NRXN1 depletion in the medial prefrontal cortex induces anxiety-like behaviors and abnormal social phenotypes along with impaired neurite outgrowth in rat

Chemicals and reagents

The mouse antibodies used were as follows: anti-NeuN (Ab104225, Abcam, dilution IF: 1:500). Rabbit antibodies used in this study were as follows: anti-NRXN1 antibody was used (GTX54845, GeneTex, dilution, IF:1:30, IHC:1:30; Santa Cruz, sc136001, WB:1:100), anti-MAP 2 (Ab32454, Abcam, dilution IF: 1:500), anti-GFP (AF5066, Beyotime, dilution IF: 1:100), and anti-GAPDH (10494, Proteintech, dilution WB 1:1000). Other antibodies were as follows: Donkey polyclonal Secondary Antibody to Mouse IgG (Alexa Fluor594, ab150108, Abcam, dilution IF 1:1000), Donkey polyclonal Secondary Antibody to Rabbit IgG (Alexa Fluor488, ab150061, Abcam), HRP-conjugated AffiniPure Goat Anti-Mouse IgG(H+L) (SA00001-1, Proteintech, dilution WB:1:5000), and HRP-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) (SA00001-2, Proteintech, dilution WB:1:5000).

Animal housing and treatment

Male Sprague Dawley (SD) rats at the prepubertal age (3 weeks) were maintained in a standard-controlled environment at 23 ± 1 °C and relative humidity of 50 ± 5% on a dark/light cycle and obtained from Charles River, Beijing Vital River Laboratory Animal Technology Co. (Beijing, China). All animals had free access to water and food. The Animal Ethics Committee of Nanjing Medical University approved the research protocol (approval no. IACUC-1711004). Before injections, animals were acclimated for 1 week. At 4 weeks old, rats were anesthetized with 10% chloral hydrate (0.35 ml/100 g) by intraperitoneal injection. The depth of anesthesia was determined by monitoring reflexes, skin color, heart rate, and respiratory rate. PFC knockdown of NRXN1 was performed in anesthetized rats by placing them on a stereotactic instrument, followed by intracerebral injection of AAV9-NRXN1-GFP (positive-sense strand: AGAUGAGCUUCCAGCUGAAdTd, viral titer: 1 × 10^12 TU/ml) targeting exon one. AAV9-NRXN1-GFP was constructed by Hanbio Biotechnology Co. (Shanghai, China). Adeno-associated viral vector containing irrelevant sequences (AAV-Nc-GFP, Hanheng Company) was used as a negative control. Twenty-seven rats were randomly divided into three groups, wild type (WT = 9), Sh-Nc (n = 9), and Sh-NRXN1 (n = 9). AAVs were injected into PFC at a 20° angle (anteroposterior = 2.6 mm; mediolateral = ±0.7 mm to bregma; dorsoventral = −3.0 mm). The infusion was performed with a 10 μl volume at 1 μL/min with a 0.5-mm flat needle, and the syringe was maintained in place for 10 min after the infusion was completed. The syringe was withdrawn gradually to prevent overflow. Animals were monitored daily following surgery and given 2 weeks to allow for full expression of the viral constructs before behavioral testing. To verify injection sites, 30-mm-thick sections were cut at the level of PFC using a cryostat. AAV infection was verified by immunohistochemical analysis of the GFP vector. Animal experiments were performed following the approved guidelines and experimental protocol of Nanjing Medical University.

Open-field test

For the open-field test (OFT), we used a black box with a bottom of 40 × 40 cm and a height of 45 cm with a 4-W cold light source above the center of the box. Animal activity was recorded using the Shanghai Jiliang Animal Behavior Analysis System (Shanghai, China). Behavioral tests were performed 2 weeks after the AAV injection. Rats were placed in the center of the open-field apparatus, and their activity under light conditions was recorded by an infrared camera system and video synthesizer. We used a computer analysis system to analyze movement trajectories. To make sure each rat’s performance did not affect the behavior the next, the box was cleaned with 75% alcohol solution after each experiment. Self-grooming behavior was also measured during OFT tests. Behavior was video-recorded for 10 min. Time spent in self-grooming and the number of bouts were manually recorded. Detailed descriptions were illustrated in the supplementary materials.

Three-chamber sociability test

The three-chamber setup (120 cm × 80 cm × 40 cm) consisted of a black acrylic floor and transparent acrylic walls separating the arena into three small compartments. An opening to the outer compartments could be closed with removable slide doors on the walls of the inner compartment. Two cylinders with a diameter of 22 cm (40 cm in height) were placed in the outer compartments for the stimulus rats during testing. One cylinder contained an unfamiliar male SD rat (age 6 weeks; “Stranger”), whereas the other remained empty. These cylinders contained acrylic bars placed 15 mm apart to allow social contact while preventing aggressive assaults. All three-chamber experiments were performed in dim light conditions (10 lux). Animals were moved to the experimental room for environmental adaptation 1 day. Before the formal experiment, each experimental animal underwent a 15 min adaptation time. Time spent exploring either cage was measured for 10 min. This is considered a test of sociability. Rat activity was recorded using the Shanghai Jiliang Animal Behavior Analysis System (Shanghai, China). Detailed descriptions were illustrated in the supplementary materials.

Primary neuronal culture

The Animal Ethics Committee of Nanjing Medical University approved all animal experiments. The PFC was dissected from SD rat embryos (E18), and cells were dissociated using trypsin and titrated through a Pasteur pipette. The neurons were plated on coverslips with poly-L-lysine in DMEM with 10% horse serum at a density of 1 × 10^5 cells/cm2. When neurons were attached to the coverslip within 24 h, the medium was changed to neurobasal media with a 2% B27 supplement.

NRXN1 knockdown in vitro

Knockdown of NRXN1 in prefrontal neurons was performed by lentivirus infection (viral titer, 1 × 10^8 TU/ml; Hanbio Biotechnology Co., Shanghai, China). In a preliminary experiment, we infected the virus with GFP markers to determine infection efficiency, and neurons were infected at a multiplicity of infection (MOI) of 10. We used viruses without fluorescent labels in formal transfection experiments. Cells were divided into three groups, a blank control group (uninfected), an Nc group (transfected with negative control lentivirus), and a lentivirus-shRNA group (transfected with target Sh-NRXN1 lentiviral vectors). Cell media was replaced with fresh Neurobasal media with a 2% B27 supplement 12 h after transfection. Knockdown efficiency was determined by Western blot, PCR, and immunofluorescence after 72 h of infection.

RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)

Using the TRIzol reagent (Invitrogen, CA, USA) and the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany), the total RNA of the neurons was isolated using the TRIzol reagent (Invitrogen, CA, USA) and the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) following the guidelines of the manufacturers. A NanoDrop 2000 system (NanoDrop Technologies, Wilmington, DE, USA) was applied to evaluate the concentration and purity of the RNA. A total of 1 μg RNA was reverse-transcribed into cDNA using the random hexamer primers, gDNA Eraser (RR047A; Perfect Real Time, Takara, Tokyo, Japan) from each sample and applying the PrimeScript™ Reverse Transcription (RT) reagent kit. The quantitative real-time PCR (qRT-PCR) was conducted on the Applied Biosciences ViiA 7 Real-Time PCR System (Invitrogen, Carlsbad, CA, USA) to measure mRNAs expression levels. The conditions were as follows: initial denaturation at 95 °C for 10 min, followed by 40 PCR cycles at 95 °C for 15 s, 60 °C for 30 s, and extension at 72 °C for 30 s, using the SYBR green method (Thermo Fisher Scientific Inc., Shanghai, China) by following the instructions of the manufacturer. The melting curve analysis was performed to confirm the selectivity of the PCR product, while the comparative cycle threshold (CT) method (2−ΔΔCt method) was applied to determine the relative expression levels. The Basic Local Alignment Search Tool (BLAST) from the National Center for Biotechnology Information (NCBI) was consulted to corroborate the specificity of the amplified result, coupled with the designing of primers with Primer 5. Primers for qPCR are exon spanning. Table S1 illustrates the primer sequences in detail.

Western blotting

Neurons (1 × 10^5 cells/cm2) were lysed in RIPA buffer (Beyotime, China). Protein extract quantification was performed using a BCA protein assay kit (Beyotime, China). Denatured protein samples were separated with 4–12% (GenScript, M00653) sodium dodecyl sulfate-polyacrylamide and then transferred onto the polyvinylidene difluoride membranes. Membranes were blocked with skim milk solution for 2 h followed by primary antibodies incubation at room temperature for 2 h. Primary antibodies for NRXN1 (Santa Cruz, sc136001) were diluted 1:100 in PBS-Tween-20. Primary antibodies for GAPDH (10494, Proteintech) were diluted at 1:5000. The membranes were washed thrice (about 10 min/wash) using Tween-20 (pH 8.0) and Tris-buffered saline (TBST). Subsequently, membranes were incubated at room temperature for 2 h, using the horseradish-peroxidase-conjugated secondary antibodies (SA00001-1; SA00001-2, Proteintech, dilution WB: 1:5000). Membranes were then washed thrice with PBST, and the enhanced chemiluminescence substrate (Millipore, USA) was used to develop the blots. The protein bands were imaged in a gel image processing system. GAPDH was used as the protein loading control. Relative expression of the target protein was determined by the corresponding ratio of the optical density values of the internal reference and the target protein band using ImageJ.

Immunofluorescence (IF)

The neurons were fixed with 4% paraformaldehyde for 5 min and then washed thrice with PBS for immunofluorescence. The neurons were permeabilized with 0.3% Triton X-100 in PBS for 5 min and then blocked with 5% goat serum for 30 min. The neurons were blocked in blocking solution and incubated with anti-MAP 2 (Ab32454, Abcam, dilution: 1:500) overnight at 4 °C. Later, the neurons were washed in PBS and stained in the dark for 30 min with a secondary antibody (Alexa Fluor488, ab150061, 1:500). After staining, neurons were washed and Hoechst stained for 5 min for nuclei staining. The labeled neurons were observed under a fluorescence microscope Imager A2 (Carl Zeiss).

Quantitation of neurite outgrowth in vitro

Collected images were counted from at least five random fields for each well, and the five random zones were selected at four corners and one field in the center. Images were taken from at least three coverslips from different wells. Assessment of neurite outgrowth was performed by counting 30 cells per group. We designed three-technique replicates per group, and at least three biological replicates were performed per set of experiments. Single neurons were analyzed using ImageJ software with the NeuronJ plug-in, with minimal or no overlapping neurite arbors with adjacent neurons. Distances from the soma perimeter to neurite tips were measured by tracing arbors. Various parameters were evaluated for each PFC neuron, including total neurite length, the number of primary neurites, and length of the longest neurite. A primary neurite was defined as a neurite that directly emerges from the cell body. Neurite branching was measured using the Sholl analysis plug-in of ImageJ. All samples were set to a polynomial fit for a best fitting degree.

Immunohistochemistry (IHC)

After their last behavioral test, the rats were deep anesthetized by 2% pentobarbital perfusion through the left ventricle using saline, followed by 4% paraformaldehyde. The removed brains were postfixed overnight in the same fixative and cryoprotection until they sank to the bottom in 30% sucrose at 4 °C. The serial 25 μm brain coronal sections cut using a cryostat microtome (Leica, Wetzlar, Germany) were mounted on gelatin-coated slides. For immunohistochemical staining, brain sections were pretreated with 0.3% hydrogen peroxide for 30 min in methanol, washed in phosphate-buffered saline (PBS), and incubated in 0.3% Triton X-100 for 10 min at 4 °C. The sections were blocked in goat serum for 1 h 22 °C and washed with PBS. Later, in the blocking solution at 4 °C, the sections were incubated for 48 h with a primary antibody NRXN1 (GTX54845, GeneTex, dilution, 1:30). The sections were again incubated in secondary antibody anti-rabbit at 37 °C for 30 min after rinsing in PBS. Sections were rinsed in PBS and visualized using the DAB (Maxvision Technology, Futian, China) as the chromogen. Other than the omission of the primary antibodies, the negative controls were prepared using the same procedure.

TMT labeling and MS-based quantitative proteomics

The MS/MS spectra data was searched against a protein sequence databases, including all entries from the Rat UniProt database (Swiss-Prot 16,768 and TrEMBL 62,460 total of 79,228 protein forms, 2015). The searches were conducted by applying a 20-ppm precursor ion tolerance and requiring each peptide N-/C termini to allow a maximum of two missed cleavages to adhere to the trypsin protease specificity. The experimental throughput for protein quantitation was enhanced using the six-plex labeling scheme allowing up to six samples to be compared in a single MS run. The six samples containing 30 μg of peptides each were labeled in duplicate using TMT-129 and 130 for the KD group and TMT-126, 127, and 128 for the Nc group. With methionine oxidation being set as the variable modification, peptide N termini and lysine residues were set as the static modifications. By applying the target-decoy database search, a false discovery rate (FDR) of 1% on the protein level was achieved with the assignment of the MS2 spectra. A percolator (64-bit version) was used for filtering. The intensity of the signal closest to the theoretical m/z value and a 0.02 m/z window centered on the theoretical m/z value of each of the six reporter ions were recorded for qualification. All intensity values were adjusted considering the potentially uneven TMT labeling and the sample handling variance for each labeled channel. The total signal intensity across all peptides quantified was summed for each TMT. Applying the Friedman test with a Benjamini-Hochberg multiple test correction with P < 0.00134 cutoffs in Scaffold 4.5 (Proteome Software), the statistical significance of the changes in protein enrichment was determined by analyzing the variance of the individual peptides across samples.

Automated capillary Western dot blot analysis

Following the manufacturer’s guidelines, the samples were prepared and analyzed (ProteinSimple, San Jose, CA, USA). Four sample volumes were denatured at 95 °C for 5 min after mixing with one fluorescent 5 × master mix volume containing 200 mM dithiothreitol (ProteinSimple). Primary antibodies against ANXA4 (Ab256456, Abcam, dilution: 1:20), GRB2 (36344s, CST, dilution1:50), ANXA1 (32943t, CST, dilution: 1:50), and GAPDH (10494, Proteintech, dilution 1:100) were diluted in antibody diluent (ProteinSimple). The primary and secondary antibodies, the biotinylated ladder, the chemiluminescent substrate, the separation matrix (12–230 kDa), the stacking matrix, and the prepared samples were transferred into an array plate of 384 wells. After adding the Simple Western assay buffers into the system tray and the insertion of capillaries, the prepared assay plate was placed into the Simple Western machine (Sally Sue; ProteinSimple). A total of 40 nL was the injection volume of each sample. The machine automatically carried out all subsequent separation, immunodetection, and analysis steps. To calculate the area under the curve (AUC) of each peak, analyze the signal peaks automatically, and visualize the Simple Western lanes, the Compass software (Atlanta, GA, USA) was used.

Statistical analysis

Data were analyzed with GraphPad Prism 7 and were used to analyze the data and calculate the mean value ± standard deviation (SD). All experiments were repeated at least three times. The identification of the normal distribution was performed using the Kolmogorov-Smirnov test, and the Levene’s test was used to check the homogeneity of variance. Unpaired t-tests were used for data analysis between groups. One-way analysis of variance (ANOVA) was used for multiple data analysis. Tukey’s post hoc test was performed to obtain P-values and adequately assess differences to determine significance following one-way ANOVA; group main effect, P < 0.05, was considered to indicate a statistically significant difference.

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