Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Wei Xiong ( wxiong@ustc.edu.cn ).

HEK293T cell line (female) was a kind gift from Dr. Xue T (USTC) and accompanied by authentication documents verifying the identity according to their short tandem repeat profiles. HEK293T cell line was cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (HyClone) supplemented with 10% fetal bovine serum (GIBCO) at 37°C and 5% CO 2 .

All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Science and Technology of China (USTC) and the Chinese Academy of Sciences (CAS). Mice were group-housed in groups of 2-5 with a 12-h light/dark cycle (lights off at 7 p.m.) and free access to food and water ad libitum under specific pathogen-free conditions. Male C57BL/6J mice were used for all experiments unless otherwise specified. Male triple knockout (TKO) mice (rod α transducin knockout, Gnat; cyclic nucleotide gated channel alpha 3 knockout, Cnga3; melanopsin knockout, Opn4) were used for sampling. The C57BL/6J mice were purchased from Beijing Vital River Laboratory Animal Technology. Genotyping of the TKO mice was performed using the primers provided in Table S1 . Experimenters were blinded to all experimental conditions until all data were obtained. Unless otherwise specified, the mice were randomly assigned to control or treatment groups in all experiments.

Method Details

UVB Irradiation 2 of UVB (7 μW/cm2, 120 min) and then placed back to their home cages before testing. The irradiation intensity of UV was detected using a UV irradiometer (Photoelectric instrument factory of Beijing normal university, Beijing, China) placed on the bottom of the acrylic chamber. The suberythematic dose was determined on the basis of previous studies ( D’Orazio et al., 2006 D’Orazio J.A.

Nobuhisa T.

Cui R.

Arya M.

Spry M.

Wakamatsu K.

Igras V.

Kunisada T.

Granter S.R.

Nishimura E.K.

et al. Topical drug rescue strategy and skin protection based on the role of Mc1r in UV-induced tanning. Fell et al., 2014 Fell G.L.

Robinson K.C.

Mao J.

Woolf C.J.

Fisher D.E. Skin β-endorphin mediates addiction to UV light. Mice were dorsally shaved 2 days before the start of radiation exposure. During the UVB irradiation experiment, mice were kept in an acrylic chamber (15 × 30 × 50 cm) with a power-adjustable UVB lamp (G15T8E, Sankyo Denki, Japan) on top. The shaved mice were exposed to 50 mJ/cmof UVB (7 μW/cm, 120 min) and then placed back to their home cages before testing. The irradiation intensity of UV was detected using a UV irradiometer (Photoelectric instrument factory of Beijing normal university, Beijing, China) placed on the bottom of the acrylic chamber. The suberythematic dose was determined on the basis of previous studies ().

Mouse Serum and Cerebrospinal Fluid (CSF) Preparation For serum collection, the mouse blood was collected into regular 1.5 mL Eppendorf tubes and stored at room temperature for 30 min, and then centrifuged for 10 min at 4500 rpm (PICO 17, Thermo Scientific, USA) at 4°C. The supernatant was transferred to a vial and stored at −20°C until analyzed by mass spectrometry (MS). To collect mouse CSF samples, the Isoflurane-anesthetized wild-type male C57BL/6J mice were mounted on a stereotaxic system (68015, RWD Life Science, China). After drilling through the skull, a 5 μL syringe (Hamilton) was lowered into the lateral ventricle (1.0 mm lateral, 0.3–0.5 mm posterior, 2.3–2.5 mm depth from bregma). CSF was withdrawn at a rate of 400 nL/min. Approximately 1 μL of CSF was collected per mouse. All mouse serum (5 μl) and CSF samples were diluted 30 folds using methanol-H 2 O solution (30:70, v/v). Atenolol (38-188 pM) was added as an internal standard for MS analysis.

Brain Slice Preparation The secondary motor cortex (M 2 ), hippocampus (HPC), central nucleus of the amygdala (CeA), dorsal striatum (DS), nucleus accumbens (NAc) and prefrontal cortex (PFC) slices were prepared from C57BL/6J mice aged P28-P30. The mice were sacrificed and then the brains were rapidly removed from the skull and immersed in ice-cold pre-oxygenated cutting solution containing sucrose 194 mM, NaCl 30 mM, NaHCO 3 26 mM, MgCl 2 1 mM, glucose 10 mM, KCl 4.5 mM, and NaH 2 PO 4 1.2 mM, pH 7.4. Coronal sections (300 μm) were cut using a Vibratome (VT 1200S, Leica, Germany). The tissue slices were incubated in a holding chamber containing oxygenated (95% O 2 and 5% CO 2 ) artificial cerebrospinal fluid (aCSF), which consisted of NaCl 124 mM, NaHCO 3 26 mM, KCl 4.5 mM, NaH 2 PO 4 1.2 mM, MgCl 2 1 mM, CaCl 2 2 mM, and glucose 10 mM (pH 7.4; osmolality, 315 mOsm/kg). For the mouse brain slices (coronal or sagittal planes) recovered from mechanical injury, they were incubated at 32°C oxygenated (95% O 2 and 5% CO 2 ) aCSF for approximately 30 min and then cooled to room temperature (21-23°C) at least 30 min before recording.

Single-Neuron MS Zhu et al., 2017 Zhu H.

Zou G.

Wang N.

Zhuang M.

Xiong W.

Huang G. Single-neuron identification of chemical constituents, physiological changes, and metabolism using mass spectrometry. 4 HCO 3 185 mM and NH 4 Cl 80 mM) by applying negative pressure. The neurons were clamped at ‒70 mV after the patched cell membrane was broken by rapidly applied negative pressure. After the electrophysiological data were recorded, the cytoplasmic chemical constituents were obtained from the assayed neuron by applying negative pressure. Only neurons with tightly held seals (> 1 GΩ) and nonruptured membranes were selected for analysis, avoiding the dilution of intracellular fluid by aCSF. Once a sufficient amount of fluid was withdrawn from the cell, the patch pipette was quickly removed from the slice and then assayed via MS. After extraction of the cell cytoplasmic chemical constituents, the capillary was coupled to the single-neuron MS, which had been fabricated as previously described (). After the brain slices recovered from mechanical injury, they were transferred to the recording chambers. The neurons were randomly chosen for the subsequent electrophysiological recording and MS analysis. We approached the neuron using a micromanipulator (MP-285, Sutter) and patched the neuron with borosilicate glass pipettes filled with pipette solution (NHHCO185 mM and NHCl 80 mM) by applying negative pressure. The neurons were clamped at ‒70 mV after the patched cell membrane was broken by rapidly applied negative pressure. After the electrophysiological data were recorded, the cytoplasmic chemical constituents were obtained from the assayed neuron by applying negative pressure. Only neurons with tightly held seals (> 1 GΩ) and nonruptured membranes were selected for analysis, avoiding the dilution of intracellular fluid by aCSF. Once a sufficient amount of fluid was withdrawn from the cell, the patch pipette was quickly removed from the slice and then assayed via MS. An AC voltage with an amplitude of 4 kV at ∼500 Hz was applied outside of the spray capillary micropipette. The tip of the spray micropipette was kept ∼5 mm away from the orifice of the MS instrument. High resolution mass measurements were analyzed using an Exactive plus MS instrument (Thermo Fisher Scientific, San Jose, CA, USA). Its main experiment parameters were set as follows: Capillary temperature, 275°C; S-lens radio frequency (RF) level, 50%; mass resolution, 70,000; maximum inject time, 10 ms; and microscan, 1. Other MS experiments were conducted with a LTQ Velos Pro. MS instrument (Thermo Fisher Scientific, San Jose, CA, USA). MS parameters were capillary temperature 275°C, S-lens RF level 42%, maximum injection time 300 ms, and microscan 1. Chemical structure were confirmed by tandem MS using LTQ Velos Pro. MS instrument based on collision-induced dissociation (CID) with helium (He) as background gas at 28% energy. Commercial electrospray ionization (ESI) source was removed before our experiments. Positive ion mode was used in our experiments.

13C-Isotope Tracing Experiments To trace HIS metabolism, 1-13C-HIS was added into aCSF at a final concentration of 3 mg/ml. After the mouse brain slices (coronal or sagittal planes) recovered from mechanical injury, they were transferred to oxygenated aCSF with or without 1-13C-HIS and incubated for 1 h at room temperature. After the mouse slices incubated with aCSF containing 1-13C-HIS, they were washed three times with oxygenated aCSF at room temperature and then transferred to the recording chambers. Other experimental procedures were the same as those for single-neuron MS. To trace UCA metabolism in vivo, we intravenously (i.v.) injected 1,2,3-13C 3 -UCA (20 mg/kg, 2.5 mg/ml) diluted in saline into the mice 30 min before serum/CSF collection and brain slices preparation. Saline is the corresponding vehicle solution. All the collected samples were analyzed using MS. All drug treatment or control groups were blinded from experimenters.

Calibration Curves of UCA Estimations of intracellular UCA levels in neurons were calculated from calibration curves with external UCA standards at concentrations ranged from 1 μM, 10 μM, 100 μM and 300 μM with three replicates per concentration point. All calibrations were obtained with artificial intracellular solution: potassium gluconate 130 mM, NaCl 6 mM, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) 11 mM, 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) 10 mM, CaCl 2 1 mM, NaOH 4 mM, MgCl 2 1 mM, Mg-ATP 2 mM and Na-GTP 0.2 mM (pH was adjusted to 7.3).

Lentivirus Production and Transduction HEK293T cells in a 10-cm plate were seeded at 70% confluence 1 day before transfection. The cells were transfected the next day at 85% confluence with 6 μg of plasmid containing Uroc1 shRNA or control shRNA, 6 μg of pRSV-Rev, 6 μg of pMDLg/pRRE and 3 μg of pMD2.G (Addgene) together with 21 μL of Lipofectamine 2000 (Invitrogen). The medium was replaced 10 h after transfection. Viral supernatant was harvested 70 h post-transfection, filtered with a 0.45-μm filter (Millipore), and centrifuged for 2.5 h at 30,000 g 4°C. After centrifugation, the supernatant was removed and the pellet was resuspended with 20 μL of PBS containing 10% FBS and 8 μg/μl polybrene and stored at −80°C.

RNA Interference Small interfering RNA (siRNA) were used for histidase knockdown. HEK293T cells were co-transfected with either targeting siRNA or control siRNA (Genepharma) and histidase overexpressing plasmid (OriGene) using lipofectamine 2000 reagent for 48 h. For knockdown of urocanase, IPPAnase or FTCD, short hairpin RNA (shRNA) plasmid constructs (Hanbio) were co-transfected with overexpressing plasmids of urocanase, IPPAnase or FTCD (OriGene) into HEK293T cells for 48 h. The subsequent western blotting analysis were performed to validate the knockdown efficacy of siRNA or shRNA. The most efficient shRNA plasmid construct was used to package AAV virus (Hanbio) for in vivo knockdown of urocanase, IPPAnase or FTCD. Several independent siRNA duplexes or shRNA plasmid constructs were used and the sequence were listed in Table S2

Synaptosomal Preparation Kamat et al., 2014 Kamat P.K.

Kalani A.

Tyagi N. Method and validation of synaptosomal preparation for isolation of synaptic membrane proteins from rat brain. 2 2 mM, MgCl 2 1 mM, glucose 5 mM, HEPES 5 mM, pH 7.4) supplemented with protease inhibitor cocktail. Tissue was homogenized using a Dounce homogenizer and the homogenate was centrifuged at 800 g, 4°C, 10 min to collect the supernatant (S1). The supernatant (S2) containing cytosol was collected for electrophoresis after the S1 was centrifuged at 10,000 g, 4°C, 20 min. The pellet (P2) containing synaptosomes were suspended in 0.8 M sucrose HEPES buffer solution and further centrifuged at 20,000 g, 4°C, 30 min. The pellet was washed in HEPES buffer twice and were suspended in lysis buffer solution (Tris 50 mM, NaCl 150 mM, 1% NP-40, 0.5% Sodium dexyocholate supplemented with protease inhibitor cocktail as the synaptosome). The synaptosomal preparation was used for western blotting analysis. Mouse brain synaptosomes were prepared as previously described (). Briefly, the mice were anaesthetized and perfused with ice-cold saline. The brain was removed and kept on ice-cold 0.32 M sucrose HEPES buffer (NaCl 145 mM, KCl 5 mM, CaCl2 mM, MgCl1 mM, glucose 5 mM, HEPES 5 mM, pH 7.4) supplemented with protease inhibitor cocktail. Tissue was homogenized using a Dounce homogenizer and the homogenate was centrifuged at 800 g, 4°C, 10 min to collect the supernatant (S1). The supernatant (S2) containing cytosol was collected for electrophoresis after the S1 was centrifuged at 10,000 g, 4°C, 20 min. The pellet (P2) containing synaptosomes were suspended in 0.8 M sucrose HEPES buffer solution and further centrifuged at 20,000 g, 4°C, 30 min. The pellet was washed in HEPES buffer twice and were suspended in lysis buffer solution (Tris 50 mM, NaCl 150 mM, 1% NP-40, 0.5% Sodium dexyocholate supplemented with protease inhibitor cocktail as the synaptosome). The synaptosomal preparation was used for western blotting analysis.

Western Blotting HEK293T cells co-transfected with overexpressing plasmids and shRNA plasmids were collected and lysed in lysis buffer solution. Mouse brain tissues were homogenized and sonicated followed by centrifugation at 10,000 g, 4°C for 30 min. The supernatants were diluted 1:1 with laemmli 2X sample buffer and denatured at 99°C for 10 min. The samples were separated on 12% SDS/PAGE gels. After electro-transferring to a 0.45 μm PVDF membrane, the membrane were blocked in 5% BSA and blotted with following antibodies: anti-HAL (Abcam, 1:500), anti-UROC1 (Novus, 1:250), anti-AMDHD1 (Sigma, 1:250) and anti-GAPDH (Millipore, 1:40000). HRP-conjugated secondary antibodies and chemiluminescent substrate (Thermo Fisher) were used to detect signals.

Brain Tissue Preparation and Immunohistochemistry Mice (N = 3) were deeply anesthetized by intraperitoneal (i.p.) injection of pentobarbital (40 mg/kg), and then perfused with chilled PBS, followed by 4% paraformaldehyde (PFA) in PBS. Brains were removed and immersed in the same fixative solution (4% PFA in PBS) overnight, then were subsequently incubated in 30% (wt/vol) sucrose in PBS for 24 h at 4°C. Sagittal brain sections 40 μm thick were prepared using a cryostat (CM 1860, LEICA Microsystems) at −20°C and collected into a 12-well plate. After a PBS wash (three times within 5 min), sections were incubated for 20 min in 3% peroxide mixed in methanol in order to inactivate the endogenous peroxidase. Sections were incubated with 0.3% Triton X-100 and 5% normal goat serum in PBS. After 40 min of blocking, the sections were incubated with rabbit anti-Histidase antibody (1:200, ab154063, Abcam), anti-urocanase antibody (1:200, NBP1-93883, Novus), or anti-IPPAnase antibody (1:200, HPA039720, Sigma) in PBS for 24 h at 4°C. On the following day, the sections were washed in PBS and incubated with biotinylated goat anti-rabbit (1:1000, PK-6101, Vector Laboratories) for 2 h. After a PBS wash, sections were reacted with avidin-biotin complex (ABC, PK6101, Vector Laboratories) for 30 min. Brain sections were washed with PBS and collected on gelatin-coated slides, air-dried. Slides were incubated with 3,3′-diaminobenzidine (DAB, SK-4100, Vector Laboratories) for 2-4 min before a distilled-water wash. Slides were dehydrated in alcohol gradient (90, 95, 100%), immersed in xylene, mounted with Neutral Balsam (E675007, Sangon Biotech). All the incubations were done at room temperature, unless mentioned otherwise.

Fluorescent in Situ Hybridization (FISH) Experiment 2 O 2 to block the endogenous peroxidase activity. After that the slides were treated with 0.5% Triton X-100/DEPC-PBS for 20 min, at last acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0). After acetylation, the sections were prehybridized for 2 h at 60°C, and then hybridized for with either scrambled or positive probe for 16 h at 60°C. After washing twice in 2 × SSC at 60°C for 15 min each, the samples were treated with 5 μg/ml RNase A at 37°C. They were sequentially washed twice in 0.2 × SSC at 60°C for 30 min each. Probes were detected by standard method using anti-Digoxigenin-POD Fab fragments (Roche) and TSA-Plus Cyanine 3 system (PerkinElmer). For FISH, the detected fragment of mouse urocanase cDNA was cloned into pEASY-T3 with the pair of primers provided in the Key Resources Table . Probes were prepared with DIG RNA Labeling Mix (Roche) by in vitro transcription with T7 or SP6 polymerases. Frozen sections with 15 μm thick were collected on PLL-coated slides, then further fixed in 4% PFA/ PBS for 10 min, subsequently washed with DEPC-PBS for 10 min, and then treated with 3% Hto block the endogenous peroxidase activity. After that the slides were treated with 0.5% Triton X-100/DEPC-PBS for 20 min, at last acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0). After acetylation, the sections were prehybridized for 2 h at 60°C, and then hybridized for with either scrambled or positive probe for 16 h at 60°C. After washing twice in 2 × SSC at 60°C for 15 min each, the samples were treated with 5 μg/ml RNase A at 37°C. They were sequentially washed twice in 0.2 × SSC at 60°C for 30 min each. Probes were detected by standard method using anti-Digoxigenin-POD Fab fragments (Roche) and TSA-Plus Cyanine 3 system (PerkinElmer). gaactggacacaacgggggaactcttggtggacctcgggtcagaccagacatcctgtcacaacccgttcaatggcggctactaccctgtgcagctcagcttctcagaggcccagagcctcatgtcctccaaccctgctgccttcaagcacctggtgcaggaaagcctgaggaggcatgtcgcagccatcaacaggttggcccaagagaagttcttcttctgggactatggtaatgccttcctcttggaggctcagagagcaggagcagatgtagagaagaaaggagccaacaagatggagttccgctacccctcctatgtccagcacattatgggggatatattctcccagggctttgggcccttccgctgggtatgcacatcaggggacccccaggacctggctgtcactgatcatctggccacatctgtactggagaaagccattgctgatggagtgaaggcatctgtgaagctacagtatatggataatatacgctggatccgagaggctgccaagcatcagctggtggtgggctcccaggcaaggatcctgtactcagaccagaaaggccgtgtggccattgctgtggccattaaccaggccatcgccagtgggaagatcaaggcaccagtggtcctgagccgtgaccaccatgatgtgagtggcactgacagccccttcagggagacttcgaatatttatgacggttctgccttctgcgcagacatggccgtgcagaacttcgtgggagatgcctgtcgtggtgccacctgggttgcacttcacaatggagggggtgtaggctggggtgaagtcatcaatgggggatttggccttgttctagatggaactgcagaggctgagcagaaagccaggatgatgctcagctgggatgtctccaatgg. Detected sequence:

Magnetic Resonance Imaging Experiments Male mice aged 8-10 weeks were used. The integrated animal/MRI facility at high magnetic field laborary allowed for the measurements to be taken without requiring the mice to be euthanized at the end of experiment, and all animals were returned to the animal facility after they had fully recovered from the experimental procedures. All magnetic resonance imaging (MRI) experiments were performed on a 9.4 Tesla 40-cm horizontal bore MR spectrometer (Agilent Technologies, Santa Clara, CA, USA). The animals were placed in a prone position on a specially designed cradle and inserted into the magnet. A home-built radiofrequency (RF) coil were used for both RF transmission and signal detection. Mice were anaesthetized with isoflurane (3.5% induction, 1.5%–2.0% maintenance in air/O 2 2:1) for the duration of the scan (approximately 2.5 h). Respiratory rate and rectal temperature were monitored throughout the experiment with a physiologic monitoring unit (model 1030; SA Instruments, Stony Brook, NY), and the body temperature was maintained at 36.5°C with a homemade heating pad. Reference T 2 -weighted images were acquired, from which the spectroscopic region of interest were determined in the cortical M 2 area as well as the HPC CA3 area. Subsequently, the i.v. injection of UCA (20 mg/kg, 2.5 mg/ml diluted in saline) began at a constant rate of 100 μl/min through the tail vein with a syringe micro-pump, and the procedure was completed within 2 min. MR spectra were acquired at 0 min, 20 min, 40 min, 60 min, 80 min and 100 min post-injection of UCA solution. 2 -weighted MR images were acquired using a fast spin echo sequence with a repetition time (TR) of 5000 ms, echo spacing = 10 ms, echo train length (ETL) = 4; additionally, localized proton spectra from the M 2 and HPC regions outlined on the T 2 -weighted images were acquired using the following parameters: effective echo time (TE) = 30 ms, field of view (FOV) = 16 × 16 mm2, matrix size = 192 × 192, slice thickness = 1 mm (10 slices, gap = 0), and bandwidth (BW) = 25 kHz. A LASER (Localization by Adiabatic Selective Refocusing) sequence ( Garwood and DelaBarre, 2001 Garwood M.

DelaBarre L. The return of the frequency sweep: designing adiabatic pulses for contemporary NMR. 3, spectral width = 4006 Hz, and Free induction decay size = 4096 with 512 averages. Typical spectra line widths for water resonance after 3D shimming were approximately 20 Hz. -weighted MR images were acquired using a fast spin echo sequence with a repetition time (TR) of 5000 ms, echo spacing = 10 ms, echo train length (ETL) = 4; additionally, localized proton spectra from the Mand HPC regions outlined on the T-weighted images were acquired using the following parameters: effective echo time (TE) = 30 ms, field of view (FOV) = 16 × 16 mm, matrix size = 192 × 192, slice thickness = 1 mm (10 slices, gap = 0), and bandwidth (BW) = 25 kHz. A LASER (Localization by Adiabatic Selective Refocusing) sequence () was used in this study. The acquisition parameters were: TR/TE = 2000/36 ms, voxel size = 1 × 1 × 2 mm, spectral width = 4006 Hz, and Free induction decay size = 4096 with 512 averages. Typical spectra line widths for water resonance after 3D shimming were approximately 20 Hz.

Electrophysiological Recording A and glycine receptors), diluted with aCSF. For mEPSC experiments, brain slices from C57BL/6J mice expressing pAAV-mCamKIIα-hChR2(H134R)-EGFP in M 2 or HPC CA3 were cut for ChR2 stimulation test. Dorsal striatum neurons or HPC CA1 neurons were recorded with single optical stimulation of GLUM2→DS or GLUCA3→CA1 projecting axons, respectively. Only neurons respond to a 473-nm laser (AniLab-Opto, 10 mW/mm2) were selected for the following tests. The mEPSCs were collected after TTX (final concentration: 0.5 μM) was added to the bath perfusion. To measure presynaptic glutamate release of M 2 -DS or HPC CA3-CA1 circuit, the multiple probability fluctuation analysis was used as previously described ( Scheuss and Neher, 2001 Scheuss V.

Neher E. Estimating synaptic parameters from mean, variance, and covariance in trains of synaptic responses. Silver, 2003 Silver R.A. Estimation of nonuniform quantal parameters with multiple-probability fluctuation analysis: theory, application and limitations. Suska et al., 2013 Suska A.

Lee B.R.

Huang Y.H.H.

Dong Y.

Schlüter O.M. Selective presynaptic enhancement of the prefrontal cortex to nucleus accumbens pathway by cocaine. Slices were transferred into the recording chamber continuously perfused at 3-4 ml/min with aCSF at 28°C. Neurons were visualized using a fixed-stage microscope (BX50WI, Olympus, Tokyo, Japan) with differential interference contrast and infrared illumination. We obtained all whole-cell voltage clamp mode recordings using borosilicate glass pipettes (5-7 MΩ) filled with internal solutions. The internal solution (pH 7.2) for excitatory postsynaptic currents (EPSC) recording contained K-gluconate 145 mM, HEPES 5 mM, Mg-adenosine triphosphate (ATP) 5 mM, Na-guanosine 5′-triphosphate (GTP) 0.2 mM, and EGTA 10 mM. The miniature excitatory postsynaptic currents (mEPSCs) were recorded with a holding potential of −70 mV and PTX (100 μM), which was used to block the inhibitory neurotransmitter receptors (GABAand glycine receptors), diluted with aCSF. For mEPSC experiments, brain slices from C57BL/6J mice expressing pAAV-mCamKIIα-hChR2(H134R)-EGFP in Mor HPC CA3 were cut for ChR2 stimulation test. Dorsal striatum neurons or HPC CA1 neurons were recorded with single optical stimulation of GLUor GLUprojecting axons, respectively. Only neurons respond to a 473-nm laser (AniLab-Opto, 10 mW/mm) were selected for the following tests. The mEPSCs were collected after TTX (final concentration: 0.5 μM) was added to the bath perfusion. To measure presynaptic glutamate release of M-DS or HPC CA3-CA1 circuit, the multiple probability fluctuation analysis was used as previously described (). Briefly, a train of five light pulses at 20 Hz frequency and 5-ms duration was used to stimulate the ChR2-expressing axons every 10 s. 50-100 evoked EPSCs were recorded at five release probability conditions. Experimental results were discarded if the series resistance varied by more than 15% or exceeded 25 MΩ.

AMPA/NMDA Ratio Measurement To evoke EPSC currents in hippocampal CA1 pyramidal neurons, Schaffer collateral was stimulated using a concentric circular electrode every 20 s. PTX (100 μM) was diluted with aCSF to block the inhibitory neurotransmitter receptors (GABA A and glycine receptors). CA1 Pyramidal neurons were voltage-clamped at −70 and +40 mV to record AMPAR-mediated and NMDAR-mediated currents respectively. For each neuron, the total 10 sweeps of AMPAR and NMDAR-mediated currents were recorded and the average value of currents were then calculated. The NMDAR-mediated current was measured at 50 ms after the stimulation. The AMPAR-mediated current was measured as the peak value. Then, the AMPA/NMDA ratio was calculated by dividing average AMPA EPSC by NMDA EPSC.

Stereotaxic Surgery, Cannula Placement and Microinjection Mice were anaesthetized with pentobarbital sodium (i.p., 0.5%, 0.01 ml/g) and placed on a stereotaxic apparatus (68015, RWD Life Science, China). Ophthalmic ointment was applied to prevent the eyes from drying. A midline scalp incision was made, and a craniotomy was drilled above each cannula implantation or injection site. For local drug infusion, guide cannula (62001, RWD) were implanted into lateral ventricles (anteroposterior, A/P: −0.3 mm; medio-lateral, M/L: −1.0 mm; dorsoventral, D/V: −1.8 mm). The glycyl-glycine (GG) was dissolved in saline (13.2 μg/μl, 0.9% NaCl). Thirty min before the experiment, infusion needles attached to 10 μL syringes were inserted into the guide cannula, and 1 μL of GG solution was delivered within 2 min. For lentiviral shRNA or pAAV-mCamKIIα-hChR2(H134R)-EGFP injection, glass micropipette attached to 10 μL syringes were placed bilaterally into the M 2 cortex (A/P: +1.3 mm; M/L: 0.7 mm; D/V: −0.8 mm) or the CA3 region of the hippocampus (A/P: −1.7 mm; M/L: 2.2 mm; D/V: 2.25 mm), and 0.5 μL of virus was infused per side at a rate of 50 nl/min using a pump (Legato130, KD Scientific). Mice were allowed to recover for 2 weeks before behavioral tests or electrophysiological recordings. For fluorescence imaging, we injected 0.5 μL of AAV-CAG-EGFP (1.6 × 108 vg/ml, Obio Technology) into the CA3 and waited 3 weeks

Immunofluorescence after Intracerebroventricular (ICV) administration of GG For NeuN immunofluorescence, after 2-h ICV injection of GG or saline, male C57BL/6J mice from both groups (N = 3 mice each group) were deeply anesthetized by pentobarbital (i.p., 40 mg/kg), and then perfused with chilled PBS, followed by 4% PFA in PBS. Brains were collected and immersed in 4% PFA in PBS overnight, then were subsequently incubated in 30% (wt/vol) sucrose in PBS for 24 h at 4°C as above described. Coronal brain sections (40 μm) were prepared using a cryostat (CM 1860, LEICA Microsystems) at −20°C and collected into a 12-well plate. After a PBS wash (three times within 5 min), sections were incubated for 20 min in PBS containing 10% goat serum and 0.3% Triton X-100.Then slices were incubated with mouse anti-NeuN antibody (1:200, Millipore) at 4°C overnight. The slices were then incubated with goat anti-mouse secondary antibody conjugated with alexa 568 (1:1000, invitrogen) for 1 h at room temperature. After washing with PBS for three times, slices were sealed for imaging. Immunofluorescence-labeled images were examined by confocal microscopy (LSM 880, Olympus).

Motor Learning Hirata et al., 2016 Hirata H.

Takahashi A.

Shimoda Y.

Koide T. Caspr3-deficient mice exhibit low motor learning during the early phase of the accelerated rotarod task. Wikgren et al., 2012 Wikgren J.

Mertikas G.G.

Raussi P.

Tirkkonen R.

Äyräväinen L.

Pelto-Huikko M.

Koch L.G.

Britton S.L.

Kainulainen H. Selective breeding for endurance running capacity affects cognitive but not motor learning in rats. We used the rotarod training system (XR1514, Xinruan, Shanghai, China) to test the motor skill learning of mice as previously described (). Before the first training sessions, the mice were habituated to stay on a stationary rod for 2 min. To assess motor learning, a total of six trials for the rotarod test were carried out using an accelerating protocol from 4 to 60 rpm in 300 s with 20-min inter-trial intervals. Three training sessions every day were performed for 2 days. After falling, the mice were immediately placed back to their home cages, and the time to fall was automatically recorded by the rotarod software. The mice were taken from the rod and the latency was recorded as 300 s, if stayed on the rotarod more than the full 300 s of a trial. The apparatus and testing area were cleaned with 75% ethanol, when the trials were finished.