Materials
Desmethylimipramine, exendin-4, LPS (serotype E. Coli 0111:B4), pargyline, catalase, L-tyrosine, ferrous ammonium sulphate, benserazide, 6 MPH4, 6-OHDA, LPS, apomorphine hydrochloride, 3-iodo-L-tyrosine, D-tyrosine and tyrosine hydroxylase were all obtained from Sigma, UK. Apomorphine and 6-OHDA were dissolved in 2.0% w/v ascorbic acid, whilst UCN solid was initially dissolved in 70% ethanol and further diluted in saline to 1 × 10-8 M stock concentration. All drugs, apart from LPS and 6-OHDA were injected in a volume of 0.1 ml/100 g body weight. Rabbit polyclonal anti-rat TH IgG was obtained from Calbiochem. Horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG was obtained from Cell Signaling, MA. Biotinylated swine anti-rabbit IgG was obtained from Dako, Denmark. ABC complex was purchased from Vector Laboratories, UK. All other reagents were of Analar or HPLC grade.
Surgical procedures
An overview of the experimental protocols used in the present series of experiments is shown in figure 1. Note that groups of rats went through all the procedures indicated from initial toxin injection to ex vivo neurochemistry and histology. Experiments were carried out in accordance with the Animals (Scientific Procedures) Act UK (1986). Male Wistar rats (210–240 g) were group housed and access to food and water was ad libitum. Care was taken to minimize animal usage and typically, tissue was used in several different assay paradigms. Prior to surgery, animals to be lesioned with 6-OHDA received pargyline (50 mg/kg, i.p.) and desmethylimipramine (25 mg/kg, i.p.), in order to maximize the selectivity of the toxin for dopaminergic neurons. Animals were anaesthetized with isofluorane (4% for induction, 1.5% for maintenance), secured in a stereotaxic frame (David Kopf, US) and given injections of 6-OHDA (8 μg/4 μl of saline with 1% ascorbic acid) injected into the right medial forebrain bundle (from bregma in mm; A -4.3, L 1.4 and V 8.2) while LPS (2 μg/2 μl saline) was injected into the substantia nigra pars compacta (SNc; from bregma in mm; A -5.2, L 2.2 and V 8.3). Seven days later rats were administered EX-4 (0.1 or 0.5 μg/kg i.p, twice daily) dissolved in 0.9% saline for a period of seven days.
Assessment of nigrostriatal lesion severity following apomorphine challenge
All animals received an apomorphine challenge (APO; 0.5 mg/kg sc.) in order to assess lesion severity as previously described [21]. Animals were placed in a circular test arena and following a short period of acclimatization, injected with the dopaminergic agonist. Contraversive turns were noted 20 min. post-injection and recorded over a 120 s observation period. Only complete, 'tight' turns were recorded. We have tested the value of observing rats for longer periods of time (up to 60 min) and recording rotations. Although, obviously, the number of rotations was considerably higher using this protocol the relative difference between groups was virtually identical to sampling for the much shorter time period and the overall methodology was far more time efficient.
In vivo microdialysis
Later that day rats were implanted with concentric dialysis probes of a construction previously described [22]. Probes were bilaterally implanted into each striatum (from bregma; A +0.2, L 3.0, and ventral 8.2) and the following day perfused with an artificial cerebrospinal fluid (aCSF) solution (composition in mM: 2.5 KCl; 125 NaCl; 1.18 MgCl2; 1.26 CaCl2) as previously used [22] but without addition of the 5-HT reuptake inhibitor citalopram. Following a 1 hour equilibration period four 30 min 'basal' samples were collected prior to infusion of 100 mM K+containing aCSF to evoke DA release. In the latter case the concentration of Na+ was decreased accordingly to maintain osmolarity of the aCSF.
Tissue dopamine assay
Animals received pargyline (50 mg/kg) 30 min prior to sacrifice. Brains were removed, striata dissected and homogenized in ice-cold phosphate buffer (pH 7.4). All homegenates were split into two equal portions, with one half of each treated with 0.2 M perchloric acid (1:10, w/v) containing ascorbic acid (0.2 μM) and EDTA (0.2 μM), to precipitate cell debris. These were then centrifuged at 9000 × g for 15 min at 4°C, supernatants passed through a syringe filter (10 μm pore size) and whole tissue dopamine levels estimated using HPLC with electrochemical detection [22]. Brain blocs containing nigra were rapidly frozen and retained for immunohistochemistry.
Ex vivo tyrosine hydroxylase assay
TH activity was measured in the remaining homogenates, using a modification of the method of Naoi et al. [23]. Aliquots were incubated with 200 μM L-tyrosine in a total reaction mixture volume of 100 μl. This consisted of the following components: 100 mM sodium acetate-acetic acid buffer (pH 6.0), 2 mM ferrous ammonium sulphate, 1 mM 6 MPH4, 10 μg of catalase and 1 mM benserazide, an inhibitor or aromatic L-amino acid decarboxylase (AADC). 6 MPH4 solution was firstly made as 10 mM in 1 M mercaptoethanol. The incubation mixture, except for tyrosine and the pteridin cofactor, was preincubated with homegenates at 37°C for 5 min, and the reaction was initiated by addition of the L-tyrosine and 6 MPH4. After incubation at 37°C for 10 min, the reaction was terminated by addition of 100 μl perchloric acid (0.1 M, containing 0.4 mM sodium metabisulphite and 0.1 mM disodium EDTA). The sample was then vortexed and left to stand on ice for 10 min, then centrifuged at 1000 × g for 10 min. The supernatant was diluted to 1 in 1000 with mobile phase, then analyzed using HPLC-ED to measure the amount of L-DOPA. As blank, a similar reaction mixture containing D-tyrosine instead of the L-isomer and 100 μM 3-iodo-L-tyrosine was used [23].
Immunohistochemistry
Slide-mounted 10 μm cryostat sections from flash-frozen rat brain blocs were removed from the freezer and allowed to equilibrate to room temperature for 30 minutes, prior to post fixation in 4% paraformaldehyde, containing 1% gluteraldehyde for 5 minutes at 0°C. Following rinsing in 0.1 M PBS for 5 minutes, sections were dehydrated through graded alcohols and endogenous peroxidase activity was blocked by incubation in 0.3% H2O2 in methanol for 10 minutes. The sections were then rehydrated and non-specific immunoreactivity was blocked with 10% swine serum in PBS for 10 minutes. Sections were then incubated in primary antibody (rabbit anti-rat TH IgG) at 1:700 in PBS for 16 hours at 4°C. After rinsing, the sections were incubated sequentially in biotinylated swine anti-rabbit antibody 1:250 in PBS (Dako, Denmark) for 30 minutes at room temperature and ABC complex following the manufacturer's instructions. Immunoreactivity was visualized through incubation in 0.5 mg/ml 3-diaminobenzidine (DAB), containing 0.009% H2O2 for 2 minutes at room temperature. The sections were counterstained in Harris haematoxylin, dehydrated, cleared and mounted for microscopic examination. Sections were viewed under light microscopy (×40 magnification). Digital images were captured using a Leica DC500 system and the manufacturer's software. For each animal, six successive nigral sections were selected from both treated and contralateral side, taken from a starting point of -5.5 mm relative to bregma, using a total of six rats per group.
Data handling and analysis
Data obtained from apomorphine challenge, whole tissue DA and TH assay studies were expressed as mean values ± S.E.M. Data were subjected to one way or two way ANOVA to identify overall trends, with a post hoc Bonferroni's multiple comparison test used to establish significant differences between the groups. Statistical analysis was performed using a proprietary software package (GraphPad Prism™). Numbers of animals used in experiments are detailed in the figure legends. In all cases, comparisons were made with respect to toxin/vehicle values. Statistical significance was set at p < 0.05.
