Mice
All mouse experiments conformed to Institutional Animal Care and Use Committee (IACUC) protocols of the Korea Brain Research Institute (KBRI). We followed all proper ethical regulations. LoxP-floxed Megf10(Megf10fl/fl) and loxP-floxed Mertk(Mertkfl/fl) mice were generated by the Stanford Transgenic, Knockout and Tumor Model Center (TKTC). Megf10tm1(KOMP)Vlcg (straight Megf10 knockout, stMegf10 KO) and Mertktm1(KOMP)Vlcg (straight Mertk knockout, stMertk KO) mice were obtained from Ben Barres’ laboratory7,9. The Megf10fl/fl and Mertkfl/fl lines were crossed together to produce double loxP-floxed Megf10 and Mertk (Megf10fl/fl;Mertkfl/fl) mice. The mouse lines were maintained through crossing with C57BL/6 mice in a standard plastic cage. A 12-hour light/12-hour dark cycle was implemented, and the temperature inside the cage was maintained between 20 °C and 23 °C. All experiments involving mutant mice were performed blindly with other littermates. All mice were randomly assigned to groups for experimentation.
Stereotaxic injection
Mice were anesthetized with isoflurane (Hana Pharm) in a sealed acrylic box and maintained under a low-flow anesthesia delivery system (SomnoSuite). For deletion of astrocytic phagocytosis receptors, either AAV5::GFAP (0.7)-Cre-eGFP-T2A-iCre-WRPE or AAV5::GFAP (0.7)-eGFP-WPRE (6.4x1012GC/ml and 1.2x1013GC/ml, respectively, purchased from Vector Biolabs) was stereotaxically injected bilaterally into the dorsal striatum (ML: ± 1.9 mm, AP: +0.79 mm from bregma, DV: -2.7 mm from the brain surface) of 7-9-week-old Megf10fl/fl;Mertkfl/fl mice. For the phagocytosis assay, an Alexa 568-labeled dextran tracer was stereotaxically injected bilaterally into either the primary/secondary motor cortex (ML: ± 0.75 mm, AP: +1.97 mm from bregma, DV: -0.5 mm from the brain surface) or thalamic central lateral (ML: ± 0.81 mm, AP: -1.5 mm from bregma, DV: -2.94 mm from the brain surface)/parafascicular nuclei (ML: ± 0.61 mm, AP: -2.3 mm from bregma, DV: -3.34 mm from the brain surface) of control or DAPS mice, respectively. For optogenetic recording, AAV2/2::hSyn-Chornos-tdTomato (2x10^12 GC/ml, purchased from BrainVTA) was injected into the motor cortex or thalamus simultaneously with AAV injection for control and DAPS production. All surgeries were performed using a stereotaxic frame (Kopf) and a Nanojector III (Drummond). A glass pipette needle (WPI) was used for the nanoinjector, and pulling of the glass pipette was performed using a motorized pipette puller (Sutter instrument). After injection, the incision on the head was closed with wound closure clips (Alzet). After the surgery, the mice were allowed to recover in a heated cage for an hour before being returned to their home cage.
Immunohistochemistry
Mice were deeply anesthetized with 1~2% isoflurane and intracardially perfused with 1X PBS (Welgene) followed by 4% paraformaldehyde (PFA, BIOSESANG) in PBS. The brains were isolated and postfixed overnight in PFA at 4 °C and then cryoprotected with 30% sucrose in PBS for 72 hours. The brains were embedded in OCT compound (Leica), and coronal sections (40 μm brain sections) were cut using cryo-stat microtomes (Leica). The sections were blocked with 10% goat serum and 0.2% Triton X-100 in 1× PBS for 1 h at room temperature (RT) and incubated for 24 h at 4 °C with primary antibodies diluted in blocking solution. The primary antibodies used were as follows: guinea pig anti-VGLUT1 (1:2000, Cat #AB5905, Millipore), chicken anti-VGLUT2 (1:500, Cat #135 416, Synaptic Systems), rabbit anti-PSD95 (1:200, Cat #D27E11, Cell Signaling), guinea pig-anti-VGAT (1:500, Cat #131 004, Synaptic Systems), rabbit anti-Gephyrin (1:500, Cat #147 008, Synaptic Systems), guinea pig-anti-S100B (1:500, Cat #287 004, Synaptic Systems), rabbit anti-MEGF10 (1:200, Cat #ABC10, Milipore Sigma) or rabbit anti-MERTK (1:200, Cat #Ab216564, Abcam).
The sections were treated with appropriate secondary antibodies conjugated with Alexa Fluor in blocking solution for 2 h at RT. The following secondary antibodies were used: AlexaFluor-405 (1:200, Cat #A31556, Invitrogen), AlexaFluor-647 (1:200, Cat #A21449), AlexaFluor-546 (1:200, Cat #A11074, Invitrogen), AlexaFluor-647 (1:200, Cat #A21245, Invitrogen), AlexaFluor-647 (1:200, Cat #706-605-148, Jackson ImmunoResearch) or AlexaFluor-568 (1:200, Cat #A21245, 1:200). The stained sections were mounted on adhesive-coated glass slides (MARIENFELD) with Vectashield Hardset Antifade Mounting Medium (Cat #H-1400). Images were acquired using confocal Nikon A1 Rsi/Ti-E, confocal STELLARIS 8 or Pannoramic scans.
Phagocytosis assay
Confocal images of bilateral injection sites in the dorsal striatum were acquired using a STELLARIS 8 (63X oil immersion optical lens) for quantification, as described below. To analyze the amount of Alexa 568-labeled dextran engulfed inside the astrocyte cytoplasm, the EGFP+ astrocyte processes were visualized, and the number of engulfed puncta was measured. The data generated were used to calculate the density of the engulfed dextran puncta (number of engulfed dextran puncta/area of EGFP+ astrocytes), the size of engulfed dextran puncta (size of engulfed dextran puncta/area of EGFP+ astrocytes), and the percentage of engulfed dextran puncta (number of engulfed dextran puncta/total projected dextran puncta) in the corticostriatal or thalamostriatal pathways. The colocalization assay was performed using the DiAna plugin33.
Quantification of synapse numbers
Confocal images of the dorsal striatum sections were acquired using a Nikon A1 Rsi/Ti-E (60x oil immersion optical lens) for quantification. All channels, including the presynaptic and postsynaptic compartments, were split using ImageJ software. The colocalization assay was performed using the ImageJ plugin. The number of excitatory/inhibitory presynaptic-only (VGLUT1&2, VGLUT1, VGLUT2 or VGAT) or postsynaptic-only puncta (PSD95 or gephyrin), as well as the number of colocalized puncta (pre- and postsynaptic together; VGLUT1&2+PSD95, VGLUT1+PSD95, VGLUT2+PSD95 or VGAT+gephyrin), were measured using the ImageJ 1.53c software colocalization assay plugin.
Whole-cell patch clamp
For whole-cell patch clamp recordings, acute brain slices were obtained from 12- to 16-week-old mice. The standard artificial cerebral spinal fluid (ACSF) consisted of (in mM) 124 NaCl, 2.5 KCl, 1.2 NaH2PO4, 24 NaHCO3, 5 HEPES, 2 CaCl2, 2 MgCl2, and 13 glucose (pH 7.3). Mice were deeply anesthetized with isoflurane and intracardially perfused with ~20 ml of slicing ACSF containing (in mM) 93 N-methyl-D-glutamine (NMDG)-Cl, 93 HCl, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 5 sodium ascorbate, 2 thiourea, 3 sodium pyruvate, 12 N-acetyl-L-cysteine (NAC), 0.5 CaCl2, 10 MgCl2, and 25 glucose (pH 7.3). Coronal slices containing the striatum (350 μm thick) were dissected using a VF-200-OZ Compresstome (Precisionary) using the slicing ACSF and recovered at 32.5 °C in recovery ACSF (in mM; 104 NaCl, 2.5 KCl, 1.2 NaH2PO4, 24 NaHCO3, 5 HEPES, 5 sodium ascorbate, 2 thiourea, 3 sodium pyruvate, 12 NAC, 2 CaCl2, 2 MgCl2, and 13 glucose; pH 7.3) for 1 h.
The slices were placed in a recording chamber and continuously perfused with oxygenated standard ACSF at a rate of 2-3 ml/min at RT. Whole-cell recordings were made with a Multipclamp 700B amplifier (Molecular Devices). The data were filtered at 5 kHz and digitized at 10-50 kHz. Borosilicate glass patch electrodes with a resistance of 3-5 MΩ were filled with pipette solution containing (in mM) 140 Cs-methanesulfonate, 7 NaCl, 0.2 EGTA, 2 MgCl2, 4 Mg-ATP, 0.3 Na2-GTP, 10 Na2-phosphocreatine, and 10 HEPES (pH 7.3, 290-300 mOsm) and used for recording spontaneous excitatory postsynaptic currents (sEPSCs). To record spontaneous inhibitory postsynaptic currents (IPSCs), pipette solutions containing (in mM) 140 CsCl, 7 NaCl, 0.2 EGTA, 2 MgCl2, 4 Mg-ATP, 0.3 Na2-GTP, 10 Na2-phosphocreatine, and 10 HEPES (pH 7.3, 290-300 mOsm) were used. Bicuculline (10 μM; Tocris), 2,3-dioxo-6-nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX, 10 μM; Tocris), and D-2-amino-5-phosphonopetanoate (D-AP5, 50 μM; Tocris) were added to inhibit GABAergic or glutamatergic synaptic transmission, respectively. Striatal medium spiny neurons (MSNs) were distinguished through membrane properties (Table 1) and delayed firing patterns via current injection (data not shown).
To selectively stimulate corticostriatal and thalamostriatal pathways, optogenetic induction was utilized with brain slices injected with AAV2/2::hSyn-Chronos-tdTomato in the motor cortex or thalamus. Preparation of coronal striatal slices (350 μm thick) and whole-cell recording from MSNs were performed as described above. To induce light-evoked EPEPs, a high-power LED (at 470 nm; X-Cite) was used to deliver blue light to the slice through the microscope (Nikon). This configuration could deliver blue light at ~2.5 mW/mm2 over a 0.22 mm2 area of the recording site using a 40X objective lens. These conditions were sufficient for eliciting stable EPSPs with a light duration of 0.5-1 ms. Bicuculline (10 μM), NBQX (10 μM), D-AP5 (50 μM), 4-aminopyridine (4-AP, 100 μM; Sigma) and tetrodotoxin (TTX, 1 μM; Alomone) were added to inhibit GABAergic synaptic transmission or confirm monosynaptic/glutamatergic synaptic transmission, respectively.
The paired-pulse ratio (PPR) of the corticostriatal and thalamostriatal pathways was measured by pairing blue light-evoked stimuli to the striatum with interstimulus intervals of 20, 50 or 100 msec. Stimulus intensity was determined by constructing an input–output relationship that plotted the amplitude of light-evoked EPSPs against stimulus intensities and then adjusted to 30–40% of the maximum amplitude of light-evoked EPSPs. After at least 10 min of stable light-evoked EPSP acquisition, the PPR was measured and calculated by dividing the amplitude of the second response by that of the first response. For optogenetic induction of corticostriatal and thalamostriatal long-term potentiation (LTP), theta-burst stimulation (TBS; 10 trains of stimuli spaced at 10 s intervals, with each train containing bursts of 4 spikes at 100 Hz and repeated 10 times at 5 Hz; Park et al., Neuron, 2014) was delivered. The data for EPSP amplitudes are presented as averages over 2 min bins. TBS-induced LTP was measured as the average EPSPs at 50-60 min. All whole-cell patch clamp recording data were analyzed by using pClamp 11.1 (Axon Instruments).
Serial order task
The serial order task (SO) was performed in the operant box (Med Associates, Inc.) for each mouse. The detailed training process for the SO task was utilized with minor modifications from published protocols21. The operant box consisted of left (L) and right (R) levers, and a food magazine was located at the middle of the levers. For effective motor sequence learning, the mice were subjected to food restriction for 7 days prior to the first training session. First, in the fixed ratio 1 (FR1) training, the association between lever and reward was established by delivering one 14 mg of sugar pellet (Bio-Serv) after each lever response. During the FR1 session, the mice received up to 50 pellets in a 60 min session. For the SO task, the mice had to perform two distinct and sequential responses (“L” then “R”). The delivery of one sugar pellet followed the correct LR sequence, and both correct and incorrect trials were followed by an 8-s intertrial interval. Daily SO training sessions lasted for up to 90 min or until the mouse received 50 pellets. The accuracy of the first step was determined by the percentage of trials that started with a correct first step (LL or LR), while the accuracy of the second step was defined as the proportion of trials that began with a correct initial step (LL or LR) and subsequently completed with a correct second step (LR).
Open field test
The open field test was performed in a square arena (nonglossy acrylic box, 300 × 300 × 280 mm, W × D × H). To start the test, the mice were placed in the center of the box and allowed to explore for 5 min. Movement was detected automatically using Noldus EthoVision 3.0 tracking software. Measurements during the test included the total distance traveled, speed, and time spent in the center/peripheral zones.
Elevated plus maze
Mice were allowed to explore an elevated platform (50 cm above the floor) consisting of two open (30 × 6 cm) and two closed arms (30 × 6 cm with a 20 cm tall opaque wall) with a central area (6 × 6 cm). To start the test, the mice were placed in the center of the maze facing the open arm and allowed to explore for 5 min. Movement was detected automatically using SMART VIDEO TRACKING Software (Panlab). Measurements during the test included the time spent in the open arms, closed arms, and center. The maze was cleaned with 70% ethanol before each trial.
Statistical analysis
All the data are expressed as the mean ± standard error of the mean (SEM). All of the statistical analyses were performed using GraphPad Prism 7 with 95% confidence. Comparisons between two groups were analyzed by two-tailed unpaired Student’s t tests.