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Behavioral responses to injections of muscimol into the subthalamic nucleus: Temporal changes after nigrostriatal lesions
Neuroscience 131 (2005) 769 –778
BEHAVIORAL RESPONSES TO INJECTIONS OF MUSCIMOL INTO THE SUBTHALAMIC NUCLEUS: TEMPORAL CHANGES AFTER NIGROSTRIATAL LESIONS A. MEHTA,a L. MENALLEDa AND M.-F. CHESSELETa,b*
include tremor, rigidity and akinesia, and develop over the course of several years (Narabayashi, 1995; Poewe and Wenning, 1998). A major consequence of dopaminergic lesions is an increase in firing in the subthalamic nucleus (Albin et al., 1989). This effect has been observed in parkinsonian patients (Hutchison et al., 1998), monkeys treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP; Bergman et al., 1994), and rats with a lesion of the nigrostriatal pathway induced by 6hydroxydopamine (6-OHDA; Ni et al., 2001). In patients and animal models of Parkinson’s disease, ablation, inhibition or high frequency stimulation of the subthalamic nucleus markedly improve akinesia and rigidity, suggesting that alterations in this brain region are crucial for parkinsonian symptoms (Bergman et al., 1990; Benazzouz et al., 1993; Kumar et al., 1998; Baron et al., 2002; Dostrovsky et al., 2002). Models of basal ganglia circuitry predict that the increased activity of subthalamic neurons after nigrostriatal degeneration is due at least in part to a decreased activity of the globus pallidus (external pallidum in primates), which provides a dense input of GABA-containing nerve terminals to the subthalamic nucleus (Albin et al., 1989; Smith et al., 1990). Data from both rats and monkeys show that nigrostriatal lesions cause a decrease in spontaneous activity in neurons of the external pallidum; the same studies, however, show a simultaneous increase in burst firing (Pan and Walters, 1988; Filion and Tremblay, 1991). Therefore, the net effect of nigrostriatal degeneration on pallido-subthalamic transmission remains unclear. Similarly, studies of changes in glutamic acid decarboxylase (Mr 67,000; GAD67) GAD mRNA expression in the globus pallidus, an index of GABAergic activity, have provided ambiguous results (Chesselet and Delfs, 1996). Previous studies from our laboratory and others have shown increased levels of mRNA encoding GAD67, one of the rate-limiting enzymes of GABA synthesis, in neurons of the globus pallidus 2–3 weeks after lesions of the nigrostriatal pathway in rats (Soghomonian and Chesselet, 1992; Kincaid et al., 1992; Delfs et al., 1995). According to a recent study (Billings and Marshall, 2004), this increase occurs both in parvalbumin-positive and parvalbuminnegative neurons of the globus pallidus, but to a larger extent in the latter. An increase in GAD67 mRNA in the external pallidum was also observed in MPTP-treated cats and monkeys (Soghomonian et al., 1994; Schroeder and Schneider, 2001). Another study, however, found no change in GAD67 mRNA in the external pallidum of MPTPtreated monkeys and in postmortem human tissue
a
Department of Neurology, University of California Los Angeles, B114, Reed Neurological Research Center, 710 Westwood Plaza, Los Angeles, CA 90095, USA b Department of Neurobiology, University of California Los Angeles, Los Angeles, CA 90095, USA
Abstract—Changes in cellular activity in the subthalamic nucleus are a cardinal feature of Parkinson’s disease and occur in rodents after lesions of the nigrostriatal pathway, a model of Parkinson’s disease. GABA-ergic neurons from the globus pallidus provide a major input to the subthalamic nucleus. Previous electrophysiological studies revealed temporal changes in the activity of pallidal neurons after nigrostriatal lesions in rats. However, little is known about the impact of these changes on GABAergic transmission in the subthalamic nucleus. We have examined the behavioral responses to a local administration of the GABA A agonist muscimol into the subthalamic nucleus. Muscimol (0.01 and 0.1 g) induced orofacial dyskinesia in normal rats; this response was blunted 2 weeks but enhanced 2 months after a unilateral lesion of the nigrostriatal pathway. The early decrease in the behavioral response occurred at a time when increased expression of mRNA for glutamic acid decarboxylase, the enzyme of GABA synthesis, and burst firing have been reported in the globus pallidus, suggesting an adaptive post-synaptic response to increased GABAergic transmission in the subthalamic nucleus. In contrast, we now show that glutamic acid decarboxylase mRNA is unchanged in the globus pallidus at the later time point, when electrophysiological changes also subside in this region. The increased behavioral response at this later time point may reflect a decreased activity in GABAergic inputs to the subthalamic nucleus. The results show time-dependent changes in behavioral responses to GABA A receptor stimulation in the subthalamic nucleus which may reflect adaptive changes in postsynaptic inhibitory responses after dopaminergic lesions. © 2005 Published by Elsevier Ltd on behalf of IBRO. Key words: globus pallidus, GABA, GABAA receptor, Parkinson’s disease, 6-hydroxydopamine, basal ganglia.
Parkinson’s disease is characterized by the progressive development of motor symptoms resulting from the degeneration of dopaminergic inputs from the substantia nigra pars compacta to the striatum. The cardinal symptoms *Corresponding author. Tel: ⫹1-310-267-1781; fax: ⫹1-310-2671786. E-mail address: [email protected] (M.-F. Chesselet). Abbreviations: ANOVA, analysis of variance; BTCP, 3H-N-[1-92benzo(b)thiophenylcyclohexyl]-piperidine; GAD67, glutamic acid decarboxylase (Mr 67,000); MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; 6-OHDA, 6-hydroxydopamine. 0306-4522/05$30.00⫹0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2004.11.036
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(Herrero et al., 1996). These differences may be due to time-dependent changes in the pallido-subthalamic pathway following nigrostriatal degeneration. Indeed, Pan and Walters (1988) showed that the burst firing activity measured in the globus pallidus of rats peaked 1 week after lesions of the nigrostriatal pathway and decreased with time, returning to control levels by 6 –9 weeks post-lesion. These data suggest that neuronal activity in the pallidosubthalamic pathway may be temporally regulated after acute nigrostriatal lesion. Changes in GABAergic inputs can induce compensatory alterations in post-synaptic receptors that influence the response of neurons to GABAergic agonists (Penney and Young 1981; Brussaard and Herbison, 2000). The present study was designed to gain insight into the temporal changes of GABAergic transmission in the subthalamic nucleus after acute dopaminergic lesions in rodents. Dyskinesia induced by the local administration of the GABA-A receptor agonist muscimol into the subthalamic nucleus were examined 2–3 weeks and 8 –9 weeks after unilateral lesions of the nigrostriatal dopaminergic neurons in adult rats. In addition, levels of expression of GAD67 mRNA in the globus pallidus were examined with quantitative in situ hybridization histochemistry and emulsion autoradiography at the later time point.
EXPERIMENTAL PROCEDURES Animals A total of 55 male Sprague–Dawley rats (270 –310 g; Charles River, Wilmington, MA, USA) separated in four cohorts were used in this study. All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and all efforts were made to reduce the number of animals. All experimental protocols were approved by the University of California Los Angeles animal care committee.
Surgery Prior to surgery, rats were maintained on a 12-h light/dark schedule with food pellets and water ad libitum. Rats were anesthetized with equithesin (prepared as per instruction of Janssen-Salbutry Laboratories, Kansas City, MO, USA; 3.0 ml/kg, i.p.) and received an unilateral infusion of either 6-OHDA (8 g/4 l) or vehicle (4 l 0.1% ascorbic acid in 0.9% saline) into the left substantia nigra pars compacta (AP⫽⫹3.4 mm, ML⫽⫹2.0 mm, both relative to interaural zero, and DV⫽7.4 mm below the surface of the cortex; Paxinos and Watson, 1998). Thirty minutes prior to surgery, all rats were pretreated with desipramine (25 mg/kg, i.p.) to prevent the uptake of 6-OHDA into noradrenergic neurons. For delivery of muscimol into the subthalamic nucleus, rats were implanted with a guide cannula for unilateral microinfusion of drug into the left subthalamic nucleus (AP⫽⫹4.9 mm, ML⫽ ⫹2.5 mm, both relative to interaural zero, and DV⫽7.4 mm below the surface of the cortex; Paxinos and Watson, 1998). Details of the surgical procedure and injection equipment have been described previously (Parry et al., 1994). Briefly, the guide shafts were made from 22-gauge cannulae cut to a length of 17 mm and the injector was made of a piece of fused silica (o.d.⫽150 m; i.d.⫽75 m) threaded through a 28-gauge internal cannula such that the tip of the fused silica extended 1 mm from the end of the internal cannula shaft. In rats with prior lesion of the nigrostriatal pathway and their sham-operated controls, this procedure was
done 12 days after the first surgery. In all cases, behavioral testing began 1 week after implanting the cannula.
Drug administration and behavioral testing No behavioral testing was conducted to assess the level of nigrostriatal lesion to avoid any confounding effect on the behavioral analysis of muscimol effects. Loss of dopaminergic nerve terminals was determined histologically at the end of the experiment as described below. In a first cohort of rats (cohort I; n⫽6), vehicle or muscimol at the concentrations of 0.001, 0.01, 0.1, 1.0 g were injected into the subthalamic nucleus following a Latin-square design so that each rat received all the doses and vehicle, in a random order. Testing was done every 4 days. For cohort II (n⫽16) and cohort III (n⫽16), each rat was treated only once, either with muscimol or vehicle. Cohort II and cohort III were examined 2–3 and 8 –9 weeks post-lesion, respectively, to avoid any confounding effect of prior treatment. On the day of behavioral testing, each rat was adapted to a quiet room in a clear plastic cylindrical chamber (30 cm diameter⫻46 cm high) for 60 min prior to muscimol (or vehicle) infusion into the subthalamic nucleus. Immediately prior to the onset of behavioral observations, the awake rat was gently hand-held and muscimol (or vehicle) was infused directly into the subthalamic nucleus via an injector placed within the surgically implanted guide cannula. A volume of 0.1 l was infused over 56 s. The injector was left in place for an additional 60 s to permit diffusion of the drug away from the injector tip. Following the local administration of drugs into the subthalamic nucleus, the rats were returned to the testing chamber and observed continuously for 60 min for bouts of orofacial movements by an observer blind to the experimental conditions. Two rats were observed during each observation period and the observer sat approximately four feet from the observation chambers. An oral bout was defined as any combination of continuous, large amplitude non-directed orofacial movements including vacuous chewing, gaping, jaw tremor, and tongue protrusion. To improve the reliability of quantification, only the number of oral bouts, not the type of oral movement, was recorded as in our previous studies (Parry et al., 1994; EberleWang et al., 1996; Mehta et al., 2000). Those oral movements that were directed toward an object or purpose, such as grooming or ingestion, were not counted. The number of oral bouts was measured over the 60 min observation period. The duration of each bout could not be accurately measured by visual observation and was not included in the analysis.
Drugs Muscimol (MW⫽114.1 g/mol) was dissolved in saline. Desipramine (MW⫽302.8 g/mol) was dissolved in d2H2O. 6-OHDA (MW⫽250.1 g/mol) was dissolved in 0.1% ascorbic acid in 0.9% saline. All drugs were purchased from Sigma-Aldrich (St. Louis, MO, USA). The pH of all solutions was in the neutral range.
Histology All rats were anesthetized with equithesin (prepared as per instruction of Janssen-Salbutry Laboratories) and killed by decapitation. Brains were removed, rapidly frozen in powdered dry ice, and stored at ⫺80 °C. Coronal sections (20 m thick) were cut through the substantia nigra pars compacta and subthalamic nucleus with a cryostat (Leica CM1800; McBain Instruments, Chatsworth, CA, USA). Tissue sections from each brain were fixed for 30 min in 4% formaldehyde and stained with Cresyl Violet for verification of lesion of the substantia nigra pars compacta and cannula placement in the subthalamic nucleus. Only data from rats in which accurate cannula placement was verified were included in the statistical analysis (Fig. 1).
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trations of ethanol, de-fatted in xylene, and air-dried. Slides were dipped in Kodak NTB3 emulsion diluted 1:1 with 300 mM ammonium acetate, and exposed in the dark until robust but not saturated signal was observed on test slides (Chesselet, 1996). Slides were developed in Kodak (Rochester, NY, USA) D-19 developer, fixed, and counterstained with eosin and hematoxylin. Two adjacent sections from each animal were processed for in situ hybridization histochemistry. The quality of hybridization was confirmed and one section from each pair was randomly chosen and analyzed quantitatively. For quantification of the hybridization signal, tissue sections were viewed under 40⫻ magnification and the image was digitized on a power Macintosh equipped with the NIH Image analysis system version 1.60. The surface area occupied by silver grains over individual cells was determined by adjusting the threshold gray value (Soghomonian and Chesselet, 1991). The average silver grain area per cell, as well as the number of cells per mm2, was calculated for each tissue section, and the data averaged for each group. Fig. 1. Photomicrograph of a Nissl-stained coronal section of a rat brain showing the tip of the injection cannula (large arrow) in the medial region of the subthalamic nucleus (STN). Scale bar⫽0.3 mm. 3
H-BTCP binding autoradiography
In order to measure the extent of the lesion of dopaminergic terminals in the striatum, the loss of dopaminergic uptake sites was determined by autoradiography with the selective ligand 3H-N-[1-92-benzo(b)thiophenylcyclohexyl]-piperidine (BTCP; Dupont NEN, Boston, MA, USA; Vignon et al., 1988), with a protocol modified from Filloux et al. (1989). Slides containing sections of the rostral striatum (see Fig. 3) were brought to room temperature from ⫺80 °C under a stream of cool air and were preincubated in ice cold 50 mM Tris–HCl buffer containing 200 mM NaCl, pH 7.0 at 4 °C for 20 min to remove endogenous dopamine. This was followed by incubation in the same buffer as above containing 2 nM 3H-BTCP at 4 °C for 90 min. Nonspecific binding was determined by the addition of 10 M GBR12909 (Research Biochemicals Inc., Natwick, MA, USA), a selective dopamine uptake inhibitor (Janowsky et al., 1986). Sections were washed four times for 8 min each in ice-cold buffer at 4 °C. After a quick dip in ice-cold distilled water, slides were dried rapidly with a stream of cool air, dried overnight, and exposed to 3H-Hyperfilm (Amersham, Chicago, IL, USA) for 30 days. Levels of binding in two adjacent sections per animal were measured and averaged to generate one value per rat.
In situ hybridization For in situ hybridization histochemistry, a separate cohort of rats (cohort IV; n⫽21; eight sham-operated and 13 rats with 6-OHDA lesions) was killed by decapitation 9 weeks post-surgery. Sections from the rostral striatum were processed for 3H-BTCP binding as described above to assess the extent of the lesion. Fresh frozen sections through the middle of globus pallidus (approximately 0.8 mm caudal to bregma; Paxinos and Watson, 1998) were prepared as above, quickly fixed (5 min) in 3% paraformaldehyde containing 0.02% of diethylpyrocarbamate and processed as previously described (Chesselet et al., 1996). Briefly, sections were acetylated with acetic anhydride, incubated 30 min in 0.1 M Tris/ glycine, dehydrated in increasing concentrations of ethanol, and air-dried. Sections were then incubated with 3 ng radiolabeled RNA probe transcribed from a cDNA donated by Allan Tobin, UCLA, Los Angeles, CA, USA, as previously described (Chesselet et al., 1987, 1996). Sections were hybridized for 3.5 h at 50 °C, washed twice in 50% formamide/2⫻ SSC at 52 °C, treated with 100 g/ml RNAse A for 30 min at 37 °C, rinsed in 50% formamide/2⫻ SSC at 52 °C and washed in 2⫻ SSC/0.05% Triton X-100 overnight. Sections were dehydrated in increasing concen-
Statistical analysis For each study, significance was defined as P⬍0.05 by two way analysis of variance (ANOVA) followed by Fisher’s PLSD for all behavioral data, or Student’s t-test, for the in situ hybridization data. All statistical analyses were performed with Statview 5.0 (SAS Institute Inc., Cary, NC, USA).
RESULTS Behavioral effects of muscimol administered into the subthalamic nucleus of intact rats Infusion of the GABAA receptor agonist muscimol (0.001, 0.01, 0.1, 1.0 g) or its vehicle (0.9% saline) into the subthalamic nucleus induced a dose-dependent increase in the total number of oral bouts observed during the 60 min test period (ANOVA, F⫽9.00, P⬍0.0001; Fig. 2A). Each oral bout was a continuous episode of non-directed mouth movements including any combination of large amplitude vacuous chewing, jaw tremor, and tongue darting. The frequency of muscimol-induced orofacial movements was dose-dependent, with a significant effect with 0.01 g, and a peak with 0.1 g of muscimol. At a dose of 0.01 g, muscimol induced a significant increase in the fourth and fifth 10-min periods post-administration while a dose of 0.1 g produced a significant increase over the entire observation period (Fisher’s PLSD, P⬍0.05; Fig. 2B). The dose-response curve exhibited an inverted U-shape, as the highest dose of 1.0 g muscimol did not induce a significant increase in oral bouts. However, similar to a previous report (Dybdal and Gale, 2000), within 20 – 40 min following administration of the high dose of muscimol, five of eight rats showed a pronounced tilting of the head contralaterally to the side of the infusion, reminiscent of a dystonic posture. This behavior was not observed following administration of the other doses. Furthermore, no other dyskinetic behaviors were observed. Verification of nigrostriatal lesions In a second set of experiments, rats received either an injection of saline (sham-operated controls) or 6-OHDA into the substantia nigra pars compacta prior to the implantation of cannulae into the subthalamic nucleus (Fig. 3C). In order to confirm adequate depletion of dopaminergic nerve terminals
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Fig. 2. Behavioral effects of several doses of muscimol injected into the subthalamic nucleus of adult rats. Rats in cohort I (n⫽6) were implanted with a cannula into one subthalamic nucleus as described in the Experimental Procedures section and received vehicle or various doses of muscimol in a Latin-square design. Orofacial dyskinesia, defined as described in Experimental Procedures, were measured continuously during 1 h following muscimol or vehicle administration. (A) Total number of oral bouts during the 1-h observation period. (B) Number of oral bouts recorded during each 10 min bins. Data are mean⫾S.E.M. * P⬍0.05 when compared with vehicle with ANOVA followed by Fisher PLSD test.
in 6-OHDA-lesioned animals, autoradiography for the selective dopamine uptake inhibitor 3H-BTCP was performed on striatal sections. Loss of 3H-BTCP binding sites has been shown to be highly correlated with decreased dopamine levels in the striatum after nigrostriatal lesions (Filloux at al., 1989; Vignon et al., 1988). The loss of dopamine uptake sites was 90% or greater in the striatum ipsilateral to the lesion in rats with unilateral lesions of the substantia nigra pars compacta (Fig. 3A, B) when compared with sham-operated con-
trols (Fig. 3A; Table 1). There was so significant difference in the level or topographical distribution of remaining 3H-BTCP sites between the cohorts. Effects of nigrostriatal lesions on muscimol-induced orofacial movements Either muscimol or vehicle was injected into the subthalamic nucleus in sham-operated controls and rats with unilateral
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Fig. 3. Depletion of dopaminergic terminals and schematic diagram of the experimental protocol. (A, B) Photomicrographs of brain sections processed for the detection of dopamine uptake sites by ligand binding autoradiography with 3H-BTCP, from a sham-operated control (A) and a rat with a unilateral lesion of the nigrostriatal dopaminergic pathway on the side indicated by the arrow (B). Scale bar⫽3.7 mm. (C) Schematic diagram showing the experimental design used for cohorts II and III in the study. Nigrostriatal lesion was created by local injection of 6-OHDA into the substantia nigra pars compacta and the GABA-A agonist muscimol was injected into the subthalamic nucleus on the same side. CP, cerebral peduncle; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; STN, subthalamic nucleus.
6-OHDA lesions (Fig. 3C). A dose of 0.01 g of muscimol was used in these experiments because it induced a submaximal increase in oral bouts in intact rats (Fig. 2A). Animals in cohort II were administered muscimol or vehicle into the subthalamic nucleus 2–3 weeks following nigrostriatal lesions. Confirming the results of the dose response experiment in intact animals (Fig. 2), 0.01 g muscimol induced a significant increase in oral bouts in sham-operated rats (n⫽8) (ANOVA, F⫽7.28, P⬍0.05; Fig. 4A). In contrast, the same
dose of muscimol did not induce an increase in oral bouts above the corresponding vehicle response in the rats with nigrostriatal lesions (n⫽8; ANOVA, F⫽0.65, N.S.; Fig. 4C). In animals in cohort III, muscimol (0.01 g) or vehicle was administered into the subthalamic nucleus 8 –9 weeks following nigrostriatal lesions. Similar to the previous two cohorts, muscimol induced a significant increase in oral bouts in sham-operated controls compared with vehicle (n⫽6; ANOVA, F⫽8.54, P⬍0.05; Fig. 4B). Unlike 2–3 weeks fol-
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Table 1. Effect of 6-OHDA lesion on dopaminergic uptake sites in the ipsilateral striatuma
Cohort II (2–3 weeks postlesion: behavior) Cohort III (8–9 weeks postlesion: behavior) Cohort IV (9 weeks post-lesion)
Sham-operated (nCi/g)
6-OHDA lesion (nCi/g)
14.93⫾1.99 (n⫽8)
1.45⫾0.59* (n⫽8)
91.2⫾3.9
15.39⫾1.92 (n⫽6)
1.28⫾0.65* (n⫽6)
91.7⫾4.2
15.16⫾1.73 (n⫽8)
1.51⫾0.50* (n⫽13)
90.0⫾3.3
% Depletion
a
Dopamine uptake sites were labeled by ligand binding autoradiography with 3H-BTCP. After exposure of the sections to film, optical densities were measured with a computer-assisted system and expressed in nCi/g of tissue with the aid of tritium standards. Non-specific binding was determined with an excess GBR12909 and subtracted from total binding values.* P⬍0.05, Student’s t-test, compared to corresponding sham.
lowing nigrostriatal lesions, however, at this time point, muscimol induced a significant increase in oral bouts in the lesioned rats (n⫽6; ANOVA, F⫽7.11, P⬍0.05; Fig. 4D). Furthermore, the overall effect of muscimol was different than in sham-operated controls (ANOVA, F⫽4.75, P⬍0.05). In contrast to the effects of the same dose of muscimol in shamoperated controls, significant differences were observed in the first and second 10-min periods following muscimol administration when compared with vehicle (Fig. 4D). At 8 –9 weeks post-lesion the low dose of muscimol (0.01 g) exhibited a response similar to the effect of the higher dose of muscimol (0.1 g) observed in intact rats (Fig. 2B). GAD67 mRNA in the globus pallidus following nigrostriatal lesions Previous reports have consistently shown an increase in GAD67 mRNA in the globus pallidus of rats 2–3 weeks following nigrostriatal lesions in rats (Kincaid et al., 1992; Soghomonian and Chesselet, 1992; Delfs et al., 1995). In view of the differences in behavioral responses to muscimol at 2–3 and 8 –9 weeks post-lesion, we sought to determine whether this effect persisted 9 weeks following nigrostriatal lesion. To avoid any confound from drug injection, the experiment was performed in a separate cohort of animals (cohort IV). Rats received an injection of 6-OHDA (n⫽13) or vehicle (n⫽8) into the substantia nigra pars compacta and were killed 9 weeks later. No drugs were administered in these animals during this period. Sections from the pallidum were processed for detection of GAD67 mRNA at the single cell level by in situ hybridization histochemistry. Unlike at 2–3 weeks post-lesion (Soghomonian and Chesselet, 1992; Kincaid et al., 1992; Delfs et al., 1995), the level of labeling for GAD67 mRNA in the pallidum of rats with nigrostriatal lesions was not different than in sham-operated controls (Table 2). Similarly, the number of pallidal neurons expressing GAD67 mRNA in rats with 6-OHDA lesions was not different from sham-operated controls (Table 2).
DISCUSSION We show that local administration of muscimol into the subthalamic nucleus of intact rats induces a dosedependent increase in non-directed orofacial movements. This effect was no longer observed 2–3 weeks following a lesion of the substantia nigra pars compacta. Muscimol, however, induced a more robust behavioral response 8 –9 weeks following nigrostriatal lesions. In contrast to previous observations 2 weeks after nigrostriatal lesions, GAD67 mRNA was not increased in neurons of the globus pallidus 9 weeks post-lesion. These results further support temporal differences in GABAergic transmission in the subthalamic nucleus following acute nigrostriatal lesions in rats. Muscimol-induced dyskinesia in rats The selectivity of muscimol strongly suggests that the increase in dyskinetic movements observed in this study is due to stimulation of GABA-A receptors. Furthermore, we have previously shown that the small volume injected (0.1 l) only diffuses within the subthalamic nucleus (Parry et al., 1994; Delfs et al., 1995), indicating that GABA-A receptors within the subthalamic nucleus likely mediated the behavioral effect we observed. We did not attempt to block this effect with a GABAergic antagonist because previous work from our laboratory (Parry and Chesselet, unpublished observation) and others (Crossman et al., 1988; Perier et al., 2002) has shown that administration of GABA-A antagonists into the subthalamic nucleus can cause dyskinetic behaviors. Hyperkinetic behavior (hemiballismus) can be induced by subthalamic lesions in humans and primates (Hyland and Forman, 1957; Hamada and DeLong, 1992). Similarly, our laboratory has previously shown that excitoxic lesions of the subthalamic nucleus in rats induce a transient increase in non-directed orofacial movements similar to those observed in the present study (Banks and Chesselet, unpublished observations). Therefore, the increase in orofacial dyskinesia after intrasubthalamic administration of muscimol is compatible with an inhibitory action of this drug on subthalamic activity. Indeed, electrophysiological and behavioral studies have shown that local administration of muscimol silences neuronal activity in the subthalamic nucleus in vivo (Robledo and Feger, 1990; Bergman et al., 1994; Levy et al., 2001; Urbain et al., 2002) and in vitro (Baufreton et al., 2001). Muscimol-induced dyskinesia in rats with nigrostriatal lesions In the present study, we used muscimol-induced orofacial dyskinesia in rats to assess changes in behavioral responses to stimulation of intrasubthalamic GABA A receptors at different times after unilateral nigrostriatal lesions. Although inhibition of the subthalamic nucleus is clearly beneficial for parkinsonian symptoms after the loss of nigrostriatal neurons (Mehta et al., 2005; Bergman et al., 1994; Levy et al., 2001), it is not surprising to observe dyskinesia in this condition. Indeed, transient dyskinesia were also observed after administration of muscimol into
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Fig. 4. Behavioral effects of muscimol injected into the subthalamic nucleus of rats with or without nigrostriatal lesions. Rats received a unilateral injection of 6-OHDA or vehicle (SHAM) into the substantia nigra pars compacta and 12 days later were implanted with a cannula into the subthalamic nucleus ipsilateral to the lesion, as described in the methods section. The rats received vehicle or muscimol (0.01 g in 0.1 l) into the subthalamic nucleus either 2–3 (left panels, animals in cohort II) or 8 –9 weeks (right panels, animals in cohort III) after the nigrostriatal lesion. Orofacial dyskinesia, defined as described in Experimental Procedures, were measured continuously during 1 h following muscimol or vehicle administration. Data are mean⫾S.E.M. of oral bouts in each 10 min bins. * P⬍0.05 when compared with vehicle with ANOVA followed by Fisher PLSD test (n⫽6 – 8).
the subthalamic nucleus of MPTP-treated monkeys (Bergman et al., 1994; Levy et al., 2001). Similarly, small adjustments in electrical output induced abnormal involuntary movements in patients bilaterally implanted with high frequency stimulators in the subthalamic nucleus, (Krack et al., 1999; Pollak et al., 2002), a procedure which may increase GABAergic transmission in the subthalamic nucleus (Windels et al., 2003; Shen et al., 2003; Bruet et al., 2003). The present data further point to a possible role of the STN in dyskinesia in general. For example, we have previously shown that local administration of dopaminergic agonists into the STN induces dyskinesia by stimulating
D1-like dopaminergic receptors, and that this effect is markedly enhanced in rats with nigrostriatal lesions (Parry et al., 1994; Mehta et al., 2000). Whether GABAergic mechanisms in the STN are involved in L-DOPA induced dyskinesia, a severe complication of the most common treatment of Parkinson’s disease deserves further investigations. The changes in the magnitude of muscimol-induced dyskinesia observed in our study suggest that responses to the stimulation of subthalamic GABA A receptors are altered after nigrostriatal lesions. Furthermore, the data suggest that these responses are initially decreased and
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Table 2. Expression of Mr 67,000 (GAD 67) mRNA in neurons of the globus pallidus 9 weeks following nigrostriatal lesionsa
Sham-operated (n⫽8) 6-OHDA-lesioned (n⫽13)
Area of silver grains per cell body (m2)
# of neurons expressing GAD67 mRNA per mm2
9.89⫾1.29 9.72⫾0.78
154.9⫾20.8 136.8⫾8.9
a
Rats (cohort IV) received a unilateral injection of vehicle (shamoperated) or 6-OHDA into the substantia nigra and were killed 9 weeks later. Sections through the globus pallidus were processed for in situ hybridization histochemistry and emulsion autoradiography, Data were analyzed as described in Experimental Procedures and represent mean⫾S.E.M. P⬎0.05 when data in rats with lesions were compared to sham-operated with Student’s t-test.
secondarily increased after an acute lesion. Indeed, 2–3 weeks after the lesion, a dose of muscimol that induces a half-maximal increase in orofacial dyskinesia in control rats, still induced dyskinesia in sham-operated controls but not in rats with a nigrostriatal lesion. Conversely, 8 –9 weeks post-surgery, the response to 0.01 g muscimol in rats with lesions was similar to the effect induced by a 10-fold higher dose of muscimol (0.1 g) in intact rats, suggesting increased sensitivity. Time-dependent changes in the pallido-subthalamic pathway The temporal changes in the behavioral response induced by intrasubthalamic muscimol administration in rats with nigrostriatal lesions suggest that GABAergic transmission may be differentially altered in this region at various times after the injection of 6-OHDA. Indeed, electrophysiological data indicate time-dependent differences in neuronal GABAergic activity in the globus pallidus, the main source of GABAergic inputs to the subthalamic nucleus (Smith et al., 1990), following nigrostriatal lesions. Thus, a decrease in spontaneous activity and an increase in burst firing were both measured in neurons of the globus pallidus following nigrostriatal lesions (Pan and Walters, 1988; Tremblay et al., 1989). The increase in pallidal burst firing activity, however, diminished with time, returning to control levels 6 –9 weeks post-lesion, while the decrease in spontaneous activity persisted (Pan and Walters, 1988). Expression of GAD67 mRNA is increased in neurons of the globus pallidus of rats 2–3 weeks after unilateral lesions of the nigrostriatal dopaminergic pathway (Soghomonian and Chesselet, 1992; Kincaid et al., 1992; Delfs et al., 1995; Billings and Marshall, 2004), an effect that may be related to the increased bursting pattern observed in pallidal neurons at that time. Indeed, lesions of the subthalamic nucleus, which provides excitatory inputs to the globus pallidus, block both the increased burst firing activity (Ni et al., 2000) and the increase in GAD67 mRNA (Delfs et al., 1996; Billings and Marshall, 2004) in the globus pallidus after nigrostriatal lesions. Accordingly, we hypothesized that changes in GAD67 mRNA would subside 8 –9 weeks after the lesion, when these electrophysiological changes are no longer observed (Pan and Walters, 1988). Indeed, we show in the present
study that the level of GAD67 mRNA in the globus pallidus returns to control levels 9 weeks following nigrostriatal lesions, an observation that reinforces the parallelism between changes in GAD67 mRNA and patterns of electrophysiological activity observed in other systems (Chesselet et al., 1993; Soghomonian and Chesselet, 2000). Together with the electrophysiological and the molecular data, the behavioral observations suggest a decrease in pallido-subthalamic GABAergic transmission, leading to increased sensitivity of the behavioral response to stimulation of GABA-A receptors in the subthalamic nucleus. Temporal changes in GAD67 mRNA gene expression in the globus pallidus were also observed in rats treated chronically with haloperidol, a preferential D2-like antagonist. In this model, levels of GAD67 mRNA show a temporal pattern of expression following treatment initiation, with an initial increased expression measured at 3–14 days, no change in expression measured at 4 weeks, and a decrease in expression measured at 8 months of chronic haloperidol treatment (Mercugliano et al., 1992; Delfs et al., 1995a,b). The data from the present study may explain differences in studies examining GAD67 mRNA expression in neurons of the external pallidum. Consistent with data in rats 2–3 weeks after nigrostriatal lesions, increased levels of GAD67 mRNA were measured in the external pallidum of MPTP-treated cats and monkeys (Schroeder and Schneider, 2001; Soghomonian et al., 1994). In both of these studies, animals were treated with MPTP until they met behavioral criteria for severe Parkinsonism and killed 7–10 days following the last MPTP injection (Schroeder and Schneider, 2001; Soghomonian et al., 1994). In contrast, in another study in which no change in GAD67 mRNA expression was detected in the external pallidum of MPTP-treated monkeys, animals were killed 90 days after the last MPTP injection (Herrero et al., 1996). Thus, levels of GAD67 mRNA in the external pallidum may be temporally regulated following MPTP intoxication, as we observed following unilateral 6-OHDA lesions in rats. Consistent with these data, no change in GAD67 mRNA was measured in the external pallidum in postmortem tissue from patients with advanced stages of idiopathic Parkinson’s disease (Herrero et al., 1996). Interestingly, GAD protein was unchanged in the STN of MPTP-treated monkeys killed more than 2 months after the last MPTP injection (Soares et al., 2004). Although a small increase in burst firing was still observed in these animals prior to kill, GABA release was decreased in their STN (Soares et al., 2004). These observations are compatible with our data showing a lack of GAD67 mRNA increase in the globus pallidus, and increased behavioral response to GABA receptor stimulation in the STN of rats 2 months after nigrostriatal lesions. Functional implications Alteration in the activity of the subthalamic nucleus is an important feature of Parkinson’s disease. Recent in vivo data demonstrate that GABA receptor stimulation in the subthalamic nucleus profoundly affects its activity, even
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superseding the effects of glutamate and dopamine receptor stimulation (Ni et al., 2001a). Our evidence of timedependent changes in GABAergic transmission to the subthalamic nucleus following nigrostriatal lesions, is consistent with observations by Ni et al. (2001a) that the activity of the subthalamic nucleus changes during the first 4 weeks following lesion of the nigrostriatal pathway in rats. The reciprocal pathway formed by the subthalamic nucleus and the globus pallidus has recently received considerable attention. This network is thought to play an important role in regulating the normal activity of the basal ganglia and in the pathophysiology of movement disorders that involve this brain system (Chesselet and Delfs, 1996; Plenz and Kitai, 1999). Together with our findings, these data suggest that both the subthalamic nucleus and the globus pallidus may be temporally regulated in unison following acute nigrostriatal degeneration. Acknowledgments—This work was supported by P50-NS38367. We thank Carole Gray and Catherine Weston for their invaluable help with the figures and manuscript, and Dr. Laurence Mignon for her comments.
REFERENCES Albin RL, Young AB, Penney JB (1989) The functional anatomy of basal ganglia disorders. Trends Neurosci 12:366 –375. Baron MS, Wichmann T, Ma D, DeLong MR (2002) Effects of transient focal inactivation of the basal ganglia in parkinsonian primates. J Neurosci 22:592–599. Baufreton J, Garret M, Dovero S, Dufy B, Bioulac B, Taupignon A (2001) Activation of GABA(A) receptors in subthalamic neurons in vitro: properties of native receptors and inhibition mechanisms. J Neurophysiol 86:75– 85. Benazzouz A, Gross C, Feger J, Boraud T, Bioulac B (1993) Reversal of rigidity and improvement in motor performance by subthalamic high-frequency stimulation in MPTP-treated monkeys. Eur J Neurosci 5:382–389. Bergman H, Wichmann T, DeLong MR (1990) Reversal of experimental parkinsonism by lesions of the subthalamic nucleus. Science 249:1436 –1438. Bergman H, Wichmann T, Karmon B, DeLong MR (1994) The primate subthalamic nucleus: II. Neuronal activity in the MPTP model of parkinsonism. J Neurophysiol 72:507–520. Billings LM, Marshall JF (2004) Glutamic acid decarboxylase 67 mRNA regulation in two globus pallidus neuron populations by dopamine and the subthalamic nucleus. J Neurosci 24:3094–3103. Bruet N, Windels F, Carcenac C, Feuerstein C, Bertrand A, Poupard A, Savasta M (2003) Neurochemical mechanisms induced by high frequency stimulation of the subthalamic nucleus: increase of extracellular striatal glutamate and GABA in normal and hemiparkinsonian rats. J Neuropathol Exp Neurol 62:1228 –1240. Brussaard P, Herbison AE (2000) Long-term plasticity of postsynaptic GABAA-receptor function in the adult brain: insights from the oxytocin neurone. Trends Neurosci 23:190 –195. Chesselet MF (1996) Quantitative analysis and interpretation of data for in situ hybridization at the cellular level. In: In situ hybridization techniques for the brain (Henderson Z, ed), pp 141–149. IBRO Handbook Series. Chichester: John Wiley & Sons. Chesselet MF, Delfs JM (1996) Basal ganglia and movement disorders: an update. Trends Neurosci 19:417– 422. Chesselet MF, MacKenzie L, Eberle-Wang K (1996). In situ hybridization with 35S- and digoxigenin-labeled RNA probes. In: In situ hybridization techniques for the brain (Henderson Z, ed), pp 79 –109.
777
Chesselet MF, Mercugliano M, Soghomonian JJ, Salin P, Qin Y, Gonzales C (1993) Regulation of glutamic acid decarboxylase gene expression in efferent neurons of the basal ganglia. Prog Brain Res 99:143–154. Chesselet MF, Weiss L, Wuenschell C, Tobin AJ, Affolter HU (1987) Comparative distribution of mRNAs for glutamic acid decarboxylase, tyrosine hydroxylase, and tachykinins in the basal ganglia: an in situ hybridization study in the rodent brain. J Comp Neurol 262:125–140. Crossman AR, Mitchell IJ, Sambrook MA, Jackson A (1988) Chorea and myoclonus in the monkey induced by gamma-aminobutyric acid antagonism in the lentiform complex: the site of drug action and a hypothesis for the neural mechanisms of chorea. Brain 111(Pt 5):1211–1233. Delfs JM, Anegawa NJ, Chesselet MF (1995a) Glutamate decarboxylase messenger RNA in rat pallidum: comparison of the effects of haloperidol, clozapine and combined haloperidol-scopolamine treatments. Neuroscience 66:67– 80. Delfs JM, Ciaramitaro VM, Parry TJ, Chesselet MF (1995) Subthalamic nucleus lesions: widespread effects on changes in gene expression induced by nigrostriatal dopamine depletion in rats. J Neurosci 15:6562– 6575. Delfs JM, Ciaramitaro VM, Soghomonian JJ, Chesselet MF (1996) Unilateral nigrostriatal lesions induce a bilateral increase in glutamate decarboxylase messenger RNA in the reticular thalamic nucleus. Neuroscience 71:383–395. Delfs JM, Ellison GD, Mercugliano M, Chesselet MF (1995b) Expression of glutamic acid decarboxylase mRNA in striatum and pallidum in an animal model of tardive dyskinesia. Exp Neurol 133:175–188. Dostrovsky JO, Hutchison WD, Lozano AM (2002) The globus pallidus, deep brain stimulation, and Parkinson’s disease. Neuroscientist 8:284 –290. Dybdal D, Gale K (2000) Postural and anticonvulsant effects of inhibition of the rat subthalamic nucleus. J Neurosci 20:6728 – 6733. Eberle-Wang K, Lucki I, Chesselet MF (1996) A role for the subthalamic nucleus in 5-HT2C-induced oral dyskinesia. Neuroscience 72:117–128. Filion M, Tremblay L (1991) Abnormal spontaneous activity of globus pallidus neurons in monkeys with MPTP-induced parkinsonism. Brain Res 547:142–151. Filloux F, Hunt MA, Wamsley JK (1989) Localization of the dopamine uptake complex using [3H]N-[1-(2-benzo(b)thiophenyl)cyclohexyl] piperidine ([3H]BTCP) in rat brain. Neurosci Lett 100:105–110. Hamada I, DeLong MR (1992) Excitotoxic acid lesions of the primate subthalamic nucleus result in transient dyskinesias of the contralateral limbs. J Neurophysiol 68:1850 –1858. Herrero MT, Levy R, Ruberg M, Javoy-Agid F, Luquin MR, Agid Y, Hirsch EC, Obeso JA (1996) Glutamic acid decarboxylase mRNA expression in medial and lateral pallidal neurons in the MPTPtreated monkey and patients with Parkinson’s disease. Adv Neurol 69:209 –216. Hutchison WD, Allan RJ, Opitz H, Levy R, Dostrovsky JO, Lang AE, Lozano AM (1998) Neurophysiological identification of the subthalamic nucleus in surgery for Parkinson’s disease. Ann Neurol 44:622– 628. Hyland HH, Forman DM (1957) Prognosis in hemiballismus. Neurology 7:381–391. Janowsky A, Berger P, Vocci F, Labarca R, Skolnick P, Paul SM (1986) Characterization of sodium-dependent [3H]GBR-12935 binding in brain: a radioligand for selective labelling of the dopamine transport complex. J Neurochem 46:1272–1276. Kincaid AE, Albin RL, Newman SW, Penney JB, Young AB (1992) 6-Hydroxydopamine lesions of the nigrostriatal pathway alter the expression of glutamate decarboxylase messenger RNA in rat globus pallidus projection neurons. Neuroscience 51:705–718. Krack P, Pollak P, Limousin P, Benazzouz A, Deuschl G, Benabid AL (1999) From off-period dystonia to peak-dose chorea: the clinical
778
A. Mehta et al. / Neuroscience 131 (2005) 769 –778
spectrum of varying subthalamic nucleus activity. Brain 122(Pt 6):1133–1146. Kumar R, Lozano AM, Kim YJ, Hutchison WD, Sime E, Halket E, Lang AE (1998) Double-blind evaluation of subthalamic nucleus deep brain stimulation in advanced Parkinson’s disease. Neurology 51: 850–855. Levy R, Lang AE, Dostrovsky JO, Pahapill P, Romas J, Saint-Cyr J, Hutchison WD, Lozano AM (2001) Lidocaine and muscimol microinjections in subthalamic nucleus reverse Parkinsonian symptoms. Brain 124:2105–2118. Mehta A, Thermos K, Chesselet MF (2000) Increased behavioral response to dopaminergic stimulation of the subthalamic nucleus after nigrostriatal lesions. Synapse 37:298 –307. Mehta A, Chesselet MF (2005) Effects of GABAA receptor stimulation in the subthalamic nucleus on motor deficits induced by nigrostriatal lesions in the rat. Exp Neurol, in press. Mercugliano M, Saller CF, Salama AI, U’Prichard DC, Chesselet MF (1992) Clozapine and haloperidol have differential effects on glutamic acid decarboxylase mRNA in the pallidal nuclei of the rat. Neuropsychopharmacology 6:179 –187. Narabayashi H (1995) The neural mechanisms and progressive nature of symptoms of Parkinson’s disease-based on clinical, neurophysiological and morphological studies. J Neural Transm Park Dis Dement Sect 10:63–75. Ni Z, Bouali-Benazzouz R, Gao D, Benabid AL, Benazzouz A (2000) Changes in the firing pattern of globus pallidus neurons after the degeneration of nigrostriatal pathway are mediated by the subthalamic nucleus in the rat. Eur J Neurosci 12:4338 – 4344. Ni ZG, Bouali-Benazzouz R, Gao DM, Benabid AL, Benazzouz A (2001a) Time-course of changes in firing rates and firing patterns of subthalamic nucleus neuronal activity after 6-OHDA-induced dopamine depletion in rats. Brain Res 899:142–147. Ni Z, Gao D, Bouali-Benazzouz R, Benabid AL, Benazzouz A (2001b) Effect of microiontophoretic application of dopamine on subthalamic nucleus neuronal activity in normal rats and in rats with unilateral lesion of the nigrostriatal pathway. Eur J Neurosci 14:373–381. Pan HS, Walters JR (1988) Unilateral lesion of the nigrostriatal pathway decreases the firing rate and alters the firing pattern of globus pallidus neurons in the rat. Synapse 2:650 – 656. Parry TJ, Eberle-Wang K, Lucki I, Chesselet MF (1994) Dopaminergic stimulation of subthalamic nucleus elicits oral dyskinesia in rats. Exp Neurol 128:181–190. Paxinos G, Watson C (1998) The rat brain in stereotaxic coordinates. San Diego, CA: Academic Press. Penney JB Jr, Young AB (1981) GABA as the pallidothalamic neurotransmitter: implications for basal ganglia function. Brain Res 207:195–199. Perier C, Tremblay L, Feger J, Hirsch EC (2002) Behavioral consequences of bicuculline injection in the subthalamic nucleus and the zona incerta in rat. J Neurosci 22:8711– 8719. Plenz D, Kitai ST (1999) A basal ganglia pacemaker formed by the subthalamic nucleus and external globus pallidus. Nature 400:677– 682.
Poewe WH, Wenning GK (1998) The natural history of Parkinson’s disease. Ann Neurol 44:S1–9 Pollak P, Krack P, Fraix V, Mendes A, Moro E, Chabardes S, Benabid AL (2002) Intraoperative micro- and macrostimulation of the subthalamic nucleus in Parkinson’s disease. Mov Disord 17(Suppl 3):S155–161. Robledo P, Feger J (1990) Excitatory influence of rat subthalamic nucleus to substantia nigra pars reticulata and the pallidal complex: electrophysiological data. Brain Res 518:47–54. Schroeder JA, Schneider JS (2001) Alterations in expression of messenger RNAs encoding two isoforms of glutamic acid decarboxylase in the globus pallidus and entopeduncular nucleus in animals symptomatic for and recovered from experimental parkinsonism. Brain Res 888:180 –183. Shen KZ, Zhu ZT, Munhall A, Johnson SW (2003) Synaptic plasticity in rat subthalamic nucleus induced by high-frequency stimulation. Synapse 50:314 –319. Smith Y, Bolam JP, Von Krosigk M (1990) Topographical and synaptic organization of the GABA-containing pallidosubthalamic projection in the rat. Eur J Neurosci 2:500 –511. Soares J, Kliem MA, Betarbet R, Greenamyre JT, Yamamoto B, Wichmann T (2004) Role of external pallidal segment in primate parkinsonism: comparison of the effects of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine-induced parkinsonism and lesions of the external pallidal segment. J Neurosci 24:6417– 6426. Soghomonian JJ, Chesselet MF (1991) Lesions of the dopaminergic nigrostriatal pathway alter preprosomatostatin messenger RNA levels in the striatum, the entopeduncular nucleus and the lateral hypothalamus of the rat. Neuroscience 42:49 –59. Soghomonian JJ, Chesselet MF (1992) Effects of nigrostriatal lesions on the levels of messenger RNAs encoding two isoforms of glutamate decarboxylase in the globus pallidus and entopeduncular nucleus of the rat. Synapse 11:124 –133. Soghomonian JJ, Chesselet MF (2000) GABA in the basal ganglia. In: GABA in the nervous system: the view at fifty years (Martin DL, Olsen RW, eds), pp 265–291. Philadelphia: Lippincott, Williams & Wilkins. Soghomonian JJ, Pedneault S, Audet G, Parent A (1994) Increased glutamate decarboxylase mRNA levels in the striatum and pallidum of MPTP-treated primates. J Neurosci 14:6256 – 6265. Tremblay L, Filion M, Bedard PJ (1989) Responses of pallidal neurons to striatal stimulation in monkeys with MPTP-induced parkinsonism. Brain Res 498:17–33. Urbain N, Rentero N, Gervasoni D, Renaud B, Chouvet G (2002) The switch of subthalamic neurons from an irregular to a bursting pattern does not solely depend on their GABAergic inputs in the anesthetic-free rat. J Neurosci 22:8665– 8675. Vignon J, Pinet V, Cerruti C, Kamenka JM, Chicheportiche R (1988) [3H]N-[1-(2-benzo(b)thiophenyl)cyclohexyl]piperidine ([3H]BTCP): a new phencyclidine analog selective for the dopamine uptake complex. Eur J Pharmacol 148:427– 436. Windels F, Bruet N, Poupard A, Feuerstein C, Bertrand A, Savasta M (2003) Influence of the frequency parameter on extracellular glutamate and gamma-aminobutyric acid in substantia nigra and globus pallidus during electrical stimulation of subthalamic nucleus in rats. J Neurosci Res 72:259 –267.
(Accepted 15 November 2004)
BEHAVIORAL RESPONSES TO INJECTIONS OF MUSCIMOL INTO THE SUBTHALAMIC NUCLEUS: TEMPORAL CHANGES AFTER NIGROSTRIATAL LESIONS A. MEHTA,a L. MENALLEDa AND M.-F. CHESSELETa,b*
include tremor, rigidity and akinesia, and develop over the course of several years (Narabayashi, 1995; Poewe and Wenning, 1998). A major consequence of dopaminergic lesions is an increase in firing in the subthalamic nucleus (Albin et al., 1989). This effect has been observed in parkinsonian patients (Hutchison et al., 1998), monkeys treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP; Bergman et al., 1994), and rats with a lesion of the nigrostriatal pathway induced by 6hydroxydopamine (6-OHDA; Ni et al., 2001). In patients and animal models of Parkinson’s disease, ablation, inhibition or high frequency stimulation of the subthalamic nucleus markedly improve akinesia and rigidity, suggesting that alterations in this brain region are crucial for parkinsonian symptoms (Bergman et al., 1990; Benazzouz et al., 1993; Kumar et al., 1998; Baron et al., 2002; Dostrovsky et al., 2002). Models of basal ganglia circuitry predict that the increased activity of subthalamic neurons after nigrostriatal degeneration is due at least in part to a decreased activity of the globus pallidus (external pallidum in primates), which provides a dense input of GABA-containing nerve terminals to the subthalamic nucleus (Albin et al., 1989; Smith et al., 1990). Data from both rats and monkeys show that nigrostriatal lesions cause a decrease in spontaneous activity in neurons of the external pallidum; the same studies, however, show a simultaneous increase in burst firing (Pan and Walters, 1988; Filion and Tremblay, 1991). Therefore, the net effect of nigrostriatal degeneration on pallido-subthalamic transmission remains unclear. Similarly, studies of changes in glutamic acid decarboxylase (Mr 67,000; GAD67) GAD mRNA expression in the globus pallidus, an index of GABAergic activity, have provided ambiguous results (Chesselet and Delfs, 1996). Previous studies from our laboratory and others have shown increased levels of mRNA encoding GAD67, one of the rate-limiting enzymes of GABA synthesis, in neurons of the globus pallidus 2–3 weeks after lesions of the nigrostriatal pathway in rats (Soghomonian and Chesselet, 1992; Kincaid et al., 1992; Delfs et al., 1995). According to a recent study (Billings and Marshall, 2004), this increase occurs both in parvalbumin-positive and parvalbuminnegative neurons of the globus pallidus, but to a larger extent in the latter. An increase in GAD67 mRNA in the external pallidum was also observed in MPTP-treated cats and monkeys (Soghomonian et al., 1994; Schroeder and Schneider, 2001). Another study, however, found no change in GAD67 mRNA in the external pallidum of MPTPtreated monkeys and in postmortem human tissue
a
Department of Neurology, University of California Los Angeles, B114, Reed Neurological Research Center, 710 Westwood Plaza, Los Angeles, CA 90095, USA b Department of Neurobiology, University of California Los Angeles, Los Angeles, CA 90095, USA
Abstract—Changes in cellular activity in the subthalamic nucleus are a cardinal feature of Parkinson’s disease and occur in rodents after lesions of the nigrostriatal pathway, a model of Parkinson’s disease. GABA-ergic neurons from the globus pallidus provide a major input to the subthalamic nucleus. Previous electrophysiological studies revealed temporal changes in the activity of pallidal neurons after nigrostriatal lesions in rats. However, little is known about the impact of these changes on GABAergic transmission in the subthalamic nucleus. We have examined the behavioral responses to a local administration of the GABA A agonist muscimol into the subthalamic nucleus. Muscimol (0.01 and 0.1 g) induced orofacial dyskinesia in normal rats; this response was blunted 2 weeks but enhanced 2 months after a unilateral lesion of the nigrostriatal pathway. The early decrease in the behavioral response occurred at a time when increased expression of mRNA for glutamic acid decarboxylase, the enzyme of GABA synthesis, and burst firing have been reported in the globus pallidus, suggesting an adaptive post-synaptic response to increased GABAergic transmission in the subthalamic nucleus. In contrast, we now show that glutamic acid decarboxylase mRNA is unchanged in the globus pallidus at the later time point, when electrophysiological changes also subside in this region. The increased behavioral response at this later time point may reflect a decreased activity in GABAergic inputs to the subthalamic nucleus. The results show time-dependent changes in behavioral responses to GABA A receptor stimulation in the subthalamic nucleus which may reflect adaptive changes in postsynaptic inhibitory responses after dopaminergic lesions. © 2005 Published by Elsevier Ltd on behalf of IBRO. Key words: globus pallidus, GABA, GABAA receptor, Parkinson’s disease, 6-hydroxydopamine, basal ganglia.
Parkinson’s disease is characterized by the progressive development of motor symptoms resulting from the degeneration of dopaminergic inputs from the substantia nigra pars compacta to the striatum. The cardinal symptoms *Corresponding author. Tel: ⫹1-310-267-1781; fax: ⫹1-310-2671786. E-mail address: [email protected] (M.-F. Chesselet). Abbreviations: ANOVA, analysis of variance; BTCP, 3H-N-[1-92benzo(b)thiophenylcyclohexyl]-piperidine; GAD67, glutamic acid decarboxylase (Mr 67,000); MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; 6-OHDA, 6-hydroxydopamine. 0306-4522/05$30.00⫹0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2004.11.036
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(Herrero et al., 1996). These differences may be due to time-dependent changes in the pallido-subthalamic pathway following nigrostriatal degeneration. Indeed, Pan and Walters (1988) showed that the burst firing activity measured in the globus pallidus of rats peaked 1 week after lesions of the nigrostriatal pathway and decreased with time, returning to control levels by 6 –9 weeks post-lesion. These data suggest that neuronal activity in the pallidosubthalamic pathway may be temporally regulated after acute nigrostriatal lesion. Changes in GABAergic inputs can induce compensatory alterations in post-synaptic receptors that influence the response of neurons to GABAergic agonists (Penney and Young 1981; Brussaard and Herbison, 2000). The present study was designed to gain insight into the temporal changes of GABAergic transmission in the subthalamic nucleus after acute dopaminergic lesions in rodents. Dyskinesia induced by the local administration of the GABA-A receptor agonist muscimol into the subthalamic nucleus were examined 2–3 weeks and 8 –9 weeks after unilateral lesions of the nigrostriatal dopaminergic neurons in adult rats. In addition, levels of expression of GAD67 mRNA in the globus pallidus were examined with quantitative in situ hybridization histochemistry and emulsion autoradiography at the later time point.
EXPERIMENTAL PROCEDURES Animals A total of 55 male Sprague–Dawley rats (270 –310 g; Charles River, Wilmington, MA, USA) separated in four cohorts were used in this study. All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and all efforts were made to reduce the number of animals. All experimental protocols were approved by the University of California Los Angeles animal care committee.
Surgery Prior to surgery, rats were maintained on a 12-h light/dark schedule with food pellets and water ad libitum. Rats were anesthetized with equithesin (prepared as per instruction of Janssen-Salbutry Laboratories, Kansas City, MO, USA; 3.0 ml/kg, i.p.) and received an unilateral infusion of either 6-OHDA (8 g/4 l) or vehicle (4 l 0.1% ascorbic acid in 0.9% saline) into the left substantia nigra pars compacta (AP⫽⫹3.4 mm, ML⫽⫹2.0 mm, both relative to interaural zero, and DV⫽7.4 mm below the surface of the cortex; Paxinos and Watson, 1998). Thirty minutes prior to surgery, all rats were pretreated with desipramine (25 mg/kg, i.p.) to prevent the uptake of 6-OHDA into noradrenergic neurons. For delivery of muscimol into the subthalamic nucleus, rats were implanted with a guide cannula for unilateral microinfusion of drug into the left subthalamic nucleus (AP⫽⫹4.9 mm, ML⫽ ⫹2.5 mm, both relative to interaural zero, and DV⫽7.4 mm below the surface of the cortex; Paxinos and Watson, 1998). Details of the surgical procedure and injection equipment have been described previously (Parry et al., 1994). Briefly, the guide shafts were made from 22-gauge cannulae cut to a length of 17 mm and the injector was made of a piece of fused silica (o.d.⫽150 m; i.d.⫽75 m) threaded through a 28-gauge internal cannula such that the tip of the fused silica extended 1 mm from the end of the internal cannula shaft. In rats with prior lesion of the nigrostriatal pathway and their sham-operated controls, this procedure was
done 12 days after the first surgery. In all cases, behavioral testing began 1 week after implanting the cannula.
Drug administration and behavioral testing No behavioral testing was conducted to assess the level of nigrostriatal lesion to avoid any confounding effect on the behavioral analysis of muscimol effects. Loss of dopaminergic nerve terminals was determined histologically at the end of the experiment as described below. In a first cohort of rats (cohort I; n⫽6), vehicle or muscimol at the concentrations of 0.001, 0.01, 0.1, 1.0 g were injected into the subthalamic nucleus following a Latin-square design so that each rat received all the doses and vehicle, in a random order. Testing was done every 4 days. For cohort II (n⫽16) and cohort III (n⫽16), each rat was treated only once, either with muscimol or vehicle. Cohort II and cohort III were examined 2–3 and 8 –9 weeks post-lesion, respectively, to avoid any confounding effect of prior treatment. On the day of behavioral testing, each rat was adapted to a quiet room in a clear plastic cylindrical chamber (30 cm diameter⫻46 cm high) for 60 min prior to muscimol (or vehicle) infusion into the subthalamic nucleus. Immediately prior to the onset of behavioral observations, the awake rat was gently hand-held and muscimol (or vehicle) was infused directly into the subthalamic nucleus via an injector placed within the surgically implanted guide cannula. A volume of 0.1 l was infused over 56 s. The injector was left in place for an additional 60 s to permit diffusion of the drug away from the injector tip. Following the local administration of drugs into the subthalamic nucleus, the rats were returned to the testing chamber and observed continuously for 60 min for bouts of orofacial movements by an observer blind to the experimental conditions. Two rats were observed during each observation period and the observer sat approximately four feet from the observation chambers. An oral bout was defined as any combination of continuous, large amplitude non-directed orofacial movements including vacuous chewing, gaping, jaw tremor, and tongue protrusion. To improve the reliability of quantification, only the number of oral bouts, not the type of oral movement, was recorded as in our previous studies (Parry et al., 1994; EberleWang et al., 1996; Mehta et al., 2000). Those oral movements that were directed toward an object or purpose, such as grooming or ingestion, were not counted. The number of oral bouts was measured over the 60 min observation period. The duration of each bout could not be accurately measured by visual observation and was not included in the analysis.
Drugs Muscimol (MW⫽114.1 g/mol) was dissolved in saline. Desipramine (MW⫽302.8 g/mol) was dissolved in d2H2O. 6-OHDA (MW⫽250.1 g/mol) was dissolved in 0.1% ascorbic acid in 0.9% saline. All drugs were purchased from Sigma-Aldrich (St. Louis, MO, USA). The pH of all solutions was in the neutral range.
Histology All rats were anesthetized with equithesin (prepared as per instruction of Janssen-Salbutry Laboratories) and killed by decapitation. Brains were removed, rapidly frozen in powdered dry ice, and stored at ⫺80 °C. Coronal sections (20 m thick) were cut through the substantia nigra pars compacta and subthalamic nucleus with a cryostat (Leica CM1800; McBain Instruments, Chatsworth, CA, USA). Tissue sections from each brain were fixed for 30 min in 4% formaldehyde and stained with Cresyl Violet for verification of lesion of the substantia nigra pars compacta and cannula placement in the subthalamic nucleus. Only data from rats in which accurate cannula placement was verified were included in the statistical analysis (Fig. 1).
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trations of ethanol, de-fatted in xylene, and air-dried. Slides were dipped in Kodak NTB3 emulsion diluted 1:1 with 300 mM ammonium acetate, and exposed in the dark until robust but not saturated signal was observed on test slides (Chesselet, 1996). Slides were developed in Kodak (Rochester, NY, USA) D-19 developer, fixed, and counterstained with eosin and hematoxylin. Two adjacent sections from each animal were processed for in situ hybridization histochemistry. The quality of hybridization was confirmed and one section from each pair was randomly chosen and analyzed quantitatively. For quantification of the hybridization signal, tissue sections were viewed under 40⫻ magnification and the image was digitized on a power Macintosh equipped with the NIH Image analysis system version 1.60. The surface area occupied by silver grains over individual cells was determined by adjusting the threshold gray value (Soghomonian and Chesselet, 1991). The average silver grain area per cell, as well as the number of cells per mm2, was calculated for each tissue section, and the data averaged for each group. Fig. 1. Photomicrograph of a Nissl-stained coronal section of a rat brain showing the tip of the injection cannula (large arrow) in the medial region of the subthalamic nucleus (STN). Scale bar⫽0.3 mm. 3
H-BTCP binding autoradiography
In order to measure the extent of the lesion of dopaminergic terminals in the striatum, the loss of dopaminergic uptake sites was determined by autoradiography with the selective ligand 3H-N-[1-92-benzo(b)thiophenylcyclohexyl]-piperidine (BTCP; Dupont NEN, Boston, MA, USA; Vignon et al., 1988), with a protocol modified from Filloux et al. (1989). Slides containing sections of the rostral striatum (see Fig. 3) were brought to room temperature from ⫺80 °C under a stream of cool air and were preincubated in ice cold 50 mM Tris–HCl buffer containing 200 mM NaCl, pH 7.0 at 4 °C for 20 min to remove endogenous dopamine. This was followed by incubation in the same buffer as above containing 2 nM 3H-BTCP at 4 °C for 90 min. Nonspecific binding was determined by the addition of 10 M GBR12909 (Research Biochemicals Inc., Natwick, MA, USA), a selective dopamine uptake inhibitor (Janowsky et al., 1986). Sections were washed four times for 8 min each in ice-cold buffer at 4 °C. After a quick dip in ice-cold distilled water, slides were dried rapidly with a stream of cool air, dried overnight, and exposed to 3H-Hyperfilm (Amersham, Chicago, IL, USA) for 30 days. Levels of binding in two adjacent sections per animal were measured and averaged to generate one value per rat.
In situ hybridization For in situ hybridization histochemistry, a separate cohort of rats (cohort IV; n⫽21; eight sham-operated and 13 rats with 6-OHDA lesions) was killed by decapitation 9 weeks post-surgery. Sections from the rostral striatum were processed for 3H-BTCP binding as described above to assess the extent of the lesion. Fresh frozen sections through the middle of globus pallidus (approximately 0.8 mm caudal to bregma; Paxinos and Watson, 1998) were prepared as above, quickly fixed (5 min) in 3% paraformaldehyde containing 0.02% of diethylpyrocarbamate and processed as previously described (Chesselet et al., 1996). Briefly, sections were acetylated with acetic anhydride, incubated 30 min in 0.1 M Tris/ glycine, dehydrated in increasing concentrations of ethanol, and air-dried. Sections were then incubated with 3 ng radiolabeled RNA probe transcribed from a cDNA donated by Allan Tobin, UCLA, Los Angeles, CA, USA, as previously described (Chesselet et al., 1987, 1996). Sections were hybridized for 3.5 h at 50 °C, washed twice in 50% formamide/2⫻ SSC at 52 °C, treated with 100 g/ml RNAse A for 30 min at 37 °C, rinsed in 50% formamide/2⫻ SSC at 52 °C and washed in 2⫻ SSC/0.05% Triton X-100 overnight. Sections were dehydrated in increasing concen-
Statistical analysis For each study, significance was defined as P⬍0.05 by two way analysis of variance (ANOVA) followed by Fisher’s PLSD for all behavioral data, or Student’s t-test, for the in situ hybridization data. All statistical analyses were performed with Statview 5.0 (SAS Institute Inc., Cary, NC, USA).
RESULTS Behavioral effects of muscimol administered into the subthalamic nucleus of intact rats Infusion of the GABAA receptor agonist muscimol (0.001, 0.01, 0.1, 1.0 g) or its vehicle (0.9% saline) into the subthalamic nucleus induced a dose-dependent increase in the total number of oral bouts observed during the 60 min test period (ANOVA, F⫽9.00, P⬍0.0001; Fig. 2A). Each oral bout was a continuous episode of non-directed mouth movements including any combination of large amplitude vacuous chewing, jaw tremor, and tongue darting. The frequency of muscimol-induced orofacial movements was dose-dependent, with a significant effect with 0.01 g, and a peak with 0.1 g of muscimol. At a dose of 0.01 g, muscimol induced a significant increase in the fourth and fifth 10-min periods post-administration while a dose of 0.1 g produced a significant increase over the entire observation period (Fisher’s PLSD, P⬍0.05; Fig. 2B). The dose-response curve exhibited an inverted U-shape, as the highest dose of 1.0 g muscimol did not induce a significant increase in oral bouts. However, similar to a previous report (Dybdal and Gale, 2000), within 20 – 40 min following administration of the high dose of muscimol, five of eight rats showed a pronounced tilting of the head contralaterally to the side of the infusion, reminiscent of a dystonic posture. This behavior was not observed following administration of the other doses. Furthermore, no other dyskinetic behaviors were observed. Verification of nigrostriatal lesions In a second set of experiments, rats received either an injection of saline (sham-operated controls) or 6-OHDA into the substantia nigra pars compacta prior to the implantation of cannulae into the subthalamic nucleus (Fig. 3C). In order to confirm adequate depletion of dopaminergic nerve terminals
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Fig. 2. Behavioral effects of several doses of muscimol injected into the subthalamic nucleus of adult rats. Rats in cohort I (n⫽6) were implanted with a cannula into one subthalamic nucleus as described in the Experimental Procedures section and received vehicle or various doses of muscimol in a Latin-square design. Orofacial dyskinesia, defined as described in Experimental Procedures, were measured continuously during 1 h following muscimol or vehicle administration. (A) Total number of oral bouts during the 1-h observation period. (B) Number of oral bouts recorded during each 10 min bins. Data are mean⫾S.E.M. * P⬍0.05 when compared with vehicle with ANOVA followed by Fisher PLSD test.
in 6-OHDA-lesioned animals, autoradiography for the selective dopamine uptake inhibitor 3H-BTCP was performed on striatal sections. Loss of 3H-BTCP binding sites has been shown to be highly correlated with decreased dopamine levels in the striatum after nigrostriatal lesions (Filloux at al., 1989; Vignon et al., 1988). The loss of dopamine uptake sites was 90% or greater in the striatum ipsilateral to the lesion in rats with unilateral lesions of the substantia nigra pars compacta (Fig. 3A, B) when compared with sham-operated con-
trols (Fig. 3A; Table 1). There was so significant difference in the level or topographical distribution of remaining 3H-BTCP sites between the cohorts. Effects of nigrostriatal lesions on muscimol-induced orofacial movements Either muscimol or vehicle was injected into the subthalamic nucleus in sham-operated controls and rats with unilateral
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Fig. 3. Depletion of dopaminergic terminals and schematic diagram of the experimental protocol. (A, B) Photomicrographs of brain sections processed for the detection of dopamine uptake sites by ligand binding autoradiography with 3H-BTCP, from a sham-operated control (A) and a rat with a unilateral lesion of the nigrostriatal dopaminergic pathway on the side indicated by the arrow (B). Scale bar⫽3.7 mm. (C) Schematic diagram showing the experimental design used for cohorts II and III in the study. Nigrostriatal lesion was created by local injection of 6-OHDA into the substantia nigra pars compacta and the GABA-A agonist muscimol was injected into the subthalamic nucleus on the same side. CP, cerebral peduncle; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; STN, subthalamic nucleus.
6-OHDA lesions (Fig. 3C). A dose of 0.01 g of muscimol was used in these experiments because it induced a submaximal increase in oral bouts in intact rats (Fig. 2A). Animals in cohort II were administered muscimol or vehicle into the subthalamic nucleus 2–3 weeks following nigrostriatal lesions. Confirming the results of the dose response experiment in intact animals (Fig. 2), 0.01 g muscimol induced a significant increase in oral bouts in sham-operated rats (n⫽8) (ANOVA, F⫽7.28, P⬍0.05; Fig. 4A). In contrast, the same
dose of muscimol did not induce an increase in oral bouts above the corresponding vehicle response in the rats with nigrostriatal lesions (n⫽8; ANOVA, F⫽0.65, N.S.; Fig. 4C). In animals in cohort III, muscimol (0.01 g) or vehicle was administered into the subthalamic nucleus 8 –9 weeks following nigrostriatal lesions. Similar to the previous two cohorts, muscimol induced a significant increase in oral bouts in sham-operated controls compared with vehicle (n⫽6; ANOVA, F⫽8.54, P⬍0.05; Fig. 4B). Unlike 2–3 weeks fol-
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Table 1. Effect of 6-OHDA lesion on dopaminergic uptake sites in the ipsilateral striatuma
Cohort II (2–3 weeks postlesion: behavior) Cohort III (8–9 weeks postlesion: behavior) Cohort IV (9 weeks post-lesion)
Sham-operated (nCi/g)
6-OHDA lesion (nCi/g)
14.93⫾1.99 (n⫽8)
1.45⫾0.59* (n⫽8)
91.2⫾3.9
15.39⫾1.92 (n⫽6)
1.28⫾0.65* (n⫽6)
91.7⫾4.2
15.16⫾1.73 (n⫽8)
1.51⫾0.50* (n⫽13)
90.0⫾3.3
% Depletion
a
Dopamine uptake sites were labeled by ligand binding autoradiography with 3H-BTCP. After exposure of the sections to film, optical densities were measured with a computer-assisted system and expressed in nCi/g of tissue with the aid of tritium standards. Non-specific binding was determined with an excess GBR12909 and subtracted from total binding values.* P⬍0.05, Student’s t-test, compared to corresponding sham.
lowing nigrostriatal lesions, however, at this time point, muscimol induced a significant increase in oral bouts in the lesioned rats (n⫽6; ANOVA, F⫽7.11, P⬍0.05; Fig. 4D). Furthermore, the overall effect of muscimol was different than in sham-operated controls (ANOVA, F⫽4.75, P⬍0.05). In contrast to the effects of the same dose of muscimol in shamoperated controls, significant differences were observed in the first and second 10-min periods following muscimol administration when compared with vehicle (Fig. 4D). At 8 –9 weeks post-lesion the low dose of muscimol (0.01 g) exhibited a response similar to the effect of the higher dose of muscimol (0.1 g) observed in intact rats (Fig. 2B). GAD67 mRNA in the globus pallidus following nigrostriatal lesions Previous reports have consistently shown an increase in GAD67 mRNA in the globus pallidus of rats 2–3 weeks following nigrostriatal lesions in rats (Kincaid et al., 1992; Soghomonian and Chesselet, 1992; Delfs et al., 1995). In view of the differences in behavioral responses to muscimol at 2–3 and 8 –9 weeks post-lesion, we sought to determine whether this effect persisted 9 weeks following nigrostriatal lesion. To avoid any confound from drug injection, the experiment was performed in a separate cohort of animals (cohort IV). Rats received an injection of 6-OHDA (n⫽13) or vehicle (n⫽8) into the substantia nigra pars compacta and were killed 9 weeks later. No drugs were administered in these animals during this period. Sections from the pallidum were processed for detection of GAD67 mRNA at the single cell level by in situ hybridization histochemistry. Unlike at 2–3 weeks post-lesion (Soghomonian and Chesselet, 1992; Kincaid et al., 1992; Delfs et al., 1995), the level of labeling for GAD67 mRNA in the pallidum of rats with nigrostriatal lesions was not different than in sham-operated controls (Table 2). Similarly, the number of pallidal neurons expressing GAD67 mRNA in rats with 6-OHDA lesions was not different from sham-operated controls (Table 2).
DISCUSSION We show that local administration of muscimol into the subthalamic nucleus of intact rats induces a dosedependent increase in non-directed orofacial movements. This effect was no longer observed 2–3 weeks following a lesion of the substantia nigra pars compacta. Muscimol, however, induced a more robust behavioral response 8 –9 weeks following nigrostriatal lesions. In contrast to previous observations 2 weeks after nigrostriatal lesions, GAD67 mRNA was not increased in neurons of the globus pallidus 9 weeks post-lesion. These results further support temporal differences in GABAergic transmission in the subthalamic nucleus following acute nigrostriatal lesions in rats. Muscimol-induced dyskinesia in rats The selectivity of muscimol strongly suggests that the increase in dyskinetic movements observed in this study is due to stimulation of GABA-A receptors. Furthermore, we have previously shown that the small volume injected (0.1 l) only diffuses within the subthalamic nucleus (Parry et al., 1994; Delfs et al., 1995), indicating that GABA-A receptors within the subthalamic nucleus likely mediated the behavioral effect we observed. We did not attempt to block this effect with a GABAergic antagonist because previous work from our laboratory (Parry and Chesselet, unpublished observation) and others (Crossman et al., 1988; Perier et al., 2002) has shown that administration of GABA-A antagonists into the subthalamic nucleus can cause dyskinetic behaviors. Hyperkinetic behavior (hemiballismus) can be induced by subthalamic lesions in humans and primates (Hyland and Forman, 1957; Hamada and DeLong, 1992). Similarly, our laboratory has previously shown that excitoxic lesions of the subthalamic nucleus in rats induce a transient increase in non-directed orofacial movements similar to those observed in the present study (Banks and Chesselet, unpublished observations). Therefore, the increase in orofacial dyskinesia after intrasubthalamic administration of muscimol is compatible with an inhibitory action of this drug on subthalamic activity. Indeed, electrophysiological and behavioral studies have shown that local administration of muscimol silences neuronal activity in the subthalamic nucleus in vivo (Robledo and Feger, 1990; Bergman et al., 1994; Levy et al., 2001; Urbain et al., 2002) and in vitro (Baufreton et al., 2001). Muscimol-induced dyskinesia in rats with nigrostriatal lesions In the present study, we used muscimol-induced orofacial dyskinesia in rats to assess changes in behavioral responses to stimulation of intrasubthalamic GABA A receptors at different times after unilateral nigrostriatal lesions. Although inhibition of the subthalamic nucleus is clearly beneficial for parkinsonian symptoms after the loss of nigrostriatal neurons (Mehta et al., 2005; Bergman et al., 1994; Levy et al., 2001), it is not surprising to observe dyskinesia in this condition. Indeed, transient dyskinesia were also observed after administration of muscimol into
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Fig. 4. Behavioral effects of muscimol injected into the subthalamic nucleus of rats with or without nigrostriatal lesions. Rats received a unilateral injection of 6-OHDA or vehicle (SHAM) into the substantia nigra pars compacta and 12 days later were implanted with a cannula into the subthalamic nucleus ipsilateral to the lesion, as described in the methods section. The rats received vehicle or muscimol (0.01 g in 0.1 l) into the subthalamic nucleus either 2–3 (left panels, animals in cohort II) or 8 –9 weeks (right panels, animals in cohort III) after the nigrostriatal lesion. Orofacial dyskinesia, defined as described in Experimental Procedures, were measured continuously during 1 h following muscimol or vehicle administration. Data are mean⫾S.E.M. of oral bouts in each 10 min bins. * P⬍0.05 when compared with vehicle with ANOVA followed by Fisher PLSD test (n⫽6 – 8).
the subthalamic nucleus of MPTP-treated monkeys (Bergman et al., 1994; Levy et al., 2001). Similarly, small adjustments in electrical output induced abnormal involuntary movements in patients bilaterally implanted with high frequency stimulators in the subthalamic nucleus, (Krack et al., 1999; Pollak et al., 2002), a procedure which may increase GABAergic transmission in the subthalamic nucleus (Windels et al., 2003; Shen et al., 2003; Bruet et al., 2003). The present data further point to a possible role of the STN in dyskinesia in general. For example, we have previously shown that local administration of dopaminergic agonists into the STN induces dyskinesia by stimulating
D1-like dopaminergic receptors, and that this effect is markedly enhanced in rats with nigrostriatal lesions (Parry et al., 1994; Mehta et al., 2000). Whether GABAergic mechanisms in the STN are involved in L-DOPA induced dyskinesia, a severe complication of the most common treatment of Parkinson’s disease deserves further investigations. The changes in the magnitude of muscimol-induced dyskinesia observed in our study suggest that responses to the stimulation of subthalamic GABA A receptors are altered after nigrostriatal lesions. Furthermore, the data suggest that these responses are initially decreased and
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Table 2. Expression of Mr 67,000 (GAD 67) mRNA in neurons of the globus pallidus 9 weeks following nigrostriatal lesionsa
Sham-operated (n⫽8) 6-OHDA-lesioned (n⫽13)
Area of silver grains per cell body (m2)
# of neurons expressing GAD67 mRNA per mm2
9.89⫾1.29 9.72⫾0.78
154.9⫾20.8 136.8⫾8.9
a
Rats (cohort IV) received a unilateral injection of vehicle (shamoperated) or 6-OHDA into the substantia nigra and were killed 9 weeks later. Sections through the globus pallidus were processed for in situ hybridization histochemistry and emulsion autoradiography, Data were analyzed as described in Experimental Procedures and represent mean⫾S.E.M. P⬎0.05 when data in rats with lesions were compared to sham-operated with Student’s t-test.
secondarily increased after an acute lesion. Indeed, 2–3 weeks after the lesion, a dose of muscimol that induces a half-maximal increase in orofacial dyskinesia in control rats, still induced dyskinesia in sham-operated controls but not in rats with a nigrostriatal lesion. Conversely, 8 –9 weeks post-surgery, the response to 0.01 g muscimol in rats with lesions was similar to the effect induced by a 10-fold higher dose of muscimol (0.1 g) in intact rats, suggesting increased sensitivity. Time-dependent changes in the pallido-subthalamic pathway The temporal changes in the behavioral response induced by intrasubthalamic muscimol administration in rats with nigrostriatal lesions suggest that GABAergic transmission may be differentially altered in this region at various times after the injection of 6-OHDA. Indeed, electrophysiological data indicate time-dependent differences in neuronal GABAergic activity in the globus pallidus, the main source of GABAergic inputs to the subthalamic nucleus (Smith et al., 1990), following nigrostriatal lesions. Thus, a decrease in spontaneous activity and an increase in burst firing were both measured in neurons of the globus pallidus following nigrostriatal lesions (Pan and Walters, 1988; Tremblay et al., 1989). The increase in pallidal burst firing activity, however, diminished with time, returning to control levels 6 –9 weeks post-lesion, while the decrease in spontaneous activity persisted (Pan and Walters, 1988). Expression of GAD67 mRNA is increased in neurons of the globus pallidus of rats 2–3 weeks after unilateral lesions of the nigrostriatal dopaminergic pathway (Soghomonian and Chesselet, 1992; Kincaid et al., 1992; Delfs et al., 1995; Billings and Marshall, 2004), an effect that may be related to the increased bursting pattern observed in pallidal neurons at that time. Indeed, lesions of the subthalamic nucleus, which provides excitatory inputs to the globus pallidus, block both the increased burst firing activity (Ni et al., 2000) and the increase in GAD67 mRNA (Delfs et al., 1996; Billings and Marshall, 2004) in the globus pallidus after nigrostriatal lesions. Accordingly, we hypothesized that changes in GAD67 mRNA would subside 8 –9 weeks after the lesion, when these electrophysiological changes are no longer observed (Pan and Walters, 1988). Indeed, we show in the present
study that the level of GAD67 mRNA in the globus pallidus returns to control levels 9 weeks following nigrostriatal lesions, an observation that reinforces the parallelism between changes in GAD67 mRNA and patterns of electrophysiological activity observed in other systems (Chesselet et al., 1993; Soghomonian and Chesselet, 2000). Together with the electrophysiological and the molecular data, the behavioral observations suggest a decrease in pallido-subthalamic GABAergic transmission, leading to increased sensitivity of the behavioral response to stimulation of GABA-A receptors in the subthalamic nucleus. Temporal changes in GAD67 mRNA gene expression in the globus pallidus were also observed in rats treated chronically with haloperidol, a preferential D2-like antagonist. In this model, levels of GAD67 mRNA show a temporal pattern of expression following treatment initiation, with an initial increased expression measured at 3–14 days, no change in expression measured at 4 weeks, and a decrease in expression measured at 8 months of chronic haloperidol treatment (Mercugliano et al., 1992; Delfs et al., 1995a,b). The data from the present study may explain differences in studies examining GAD67 mRNA expression in neurons of the external pallidum. Consistent with data in rats 2–3 weeks after nigrostriatal lesions, increased levels of GAD67 mRNA were measured in the external pallidum of MPTP-treated cats and monkeys (Schroeder and Schneider, 2001; Soghomonian et al., 1994). In both of these studies, animals were treated with MPTP until they met behavioral criteria for severe Parkinsonism and killed 7–10 days following the last MPTP injection (Schroeder and Schneider, 2001; Soghomonian et al., 1994). In contrast, in another study in which no change in GAD67 mRNA expression was detected in the external pallidum of MPTP-treated monkeys, animals were killed 90 days after the last MPTP injection (Herrero et al., 1996). Thus, levels of GAD67 mRNA in the external pallidum may be temporally regulated following MPTP intoxication, as we observed following unilateral 6-OHDA lesions in rats. Consistent with these data, no change in GAD67 mRNA was measured in the external pallidum in postmortem tissue from patients with advanced stages of idiopathic Parkinson’s disease (Herrero et al., 1996). Interestingly, GAD protein was unchanged in the STN of MPTP-treated monkeys killed more than 2 months after the last MPTP injection (Soares et al., 2004). Although a small increase in burst firing was still observed in these animals prior to kill, GABA release was decreased in their STN (Soares et al., 2004). These observations are compatible with our data showing a lack of GAD67 mRNA increase in the globus pallidus, and increased behavioral response to GABA receptor stimulation in the STN of rats 2 months after nigrostriatal lesions. Functional implications Alteration in the activity of the subthalamic nucleus is an important feature of Parkinson’s disease. Recent in vivo data demonstrate that GABA receptor stimulation in the subthalamic nucleus profoundly affects its activity, even
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superseding the effects of glutamate and dopamine receptor stimulation (Ni et al., 2001a). Our evidence of timedependent changes in GABAergic transmission to the subthalamic nucleus following nigrostriatal lesions, is consistent with observations by Ni et al. (2001a) that the activity of the subthalamic nucleus changes during the first 4 weeks following lesion of the nigrostriatal pathway in rats. The reciprocal pathway formed by the subthalamic nucleus and the globus pallidus has recently received considerable attention. This network is thought to play an important role in regulating the normal activity of the basal ganglia and in the pathophysiology of movement disorders that involve this brain system (Chesselet and Delfs, 1996; Plenz and Kitai, 1999). Together with our findings, these data suggest that both the subthalamic nucleus and the globus pallidus may be temporally regulated in unison following acute nigrostriatal degeneration. Acknowledgments—This work was supported by P50-NS38367. We thank Carole Gray and Catherine Weston for their invaluable help with the figures and manuscript, and Dr. Laurence Mignon for her comments.
REFERENCES Albin RL, Young AB, Penney JB (1989) The functional anatomy of basal ganglia disorders. Trends Neurosci 12:366 –375. Baron MS, Wichmann T, Ma D, DeLong MR (2002) Effects of transient focal inactivation of the basal ganglia in parkinsonian primates. J Neurosci 22:592–599. Baufreton J, Garret M, Dovero S, Dufy B, Bioulac B, Taupignon A (2001) Activation of GABA(A) receptors in subthalamic neurons in vitro: properties of native receptors and inhibition mechanisms. J Neurophysiol 86:75– 85. Benazzouz A, Gross C, Feger J, Boraud T, Bioulac B (1993) Reversal of rigidity and improvement in motor performance by subthalamic high-frequency stimulation in MPTP-treated monkeys. Eur J Neurosci 5:382–389. Bergman H, Wichmann T, DeLong MR (1990) Reversal of experimental parkinsonism by lesions of the subthalamic nucleus. Science 249:1436 –1438. Bergman H, Wichmann T, Karmon B, DeLong MR (1994) The primate subthalamic nucleus: II. Neuronal activity in the MPTP model of parkinsonism. J Neurophysiol 72:507–520. Billings LM, Marshall JF (2004) Glutamic acid decarboxylase 67 mRNA regulation in two globus pallidus neuron populations by dopamine and the subthalamic nucleus. J Neurosci 24:3094–3103. Bruet N, Windels F, Carcenac C, Feuerstein C, Bertrand A, Poupard A, Savasta M (2003) Neurochemical mechanisms induced by high frequency stimulation of the subthalamic nucleus: increase of extracellular striatal glutamate and GABA in normal and hemiparkinsonian rats. J Neuropathol Exp Neurol 62:1228 –1240. Brussaard P, Herbison AE (2000) Long-term plasticity of postsynaptic GABAA-receptor function in the adult brain: insights from the oxytocin neurone. Trends Neurosci 23:190 –195. Chesselet MF (1996) Quantitative analysis and interpretation of data for in situ hybridization at the cellular level. In: In situ hybridization techniques for the brain (Henderson Z, ed), pp 141–149. IBRO Handbook Series. Chichester: John Wiley & Sons. Chesselet MF, Delfs JM (1996) Basal ganglia and movement disorders: an update. Trends Neurosci 19:417– 422. Chesselet MF, MacKenzie L, Eberle-Wang K (1996). In situ hybridization with 35S- and digoxigenin-labeled RNA probes. In: In situ hybridization techniques for the brain (Henderson Z, ed), pp 79 –109.
777
Chesselet MF, Mercugliano M, Soghomonian JJ, Salin P, Qin Y, Gonzales C (1993) Regulation of glutamic acid decarboxylase gene expression in efferent neurons of the basal ganglia. Prog Brain Res 99:143–154. Chesselet MF, Weiss L, Wuenschell C, Tobin AJ, Affolter HU (1987) Comparative distribution of mRNAs for glutamic acid decarboxylase, tyrosine hydroxylase, and tachykinins in the basal ganglia: an in situ hybridization study in the rodent brain. J Comp Neurol 262:125–140. Crossman AR, Mitchell IJ, Sambrook MA, Jackson A (1988) Chorea and myoclonus in the monkey induced by gamma-aminobutyric acid antagonism in the lentiform complex: the site of drug action and a hypothesis for the neural mechanisms of chorea. Brain 111(Pt 5):1211–1233. Delfs JM, Anegawa NJ, Chesselet MF (1995a) Glutamate decarboxylase messenger RNA in rat pallidum: comparison of the effects of haloperidol, clozapine and combined haloperidol-scopolamine treatments. Neuroscience 66:67– 80. Delfs JM, Ciaramitaro VM, Parry TJ, Chesselet MF (1995) Subthalamic nucleus lesions: widespread effects on changes in gene expression induced by nigrostriatal dopamine depletion in rats. J Neurosci 15:6562– 6575. Delfs JM, Ciaramitaro VM, Soghomonian JJ, Chesselet MF (1996) Unilateral nigrostriatal lesions induce a bilateral increase in glutamate decarboxylase messenger RNA in the reticular thalamic nucleus. Neuroscience 71:383–395. Delfs JM, Ellison GD, Mercugliano M, Chesselet MF (1995b) Expression of glutamic acid decarboxylase mRNA in striatum and pallidum in an animal model of tardive dyskinesia. Exp Neurol 133:175–188. Dostrovsky JO, Hutchison WD, Lozano AM (2002) The globus pallidus, deep brain stimulation, and Parkinson’s disease. Neuroscientist 8:284 –290. Dybdal D, Gale K (2000) Postural and anticonvulsant effects of inhibition of the rat subthalamic nucleus. J Neurosci 20:6728 – 6733. Eberle-Wang K, Lucki I, Chesselet MF (1996) A role for the subthalamic nucleus in 5-HT2C-induced oral dyskinesia. Neuroscience 72:117–128. Filion M, Tremblay L (1991) Abnormal spontaneous activity of globus pallidus neurons in monkeys with MPTP-induced parkinsonism. Brain Res 547:142–151. Filloux F, Hunt MA, Wamsley JK (1989) Localization of the dopamine uptake complex using [3H]N-[1-(2-benzo(b)thiophenyl)cyclohexyl] piperidine ([3H]BTCP) in rat brain. Neurosci Lett 100:105–110. Hamada I, DeLong MR (1992) Excitotoxic acid lesions of the primate subthalamic nucleus result in transient dyskinesias of the contralateral limbs. J Neurophysiol 68:1850 –1858. Herrero MT, Levy R, Ruberg M, Javoy-Agid F, Luquin MR, Agid Y, Hirsch EC, Obeso JA (1996) Glutamic acid decarboxylase mRNA expression in medial and lateral pallidal neurons in the MPTPtreated monkey and patients with Parkinson’s disease. Adv Neurol 69:209 –216. Hutchison WD, Allan RJ, Opitz H, Levy R, Dostrovsky JO, Lang AE, Lozano AM (1998) Neurophysiological identification of the subthalamic nucleus in surgery for Parkinson’s disease. Ann Neurol 44:622– 628. Hyland HH, Forman DM (1957) Prognosis in hemiballismus. Neurology 7:381–391. Janowsky A, Berger P, Vocci F, Labarca R, Skolnick P, Paul SM (1986) Characterization of sodium-dependent [3H]GBR-12935 binding in brain: a radioligand for selective labelling of the dopamine transport complex. J Neurochem 46:1272–1276. Kincaid AE, Albin RL, Newman SW, Penney JB, Young AB (1992) 6-Hydroxydopamine lesions of the nigrostriatal pathway alter the expression of glutamate decarboxylase messenger RNA in rat globus pallidus projection neurons. Neuroscience 51:705–718. Krack P, Pollak P, Limousin P, Benazzouz A, Deuschl G, Benabid AL (1999) From off-period dystonia to peak-dose chorea: the clinical
778
A. Mehta et al. / Neuroscience 131 (2005) 769 –778
spectrum of varying subthalamic nucleus activity. Brain 122(Pt 6):1133–1146. Kumar R, Lozano AM, Kim YJ, Hutchison WD, Sime E, Halket E, Lang AE (1998) Double-blind evaluation of subthalamic nucleus deep brain stimulation in advanced Parkinson’s disease. Neurology 51: 850–855. Levy R, Lang AE, Dostrovsky JO, Pahapill P, Romas J, Saint-Cyr J, Hutchison WD, Lozano AM (2001) Lidocaine and muscimol microinjections in subthalamic nucleus reverse Parkinsonian symptoms. Brain 124:2105–2118. Mehta A, Thermos K, Chesselet MF (2000) Increased behavioral response to dopaminergic stimulation of the subthalamic nucleus after nigrostriatal lesions. Synapse 37:298 –307. Mehta A, Chesselet MF (2005) Effects of GABAA receptor stimulation in the subthalamic nucleus on motor deficits induced by nigrostriatal lesions in the rat. Exp Neurol, in press. Mercugliano M, Saller CF, Salama AI, U’Prichard DC, Chesselet MF (1992) Clozapine and haloperidol have differential effects on glutamic acid decarboxylase mRNA in the pallidal nuclei of the rat. Neuropsychopharmacology 6:179 –187. Narabayashi H (1995) The neural mechanisms and progressive nature of symptoms of Parkinson’s disease-based on clinical, neurophysiological and morphological studies. J Neural Transm Park Dis Dement Sect 10:63–75. Ni Z, Bouali-Benazzouz R, Gao D, Benabid AL, Benazzouz A (2000) Changes in the firing pattern of globus pallidus neurons after the degeneration of nigrostriatal pathway are mediated by the subthalamic nucleus in the rat. Eur J Neurosci 12:4338 – 4344. Ni ZG, Bouali-Benazzouz R, Gao DM, Benabid AL, Benazzouz A (2001a) Time-course of changes in firing rates and firing patterns of subthalamic nucleus neuronal activity after 6-OHDA-induced dopamine depletion in rats. Brain Res 899:142–147. Ni Z, Gao D, Bouali-Benazzouz R, Benabid AL, Benazzouz A (2001b) Effect of microiontophoretic application of dopamine on subthalamic nucleus neuronal activity in normal rats and in rats with unilateral lesion of the nigrostriatal pathway. Eur J Neurosci 14:373–381. Pan HS, Walters JR (1988) Unilateral lesion of the nigrostriatal pathway decreases the firing rate and alters the firing pattern of globus pallidus neurons in the rat. Synapse 2:650 – 656. Parry TJ, Eberle-Wang K, Lucki I, Chesselet MF (1994) Dopaminergic stimulation of subthalamic nucleus elicits oral dyskinesia in rats. Exp Neurol 128:181–190. Paxinos G, Watson C (1998) The rat brain in stereotaxic coordinates. San Diego, CA: Academic Press. Penney JB Jr, Young AB (1981) GABA as the pallidothalamic neurotransmitter: implications for basal ganglia function. Brain Res 207:195–199. Perier C, Tremblay L, Feger J, Hirsch EC (2002) Behavioral consequences of bicuculline injection in the subthalamic nucleus and the zona incerta in rat. J Neurosci 22:8711– 8719. Plenz D, Kitai ST (1999) A basal ganglia pacemaker formed by the subthalamic nucleus and external globus pallidus. Nature 400:677– 682.
Poewe WH, Wenning GK (1998) The natural history of Parkinson’s disease. Ann Neurol 44:S1–9 Pollak P, Krack P, Fraix V, Mendes A, Moro E, Chabardes S, Benabid AL (2002) Intraoperative micro- and macrostimulation of the subthalamic nucleus in Parkinson’s disease. Mov Disord 17(Suppl 3):S155–161. Robledo P, Feger J (1990) Excitatory influence of rat subthalamic nucleus to substantia nigra pars reticulata and the pallidal complex: electrophysiological data. Brain Res 518:47–54. Schroeder JA, Schneider JS (2001) Alterations in expression of messenger RNAs encoding two isoforms of glutamic acid decarboxylase in the globus pallidus and entopeduncular nucleus in animals symptomatic for and recovered from experimental parkinsonism. Brain Res 888:180 –183. Shen KZ, Zhu ZT, Munhall A, Johnson SW (2003) Synaptic plasticity in rat subthalamic nucleus induced by high-frequency stimulation. Synapse 50:314 –319. Smith Y, Bolam JP, Von Krosigk M (1990) Topographical and synaptic organization of the GABA-containing pallidosubthalamic projection in the rat. Eur J Neurosci 2:500 –511. Soares J, Kliem MA, Betarbet R, Greenamyre JT, Yamamoto B, Wichmann T (2004) Role of external pallidal segment in primate parkinsonism: comparison of the effects of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine-induced parkinsonism and lesions of the external pallidal segment. J Neurosci 24:6417– 6426. Soghomonian JJ, Chesselet MF (1991) Lesions of the dopaminergic nigrostriatal pathway alter preprosomatostatin messenger RNA levels in the striatum, the entopeduncular nucleus and the lateral hypothalamus of the rat. Neuroscience 42:49 –59. Soghomonian JJ, Chesselet MF (1992) Effects of nigrostriatal lesions on the levels of messenger RNAs encoding two isoforms of glutamate decarboxylase in the globus pallidus and entopeduncular nucleus of the rat. Synapse 11:124 –133. Soghomonian JJ, Chesselet MF (2000) GABA in the basal ganglia. In: GABA in the nervous system: the view at fifty years (Martin DL, Olsen RW, eds), pp 265–291. Philadelphia: Lippincott, Williams & Wilkins. Soghomonian JJ, Pedneault S, Audet G, Parent A (1994) Increased glutamate decarboxylase mRNA levels in the striatum and pallidum of MPTP-treated primates. J Neurosci 14:6256 – 6265. Tremblay L, Filion M, Bedard PJ (1989) Responses of pallidal neurons to striatal stimulation in monkeys with MPTP-induced parkinsonism. Brain Res 498:17–33. Urbain N, Rentero N, Gervasoni D, Renaud B, Chouvet G (2002) The switch of subthalamic neurons from an irregular to a bursting pattern does not solely depend on their GABAergic inputs in the anesthetic-free rat. J Neurosci 22:8665– 8675. Vignon J, Pinet V, Cerruti C, Kamenka JM, Chicheportiche R (1988) [3H]N-[1-(2-benzo(b)thiophenyl)cyclohexyl]piperidine ([3H]BTCP): a new phencyclidine analog selective for the dopamine uptake complex. Eur J Pharmacol 148:427– 436. Windels F, Bruet N, Poupard A, Feuerstein C, Bertrand A, Savasta M (2003) Influence of the frequency parameter on extracellular glutamate and gamma-aminobutyric acid in substantia nigra and globus pallidus during electrical stimulation of subthalamic nucleus in rats. J Neurosci Res 72:259 –267.
(Accepted 15 November 2004)