Animals and reagents
Experiments were performed with 8-week-old C57BL/6J mice (RRID: MGI: 5657312) and 12-hour-old Sprague-Dawley rats (RRID: MGI: 5651135) supplied by the Animal Center of the Academy of Military Medical Science (Beijing, China). C57BL/6J mice were group-housed in a temperature-controlled environment (22 ± 2°C) with a 12/12-h light/dark cycle and access to food and water ad libitum. All mice were group-housed for 3 days prior to use and were handled daily throughout the experiment to minimize the effects of handling stress. All tests were performed and recorded between 9:00 and 15:00 during the lights-on cycle. Animal experiments (Ethics approval No. LACUC-DWZX-2020-517) were approved by the Beijing Local Committee on Animal Care and Use and were performed according to the Guide for the Care and Use of Experimental Animals produced by the Beijing Local Committee and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, revised 1985). Efforts were made to minimize the number of animals used for each experiment.
The following reagents were used. Oxotremorine M (Oxo-M, Cat. #O100) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Roscovitine (ROSC, Cat. #T6259) was purchased from Target Mol (Shanghai, China). Muscarinic toxin 3 (MT3, Cat. #4410-s) and muscarinic toxin 7 (MT7, Cat. #4340-s) were obtained from the Peptide Institute (Tokyo, Japan). All reagents were dissolved or diluted in normal saline as vehicle and the solutions were adjusted to a pH range of 6.5 to 7.0.
Primary cultures of MSNs
According to methods described previously [17], primary MSN cultures were prepared from neonatal (12-h-old) Sprague-Dawley rats. In brief, the striatum was dissected in ice-cold PBS (135 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8 mM K2HPO4, pH 7.2). The tissue was then digested in 0.25% trypsin for 30 min at 37°C until completely triturated. Next, the cells were suspended in neurobasal medium (Gibco, Thermo Scientific, MA, USA, Cat. #A3582901) containing 1% L-glutamine (Gibco, Thermo Scientific, Cat. #35050061) and 2% B-27 serum-free supplement (Gibco, Thermo Scientific, Cat. #17504044) and plated at equal density in 15-mm-diameter dishes (NEST Biotechnology Co. Ltd., CA, USA, Cat. #801002) pre-coated with 0.1 mg/mL poly-D-lysine (Sigma-Aldrich). The cells were incubated at 37°C in a 5% CO2 atmosphere.
Lentiviral M4-shRNA transduction and knockdown of M4 in isolated MSNs
A pGMLV-SC5 lentivirus shRNA expression system encoding a cholinergic receptor, muscarinic 4 shRNA (Chrm4-shRNA) was constructed as previously described [17]. The sequence inserted into PGMLV-hU6-MCS-CMV-ZsGreen1-PGK-Puro-WPRE to generate Chrm4-shRNA was 5′-GATCCGCAAAGTGACTCGGACAATCTTTCAA GAGAAGATTGTCCGAGTCACTTTGTTTTTTG-3′. The shRNA sequence 5′-TTC TCCGAACGTGTCACGT-3′ was used as a negative control (NC). Primary MSNs were plated onto 0.1% polyethylene poly-L-lysine-coated 15-mm-diameter dishes or 6-well plates (NEST Biotechnology Co. Ltd., Cat. #703001). After culture for 24 h, cells were transduced with Chrm4-shRNA or NC shRNA lentiviral vectors and incubated at 37°C in a 5% CO2 atmosphere for 96 h. Cells transduced with Chrm4-shRNA are referred to as M4-knockdown (M4-KD) MSNs.
Knockout of the M4 gene within the DMS by lentiviral delivery of CRISPR/Cas9
Three single-guide RNAs (sgRNAs) targeting the M4 gene were designed and assessed for off-target activities using MIT CRISPR Design. The sgRNAs were synthesized by Jikai Gene Company (Shanghai, China). Each sgRNA was cloned into a lentiviral (LV)-sgRNA-CAS9 vector containing a U6 promoter multicloning site and P2A promoter-driven enhanced green fluorescent protein (eGFP) and then packaged into the GV393 vector. A vector expressing a sgRNA targeting a non-specific sequence (5′-CGCTTCCGCGGCCCGTTCAA-3′) was used as a negative control (NC). The specific sequence used for targeted editing of M4 was (5′-CACCGTGCCA GCATCGCTCGTAACC-3′) (for cloning details, see Additional file: supplementary cloning construction). C57BL/6J mice were anesthetized with 5% chloral hydrate (5 mL/kg, i.p.) then mounted on a stereotaxic apparatus (Kopf Instruments, Beijing, China). We injected 3 µL GV393 LV/Cas9-sgRNA-eGFP containing the targeting or NC sgRNA into the DMS at three sites within the DMS (site 1: +1.0 mm AP, ±2.0 mm ML, −3.5 mm DV; site 2: +1.2 mm AP, ±2.1 mm ML, −3.5 mm DV; and site 3: +0.7 mm AP, ±2.2 mm ML, −3.5 mm DV; according to the stereotaxic mouse atlas of Paxinos and Franklin, 2001). After injection, needles were left in place for 5 min to allow the solutions to fully diffuse.
Microinjection and drug treatment
Oxo-M was dissolved in vehicle and the final dose used was 0.1 µM. Mice received intracerebroventricular (i.c.v) injection of Oxo-M (2.0 µL) or vehicle (2.0 µL) at the following coordinates: +1.0 mm AP, ±0.2 mm ML, −2.5 mm DV. The solutions were injected bilaterally through a microinjection needle (30 gauge) that extended 1 mm beyond the tip of the guide cannula. Each microinjection needle was attached to a 10-µL Hamilton microsyringe through polyethylene tubing (PE-10). Infusions were controlled by an infusion pump (Model Bi2000e Insight Equipment, Sao Paulo, Brazil), programmed to deliver solution at a constant speed of 0.5 µL per min. The microinjection needle was kept in place for an additional minute to allow for drug diffusion. The mice were allowed to move freely during drug administration.
Behavioral tests
Mice were screened 3 days before lentivirus or drug injection, and those with abnormal motor behavior were excluded. Behavioral tests were performed 14 days after lentivirus injection. Mice were transferred to a behavioral testing room (22±1°C) and habituated for at least 5 min before behavioral testing. All behavioral experiments were performed at fixed times (autonomic movement test: 9:00–12:00; rotarod and forced swim test: 12:00-14:00). The testing apparatus was cleaned with a hypochlorous acid solution.
Locomotor activity measurements
Mice were individually transferred from their home cage to a plastic open field apparatus (60 cm × 60 cm × 60 cm; Xingruan Information Technology Co. Ltd., Beijing, China). The apparatus was virtually divided into peripheral, intermediate, and central zones. The test was started by placing the animal in the center of the open field illuminated by a dim light (5 lx). Each mouse was placed in the locomotor activity box for 60 min and was recorded by a video camera. The total distance and the distance traveled in the central zone (referred to hereafter as central distance) was recorded and analyzed by ANY-Maze automated video tracking software (Stoelting Co., Wood Dale, IL, USA; RRID: SCR_014289).
Rotarod test
The rotarod system (Xingruan Information Technology Co. Ltd, China) for assessing locomotor skills measures the time that an animal maintains balance on a moving rod. Animals were first conditioned on a stationary rod for 30 s and during this time any animal that fell was placed back on the rotarod. Animals were next conditioned at a constant speed of 5 rpm for a period of 5 min. Twenty-four hours after conditioning, animals were placed on the rod and timed for 30 min to assess their locomotor skill. The rod speed started at 5 rpm and was increased at 0.1 rev/s; the time before falling off the rod (fall off duration) was recorded by ANY-Maze automated video tracking software (Stoelting Co. Ltd., Sheboygan, IL, USA).
Forced swimming test
The test procedure was previously standardized and validated [22]. Mice were individually forced to swim in an open cylindrical container (diameter 10 cm, height 25 cm) containing 19 cm of water (depth) at 25 ± 1°C. Each mouse was considered to be immobile when it ceased struggling and remained floating motionless in the water. The total duration of immobility was recorded during a 10-min period by ANY-Maze automated video tracking software (Stoelting Co. Ltd, China).
Immunocytochemistry and confocal microscopy analysis
For immunocytochemistry, cells were fixed in methanol for 20 min and blocked with 5% BSA (ZSGB-Bio, Wuhan, China) in PBS for 1 h, then incubated with primary antibodies overnight at 4°C. The cells were then washed before incubation with the corresponding secondary antibody for 1 h at room temperature.
For immunohistochemistry, tissues were dissected and postfixed overnight at 4°C in 4% paraformaldehyde and then cryoprotected with 30% sucrose in 0.1 M PBS for 2 days. Sections were cut at 30 µm on a cryostat microtome (CM1950, Leica, Wetzlar, Germany). Free-floating sections were permeabilized with 0.3% Triton X-100 in PBS for 30 min at room temperature. After blocking with 10% normal goat serum for 2 h, sections were incubated with primary antibodies overnight at 4°C. Sections were then incubated with secondary antibodies for 2 h at room temperature.
The primary and secondary antibodies are shown in Additional file: supplementary Table 1. The nuclear layers were stained with DAPI (Sigma-Aldrich). Images were obtained with a laser confocal microscope (LSM 8000, Carl Zeiss, Oberkochen, Germany). ImageJ imaging software (National Institutes of Health, MD, USA) was used to quantify the fluorescence intensity.
Western blotting
Primary cell cultures or dorsal striatum tissue were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 5 mM NaF, 0.2% SDS, and 1% NP-40, pH 8.0) containing a mix of protease and phosphatase inhibitors (Google Biological Technology Co., Ltd., Wuhan, China) before being centrifuged at 4°C, 12,000 ×g, for 30 min. The supernatants were collected, and protein concentration measured using a commercial BCA kit (Google Biological Technology Co., Ltd.). Equal amounts of protein were separated on 10% SDS-PAGE gradient gels, transferred to nitrocellulose membranes, and incubated with primary antibodies diluted in TBST (150 mM NaCl, 40 mM Tris-HCl, pH 7.4, 0.1% Tween-20) overnight at 4°C. After washing, membranes were incubated with secondary antibodies coupled to horseradish peroxidase. The primary and secondary antibodies are shown in Additional file: supplementary Table 2. Immunocomplexes were detected by chemiluminescence (ECL, Pierce, USA), imaged using an imaging instrument (GENE Co. Ltd., Beijing, China) and analyzed using Image J. Analyses were performed in duplicate and the mean value was calculated.
Whole-cell patch clamp electrophysiology in the DMS.
Mice were anesthetized by injection with pentobarbital sodium (60 mg/kg, i.p.), then decapitated and the brains removed. Coronal slices (300 µm thick) containing the striatum (0.2-1.3 mm posterior to bregma) were prepared in ice-cold ACSF (as follows: 124 mM NaCl, 2.8 mM KCl, 1.25 mM NaH2PO4, 2 mM CaCl2, 1.25 mM MgSO4, 26 mM NaHCO3, 10 mM glucose, pH 7.5, bubbled with 95% O2/5% CO2) using a vibrating tissue slicer (MA752, Campden Instruments, USA) as previously described [23]. Slices were submerged for 30 min at 32°C in protective media containing 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM Hepes, 25 mM D-glucose, 5 mM sodium ascorbate, 2 mM thiourea, 3 mM sodium pyruvate, 10 mM MgSO4, 0.5 mM CaCl2 (pH 7.3) at 295-300 mOsm/L. Following this recovery period, slices were transferred to ACSF containing 126 mM NaCl, 2.5 mM KCl, 26.2 mM NaHCO3, 1.25 mM NaH2PO4, 2 mM CaCl2, 1.5 mM MgSO4, 10 mM D-glucose and 5 mM sodium ascorbate.
Patch pipets were prepared from borosilicate glass (Sutter Instrument Company, Novato, CA, USA) using a P-97 Flaming/Brown micropipette puller (Sutter Instrument) and had resistance of 6-8 MΩ when filled with the following intracellular solution: 130 mM CsCl, 10 mM NaCl, 0.25 mM CaCl2, 2 mM MgCl2, 5 mM EGTA, 10 mM Hepes, 10 mM glucose, 2 mM Mg-ATP, and 0.3 mM Na2-GTP. The pH of the pipette solution was adjusted to 7.3 with 1 mM CsOH and osmolarity was adjusted to 285-290 mOsm/L. A low-power objective (4×) was used to identify the striatum and a 40× water immersion objective (NIR Apo, Nikon, Tokyo, Japan), coupled with infrared differential interference contrast microscopy and a charge coupled device camera, was used to visualize individual neurons. Cells in the dorsolateral striatum up to approximately 50 µm beneath the slice surface were patched and monitored. Recordings in normal current-clamp or voltage-clamp mode were acquired at room temperature using the Digidata 1440 A interface (Molecular Devices, Sunnyvale, CA, USA) with an Axon 200B amplifier (Molecular Devices) and Clampex 10.2 software (Molecular Devices). After formation of a tight seal (>1 GΩ), fast and slow capacitance compensation was performed. During the whole-cell recording, series resistance was compensated (80%-90%) and monitored periodically. Neurons were excluded from the analysis when their series resistance was above 50 GΩ or changed by more than 25% during the experiment. Data were filtered at 2 kHz and acquired at a sampling rate of 10 kHz. MSNs were identified by previously determined membrane characteristics and firing patterns [24].
For miniature excitatory postsynaptic current (mEPSC) recordings, oxygenated ACSF containing both the GABA receptor antagonist bicuculline (10 µM; Sigma) and the voltage-gated sodium channel blocker tetrodotoxin (TTX; 1 µM; Abcam Biochemicals, UK) was applied to the bath to abolish inhibitory postsynaptic current events and action potentials, respectively. Slices were perfused with this solution at 25°C for at least 15 min following establishment of electrical access. Access resistances were <15 MΩ. mEPSCs were recorded from MSNs held at 70 mV in gap-free mode. After 5 min of stable baseline recordings, Oxo-M was bath applied to slices for 5 min and recording performed for at least 3 min.
Whole-cell patch clamp electrophysiology in primary MSN cultures
Whole-cell patch clamp recordings were performed on primary MSNs from the striatum of newborn Sprague-Dawley rats cultured for 14 to 16 days in vitro. Borosilicate patch pipettes (6-8 MΩ) were filled with an internal solution containing the following: 130 mM CsCl, 10 mM NaCl, 0.25 mM CaCl2, 2 mM MgCl2, 5 mM EGTA, 10 mM Hepes, 10 mM glucose, 2 mM Mg-ATP, 0.3 mM Na2-GTP (pH 7.35). Cultures were continuously superfused with an external solution: 100 mM NaCl, 26 mM NaHCO3, 2.5 mM KCl, 11 mM glucose, 2.5 mM CaCl2, 1.3 mM MgSO4, 1.0 mM NaH2PO4. For mEPSC recordings, the bath solution contained TTX (1 µM) and bicuculline (10 µM). Cells were held at 60 mV. The time course of the experiments, data collection and analysis were the same as for slice electrophysiology.
Experimental overview
For characterization of M4-mediated cholinergic modulation of behavior, we directly injected Oxo-M (0.1 µM, 2 µL) into the lateral ventricle of wild-type (WT), NC and DMS-M4-KD mice once daily for 3 consecutive days. A control group received daily saline injections. After Oxo-M injection, one set of mice was immediately used for evaluation of behavioral responses. Another set of mice was killed by decapitation 2 h after the final Oxo-M injection to assess biochemical changes in the DMS using immunohistochemistry, confocal microscopy and western blot analyses. For characterization of M4-mediated cholinergic inhibition of neuronal electrophysiological activity, tissues from the DMS were dissected for whole-cell patch clamp electrophysiology analyses.
Data analysis
For behavioral experiments and biochemistry assays, including immunocytochemistry and western blotting, statistical analysis was performed by one-way ANOVA or two-way ANOVA (with or without repeated measures) followed by Tukey’s multiple comparisons test using GraphPad Prism 7.0 (GraphPad, San Diego, CA). For electrophysiology, statistical analysis was performed using Clampex 10.2 software (Molecular Devices). The two-sample Kolmogorov–Smirnov test was used to compare the cumulative distributions of frequency of the two groups. Data are expressed as the mean ± S.E.M. “n=8” is the number of animals used, “n=10” is the total number of ROIs in immunocytochemistry staining and patch clamp electrophysiology (cells or tissue sections), and “n=3” the number of independent western blotting experiments performed, unless otherwise stated. P values below 0.05 were considered significant.