The subjects of this study were 8 weeks-old adult male albino Sprague-Dawley rats (Orient Animal Corp., Gyungki-do, Korea) weighing 280–300 g each. They were group-housed with three per cage, under a reversed 12: 12 h light-dark cycle with light on from 08: 00–20: 00 h. All of the rats had free access to food and water in the barrier system with regulated temperature (23±1°C), humidity (50±1°C), and ventilation (9–12 times per hour). All animals were handled daily for at least 7 days prior to the experiment. The care and treatment of the animals were provided in accordance to the Guide for the Care and Use of Laboratory Animals (NIH). All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Catholic University. All experiments were carried out in accordance with the Guidelines for Animal Experimentation.

The rats were divided into three groups (n=6 per group): 1) sham group (no-treatment), 2) 6-OHDA-lesioned group, and 3) 6-OHDA-lesioned plus DBS group. To assess the efficacy of the 6-OHDA lesion, which induces Parkinsonian-like motor deficits, all rats were tested for apomorphine (Sigma-Aldrich Inc., St. Louis, MO, USA)-induced (0.05 mg/kg, s.c) rotational behavior at 21 days after the exposure to 6-OHDA. The results were expressed as ipsilateral turns/min. The rats exhibiting a vigorous rotational response (>100 total turns in a period of 45 minutes) to apomorphine (0.05 mg/kg, s.c.) were selected for further study (Fig. 1). It has previously been demonstrated that the rats meeting this criterion have greater than 95% depletion of striatal dopamine 19).

For medial forebrain bundle (MFB) lesioning, all of the rats were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and secured in Kopf stereotaxic apparatus (Phymep, Paris, France). We treated 12 rats with the injections of 3 ug of 6-hydroxydopamine (6-OHDA; Sigma-Aldrich, USA; 8 ug/2 uL in 0.02% ascorbic acid in normal saline, Sweden AB) into the right MFB, at the flow rate of 2 uL/min. The stereotaxic coordinates of the injection site at Bregma were: anteroposterior, −4.4 mm; lateral, −1.2 mm; and dorsoventral, −7.8 mm. All stereotaxic co-ordinates cited here are according to the stereotaxic atlas of Paxinos20). The rats were kept warm after the injections and were allowed to recover from anesthesia. They were returned to the animal facility for 3 weeks to allow the degeneration of dopaminergic neurons induced by the neurotoxin. After they stabilized, they were processed for microdialysis experiments.

The rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). Using aseptic techniques, a guide cannula (CMA/12, CMA Microdialysis, Solna, Sweden) aimed to terminate in the dorsal striatum (anteroposterior, 1.0 mm; lateral, −3.2 mm and dorsoventral, −6.0 mm to Bregma) was stereotaxically implanted and attached to the skull using skull screws and dental cement. The cannula was then closed with a tight-fitting stainless-steel obturator. The microdialysis was performed with a 4 mm microdialysis probe (CMA/12, CMA Microdialysis), connected to a syringe pump via dual liquid swivel, that was inserted into the guide cannula which is then perfused with an artificial cerebrospinal fluid (145 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl₂, 1.0 mM MgCl₂, 2.0 mM Na₂HPO₄; pH 7.4) at a constant rate of 1.0 mL/min. Dialysate samples were collected during 20 minutes of sampling intervals via outlet tubing connected to a microfraction collector (CMA Microdialysis). The position of the probes was verified by the histological examination at the end of the experiments. The rats suffering from bleeding was excluded from the analysis.

Concentric bipolar platinum/iridium stimulating electrodes (style CB-BFE75, FHC, Inc., Bowdoin, ME, USA) were used. The stereotaxic co-ordinates of the stimulation site (STN) at Bregma were: anteroposterior, −3.6 mm; lateral, −2.5 mm; and dorsoventral, −8.0 mm. Stimuli were delivered for 20 minutes, with a programmable pulse generator (Iso-Flex/Master-8; AMPI, Israel) and a stimulus isolation unit. Stimulation parameters corresponded to those used in the clinical practice (frequency 130 Hz, 60 us rectangular pulse width and 200 uA intensity). At the end of each experiment, a mechanical lesion created by stimulating electrode in the STN, so that the position of the electrode could be checked at the end of experiments.

The dialysate (injection volume 30 μL) was assayed for GABA and glutamate using high performance liquid chromatography (HPLC) coupled to a fluorescence detector. The detector was equipped with a high performance analytical cell (ESA model 5014), which is tailored for use in microdialysis applications. The mobile phase was composed of 75 mM monobasic sodium phosphate, 0.1 mM ethylenediaminetetraacetic acid, 1.4 mM octanesulfonic acid, and 10% acetonitrile, adjusted to pH 3.2 with HPLC grade phosphoric acid. The separation of monoamine metabolites was performed on Waters Nova-Pak C-18 column (4 mm, 150×3.9 mm). The flow rate of the system was 0.8 mL/min. GABA and glutamate in dialysates were expressed as percentages of three baseline samples collected immediately before DBS.

After the microdialysis processes, all of the rats were deeply anesthetized with sodium pentobarbital (80 mg/kg, i.p.). The rats were intracardially perfused with 100 mL of saline followed by 500 mL of 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS) (pH 7.0). The brains were removed, postfixed overnight, and cryoprotected in 20% sucrose in 0.1 M PBS at 4°C. The brains were cut via cryostat sectioning into 30 um coronal sections at the level of the striatum and substantia nigra (Leica, Germany). The tissue was stored at −20°C for histochemistry and these slices were histochemically processed as free-floating sections.

The brain sections were washed in PBS containing 0.3% Triton X-100. The primary mouse monoclonal antibodies against tyrosine hydroxylase were used (TH, concentration 1: 2000; Zymed Laboratories INC., San Francisco, CA, USA). The primary antibodies were diluted with blocking solution (10% bovine serum in phosphate buffered saline, pH 7.4) and the tissues were incubated for overnight at 4°C with constant agitation. After rinsing in PBS, the sections were incubated for 2 hours at room temperature in biotinylated anti-mouse (Santa Cruz Biotechnology Inc., San Francisco, CA, USA) diluted 200X in phosphate buffered saline tween and 2% normal goat blocking serum. The sections were placed in the mouse ABC staining system (Santa Cruz Biotechnology Inc.) for 2 hours at room temperature. Following further rinsing with PBS, the tissue was developed using diaminobenzadine (Sigma-Aldrich Inc.) as the chromogen with nickel intensification. The sections were mounted on slides, air-dried, and cover slipped for microscopic observation.

The results were analyzed using repeated measures analysis of variance (ANOVA) for multiple comparisons where necessary as means±SE (standard error). Repeated measures ANOVA was used to determine the significance of any changes in the HPLC analysis of GABA and glutamate results and in histochemical data, among the different groups; this was followed by the Tukey technique. The criterion for statistical significance was considered as p values <0.05.

As illustrated in Fig. 2, the average and SE of baseline glutamate level in the striatum corresponding to 100% was 543±72 nM (sham group), 491±87 nM (6-OHDA group) and 632±83 nM (6-OHDA plus group), respectively. There is a statistical significance in three groups (F2, 15=7.641, p<0.01). Post-hoc comparisons indicated that the striatal glutamate contents in the 6-OHDA plus DBS group were significantly high compared to the sham (p<0.01) and 6-OHDA group (p<0.01). Post-hoc comparisons also indicated that the striatal glutamate contents in the sham group were significantly high compared to the 6-OHDA group (p<0.05). HPLC analysis demonstrated a tendency of an increase in extracellular striatal glutamate during DBS and a decrease after DBS. The maximal peak of extracellular glutamate (110±8%) is shown at 20 minutes after DBS start time in 6-OHDA plus DBS group. The concentration of extracellular glutamate progressively declined to basal value in 20 minutes after DBS. However, it showed a tendency of maintaining the extracellular glutamate to high concentration state even after DBS, in the 6-OHDA plus DBS group compared to the 6-OHDA group.

As illustrated in Fig. 3, the average and SE of baseline GABA level in the striatum corresponding to 100% was 67±15 nM (sham group), 48±19 nM (6-OHDA group) and 75±17 nM (6-OHDA plus group), respectively. There is a statistical significance in the three groups (F2, 15=5.826, p<0.05). Post-hoc comparisons indicated that the striatal GABA contents in the 6-OHDA plus DBS group were significantly high compared to the 6-OHDA group (p<0.01). However, there was no statistical significance of GABA contents between the sham and 6-OHDA plus DBS group (p>0.05). Post-hoc comparisons also indicated that the striatal GABA contents in the sham group were significantly high compared to the 6-OHDA group (p<0.01). HPLC analysis demonstrated a tendency of an increase in extracellular striatal GABA during DBS and a decrease after DBS. The maximal peak of extracellular GABA (114±6%) is shown at 40 minutes after DBS start time, in 6-OHDA plus DBS group. The concentration of extracellular GABA gradually declined to basal value in 40 minutes after DBS. However, it showed the tendency of maintaining the extracellular GABA to high concentration state even after DBS, in the 6-OHDA plus DBS group compared to the 6-OHDA group.

At the end of the experiment, histologic evaluation of the location of stimulating electrode tip in STN was examined by cresyl violet stained section (Fig. 4). As illustrated in Fig. 5, dopamine depletion was severe in the substantia nigra and striatum of 6-OHDA group. Optical density measurement of TH immunoreactivity in the substantia nigra and striatum in 6-OHDA group showed a mean depletion of 90% compared to the unlesioned side.