Adeno-associated virus production
The D2Rs and GFP coding sequences were cloned into AAV genomes under the control of the chicken β-actin/cytomegalovirus (CBA/CMV) promoter for ubiquitous and robust expression. AAV 2/9 was produced via triple-transfection of HEK 293 T cells with the genome and helper plasmids. Virus was recovered from cells using freeze-thaw cycles, purified using an iodixanol gradient (Optiprep Density Gradient, Sigma-Aldrich, St. Louis, MO), followed by buffer exchange and concentration using concentrator columns (Orbital Biosciences, Topsfield, MA) as described previously . The viral titer was determined using digital droplet PCR (ddPCR) and normalized to 1 × 1013 vector genomes (vg)/ml using Balanced Salt Solution (Sigma-Aldrich, St. Louis, MO).
Animals and surgeries
Studies were performed using adult male Fischer F344 rats (200-220 g upon arrival; Charles River, Wilmington, MA) in accordance with the guidelines of Michigan State University (AUF 06/16–093-00), Binghamton University (AUF# 779–17), and Rosalind Franklin University (AUF# A3279–01) Institutional Animal Care & Use Committees. All work was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Rats were housed two per cage prior to behavioral testing, and then separated and individually housed with environment enrichment during behavior studies for the remainder of the experiments. The animals were housed in a light-controlled (12 h light/dark cycle) and temperature-controlled (22 ± 1 °C) room, and had free access to standard lab chow and water.
All 6-OHDA and vector surgeries were performed under 2% isoflurane. After being anesthetized, animals were placed in a stereotaxic frame and were injected using a glass capillary needle fitted to a Hamilton syringe (Hamilton, Reno, NV) . Three weeks following lesion surgery, animals were tested for spontaneous forepaw use (cylinder test) to estimate lesion efficacy. Vector treatment groups were normalized using forepaw deficits in order to ensure equal lesions between the treatment groups.
For lesion surgeries 5 mg/ml 6-OHDA hydrobromide (Sigma-Aldrich, St. Louis, MO) was prepared in 0.2 mg/ml ascorbic acid immediately prior to the injections. Animals received 2 μl injections of 6-OHDA into the medial forebrain bundle (MFB) (from bregma: Anterior Posterior (AP) – 4.3 mm, Medial Lateral (ML) + 1.6 mm, Dorsal Ventral (DV) -8.4 mm from skull) and the SNc (from bregma: AP -4.8 mm, ML + 1.7 mm, DV -8.0 mm from skull), for a total of 10 μg 6-OHDA per site and 20 μg per animal. The glass needle was lowered to the site and the injection started after 30 s. 6-OHDA was injected at a rate of 0.5 μl/minute. The needle was removed 2 minutes after the injection was finished and cleaned between each injection.
Vector delivery was performed 3 weeks following the 6-OHDA lesion via stereotaxic delivery . A subset of animals (N = 7) destined for electrophysiological measures did not receive a 6-OHDA lesion. Using the same procedure as described for the lesion surgeries, animals received a single midline 2 μl injection of virus (AAV2/9-DRs, 1 × 1013 vg/ml; AAV2/9-GFP, 1 × 1013 vg/ml) to the DRN (from bregma: AP -7.8, ML -3.1, DV -7.5 from skull). The stereotaxic arm was positioned in a 30° lateral angle in order to avoid the cerebral aqueduct.
Parkinsonian and vector-injected animals used for in vivo microdialysis were shipped to Binghamton University 2 weeks following the vector surgeries. Following quarantine, rats were acclimated to the colony room and habituated to handling for 1 week. Rats were then tested for baseline forepaw adjusting steps. Thereafter, microdialysis cannulation surgery was performed under 2–3% isoflurane in oxygen with the tooth bar set to 5 mm below the interaural line. Five minutes before surgery and 24 h after surgery rats received an injection of Buprinex (0.03 mg/kg, i.p.). A unilateral dorsal striatal-directed cannula (CMA 12 Elite; Stockholm, Sweden) was implanted ipsilateral to lesion (from bregma AP: 1.2 mm; ML: − 2.8 mm; DV: − 3.7 mm). The cannula was fixed in place by four jeweler’s screws, jet liquid, and dental acrylic (Lang Dental, Wheeling, IL). Two weeks following cannulation surgery, rats underwent behavioral testing.
Non-lesioned rats used for electrophysiological recordings of the DRN were shipped to Rosalind Franklin University 2 weeks following the vector surgeries and housed for an additional 4–8 weeks prior to electrophysiological recordings. Burr holes (~ 1 mm in diameter) were drilled in the skull overlying the DRN. Prior to experimentation all animals were anesthetized with urethane (1.5 g/kg i.p.) and placed in a stereotaxic apparatus. Bipolar stimulating/recording electrodes were implanted in the frontal cortex and DRN on the right side using a micromanipulator (coordinates from Bregma: AP: 3.2 mm; ML: 0.8 mm lateral; DV: 4.4 mm ventral (frontal cortex) or AP: 7.8 mm; ML 3.1 mm; DV: 7.5 mm with the manipulator angled 30 degrees toward Bregma) as previously described .
Abnormal involuntary movement (AIM) ratings and drug treatments
Animals were allowed to recover for 3 weeks following vector injections, and to allow for peak expression of the viral transgene . After this time, L-DOPA treatment and abnormal involuntary movement (AIM) scale ratings began (see time line in Fig. 1a). As described previously, the AIM rating scale can be used to evaluate the severity of LID and has been adapted for animal use [44, 71]. Briefly, AIMs are evaluated by scoring the level of dystonia of the limbs and body, hyperkinesia of the forelimbs, and orolingual movements. Each AIM is given two numerical scores—one indicating the intensity (0 = absent, 1 = mild, 2 = moderate, or 3 = severe) and frequency (0 = absent, 1 = intermittently present for < 50% of the observation period, 2 = intermittently present for > 50% of the observation period, or 3 = uninterruptable and present through the entire rating period) . Each AIM is given a severity score by multiplying the intensity and frequency, and the total AIM score is a sum of all the behaviors severities. An animal is considered non-dyskinetic with a score of ≤4, as non-dyskinetic parkinsonian rats can display low level AIMs from exhibiting normal chewing behavior and a mild parkinsonian dystonic posture .
Animals received subcutaneous injections of L-DOPA/benserazide (Sigma-Aldrich, St. Louis, MO) three times per week and were rated using the AIM scale in 25-min intervals post-injection until all LID behavior had subsided. L-DOPA doses ranged between 2 mg/kg-12 mg/kg (Fig. 1a). Benserazide doses (12 mg/kg) remained constant for all L-DOPA injections. The same injection and rating paradigm was used for AIM evaluations with the non-selective DA agonist apomorphine (0.1 mg/kg, R&D Systems, Minneapolis, MN), the D2/D3 receptor agonist quinpirole (0.2 mg/kg, Sigma-Aldrich, St. Louis, MO) and the D1 receptor agonist SKF-81297 (0.8 mg/kg, Sigma-Aldrich, St. Louis, MO). DA agonist doses were selected based on doses known to induce AIMs in parkinsonian rats [9, 42]. Peak AIM scores of DA agonists were determined based off the highest average AIM scores of control animals during the rating period.
Parkinsonian motor evaluation
To assess whether D2Rs viral therapy affects the anti-parkinsonian properties of L-DOPA therapy, we evaluated parkinsonian motor behavior on and off L-DOPA using the cylinder task and the forepaw adjusting steps (FAS) test. Rats with significant lesions perform poorly on both these tests, with impairment to the forepaw contralateral to the lesion that is alleviated with L-DOPA treatment [18, 68]. The cylinder task was conducted as previously reported . Animals were placed in a clear Plexiglas cylinder on top of a light box for 5 to 7 minutes while being recorded. Each animal was rated by counting ~ 20 weight-bearing forepaw placements on the cylinder (contralateral to the lesion, ipsilateral to the lesion, both) to determine the percentage use of the forepaw contralateral to the lesion, which is derived by dividing the sum of contralateral touches and half of both forepaw touches by the total forepaw touches, and multiplying this number by 100. Trials were performed following the initial L-DOPA treatment (AIM evaluation) period, and tested either off L-DOPA or, on the following day, 50 min after receiving a 6 mg/kg L-DOPA injection (12 mg/kg benserazide).
The FAS test was performed as described previously . Briefly, rats were restrained by an experimenter so that only one forepaw was free to touch the counter. Rats were then dragged laterally along a 90 cm distance over 10 s while a trained rater blind to the experimental condition counted the number of steps. Data are represented as forehand percent intact, which are derived by taking the number of steps taken by the contralateral forehand and dividing it by the ipsilateral forehand, and then multiplying this number by 100. The test was performed over 2 days either off L-DOPA or 60 min following an 8 mg/kg or 12 mg/kg L-DOPA injection.
Two hours following the final L-DOPA administration, animals from the AIM experimentation were sacrificed via sodium pentobarbital overdose and intracardially perfused with Tyrode’s solution (137 mM sodium chloride, 1.8 mM calcium chloride dihydrate, 0.32 mM sodium phosphate monobasic dihydrate, 5.5 mM glucose, 11.9 mM sodium bicarbonate, 2.7 mM potassium chloride). Brains were rapidly removed and coronally hemisected, with the rostral portion of the left and right striatum dissected out and flash frozen in liquid nitrogen for biochemical analysis. The caudal portion of the brain was postfixed for 72 h in 4% paraformaldehyde (PFA) in phosphate-buffered saline and then cryoprotected by saturation in 30% sucrose. Brains were frozen and sectioned coronally at 40 μm thickness using a sliding microtome into free floating sections and stored in cryoprotectant (30% ethylene glycol, 0.8 mM sucrose in 0.5× tris-buffered saline) until further use.
A 1:6 series of free-floating tissue was stained immunohistochemically for TH (MAB318, MilliporeSigma, Burlington, MA), D2R (AB5084P, MilliporeSigma, Burlington, MA), GFP (AB290, Abcam, Cambridge, United Kingdom), IBA1 (019–19,741, Wako Life Sciences, Richmond, VA), or 5-HT (NT-102, Protos Biotech, New York, NY) using methods previously reported . Sections were washed in 1× Tris-buffered saline (TBS) with .25% Triton x-100, incubated in 0.3% H2O2 for 30 min, and rinsed and blocked in 10% normal goat serum for 2 h. Tissue was incubated in primary antibody (TH 1:4000, D2R 1:1000, GFP 1:20,000, IBA1 1:4000, 5-HT 1:10,000) overnight at room temperature. After washing, tissue was incubated in secondary antibody (biotinylated horse anti-mouse IgG 1:500, BA-2001; Vector Laboratories, Burlingame, CA; biotinylated goat anti-rabbit IgG 1:500, AP132B, Millipore-Sigma, Burlington, MA) followed by the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Tissue staining was developed with 0.5 mg/ml 3,3′ diaminobenzidine (DAB, Sigma-Aldrich, St. Louis, MO) and 0.03% H2O2. Sections were mounted on glass slides, dehydrated, and coverslipped with Cytoseal (ThermoFisher, Waltham, MA).
Tissue for immunofluorescence dual labeling of D2Rs or GFP with SERT (340–004, Synaptic Systems, Goettingen, Germany) were washed with 1× TBS with 0.25% Triton x-100, blocked in 10% normal goat serum for 2 h, and probed with primary antibody overnight (D2Rs 1:1000, GFP 1:20,000, SERT 1:300). Tissue was incubated with secondary antibody (A11008 1:500, A11076 1:500; ThermoFischer, Waltham, MA) in the dark for 2 hours, and washed in TBS before being mounted and coverslipped with Vectashield Hardset Antifade Mounting Medium (Vector Laboratories, Burlingame, CA).
Images were taken on a Nikon Eclipse 90i microscope with a QICAM fast 1394 camera (fluorescence; QImaging, Surrey, British Columbia, Canada) or a Nikon D-1 camera (brightfield microscopy; Nikon, Tokyo, Japan). The figures were made using Photoshop 7.0 (Adobe, San Jose, CA) with the brightness, sharpness, and saturation adjusted only as needed to best represent the staining as it is viewed directly under the microscope.
In vivo microdialysis
As outlined above, a separate cohort of parkinsonian rats treated with GFP or D2Rs were utilized for in vivo microdialysis. The night before the procedure, striatal probes (CMA 12 Elite; membrane length = 3 mm; 20,000 Da; Stockholm, Sweden) were inserted into the guide cannula so that they extended from bregma DV: − 3.7 to − 6.7 mm within the dorsal striatum. Rats underwent microdialysis at least 2 days following the last L-DOPA administration. During microdialysis, rats received intrastriatal infusion of filtered artificial cerebrospinal fluid (aCSF) (128 mM NaCl, 2.5 mM KCl, 1.3 mM CaCl2, 2.1 mM MgCl2, 0.9 mM NaH2PO4, 2.0 mM Na2HPO4, and 1.0 mM glucose, pH 7.4). Dialysate samples were collected every 20 min. Briefly, rats were habituated to microdialysis for 1 h. Fifty minutes into the procedure, rats received a subcutaneous injection of L-DOPA vehicle, which consisted of 0.9% NaCl, and 0.1% ascorbate. Rats then underwent baseline testing for 1 hour to determine baseline levels of monoamines prior to L-DOPA treatment. After that a new collection tube was used and 10 minutes later rats received an injection of L-DOPA (12 mg/kg + 12 mg/kg Benserazide, s.c.). Samples were taken every 20 min for 3 h. Following the procedure, rats were removed from the microdialysis bowl and striatal probes were replaced with a dummy probe. At least 2 days after microdialysis, rats were sacrificed via rapid decapitation, the anterior striatum was taken for verification of cannula placement, the posterior striatum was taken for HPLC, and the hindbrain was placed in 4% PFA for 3 days before being placed in 30% sucrose in phosphate-buffered saline (PBS). Brains were shipped on ice in a 50 mL conical containing 30% sucrose in 0.1 M PBS to MSU.
High-performance liquid chromatography for monoamine tissue analysis
Striatal tissue and in vivo microdialysis samples were analyzed using HPLC. Reverse-phase HPLC was performed on striatal tissue samples as previously described [38, 52]. Briefly, tissue samples were homogenized in ice-cold perchloric acid (0.1 M) with 1% ethanol and 0.02% ethylenediaminetetraacetic acid (EDTA). Homogenate was spun at 4 °C for 45 min at 14,000 g. Supernatant was removed and, using an ESA solvent delivery system (Model 542; Chelmsford, MA, USA) ESA autoinjector (Model 582), analyzed for levels of norepinephrine, 3,4-dihydroxyphenylacetic acid (DOPAC), DA, 5-hydroxyindoleacetic acid (5-HIAA), and 5-HT. Monoamines and metabolites were detected as a generated current as a function of time by EZCHROM ELITE software via a Scientific Software, Inc. (SS240x) Module. Data are displayed as peaks for monoamines and metabolites, which are compared to a standard curve made from monoamine and metabolite samples of known concentrations ranging from 1e-6 to 1e-9. Values were then normalized to tissue weight and lesion deficits are reported as percent depletion, which is equal to 100 (1 – M Lesion/ M Intact).
Dialysate samples were analyzed via reverse-phase HPLC on an Eicom HTEC-500 System (Amuza Inc., San Diego, CA). Briefly, 10 μL of each dialysate sample was analyzed for NE, DA, and 5-HT using an Eicompak CAX column maintained at 35 °C with a flow rate of 250 μL/min. Mobile phase (75 mM Ammonium acetate, 9.36 mM acetic acid, 1.33 mM EDTA, 0.94 mM Methanol, 50 mM sodium sulfate). Samples were compared to known concentrations of monoamines (100, 10, 1, 0.1, and 0.05 ng/μL dissolved in a potassium phosphate buffer (0.1 mM potassium phosphate monobasic, 0.1 mM ethylenediaminetetraacetic acid, 0.02 mM phosphoric acid), resulting in a final value of monoamine in ng/μL.
Total enumeration of TH+ and 5-HT+ neurons
Lesion severity was determined using total enumeration of TH-positive neurons in three representative sections within the SNc identified by the presence and proximity to the medial terminal nucleus (MTN) of the accessory optic tract at levels equivalent to − 5.04 mm, − 5.28 mm and − 5.52 mm relative to bregma according to our previously validated method . Briefly, the intact and lesion SNc were quantified for all TH immunoreactive cells using a 20× objective and MicroBrightfield StereoInvestigator software (MicroBrightfield Bioscience, Williston, VT). The total number of TH cells on the intact and lesioned hemispheres were averaged, and lesion efficacy was derived by dividing the lesioned hemisphere average by the intact hemisphere average and multiplying that value by 100.
Total number of 5-HT positive neurons in the DRN were also quantified with total enumeration . Three sections of the DRN were quantified for all 5-HT immunoreactive cells under a 20× objective using MicroBrightfield StereoInvestigator (MicroBrightfield Bioscience, Williston, VT). The total number of from all three sections per animal were summed to give a total number of 5-HT neurons.
Recording microelectrodes were manufactured from 2.0 mm OD borosilicate glass capillary tubing and filled with sodium chloride (2 M) solution. Electrode impedance was 5-15 MΩ. The signal to noise ratio for all recordings was > 4:1. The level of urethane anesthesia was periodically verified via the hind limb compression reflex and maintained using supplemental administration as previously described [59, 66]. Temperature was monitored using a rectal probe and maintained at 37C° using a heating pad (Vl-20F, Fintronics Inc., Orange, CT). Electrical stimuli (duration = 500 μs, intensity = 1000 μA) were generated using a Grass stimulator and delivered in single pulses (0.5 Hz) while searching for cells . Once isolated, recordings consisted of basal (pre-drug), saline vehicle, and drug-treatment-(see below) induced changes in spike activity recorded in a series of 3 min duration epochs.
All compounds and physiological 0.9% saline were prepared daily and administered intravenously (i.v.) through the lateral tail vein to enable rapid examination of potential acute effects of vehicle or drug on DRN neuronal activity. The selective 5-HT1A agonist 8-OH-DPAT (5 μg/kg, i.v.), the selective 5-HT1A antagonist WAY100635 (100 μg/kg, i.v.), and the D2R agonist Quinpirole (500 μg/kg, i.v.) were dissolved in vehicle and administered systemically to either BFP or D2Rs rats. DRN 5-HT neuron activity was recorded prior to and immediately following drug administration as described above.
Statistical analysis was performed using Statview (version 5.0) or in SPSS version 23 with α set to 0.05. All graphs were created in GraphPad Prism version 7.0 (GraphPad Software, La Jolla, CA) or Excel (Microsoft, Redmond, WA). Lesion status was evaluated using unpaired, one-tailed t-tests. AIMs were evaluated using a non-parametric Mann-Whitney U test, with p ≤ 0.05 being considered statistically significant. Bonferroni post-hoc tests were employed when significant main effects were detected. Cylinder and FAS data for forehand and backhand stepping were submitted to a mixed model ANOVA with within-subjects factors of treatment (2: Baseline, L-DOPA) and between-subjects factors of vector (GFP, D2R). Overall percent intact values for FAS were determined by taking the overall number of right paw steps divided by the number of left paw steps and multiplying the quotient by 100. Similarly, overall percent intact values were analyzed via a repeated-measures ANOVA with within-subjects factor of treatment and between subjects factor of vector. Monoamine content (as determined by HPLC) was submitted to a mixed-model ANOVA with within-subjects factor of treatment (2: Vehicle, L-DOPA) and between-subjects factor of vector. Fisher’s least significant difference (LSD) post-hocs and planned paired-samples t-tests were employed as appropriate to clarify significant effects. Additionally, independent-samples t-tests were employed to reveal effects of vector on the timing of DA, NE, and 5-HT efflux. HPLC values for striatal tissue were submitted to a mixed-model ANOVA with within-subjects factor of side and between-subjects factor of vector. Subsequently, since DA depletion did not vary as a function of vector, values for each monoamine for each side were collapsed across treatments and compared via paired-samples t-tests. For electrophysiology experiments, the difference between the spontaneous and evoked electrophysiological activity of identified DRN-5-HT neurons across groups was determined and served as the dependent variable for our analyses. A two-way repeated measures ANOVA (GFP vs. gene therapy (ectopic expression of the DA D2 AR in 5-HT DR neurons)) × 2 (vehicle vs. drug treatment) with α set to 0.05 and all “n’s” adequately powered for electrophysiological studies was conducted using Sigma Stat software (San Jose, CA), and the potential two-way interaction effect was examined to determine how treatment effects differ as a function of drug treatment or gene therapy .