Robbins, 1999). In
Stimulation
STN-HFS was performed with an isolated stimulator (Coulbourn Instruments, Allentown, PA, USA). Implanted electrodes were connected to the stimulator via an isolated cable system hanging from the ceiling of the behavioral room. A swivel and a minimal resistance hairspring connected the cable system to the implanted electrodes and allowed the rat to freely turn and move on the entire platform without being constricted or tangled up by the cable system during stimulation or sham-stimulation. For
Histology
After the 13th (HFS experiment) or the 15th (pharmacological inactivation experiment) session, rats were anaesthetized with chloral hydrate (50 mg/kg, Merck, Darmstadt, Germany) and perfused transcardially with 0.1 M phosphate buffered saline, followed by ice-cold 4% paraformaldehyde. Brains were removed and postfixed overnight in the same fixative and then stored at 4 °C in 30% sucrose. 40 μm frozen coronal sections were cut using a cryostat. For histological examination, every second section
Statistical Analysis
Phase I: For comparisons between the performance of the two groups (QNP and control) on the last session (10th – baseline) of phase I, t-tests were performed. Phase II: For comparisons between treatment conditions within a group (10th, 12th to 15th test), one way repeated measures analysis of variance (ANOVA) was performed, followed by the Holm Sidak post hoc test for pair wise multiple comparisons, when appropriate. A probability level (p) of less than 0.05 was considered statistically
Anatomical
Figs. 1A and B present a photomicrograph of a coronal section taken from representative rats implanted with an electrode and with a cannula, respectively. The only visible damage in these rats was the electrode/cannulae tracks toward the STN. Figs. 1C and D present a schematic reconstruction of electrodes and cannulae tips, respectively, in the STN. Four QNP-treated rats from the STN-HFS group and 2 QNP-treated rats from the muscimol group were excluded due to inappropriate localization or
QNP-induced Compulsive Checking Behavior
QNP treatment over a total of 10 injections induced compulsive checking behavior as demonstrated with the three performance criteria of compulsive checking introduced by Szechtman et al. (1998). In particular: 1. QNP-treated rats revisited their HB significantly more often than did saline-treated animals (Fig. 2A, P < 0.05). This was also true when taking into account the higher total number of visits to locales in QNP-treated rats compared to control rats. Thus, the ratio of observed to expected
Control rats
HFS of the STN had no effect on the behavior of control rats (Table 1). Specifically, HFS did not affect compulsive checking behavior as measured in the frequency of returns and the return time to the HB as well as in the number of locales visited before coming back to the HB. In addition, HFS did not affect the general amount of locomotion exhibited by control rats.
QNP rats
Fig. 3B presents the total distance traveled by QNP-treated rats on sessions 10, 12 and 13. As can be seen, STN-HFS did not affect locomotion in QNP treated rats (F(2,29) = 2.61, P = 0.10, Fig. 3B).
Fig. 4 presents the different measures of compulsive checking of QNP-treated rats on sessions 10 (no stimulation), 12 (during STN-HFS) and 13 (no stimulation). As can be seen, STN-HFS transiently attenuated QNP-induced compulsive checking in the four measures: 1. QNP-treated rats under HFS (session 12)
Control rats
Microinjections of muscimol into the STN of control rats resulted in a dose-dependent effect on locomotion (Table 1). Specifically, the lowest dose tested (0.001 μg per side) had no effect, whereas the higher doses tested (0.01 and 0.005 μg per side) differentially decreased the total distance traveled, the number of visits to the HB and the return time to the HB. Notably, muscimol at the three doses tested did not affect behavioral measures that are not dependent on the level of locomotion,
QNP rats
Fig. 3C presents the total distance traveled by QNP-treated rats following intra-STN injection of muscimol. As can be seen, the highest dose of muscimol (0.01 μg per side) significantly decreased locomotion whereas the two lowest doses (0.005 and 0.001 μg per side) had no effect on locomotion (F(4,49) = 5.64, P < 0.05).
Fig. 5 presents the effects of muscimol (0.01, 0.005 and 0.001 μg per side) on compulsive checking in QNP-treated rats. In general, the effects of muscimol on compulsive checking
Discussion
The present study assessed the effects of HFS and of pharmacological inactivation of the STN in the QNP rat model of OCD. As has previously been reported (Szechtman et al., 1998, Szechtman et al., 2001), 10 injections of QNP (given twice a week), led to the emergence of compulsive checking in QNP-treated rats. Specifically, QNP-treated rats revisited their HB excessively often and rapidly compared to other locales and to saline-treated controls, and stopped at only a few other locales before
Conclusion
The present study demonstrated that acute HFS of the STN has a specific and selective anti-compulsive effect in a rat model of OCD. Although the extrapolation from an animal model to the clinical condition is problematic, this finding adds to previous reports of alleviation of obsessive-compulsive symptoms in PD patients, and supports the possibility that STN-HFS may be effective in alleviating symptoms in OCD patients. To assess the full implication of these data, further studies will be
Disclosure/Conflict of interest
There is no conflict of interest, financial or otherwise, related directly or indirectly to the submitted work for all authors.
Acknowledgment
We wish to thank C. Koelske and R. Winter for their excellent technical assistance. This study was supported by GIF grant (851/2004). C.W. is a Rahel-Hirsch Fellow of the Humboldt University, Berlin, Germany.
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denotes equal contribution as first authors.