Animals
Eight-week-old male C57BL/6 mice, initially weighing 18–20 g (Vital River Laboratories, Beijing, China), were group-housed in a controlled environment at 18–22 °C and 40–60% humidity, with a 12/12 h light/dark cycle and access to food and water ad libitum. Enrichment was provided with nestlet shredding. Cages (32 × 22 × 17 cm, eight mice per cage) were changed every week by designated facility staff. All mice were group-housed for 1 week prior to use in experiments and handled daily to minimize the effects of handling stress. All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) and approved by the Animal Care and Use Committees of the Beijing Institute of Pharmacology and Toxicology. Efforts were made to minimize the number of animals used for each experiment.
In vivo KD of the Cdk5 gene in the mouse DS
To efficiently KD mouse CDK5 in the DS, Cdk5-targeting single-guide RNAs (sgRNAs) were designed (Fig. 1A). Lentiviral (LV) vectors (p24 stock solutions ranging between 200 and 300 ng/µL and approximately 2 × 108 transducing units/mL) co-expressing a P2A promoter-driven enhanced green fluorescent protein (eGFP) and a U6 promoter-driven small single-guide RNA (sgRNA), either specifically directed against Cdk5 mRNA (LV-Cdk5-sgRNA) or a negative control (LV-NC-sgRNA), were produced by GeneChem Co., Ltd (Shanghai, China). The sequence used for targeted editing of Cdk5 was 5ʹ-TCAGCTTCTTGTCACTATGC-3ʹ, and the non-specific NC sequence was 5ʹ-CGCTTCCGCGGCCCGTTCAA-3ʹ. Standard procedures and LV/Cas9-sgRNA validation in vivo were reported previously [21]. Briefly, HEK293T cells were plated in 6-well plates (5 × 105 cells/well) and cultured in Dulbecco’s Modified Eagle Medium with 10% fetal bovine serum at 37 °C in 5% CO2. Once cells were completely attached, they were transduced with 10 μL LV-Cdk5-sgRNA or LV-NC-sgRNA (2 × 108 pfu/mL). The medium was replaced after 48 h, and the cells were further incubated until 48 h as required. For in vitro validation, C57BL/6J mice were anesthetized with pentobarbital sodium [50 mg/kg, intraperitoneal (i.p.), Sigma-Aldrich, St. Louis, MO, USA] and mounted on a stereotaxic apparatus (Zhongshidichuang Science and Technology Development Co., Ltd, Beijing, China). Mice were randomly divided into wild-type (WT), LV-NC-sgRNA (CDK5-NC), and LV-Cdk5-sgRNA (CDK5-KD) groups, and the respective LV vectors were bilaterally microinjected into the DS. For microinjection, we used the following two bilateral sites in the DS (1.5 µL per site, 3 µL total) with stereotaxic coordinates (in mm) according to the modified stereotaxic mouse atlas of Paxinos and Franklin [27]: (1) anterior/posterior (A/P) + 1.1, medial/lateral (M/L) ± 1.2, dorsal/ventral (D/V) − 3.2 and (2) A/P + 0.9, M/L ± 2.0, D/V − 3.2. The delivery rate was 0.33 µL/min using a UMP3 microsyringe injector and Micro4 controller (World Precision Instruments, Sarasota, FL, USA). The needles were left in place for 3 min to allow for full delivery of the solutions and then slowly retracted. Next, the scalp was sutured with 7-0 Vicryl sutures (ETHICON Co., Ltd, New Jersey, USA).
Experimental design and behavioral tests
Mice were screened 3 days before use, and mice with abnormal behavior were excluded. Mice were randomly divided into WT, DS-CDK5-NC, and DS-CDK5-KD groups, with 30 mice in each group. Fourteen days after LV transduction, 20 mice from each group were selected for behavioral testing. After the behavioral tests were completed, mice were deeply anesthetized with ether and sacrificed by decapitation for immunoassays (immunofluorescent and western blot). The remaining mice were used for Golgi staining, electrophysiology assays, and anterograde labeling. All behavioral experiments were stared at 9:00 am. Each behavioral test was performed on the same animals with an interval of 24 h between tests. The testing apparatus was cleaned with a hypochlorous acid solution between subjects. The experimenters were blinded to grouping and drug treatment.
Locomotor activity test
A locomotor activity test was used to assess spontaneous locomotor activity and arousal in mice. The method used was similar to a previously published protocol [28]. Briefly, animals were placed individually in a square arena (50 × 50 × 80 cm) with a Plexiglas floor and walls (Zhongshidichuang Science and Technology Development Co., Ltd) and allowed to move freely. After a 5-min habituation period, all animal locomotor activities were recorded with AnyMaze software (Stoelting Inc., Wood Dale, USA), and the distance traveled during a 60-min period was recorded and analyzed.
Wheel-running behavioral test
Mice were housed in temperature-controlled circadian cabinets (Zhongshidichuang Science and Technology Development Co., Ltd) within polypropylene cages (33.2 × 15 × 13 cm) containing a metal running wheel (35-cm diameter). The experiment started at 6:00 am and ended at 6:00 pm on the next day (total 48 h; LD condition: 12 h-light/12 h dark cycle × 2; DD condition: 48 h dark). Mice were acclimatized to the running wheel for 1 h prior to assessment. Rest/activity behavior were monitored with a ClockLab data collection system (Version 3.603, Actimetrics, Wilmette, IL, USA) by determining the number of electrical closures triggered by wheel rotations. The number and duration of wheel running by mice were the main indicators. The α/ρ ratio is the ratio of wheel running duration (α) to wheel stopping duration (ρ). Cage changes were performed in 48-h intervals. Wheel-running activity was monitored over 48 h and analyzed using ClockLab Analysis software (Actimetrics Software).
Golgi staining and dendritic spine measurements
Golgi-Cox staining was performed using a Rapid Golgi Stain Kit (FD Neuro Technologies, Ellicott City, MD, USA) following the manufacturer’s instructions. Briefly, brains were quickly removed, rinsed, and incubated in a mix of solution A/B for 14 days in the dark at room temperature. Then, solution A/B was changed to solution C for 3 days. Coronal sections of the DS (200 μm thick) were cut (ranging from 0.7–1.2 mm anterior to bregma; two sections per animal) on a freezing microtome (Leica, Wetzlar, Germany) and mounted onto gelatinized slides. After sections were dried in the dark, they were reacted in solutions D and E for 10 min and dehydrated sequentially in 50%, 75%, 95%, and 100% ethanol. Finally, sections were cleared in xylene and cover slipped with resinous mounting medium.
For dendritic spine measurements, the DS region was identified at low power (100× magnification), and MSNs were traced at 250× (final magnification) using the camera lucida technique on an Olympus light microscope (Model BX51) equipped with a drawing attachment and the 8-bit ImageJ plugin Neuron J. Dendrite length and branching were measured by Sholl’s analysis of ring intersections. A series of concentric rings at 20 µm increments printed on a transparent grid was centered over the soma. The total number of intersections between each ring and dendritic branches was counted and converted to estimates of dendrite length as a function of distance from the soma (i.e., for each 20-µm segment) and overall dendrite length. Spine density was measured manually in the stacks using the ImageJ Plugin Cell Counter. Three to five dendrite segments per slice, with each dendrite segment ranging from 20 to 30 μm in length, were used for spine density analysis. Spines were marked in the appropriate focal plane, preventing any double counting of spines.
Immunohistochemistry
After the last behavioral test, mice were euthanized, and striatum sections were prepared for immunohistochemistry. Briefly, striatal sections (30 μm) were cut with a vibratome (VT1000 S, Leica) and collected in phosphate-buffered saline (PBS) as free-floating sections. Sections were rinsed three times in PBS and permeabilized and blocked in PBS containing 0.3% Triton X-100 and 5% normal goat serum (Pierce Biotechnology, Rockford, IL, USA) for 1 h at room temperature. Sections were then washed in PBS and incubated overnight at 4 °C with an anti-CDK5 antibody (1:400; Cat# ab40773, Abcam, Cambridge, England) and anti-GAD antibody (1:400; Cat# ab26116, Abcam), which was detected with an anti-rabbit Alexa Fluor 594 secondary antibody (1:200; Absorption Peak: 590 nm, Emission Peak: 617 nm; Cat# ZF-0516, ZSGB Biotech, Jiangsu, China) and anti-mouse Alexa Fluor 488 secondary antibody (1:200; Absorption Peak: 493 nm, Emission Peak: 519 nm; Cat# ZF-0512, ZSGB Biotech). To identify nuclei, sections were counterstained in mounting medium containing 4ʹ,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). Finally, sections were mounted and examined using a fluorescence microscope (Zeiss, Oberkochen, Germany or Leica, Germany).
Western blotting
Western blot assays were used to measure protein levels. After the last behavioral test, mice were euthanized with pentobarbital sodium salt (50 mg/kg, i.p.). The striatum was dissected and homogenized in radioimmunoprecipitation assay lysis buffer [10 mM PBS, pH 7.4, containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and complete protease inhibitor cocktail (Roche, Basel, Switzerland)]. Tissue dissection was performed in the same time window for all groups. For western blotting, solubilized proteins (50 µg per sample) were resolved on SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat milk diluted in TBST at room temperature for 1 h and then incubated overnight at 4 °C with the following primary antibodies: anti-CDK5 mouse monoclonal antibody (1:1000; Cat# ab40773, Abcam), anti-MAP2 rabbit polyclonal antibody (1:1000; Cat# ab183830, Abcam), anti-synapsin 1 mouse monoclonal antibody (1:1000; Cat# MAN894, Millipore), anti-PSD-95 rabbit polyclonal antibody (1:500 dilution, Cat# ab12093, Abcam), anti-phospho-Tau (Ser202) rabbit polyclonal antibody (1:800; Cat# 11834s, Cell Signaling Technology, MA, USA), anti-phospho-Tau (Thr181) rabbit polyclonal antibody (1:800; Cat# 12885s, Cell Signaling Technology), anti-Tau mouse monoclonal antibody (1:1000; Cat# MAB3420, Millipore), and anti-β-actin mouse monoclonal antibody (1:1000; AF0003, Beyotime). The membranes were then washed with TBST and incubated with the appropriate peroxidase-conjugated secondary goat anti-rabbit/mouse IgG (1:2000; Cat# AB-2301/ZB-2305, ZSGB-Bio, Beijing, China) for 1 h at room temperature. An ECL detection kit (Thermo Fisher Scientific, Waltham, MA, USA) was used, and immunoreactive bands were quantified on an imaging system (ProteinSimple, San Jose, CA, USA). β-actin was used as the loading control.
Anterograde labeling
To characterize the dopaminergic projections to different brain regions, adeno-associated virus (AAV)-hSyn-mCherry-IRES-WGA-Cre was packaged (SunBio Biomedical Technology Co. Ltd., Shanghai). On day 14 after Cdk5-sgRNA injection, the AAV-hSyn-mCherry-IRES-WGA-Cre was bilaterally microinjected into the center of the original virus injection points (coordinates: A/P + 1.0 mm, M/L + 1.8 mm, D/V − 3.2 mm). Animals were anesthetized throughout surgery with pentobarbital sodium (50 mg/kg, i.p.). Following craniotomy, a microsyringe was lowered into the brain using the above coordinates, and 500 nL of AAV-hSyn-mCherry-IRES-WGA-Cre were injected over a 10-min period [29]. Thirty days later, mice were deeply anesthetized and immediately perfused transcardially with normal saline followed by 4% paraformaldehyde. Brains were removed, post-fixed overnight in the same solution, cryoprotected by immersion in 30% sucrose, and then frozen in dry ice-cooled methyl butane. Serial coronal cryostat sections (40 μm) through the whole brain were cut with a vibratome (VT1000 S, Leica), rinsed in PBS, counterstained with the nuclear dye DAPI (0.2 mL; Zsbio, Beijing, China), and mounted on slides. Images were captured with a microscope slide scanner (Pannoramic SCAN II, 3DHistech, Ltd., Budapest, Hungary). Coronal sections of the cortex, striatum, thalamus, and basolateral amygdala (BLA) were confirmed in the microscope slide scanner. CaseViewer 2.3 software (3DHistech, Ltd.) was used for image observation and analysis.
Electrophysiology
Mice were anesthetized with pentobarbital sodium (50 mg/kg, i.p.), and brains were removed. Striatal slices (300 μm) were cut in ice-cold cutting solution [(in mM) 124 NaCl, 2.8 KCl, 1.25 NaH2PO4, 2 CaCl2, 1.25 MgSO4, 26 NaHCO3, 10 glucoses; pH 7.3; bubbled with 95% O2/5% CO2] using a vibrating tissue slicer (MA752, Campden Instruments, Loughborough, UK). Coronal slices were submerged for 30 min at 32 °C in artificial cerebrospinal fluid (ACSF) [(in mM) 126 NaCl, 2.5 KCl, 26.2 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1.5 MgSO4, 10 d-glucose, 5 sodium ascorbate; pH 7.3; bubbled with 95% O2/5% CO2] at 295–300 mOsm/L. All reagents were from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated. Following this recovery period, slices were transferred to room-temperature ACSF until recording.
Patch pipettes were prepared from borosilicate glass (Sutter Instrument Company, Novato, CA, USA) using a P-97 Flaming/Brown micropipette puller (Sutter Instrument Company) and had a resistance of 6–8 MΩ when filled with the following intracellular solution [(in mM) 130 CsCl, 10 NaCl, 0.25 CaCl2, 2 MgCl2, 5 EGTA, 10 HEPES, 10 glucose, 2 Mg-ATP, 0.3 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 DS region, and a 40× water immersion objective (NIR Apo, Nikon, Japan) coupled to an infrared differential interference contrast microscope with a fluorescence system and a CCD camera was used to visually identify, patch, monitor, and record CDK5-KD neurons in the DS. MSNs were identified according to previously determined membrane characteristics and firing properties [30]. Recording in normal current-clamp or voltage-clamp modes was performed with a Digidata 1440A digitizer, an Axon 200B amplifier, and Clampex 10.2 software (all from Molecular Devices, San Jose, CA, USA) at room temperature. Fast and slow capacitance compensation were performed after tight-seal (> 1 GΩ) formation. During whole-cell recordings, series resistance was monitored and compensated (80–90%) periodically. When the series resistance of a neuron was above 50 GΩ or changed by more than 25%, it was excluded from further analysis. Data were filtered at 2 kHz and acquired at a sampling rate of 10 kHz. Access resistance and leak currents were monitored, and recordings were rejected if these parameters changed significantly during data acquisition.
For miniature excitatory postsynaptic current (mEPSC) recordings, the oxygenated ACSF contained the GABA receptor antagonist (+)-bicuculline (10 μM; Sigma-Aldrich) and the voltage-gated sodium channel blocker tetrodotoxin (TTX) (1 μM; Abcam) to abolish inhibitory postsynaptic current (IPSC) events and action potentials. For spontaneous IPSC (sIPSC) recordings, the oxygenated ACSF contained the competitive NMDA receptor antagonists 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) (20 μM; Sigma-Aldrich) and l-(+)-2-amino-5-phosphonopentanoic acid (l-AP5) (50 μM; Tocris, Ellisville, MO, USA), as well as TTX (1 μM) to abolish excitatory postsynaptic current events and action potentials. The sections were superfused with the oxygenated ACSF solutions at a rate of 1.2 mL/min. Spontaneous activity was recorded 5 min after the whole-cell mode was obtained for at least 3 min. Data were analyzed using Clampfit 10.2 (Molecular Devices), OriginPro 2018 (Origin Lab, Washington, MA, USA), and/or GraphPad Prism 7 (GraphPad, San Diego, CA, USA).
Statistical analysis
GraphPad Prism 8.0 software (GraphPad Software, Inc, La Jolla, CA, USA) was used for behavior analyses, western blotting, fluorescence imaging, and Golgi staining. Clampex 10.2 software (Molecular Devices, Union City, CA, USA) was used for electrophysiology analysis, and the Kolmogorov–Smirnov test was used to compare the cumulative distributions of frequency and amplitude between groups. Differences between groups were determined by one-way analysis of variance followed by Tukey’s multiple comparisons test. All results are expressed as the mean ± standard error of the mean. The number of samples/subjects per experiment is noted in the corresponding figure legend. A p-value < 0.05 was considered statistically significant.