Lrp4LacZ/+mice were described before; in brief words, β-galactosidase (β-gal) protein cassette, including stop code and a polyadenylation termination signal, was inserted into the downstream of Lrp4 promoter.Lrp4ECD/ECD mice (JAX stock #013157) were described before, which introduced a stop codon just upstream of Lrp4 TMD[6, 7, 18]. Mice were housed in a room 12-h light/dark cycle, 23-25°C, with ad libitum access to rodent chow diet and water. Experiments involving animals were conducted according to the “guidelines for the care and use of experimental animals” issued by Nanchang University, following the directive 2010/63/EU to protect animals used for scientific purposes. For in vivo experiment, surgery was executed with sodium pentobarbital anesthesia (50 mg/kg, ip injection), and all efforts were made to minimize suffering . Male mice were utilized for the experiments, and after terminal experiments, the mice were euthanized by carbon dioxide inhalation.
Western blotting was performed as described previously with minor modifications. In brief, total proteins were extracted by RIPA buffer (150 mM NaCl, 1.0% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 50 mM pH 8.0 Tris), supplementary with phenyl methane sulfonyl fluoride (PMSF) and proteinase inhibitor mix before using. After electrophoresis, samples were transferred to the PVDF membrane (Millipore, USA) with transfer buffer (25 mM Tris, 192 mM Glycine, 20%(v/v) Methanol). The membrane was blocked by blocking buffer(5%(m/v) Skim-milk, 20 mM Tris, 150 mM NaCl, 0.1%(v/v) Tween20) for 2 h and was washed 3 times with washing buffer (20 mM Tris, 150 mM NaCl, 0.1%(v/v) Tween20). Anti-LRP4 (Rabbit-anti-mouse, Lab produced, 1:1000) and anti-α-tubulin (mouse monoclonal, SC-23948, Santa Cruz Biotechnology, 1:1000) primary antibody was added and incubated at 4°C overnight. The HRP-labeled secondary antibody (Goat anti-Mouse IgG 31431, Goat anti-Rabbit IgG, 31466, Thermo Fisher Scientific,1:2000) was added to incubate at room temperature for 2h and then washed 3 times. LuminataTM Crescendo Western HRP Substrate was added. Immunoreacted bands were captured by an enhanced chemiluminescence system (BIO-RAD, USA).
Quantitative real-time PCR (qPCR)
Total RNA was isolated from mice brain tissues according to the manufacturer’s instructions of TRIzol Reagent (Invitrogen), and complementary DNA (cDNA) was synthesized following the manufacturer’s protocol of High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, 4368814). The qPCR primer sets as below: Lrp4 (5′- GTGTGGCAGAACCTTGACAGTC-3′ and 5′-TACGGTCTGAGCCATCCATTCC-3′), and Gapdh (5′-CATCACTGCCACCCAGAAGACTG-3′ and 5′-ATGCCAGTGAGCTTC CCGTTCAG-3′). qPCR was carried out by the StepOnePlus Real-Time PCR system (Applied Biosystems) using the mix. Expression levels of mRNA were normalized to the reference gene Gapdh using the ΔCT method.
In behavioral tests, the activity levels of the mice were evaluated at P50. The open field (40 × 40 × 20cm) was used to measure the mice’s moving distance over 10 min. The data were recorded using a video camera, and the data were analyzed using the behavior analysis software ANY-maze (Stoelting Co., Wood Dale, IL, USA).
Buried food-seeking test
Mice were food-deprived for 2 d, trained for 2 d, and tested continuously 3 d, with ad libitum access to enough water all the time. Food was randomly placed in the box and was buried under padding for 0.5cm in testing trials. The mice seizing the food with their front paws and biting with mouth were regarded as finding the food. The time was recorded when the mice were placed in the container and found the food.
X-gal(5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside), the inert chromogenic substrate for β-gal, hydrolyzes X-gal into colorless galactose and 4-chloro-3-brom-indigo, forming an intense blue precipitate. Mice brains were fixed for 8-10 h in 2%(m/v) paraformaldehyde (PFA) at room temperature and then were transferred into 30% sucrose solution at 4°C. The brain slices were added PBS (phosphate-buffered sodium, pH 7.4) in a wet box, washing the slices at room temperature 3 times with PBS. After rinsing for 10 min, adding dye solution, putting the slices at 37°C for 8 h. After the reaction, brain slices were washed 3 times with PBS.
Immunohistochemistry co-staining with X-gal
X-gal-stained brain slices were immersed in blocking solution (10%(v/v) donkey serum, 1%(m/v) calf serum albumin, 0.5%(v/v) Triton X-100 in PBS) for 2 h. Then the slices were rinsed with PBS at room temperature 3 times. Incubating the brain slices with the primary antibody (Rabbit anti-GFAP antibody, Z0334, Dako,1:1000) at 4°C overnight. The slices were incubated with a secondary antibody (Alexa Fluor® 488 Goat anti-rabbit, A32731TR, Thermo Fisher Scientific, 1:1000) at room temperature for 2 h. Brain slices were washed with PBS 3 times, and then the images were captured by a microscope (Olympus FSX100).
Nuclear fast red counterstaining
Put the X-gal-stained or co-stained brain slices into a vitreous tank containing nuclear fast red staining solution for 5 min. Slides with the slices were put into glass tanks containing 50%, 75%, and 90%ethanol in sequence, each for 4 min. The slides were transferred into 100% ethanol 2 times. Then the slides were put into xylene for 5 min. At last, the slides were sealed with mounted in Hydro mount (National Diagnostics). Images were captured by an inverted fluorescence microscope (Olympus FSX100).
The brain slices were rinsed with PBS at room temperature and were immersed with antibody blocking solution (0.5%(v/v) Triton X-10010%(v/v) donkey serum, 1%(m/v) calf serum albumin, in PBS) at room temperature for 2 h. And then, the slices were rinsed with PBS at room temperature 3 times. The slices were incubated with primary antibody anti-NeuN (mouse monoclonal, MAB377, Merck Millipore, 1:1000) at 4°C overnight. The slices were washed with PBS at room temperature 3 times, each time for 10 min. The secondary antibody (Alexa Fluor® 568 Goat anti-Mouse A-11019, Thermo Fisher Scientific, 1:1000) was added, and then the slices were incubated at room temperature for 2 h in the dark. After washing 3 times with PBS, samples were mounted in a Hydro mount (National Diagnostics).
Golgi staining was performed following the FD Rapid Golgi Stain™ Kit (FD Neuro Technologies, PK-401, USA). Staining solution D and solution E were mixed with ultra-pure water in a ratio of 1:1:2. Dying at room temperature for 10 min. Slides with the slices were washed in ultra-pure water twice, then put into the plate hole containing 50%, 75%, 90%, and 100% ethanol in sequence, each for 4 min. Dehydration for 3 times, then the slides were put into xylene for 1 h and mounted in Hydro mount (National Diagnostics). Images were captured by an Olympus fluorescence microscope (FSX100), and dendritic spines were counted with image J.
The electrophysiological recording was performed as previously described[21, 22]. Brain sections of 300μm thickness were cut with a vibratome (Leica, VT1000S) in oxygenated (95% O2, 5% CO2) sectioning buffer (120 mM Choline-Cl, 2.5 mM KCl, 0.5 mM CaCl2, 7 mM MgCl2, 1.25 mM NaH2PO4, 26 mM NaHCO3, and 25 mM glucose)at 4°C. Slices were then placed into the oxygenated artificial cerebrospinal fluid(ACSF) (124 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 2 mM MgSO4, 1.25 mM NaH2PO4, 26 mM NaHCO3, and 10 mM glucose) at 34°C for 30 min and recovery at room temperature (25 ± 1°C) for more than 1 h before recording. Slices were transferred to a recording chamber under perfusion ACSF (2 ml/min, 32-34°C). Pyramidal neurons in the piriform cortex were visualized with infrared optics using an upright fixed microscope equipped with a 40x water-immersion lens (FN1, Nikon) and CCD monochrome video camera (IR-1000, DAGE-MTI). Patch pipettes (resistance of 3–5 MΩ) were prepared by a horizontal pipette puller (P-1000; Sutter Instruments). For spontaneous excitatory postsynaptic current (sEPSC) recording, pyramidal neurons were held at -70 mV in the present of 20 µM bicuculline methiodide (BMI), with the pipette solution (125 mM K-gluconate, 5 mM KCl, 10 mM HEPES, 0.2 mM EGTA, 1 mM MgCl2, 4 mM Mg-ATP, 0.3 mM Na-GTP and 10 mM phosphocreatine, pH 7.35, 290 mOsm). For miniature excitatory postsynaptic current (mEPSC) recording, 20 μM BMI and 1 μM TTX were added into the perfusion ASCF to block GABA receptor-mediated currents and action potentials.
Data were statistically analyzed with GraphPad Prism 6.0 software systems, and the values were expressed as means ± standard error (means ± SEM). One-way ANOVA (Fig 1C), two-way ANOVA (Fig 3B), and t-test analyzed the normality distributed data. All tests were two-sided. * p< 0.05, ** p < 0.01.