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Mouse models

All animal experiments were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Chancellor’s Animal Research Committee at the University of California, Los Angeles (UCLA). All mice were housed with food and water available ad libitum in a 12-hour light–dark environment at temperatures of 20–22 °C with 40–60% humidity. All mice were healthy with no obvious behavioural phenotype, were not involved in previous studies and were euthanized during the light cycle. Data for most experiments were collected from adult mice aged 9–15 weeks old, but to characterize µ-crystallin expression during development and ageing, mice were used between P0 and 22 months old. For experiments, both male and female mice were used. C57Bl/6NTac mice were maintained as an in-house breeding colony or purchased from Taconic Biosciences. CAG-Cas9 transgenic mice (B6J.129(Cg)-Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J, JAX stock 026179) and SAPAP3−/− mice (B6.129-Dlgap3tm1Gfng/J, JAX stock 008733) were purchased from the Jackson Laboratory and maintained as breeding colonies at UCLA. SAPAP3−/− mice were used at six months of age. Tg(Crym-EGFP)GF82Gsat (strain 012003-UCD) reporter mice were obtained from MMRRC and maintained at UCLA.

sgRNA design and molecular cloning

To design sgRNAs for CRISPR–Cas9 knockout with minimal off-target effects, six target sequences (crRNAs) for the Crym gene were designed using the web tool CHOPCHOP ( Control target sequences for EGFP were designed using the same method. To select the three most efficient crRNA sequences for Crym and the most efficient crRNA sequence for EGFP, in vitro knockout efficiency was assessed using the Guide-it Complete sgRNA Screening System (Takara Bio 632636) by following the manufacturer’s instructions. The genomic sequence of Crym was obtained from the NCBI GenBank database (gene ID: 12971) and the genomic template was obtained by PCR amplification using the following primers: forward 5′-AGGTGGAACCAGAAAGTCCTCT-3′ and reverse 5′- GCACTTGGTGTATCTGAGCGTG-3′. An original vector containing a U6 promotor followed by two Bbs1 restrictions sites and by the tracrRNA sequence for SpCas9 (pZac2.1-U6-Bbs1-Bbs1-tracrRNA-GfaABC1D-mCherry-SV40) was created. To insert the three crRNAs for Crym, each sequence was separately inserted in chaperone vectors to form three constructs with an sgRNA controlled by the U6 promotor. The three U6-sgRNAs were then combined in the same vector using a BglII restriction site and the In-Fusion cloning kit (Takara Bio 638943). The three crRNA sequences for Crym were: 5′-AAGTTAGTCACCTTCTATGA-3′, 5′-GCACCGATGCCTGATGGGAG-3′ and 5′-GGCAGGCGGCGAGATGAAGC-3′. The crRNA sequence for EGFP was 5′-CACCGGGGCGAGGAGCTGTTCACCGGTT-3′. The U6 sgRNA plasmids for Crym and the EGFP control have been deposited at Addgene in the Khakh laboratory repository (Addgene 200067 and 200068). The fully sequenced plasmids were sent to the UPenn Vector Core or Vigene Biosciences to generate AAV2/5 serotype for each construct, yielding a concentration higher than 1.0 × 1013 genome copies per ml (gc ml−1). The cloning and sequencing strategies were designed with SnapGene software (v.7.0.3, Insightful Science).

Stereotaxic microinjections of AAVs

All surgical procedures were conducted under general anaesthesia using continuous isoflurane (induction at 5%, maintenance at 1–2% v/v). The depth of anaesthesia was monitored continuously and adjusted when necessary. After the induction of anaesthesia, the mice were fitted into a stereotaxic frame with their heads secured by blunt ear bars and their noses placed into a veterinary grade anaesthesia and ventilation system (VetEquip). Mice were administered with 0.1 mg per kg of buprenorphine (Buprenex, 0.1 mg ml−1) subcutaneously before surgery. The surgical incision site was then cleaned three times with 10% povidone iodine and 70% ethanol (v/v). Skin incisions were made, followed by craniotomies of 2–3 mm in diameter above the left frontal or parietal cortex using a small steel burr (Fine Science Tools) powered by a high-speed drill (K.1070, Foredom). Saline (0.9%) was applied onto the skull to reduce heating caused by drilling. Bilateral viral injections were performed by using a stereotaxic apparatus (David Kopf Instruments) to guide the placement of beveled glass pipettes (1B100-4, World Precision Instruments). For the central striatum, the coordinates were 0.8 mm anterior to bregma, 1.85 mm lateral to midline and 2.9 mm from the pial surface. For the lOFC, the coordinates were 2.8 mm anterior to bregma, 1.5 mm lateral to midline and 1.6 mm from the pial surface. For M1, the coordinates were 1.8 mm anterior to bregma, 1.8 mm lateral to midline and 0.6 mm from the pial surface. AAVs were injected by using a syringe pump (Pump11 PicoPlus Elite, Harvard Apparatus). After AAV microinjections, glass pipettes were left in place for at least 10 min before being slowly withdrawn. Surgical wounds were closed with external 5-0 nylon sutures. After surgery, mice were allowed to recover overnight in cages placed partially on a low-voltage heating pad. Buprenorphine was administered twice a day for up to two days after surgery. In addition, trimethoprim sulfamethoxazole was provided in food to the mice for one week to prevent infection.

AAV-injected mice were used for experiments three weeks after surgery. The viruses used were: 0.5 μl or 0.3 μl of AAV2/5 U6-sgRNA-Crym(×3)-GfaABC1D-mCherry virus (3 × 1013 gc ml−1) (Addgene 200067), 0.5 μl or 0.3 μl of AAV2/5 U6-sgRNA-GFP-GfaABC1D-mCherry virus (3.8 × 1013 gc ml−1) (Addgene 200068), 0.3 μl of AAV9-CaMK11a-hChR2(H134R)-mCherry (2.1 × 1013 gc ml−1) (Addgene 26975), 0.3 μl of AAV1-hSyn-Chronos-GFP (1.4 × 1013 gc ml−1) (Addgene 59170), 0.3 μl of retrograde AAV-hSyn-hM4D(Gi)-mCherry (2.4 × 1013 gc ml−1) (Addgene 50475), 0.3 μl of retrograde AAV-hSyn-mCherry (2.4 × 1013 gc ml−1) (Addgene 114472), 0.3 μl AAV2/5-GfaABC1D-Rpl22-HA (2.1 × 1013 gc ml−1) (Addgene 111811), 0.5 μl AAV2/5 GfaABC1D-Crym-EGFP (2.5 × 1013 gc ml−1) (Addgene 200080), 0.5 μl AAV2/5 GfaABC1D-Crym-BioID2-HA (3.1 × 1013 gc ml−1) (Addgene 200070), 0.5 μl AAV2/5 GfaABC1D-jGCaMP8m (Addgene 213010), 0.5 μl AAV2/5 GfaABC1D-tdTomato (Addgene 44332). All AAVs are listed in Supplementary Table 1.


Frozen sections

For transcardial perfusion, mice were euthanized with isoflurane and perfused with 0.1 M phosphate-buffered saline (PBS) followed by 10% buffered formalin (Fisher SF100-20). Heparin (50 units) was injected into the heart to prevent blood clotting. After gentle removal from the skull, the brain was post-fixed in 10% buffered formalin overnight at 4 °C. For the characterization of striatal µ-crystallin expression during development, P0 and P7 brains were directly post-fixed in 10% buffered formalin without transcardial perfusion. The tissue was cryoprotected in 30% sucrose PBS solution for at least 48 h at 4 °C until use. Forty-micrometre coronal sections were prepared using a cryostat microtome (Leica) at −20 °C and processed for immunohistochemistry. Sections were washed three times in 0.1 M PBS for 10 min each, and then incubated in a blocking solution containing 10% NGS in 0.1 M PBS with 0.2% Triton-X 100 for one hour at room temperature with agitation. Sections were then incubated with agitation in primary antibodies diluted in blocking solution overnight at 4 °C. The following primary antibodies were used: mouse anti-µ-crystallin (1:250, Santa Cruz, sc-376687), chicken anti-GFP (1:1,000; Abcam ab13970), mouse anti-NeuN (1:500; Millipore MAB377), guinea pig anti-NeuN (1:500; Synaptic Systems, 266004), rabbit anti-DARPP-32 (1:1,000, Abcam, ab40801), rabbit anti-S100β (1:1,000; Abcam ab41548), rabbit anti-cFOS (1:1,000; Synaptic Systems 226008), rabbit anti-RFP (1:1,000; Rockland 600–401-379), rabbit anti-mCherry (1:1,000; Abcam, ab167453), rabbit anti-opioid receptor, µ, pain (MOR1) (1:200, Millipore Sigma, AB5511), chicken anti-calbindin D-28K (1:200, Novus Biologicals, NBP2-50028), chicken anti-GFAP (1:1,000; Abcam, ab4674) and rabbit anti-HA tag (1:1,000; Abcam, ab9110), guinea pig anti-RFP (1:1,000, Synaptic Systems, 390004), rabbit anti-SOX9 (1:500, EMD Millipore, AB5535), rabbit anti-OLIG2 (1:500, EMD Millipore AB9610), rabbit anti-KIR4.1 (1:500, Alomone Labs, APC-035), rabbit anti-ATP1a2 (1:300, Proteintech, 16836-1-AP), rabbit anti-GLT1 (1:500, Synaptic Systems, 250203), mouse anti-GABA (1:500, Abcam, ab86163), rabbit anti-MAOB (1:500, Thermo Fisher Scientific PA5-28338), rabbit anti-GAT3 (1:250, gift from the N. Brecha laboratory at UCLA), rabbit anti-CAPZB (1:250, Thermo Fisher Scientific PA5-83196). The next day, the sections were washed three times in 0.1 M PBS for 10 min each before incubation at room temperature for two hours with secondary antibodies diluted in 0.1 M PBS. Alexa-conjugated (Molecular Probes) secondary antibodies were used at a 1:1,000 dilution (Alexa Fluor 405 goat anti-mouse (A31553), Alexa Fluor 488 goat anti-chicken (A11039), Alexa Fluor 488 goat anti-rabbit (A11008), Alexa Fluor 488 goat anti-mouse (A11010), Alexa Fluor 546 goat anti-mouse (A11030), Alexa Fluor 546 goat anti-rabbit (A11001), Alexa Fluor 647 goat anti-rabbit (A21244), streptavidin and Alexa Fluor 488 conjugate (S11223)). The sections were rinsed three times in 0.1 M PBS for 10 min each before being mounted on microscope slides and sealed in fluoromount-G. Fluorescence images were taken using UPlanSApo 10× 0.4 NA, UPlanFLN 40× 1.30 NA oil immersion or PlanApo N 60× 1.45 NA oil immersion objective lenses on a confocal laser-scanning microscope (Fluoview FV3000; Olympus). Laser settings were kept the same within each experiment. Images represent maximum intensity projections of optical sections with a step size of 1 μm or 5 µm for the entire brain or striatum images. Images were processed with Image J (NIH v.2.1). Cell counting was done on maximum intensity projections using the Cell Counter plug-in; only cells with soma completely within the region of interest (ROI) were counted.

Acute sections

Acute sections were used for biocytin staining to assess MSN morphology, spine density and spine head width. Fresh brain slices (300 μm) were placed into 10% buffered formalin overnight at 4 °C and processed as for IHC. Sections were washed three times in 0.1 M PBS with 2% Triton-X 100 for 5 min each, and then incubated in a blocking solution containing 10% NGS in 0.1 M PBS with 1% Triton-X 100 for one hour at room temperature with agitation. Sections were then incubated at room temperature with streptavidin-conjugated Alexa 647 (1:250, S11223) diluted in 0.1 M PBS with 0.4% Triton-X 100 for 3 h. The sections were rinsed three times in 0.1 M PBS for 10 min each before being mounted on microscope slides in fluoromount-G. Images were obtained in the same way as for the IHC for frozen sections, except with a step size of 0.33 μm. For the quantification of spine density, we analysed only spines on dendritic shafts that were parallel to the imaging plane to minimize the possibility of rotational artefacts. Spine density was calculated by dividing the number of spines by the length of the dendritic segment. For quantification of spine head width, a line ROI across the maximum diameter of the spine was made and a profile that has a single peak was obtained. The MSN morphology was determined using a Sholl analysis plug-in with ImageJ (NIH v.2.1).

TUNEL assay

To assess apoptosis, a TUNEL assay was performed on frozen sections in control and Crym KO mice using the TUNEL Andy Fluor 647 Apoptosis Detection Kit (ABP Biosciences, A052). In brief, DNA strand breaks with 3’-hydroxyl ends are labelled with biotin-11-dUTP in the presence of terminal deoxynucleotidyl transferase (TdT). Once incorporated into the DNA, biotin is detected using HRP- or fluorophore-labelled streptavidin. The experiment was performed following the manufacturer’s instructions.

Retrograde tracing and afferent projections

For retrograde tracing experiments, 300 nl of cholera toxin subunit B, Alexa Fluor 647-conjugated (CTB-647) (Thermo Fisher Scientific C34778) was injected in the central striatum of CAG-Cas9-GFP mice. Ten days after injection, frozen sections were prepared as described above. The number of CTB-647+ cell bodies per mm2 were manually counted in regions of the prefrontal cortex and motor cortex delineated by the Paxinos Brain Atlas. To visualize the projections from the lOFC and M1, 0.3 µl of AAV9-CaMKIIa-hChR2(H134R)-mCherry and 0.3 µl of AAV1-Syn-Chronos-GFP were injected in the lOFC and M1 respectively. Four weeks after injection, mCherry and GFP fluorescence in the striatum were delineated with a threshold method and the intensity of µ-crystallin was quantified in each ROI.

Astrocyte morphology

Astrocyte morphology was assessed using Lucifer Yellow iontophoresis. In brief, control or Crym KO mice were transcardially perfused with 10 ml of 35 °C Ringer’s solution (135 mM NaCl, 14.7 mM NaHCO3, 5 mM KCl, 1.25 mM Na2HPO4, 2 mM CaCl2, 1 mM MgCl2 and 11 mM d-glucose, bubbled with 95% O2 and 5% CO2) with 0.02% lidocaine followed by 50 ml of 10% buffered formalin (Thermo Fisher Scientific SF100-20). Brains were lightly post-fixed at room temperature for 1.5 h and then washed three times in ice-cold 0.1 M PBS for 10 min. Coronal sections (100 μm) were cut using a Pelco Vibrotome 3000 and then placed in ice-cold PBS for the duration of the experiment. Ten milligrams of Lucifer yellow CH di-Lithium salt (Sigma L0259) was dissolved in 1 ml 5 mM KCl solution and filtered before use. Sharp (around 200 MOhm) glass electrodes were pulled from borosilicate glass capillaries with filament (outer diameter 1.0 mm; inner diameter 0.58 mm). Electrodes were gravity filled with Lucifer yellow solution. Sections were transferred to a PBS solution at room temperature for filling. Astrocytes were identified using mCherry expression and then impaled with the sharp electrode. Lucifer yellow was injected into the cell by applying around 1 V for 10 min. After the astrocyte was filled, the electrode was removed completely. The sections were fixed with 4% paraformaldehyde and the filled astrocyte was imaged with UPlanFLN 40× 1.30 NA oil immersion or PlanApo N 60× 1.45 NA oil immersion objective lenses on a confocal laser-scanning microscope (Fluoview FV3000; Olympus) at a digital zoom of two to three and a 0.3-µm confocal z-step. Territory area and soma area were generated using threshold reconstruction with Image J (NIH v.2.1).

Dual in situ hybridization with RNAscope and IHC

Fixed-frozen tissue was processed as described above. Serial coronal sections (14 μm) containing striatum were prepared using a cryostat microtome (Leica) at −20 °C and mounted immediately onto glass slides. In situ hybridization was performed using Multiplex RNAscope (ACDBio 320851). Sections were washed for at least 15 min with 0.1 M PBS, and then incubated in 1× Target Retrieval Reagents (ACDBio 322000) for 5 min at 95–100 °C. After washing with ddH2O twice for 1 min each, they were dehydrated with 100% ethanol for 2 min and dried at room temperature. Then, the sections were incubated with Protease Pretreat solution (ACDBio 322340) for 30 min at 40 °C. The slides were washed with ddH2O twice for 1 min each and then incubated with probe(s) for 2 h at 40 °C. The following probes were used: Mm-Crym-C3 (ACDBio 466131-C3), Mm-Drd1-C3 (ACDBio 461901-C3) and Mm-Drd2-C3 (ACDBio 406501-C3). The sections were incubated in AMP 1-FL for 30 min, AMP2-FL for 15 min, AMP3-FL for 30 min and AMP4-FL for 15 min at 40 °C with washing in 1× wash buffer (ACDBio 310091) twice for 2 min each before the first incubation and in between incubations. All the incubations at 40 °C were performed in the HybEZ Hybridization System (ACDBio 310010). Slices were washed in 0.1 M PBS three times for 10 min each, followed by IHC that was performed as described above except with antibody dilutions. The following primary antibodies were used: mouse anti-µ-crystallin (1:100, Santa Cruz, sc-376687), rabbit anti-DARPP-32 (1:500, Abcam, ab40801) and rabbit anti-S100β (1:500; Abcam ab41548). Images were obtained in the same way as for the IHC described above, and were processed with Image J (NIH v.2.1). Astrocyte area was demarcated by drawing an 80-µm-diameter circle around the soma (the average size of striatal astrocytes based on our previous experiments). Then, the percentage of D1 and D2 positive neurons was quantified within this area.

Pharmacological treatments

To reduce stress, mice were handled and habituated three days before beginning the treatments. For fluoxetine experiments, six-month-old SAPAP3−/− mice were treated with an intraperitoneal (i.p.) injection of fluoxetine (Tocris 0927) at 10 mg per kg (dissolved in 0.9% sodium chloride) for seven consecutive days. Three weeks after AAV microinjection, striatal Crym KO mice were treated with an i.p. injection of fluoxetine at 10 mg per kg for 21 consecutive days. For Gi activation, mice were treated with a single i.p. injection of deschloroclozapine (DCZ, Tocris 7193) at 1 µg kg−1 for one day (grooming test), two days (open-field test), three days (marble-burying test), seven days (novel object recognition test) or eight days (IHC). For both experiments, untreated mice received the same amount of 0.9% sodium chloride (vehicle). Behavioural tests or euthanasia were performed one to two hours after treatments.

Behavioural tests

Behavioural tests were performed during the light cycle between 09:00 and 18:00, three to five weeks after AAV injection. Both male and female mice were used in behavioural tests. The mice were randomly allocated to a group as they became available and of age in the breeding colony. All of the experimental mice were transferred to the behaviour testing room at least 30 min before the tests to allow them to acclimatize to the environment and to reduce stress. The temperature and humidity of the experimental rooms were kept at 23 ± 2 °C and 55 ± 5%, respectively. Background noise (65 ± 2 dB) was generated by a white noise generator (San Diego Instruments). The brightness of the experimental room was kept at less than 20 lux.

Open-field test

The open-field chamber consisted of a square arena (28.5 × 30 cm) enclosed by walls made of translucent polyethylene (15 cm tall). The locomotor activity of mice was recorded for 10 or 30 min using a camera located above the open-field chamber. The open-field behaviours were analysed with an automated video tracking software ANY-maze (v.6.3, Stoelting).

Rotarod test

Mice were held by the tails and placed on the rod (3 cm diameter) of a single lane rotarod apparatus (ENV-577 M, Med Associates), facing away from the direction of rotation. The rotarod was set with a start speed of 4 rpm. Acceleration started 10 s later and was set to 20 rpm with a maximum speed 40 rpm per min. Each mouse received five trials 30 min apart for five consecutive days and the latency to fall was recorded for each trial. The average latency to fall was used as a measurement for motor coordination.

Footprint test

A 1-m-long runway (8 cm wide) was lined with paper. Each mouse had its hind paws painted with non-toxic ink and was placed at an open end of the runway. The mice were allowed to walk to the other end with a darkened box. For the gait analysis, stride length and width were measured and averaged for both left and right hindlimbs over five steps.

Self-grooming behaviour

Mice were placed individually into plastic cylinders (15 cm in diameter and 35 cm tall), and allowed to habituate for 20 min. Self-grooming behaviour was recorded for 10 min. A timer was used to assess the cumulative time spent in self-grooming behaviour, which included paw licking; unilateral and bilateral strokes around the nose, mouth and face; paw movement over the head and behind the ears; body fur licking; body scratching with hind paws; tail licking; and genital cleaning. The number of self-grooming bouts and rearing bouts was also counted. Separate grooming bouts were considered when the pause was more than 5 s or if behaviours other than self-grooming occurred. The self-grooming microstructure was not assessed.

Spray test

A standard spray bottle was filled with distilled water and the nozzle was adjusted to the ‘misting’ mode. Mice were held by the tails on the bench and sprayed four times from 30 cm away to be adequately covered with mist. Mice were placed individually into the plastic cylinders and grooming behaviour was recorded for 10 min after the spray and then analysed as described in the preceding section.

Marble-burying and digging test

Fresh, unscented soft wood chip bedding was added to polycarbonate cages (21 × 43 × 20.5 cm) to a depth of 5 cm. Fifteen sanitized glass marbles were gently placed on the surface of the bedding in five rows of three marbles. Mice were allowed to remain in the cage undisturbed for 30 min. A marble was scored as buried if two-thirds of its surface area was covered by bedding. For the digging test, the same experiment without the marbles was performed. The latency to start digging, the total digging duration and the duration of digging bouts were also counted.

Novel object recognition

On day 1 and day 2, mice were placed in an empty open chamber (28.5 × 30 cm) for 10 min for habituation. On day 3 (training day), mice were placed in the same open chamber containing two identical objects evenly spaced apart; the trial was video recorded for 10 min. For the novel object recognition test, at day 4 (testing day), 24 h after training, mice were placed in the same open chamber, with one of the two objects replaced with a novel object. The trial was video recorded for 10 min. Time exploring around the objects was manually measured. The recognition index was calculated as follows: (time exploring the novel object – time exploring the familiar object)/(time exploring both objects) – 50.

Lickometer test

Mice were placed in a lickometer system (Stoelting) to measure licking behaviour (20 × 20 cm). In brief, an electrical signal was generated from the tongue touching the sipper tube, with each lick recorded as an event using the ANY-maze software (v.6.3, Stoelting). Mice were placed in the chamber for 30 min during four consecutive days and placed back to their home cage at the end of each session. Before the fourth day, mice were water deprived for 12–16 h before the session. The total number of licks, the total duration of drinking and the latency to start drinking were measured by the software.

Elevated plus maze

The elevated plus maze consisted of arms that were 30 × 7 cm with closed-arm walls with a height of 20 cm. The maze was elevated 65 cm above floor level and was placed in the centre of the room away from other stimuli. Mice were placed in the centre of the maze facing an open arm. Mice were recorded for 10 min using a camera (Logitech) located above the maze. ANY-maze video analysis software was used to quantify the time spent in open arms and time spent in close arms.

Food consumption

Three weeks after AAV injections, 200 g of food was placed in the home cage for 72 h. The remaining food was weighed and divided by the number of days and the number of mice to calculate the consumption in gram per day per mouse.


Mice were weighed the day before the AAVs injection and every week for four weeks after injection.

Brain-slice preparation and electrophysiological recordings in the striatal slices

Coronal or sagittal striatal slices were prepared from 6–12-week-old Crym KO mice, control or Crym-GFP mice. In brief, mice were deeply anaesthetized with isoflurane and decapitated with sharp shears. The brains were placed and sliced in ice-cold modified artificial CSF (aCSF) containing the following: 194 mM sucrose, 30 mM NaCl, 4.5 mM KCl, 1 mM MgCl2, 26 mM NaHCO3, 1.2 mM NaH2PO4 and 10 mM d-glucose, and saturated with 95% O2 and 5% CO2. A vibratome (DSK Microslicer; Ted Pella) was used to cut 300-µm brain sections. The slices were allowed to equilibrate for 30 min at 33 °C in normal aCSF containing: 124 mM NaCl, 4.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 26 mM NaHCO3, 1.2 mM NaH2PO4 and 10 mM d-glucose, continuously bubbled with 95% O2 and 5% CO2. Slices were then stored at 21–23 °C in the same buffer until use. All slices were used within 6 h of slicing. The brain-slice recordings were performed at room temperature (21–23 °C). Whole-cell patch-clamp recordings were made from astrocytes or MSNs in the central striatum using patch pipettes with a typical resistance of 4–6 MΩ. MSNs were morphologically and electrophysiologically identified. Astrocytes were identified by mCherry or EGFP expression. The intracellular solution for MSN membrane properties, EPSCs, evoked EPSCs and astrocyte recordings comprised the following: 135 mM potassium gluconate, 3 mM KCl, 10 mM HEPES, 1 mM EGTA, 0.3 mM Na-ATP, 4 mM Mg-ATP, 0.1 mM CaCl2 and 8 mM Na2-phosphocreatine, with the pH adjusted to 7.3. The intracellular solution for IPSC recordings comprised the following: 138 mM KCl, 10 mM HEPES, 1 mM EGTA, 0.3 mM Na-ATP, 4 mM Mg-ATP, 0.1 mM CaCl2, 8 mM Na2-phosphocreatine and 3 mM QX314, with the pH adjusted to 7.3. To evoke EPSCs specifically from the M1 or lOFC projections, Channelrhodopsin 2 was injected bilaterally in one of these two areas and afferents in the central striatum were stimulated with an LED light source (Lambda TLED+, Sutter Instrument). To assess input–output function, test stimuli were applied at increasing intensities ranging from 0 to 3 mW. Stimulation intensities were set to evoke responses at 50–60% maximal amplitude to induce paired-pulse responses. Paired pulses were delivered at five different interpulse intervals: 40, 60, 80, 100 and 160 ms apart. To isolate mEPSCs, MSNs were voltage-clamped at −70 mV and pre-incubated with 0.3 µM tetrodotoxin (TTX) before recording. To isolate sIPSCs, MSNs were voltage-clamped at −60 mV and pre-incubated in the presence of 10 µM 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) for 10 min before recording. mIPSCs were recorded after incubation with 10 µM CNQX and 0.3 µM TTX for 10 min. To access tonic GABA currents, 25 µM bicuculline was bath-applied in the presence of 10 µM CNQX, 0.3 µM TTX. In some cases, 1 mg ml−1 biocytin (Tocris, 3349) was added to the intracellular solution to subsequently visualize patched neuron. All recordings were performed at room temperature, using pCLAMP11.2 (Axon Instruments, Molecular Devices) and a MultiClamp 700B amplifier (Axon Instruments, Molecular Devices). Cells with Ra that exceeded 25 MΩ were excluded from analysis. Analysis was performed using ClampFit 10.7 software.

Calcium imaging of astrocytes in brain slices

Slice preparation was performed as above. Cells for all of the experiments were imaged using a confocal microscope (Fluoview 1000; Olympus) with a 40× water-immersion objective lens with 0.8 NA and a digital zoom of two to three. We used the 488-nm line of an Argon laser, with the intensity adjusted to 5–10% of the maximum output of 10 mW. The emitted light pathway consisted of an emission high-pass filter (505–525 nm) before the photomultiplier tube. Astrocytes were typically around 25 μm below the slice surface and were scanned at one frame per second for imaging sessions. For pharmacological activation of endogenous G-protein-coupled receptors, agonists were dissolved in water. Stock solutions were diluted in aCSF immediately before use. Analyses of time-lapse image series were performed using Image J v.2.1 (NIH). The XY drift was corrected using Image J; cells with Z drift were excluded from analyses. Time traces of fluorescence intensity were extracted from the ROIs and converted to ΔF/F values. For microdomains, GECIquant software (v.1.0) was used. Events were identified on the basis of amplitudes that were at least twofold above the baseline noise of the ΔF/F trace. Extracted calcium signals were analysed using OriginPro 2017 (OriginLab, v.9.4.2).

Striatal scRNA-seq

scRNA-seq was performed to profile the whole striatum of four adult mice using a protocol that preferentially captures non-neuronal cells23. Male mice at 7–8 weeks old were anaesthetized and decapitated. The brain was immediately dissected out and was sectioned on a vibratome (Microslicer DTK-Zero 1; Ted Pella) into 400-μm slices in ice-cold aCSF + trehalose (ACSF-T) (124 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 24 mM NaHCO3, 5 mM HEPES, 13 mM glucose, 2 mM MgSO4, 2 mM CaCl2 and 14.6 mM trehalose, pH adjusted to 7.3–7.4) oxygenated with 95% O2/5% CO2. The slices containing the striatum were immediately transferred to an oxygenated recovery solution (93 mM N-methyl-d-glucamine, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 10 mM MgSO4, 0.5 mM CaCl2, 5 mM sodium ascorbate, 2 mM thiourea, 3 mM sodium pyruvate and 45 μM actinomycin D, with a pH of 7.3–7.4) for 15 min on ice. The striatum was dissected out under a dissecting microscope in ice-cold ACSF-T and cut into small pieces (less than 1 mm in all dimensions). Tissue was then transferred to a Petri dish for digestion with ACSF-T containing 1 mg ml−1 pronase (Sigma-Aldrich, P6911) and 45 μM actinomycin D (Sigma-Aldrich, A1410) and incubated at 34 °C for 20 min with aerated carbogen. The digested tissue was transferred to ice-cold oxygenated ACSF-T containing 1% fetal bovine serum and 3 μM actinomycin D. The tissue was dissociated sequentially by gentle trituration through glass Pasteur pipettes with polished tip openings of 500 μm, 300 μm and 150 μm diameter. Actinomycin D was added to the recovery solution at 45 μM, the pronase solution at 45 μM and trituration solution at 3 μM to prevent stress-induced transcriptional alterations. To increase the yield of glial cells23, filters with various pore sizes (70 μm, 40 μm and 20 μm) were tested, and a 20-μm filter gave the highest yield and therefore was chosen. The dissociated cells were filtered through a 20-μm filter and washed with ice-cold ACSF-T. To remove myelin, the cell pellet was resuspended in PBS and processed with a debris removal kit (Miltenyi Biotec, 130-109-398). The cell density was calculated and isolated cells were diluted to 1,000 cells per microlitre and processed with the 10X Genomics platform within 10 min. Single-cell libraries were generated and sequenced on the Illumina NextSeq500 sequencer.

scRNA-seq analysis

Sequence reads were processed and aligned to the mouse genome (mm10) using CellRanger 3.0. Striatal cells with fewer than 300 genes and genes expressed in more than 3 cells were used for the subsequent analysis in R. Basic processing and visualization of the scRNA-seq data were performed with the Seurat package (v.4.0.5) in R (v.4.0.3). Scrublet was embedded in the Seurat pipeline to remove doublets. Our initial dataset contained 64,836 cells. Data across batches were integrated with canonical correlation analysis (CCA), and annotated with mouse ID and age with a metadata column. The transcript expression was normalized for each cell by the total expression, and multiplied by a scale factor of 10,000, and log-transformed. Next, principal component analysis (PCA) was performed, and the top 30 principal components (PCs) were stored. Clusters were identified with the FindClusters() function by use of the shared nearest neighbour modularity optimization with a clustering resolution set to 0.08. Cell-class clusters were then annotated on the basis of the expression of cell lineage marker genes. Astrocyte cell class was further analysed. Astrocytes were subset, CCA integrated, scaled and normalized; this was followed by PCA analysis, and shared nearest neighbour modularity optimization with a clustering resolution of 0.08 was performed. Six molecular subclusters were identified. Astrocyte cells that were Crym+ were defined if the Crym transcript expression level was greater than 0.25 (2,126 cells), and Crym cells were defined if the Crym transcript expression level was less than 0.25 (2,093 cells). MAST (model-based analysis of single-cell transcripts) comparison identified differentially expressed genes (FDR < 0.05) in Crym+ and Crym cells with a threshold criteria of 0.1 (that is, 10% of cells). Genes were considered commonly expressed if they were expressed in all astrocyte cells, but were not differentially expressed in Crym+ astrocytes. QIAGEN ingenuity pathway analysis (IPA) was performed to identify canonical pathways associated with these genes (made available by QIAGEN Digital Insights).

RNA-seq experiments using the RiboTag method

CAG-Cas9-GFP mice (6–8 weeks old) were bilaterally injected in the central striatum with astrocytic RiboTag (AAV2/5-GfaABC1D-Rpl22-HA) in conjunction with AAVs expressing sgRNAs for Crym (AAV2/5 U6-sgRNA-Crym(×3)-GfaABC1D-mCherry) or for EGFP (AAV2/5 U6-sgRNA-GFP-GfaABC1D-mCherry). RNA was immunoprecipitated from astrocytes or neurons at 10–12 weeks old as previously described33. In brief, freshly dissected striatum tissues were collected and individually homogenized in 1 ml homogenization buffer (50 mM Tris pH 7.4, 100 mM KCl, 12 mM MgCl2, 1% NP-40, 1 mM dithiothreitol (DTT), 1× protease inhibitors, 200 U ml−1 RNAsin, 100 µg ml−1 cyclohexamide and 1 mg ml−1 heparin). RNA was extracted from 20% of cleared lysate as Input (200 µl). The remaining lysate (800 µl) was incubated with 5 µl of mouse anti-HA antibody (Biolegend 901514) with rocking for 4 h at 4 °C, followed by the addition of 200 µl of magnetic beads (Pierce 88803) and overnight incubation with rocking at 4 °C. The beads were washed three times in high-salt solution (50 mM Tris pH 7.4, 300 mM KCl, 12 mM MgCl2, 1% NP-40, 1 mM DTT and 100 µg ml−1 cyclohexamide). RNA was purified from the immunoprecipitated and corresponding input samples (RNeasy Plus Micro, QIAGEN 74034). RNA concentration and quality were assessed with Agilent 2100 Bioanalyzer. RNA samples with an RNA integrity number greater than seven were processed with the Ribo-Zero Gold kit (Epicentre) to remove ribosomal RNA. Sequencing libraries were prepared using the Illumina TruSeq RNA sample preparation kit following the manufacturer’s protocol. After library preparation, amplified double-stranded cDNA was fragmented into 125-bp (Covaris-S2) DNA fragments, which were (200 ng) end-repaired to generate blunt ends with 5’-phosphates and 3’-hydroxyls and adapters ligated. The purified cDNA library products were evaluated using the Agilent Bioanalyzer and diluted to 10 nM for cluster generation in situ on the HiSeq paired-end flow cell using the CBot automated cluster generation system. Samples in each experiment were multiplexed into a single pool to avoid batch effects and 69-bp paired-end sequencing was performed using an Illumina HiSeq 4000 sequencer (Illumina). A yield of between 51 and 108 million reads was obtained per sample. Quality control was performed on base qualities and the nucleotide composition of sequences. Alignment to the Mus musculus (mm10) refSeq (refFlat) reference gene annotation was performed using the STAR spliced read aligner (v.2.7.5c) with default parameters. Further quality control was performed after the alignment to examine: the degree of mismatch rate, mapping rate to the whole genome, repeats, chromosomes, key transcriptomic regions (exons, introns, UTRs and genes), insert sizes, AT/GC dropout, transcript coverage and GC bias. Between 75% and 94% (average 87.6%) of the reads mapped uniquely to the mouse genome. Total counts of read fragments aligned to candidate gene regions were derived using the HTSeq program ( with mouse mm10 refSeq (refFlat table) as a reference and used as a basis for the quantification of gene expression. Only uniquely mapped reads were used for subsequent analyses. Differential expression analysis was conducted with R Project and the Bioconductor package limmaVoom. Weighted gene co-expression network analysis (WGCNA) was performed using an R package. Modules of genes that highly correlated with HD samples were selected. RNA-seq data have been deposited in the Gene Expression Omnibus (GEO) repository (

Metabolomics experiments

Four to six weeks after AAV injection, mice were decapitated and striata were dissected and flash-frozen. Five to eight milligrams of each tissue sample was extracted using a Folch-like method (water, methanol and chloroform) and a bead-based mechanical tissue disruptor. The polar phase was dried and derivatized for GC–MS analysis as previously described50. The results were scaled against calibrated standards and normalized to the frozen weight of the starting material to obtain nmol per mg values.

Proteomic experiments

In vivo BioID2 protein biotinylation

A detailed protocol is available in a previous report48. In brief, three weeks after AAV microinjection with Crym-BioID2 AAV, mice were treated with a subcutaneous injection of biotin at 24 mg per kg (Millipore Sigma RES1052B-B7) dissolved in sterile 0.1 M PBS once per day for seven consecutive days. The mice were processed 16 h after the last biotin injection.

Western blot of in vivo BioID2 samples

Mice were decapitated and the striata were dissected and homogenized with a dounce and pestle in ice-cold RIPA buffer (150 mM NaCl, 50 mM Tris pH 8.0, 1% Triton-X, 0.5% sodium deoxycholate, 0.1% SDS and Halt protease inhibitor (Thermo Fisher Scientific 78429). The homogenate was incubated at 4 °C while spinning for one hour. The homogenate was sonicated and then centrifuged at 4 °C for 10 min at 15,600g. The clarified lysate was collected and the protein concentration was measured using the BCA protein assay (Thermo Fisher Scientific). The samples were then mixed with 2× Laemmli solution (BioRad) containing β-mercaptoethanol. The samples were boiled at 95 °C for 10 min before being electrophoretically separated by 10% SDS–PAGE (30 μg protein per lane) and transferred onto a nitrocellulose membrane (0.45 μm). The membrane was incubated with agitation in a solution containing 5% BSA, 0.1% Tween-20 and 0.1 M PBS for 1 h. The membrane was probed with streptavidin–HRP (Sigma RABHRP3) at 1:250 for two hours. The membrane was then treated with the Pierce chemiluminescence solution for 1 min and imaged. The blot was incubated overnight at 4 °C with rabbit anti-β-actin (1:1,000; Abcam ab8227). IR-dye 800CW anti-rabbit (1:10,000; Li-Cor) secondary was used and images were acquired on a Li-Cor odyssey infrared imager. Signal intensities at the expected molecular weight were quantified using Image J (v.2.1). The streptavidin signal levels were normalized to β-actin by dividing the streptavidin signal intensity by the β-actin signal intensity. Thirty micrograms of protein was loaded into each gel well.

In vivo pull-down of BioID2 biotinylated proteins

The purification of biotinylated proteins was performed as previously described48. Each AAV Crym-BioID2 probe and its counterpart AAV Crym-GFP control were injected into the striatum of six-week-old C57/Bl6NTac mice. Three weeks after AAV microinjection, biotin (Millipore Sigma RES1052B-B7) was subcutaneously injected at 24 mg per kg for seven consecutive days. All mice were processed 16 h after the last biotin injection. Eight mice were used for each biotinylated protein purification and each purification was performed independently five times. Mice were decapitated and striata were microdissected. Striata from four mice were dounce homogenized with 600 μl of lysis buffer 1 (1 mM EDTA, 150 mM NaCl and 50 mM HEPES pH 7.5, supplemented with Halt protease inhibitor (Thermo Fisher Scientific 78429). Immediately after homogenization, 600 μl of lysis buffer 2 (2% sodium deoxycholate, 2% Triton-X, 0.5% SDS, 1 mM EDTA, 150 mM NaCl and 50 mM HEPES pH 7.5) was added. The lysed samples were sonicated for 5 min at 60% power and then centrifuged at 15,000g for 15 min at 4 °C. The resulting supernatant was then ultracentrifuged at 100,000g for 30 min at 4 °C. SDS was added to the supernatant for a final concentration of 1%. The sample was then boiled at 95 °C for 5 min. Sample was cooled on ice and incubated with 35 μl of equilibrated anti-pyruvate carboxylase (5 μg; Abcam 110314) conjugated agarose beads (Pierce 20398) for four hours at 4 °C while rotating. Subsequently, the sample was centrifuged at 2,000 rpm for 5 min at 4 °C and the supernatant was incubated with 80 μl NeutrAvidin agarose at 4 °C overnight while rotating. The NeutrAvidin beads were then washed twice with 0.2% SDS, twice with wash buffer (1% Na deoxycholate, 1% Triton-X and 25 mM LiCl), twice with 1 M NaCl and five times with 50 mM ammonium bicarbonate. Proteins bound to the agarose were then eluted in elution buffer (5 mM biotin, 0.1% RapiGest SF surfactant and 50 mM ammonium bicarbonate) at 60 °C for a minimum of 2 h. The final protein concentration was measured by BCA.

Analysis of biotinylated proteins and bulk mouse tissue using mass spectrometry

Protein samples were subjected to reduction using 5 mM Tris (2-carboxyethyl) phosphine for 30 min, alkylated by 10 mM iodoacetamide for another 30 min and then digested sequentially with Lys-C and trypsin at a 1:100 protease-to-peptide ratio for 4 and 12 h, respectively. The digestion reaction was terminated by the addition of formic acid to 5% (v/v) with centrifugation. Each sample was then desalted with C18 tips (Thermo Fisher Scientific 87784) and dried in a SpeedVac vacuum concentrator. The peptide pellet was reconstituted in 5% formic acid before analysis by liquid chromatography–tandem mass spectrometry (LC–MS/MS).

Tryptic peptide mixtures were loaded onto a 25 cm long, 75 μm inner diameter fused-silica capillary, packed in-house with bulk 1.9 μM ReproSil-Pur beads with 120-Å pores. Peptides were analysed using a 140-min water–acetonitrile gradient delivered by a Dionex Ultimate 3000 UHPLC (Thermo Fisher Scientific), operated initially at a flow rate of 400 nl per min with 1% buffer B (acetonitrile solution with 3% dimethyl sulfoxide (DMSO) and 0.1% formic acid) and 99% buffer A (water solution with 3% DMSO and 0.1% formic acid). Buffer B was increased to 6% over 5 min, at which time the flow rate was reduced to 200 nl per min. A linear gradient from 6–28% B was applied to the column over the course of 123 min. The linear gradient of buffer B was then further increased to 28–35% for 8 min followed by a rapid ramp up to 85% for column washing. Eluted peptides were ionized using a Nimbus electrospray ionization source (Phoenix S&T) by application of a distal voltage of 2.2 kV.

The spectra were collected using data-dependent acquisition on an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) with an MS1 resolution of 120,000, followed by sequential MS2 scans at a resolution of 15,000. Data generated by LC–MS/MS were searched using the Andromeda search engine integrated into the MaxQuant bioinformatic pipelines against the Uniprot M. musculus reference proteome (UP000000589 9606) and then filtered using a ‘decoy’ database-estimated FDR < 1%. Label-free quantification (LFQ) was performed by integrating the total extracted ion chromatogram of peptide precursor ions from the MS1 scan. These LFQ intensity values were used for protein quantification across samples. Statistical analysis of differentially expressed proteins was done using the Bioconductor package ArtMS. To generate a list of proteins with high confidence, all mitochondrial proteins including carboxylases and dehydrogenases were manually filtered because they are artefacts of endogenously biotinylated proteins. Proteins with a log2-transformed fold change (log2FC) > 1 and FDR < 0.05 versus GFP controls were considered putative hits and used for subsequent comparisons between subcompartments and cell types. To account for variations in pull-down efficiency, all proteins and their LFQ values were normalized to pyruvate carboxylase (Uniprot ID Q05920). Downstream analysis was conducted only on proteins with non-zero LFQ values in three or more experimental replicates. Data analysis for whole bulk tissue analyses was performed identically, except that samples were normalized by median intensity. The gene ontology (GO) enrichment analysis for cellular compartments and biological function was performed using the PANTHER overrepresentation test (GO ontology database released 2020-01-01) with FDR < 0.05 with all M. musculus genes used as a reference, and with STRING ( with a confidence score of 0.5 and with all M. musculus genes used as a reference. The GO pathway analysis for the Crym-BioID2 interactome was done with Enrichr (

Protein networks and protein–protein interaction analysis

Network figures were created using Cytoscape (v.3.8) with nodes corresponding to the gene name for proteins identified in the proteomic analysis. A list of protein–protein interactions from published datasets was assembled using STRING. In all networks, node size is proportional to the fold enrichment over GFP control. To identify interactors of µ-crystallin protein, Significance Analysis of INTeractome (SAINTexpress) was used with an FDR cut-off of 0.05. The Bioconductor artMS package was used to reformat the MaxQuant results to make them compatible with SAINTexpress.


The PLA detects native interacting proteins within about 40 nm of each other. Fixed-frozen tissue was processed as described in previous sections. Serial coronal sections (20 μm) containing striatum sparsely labelled with astrocyte-specific AAV2/5 GfaABC1D-tdTomato were prepared using a cryostat microtome (Leica) at −20 °C and mounted immediately onto glass slides. PLAs were performed using the Sigma-Aldrich Duolink PLA fluorescence protocol (Sigma-Aldrich DUO92101 and DUO92013). Sections were baked for 30 min at 60 °C. Sections were washed for at least 15 min in 0.1 M PBS. After washing, sections were incubated in 1× citrate pH 6.0 antigen retrieval buffer (Sigma, C999) for 10 min at 90 °C. After washing three times in 0.2% Triton-X in PBS (PBS-T), the sections were blocked for 45 min at room temperature with 5% donkey serum (Sigma D9663) in PBS-T. Sections were then incubated with the following primary antibodies overnight at 4 °C: rabbit anti-USP9X (1:250, Proteintech 55054-1-AP); rabbit anti-MAPT (1:250, Thermo Fisher Scientific PA-10005); mouse anti-µ-crystallin (1:125, Santa Cruz sc-376687); and guinea pig anti-RFP (1:500; Synaptic Systems 390004). Sections were then incubated with PLA probe cocktail containing the anti-rabbit PLUS primer probe (DUO92002) and the anti-mouse MINUS primer probe (DUO92004) for 1 h at 37 °C. The sections were washed twice in 1× wash buffer A (DUO82049). Sections were then incubated with ligation solution containing ligase for 30 min at 37 °C. Sections were once again washed twice with 1× wash buffer A and then incubated with amplification solution containing DNA polymerase for at least 3 h at 37 °C. Sections were then washed twice in 1× wash buffer B (DUO82049) and then washed in 0.01× wash buffer B. To amplify the tdTomato signal, sections were then incubated with donkey anti-guinea pig Cy3 (1:500; Jackson ImmunoResearch 706-165-148) for 45 min at room temperature. Sections were washed twice with PBS and then coverslips were mounted with DuoLink mounting medium with DAPI (DUO82040). Images were obtained in the same way as for IHC (described above) with a step size of 1 μm. Images were processed using Image J (v.2.1). Astrocyte territories were identified from tdTomato fluorescence, and the number of puncta within each territory was measured. Two negative controls were performed. The first was the same experiment as described above, but for astrocytes located in the dorsolateral striatum that lack μ-crystallin. The second was an independent experiment without the anti-rabbit PLUS primer probe.

Statistical analysis

Statistical tests were run in OriginPro 2017 (OriginLab, v.9.4.2). Summary data are presented as mean ± s.e.m. Sample sizes were not determined in advance and were based on past studies that are cited at the relevant sections of the manuscript and methods. Statistical tests were chosen as described below. All replicates were biological, not technical. Blinding was not done. For each set of data to be compared, we used OriginPro to determine whether the data were normally distributed or not. If they were normally distributed, we used parametric tests. If the data were not normally distributed, we used non-parametric tests. Paired or unpaired Student’s t-test, Wilcoxon signed-rank test, or Mann–Whitney tests were used for statistical analyses with two samples (as appropriate). One-way ANOVA, two-way ANOVA or repeated two-way ANOVA tests followed by Tukey’s post-hoc test were used for statistical analyses with more than three samples. Significant differences were defined as P < 0.05 and are indicated as such throughout.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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