Cell culture

COS-7 cells (ATCC Cat# CRL-1651, RRID: CVCL_0224) were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, and 1% antibiotics in a humidified incubator under 5% CO2 at 37 °C.

Cell binding assay

The interaction between Cbln1-HA (R&D Systems, Minneapolis, MN, USA) and GluD1 was assessed using an in vitro binding assay as previously described with minor modifications [19]. COS-7 cells were cultured on poly l-lysine-coated coverslips and transfected with the rat pCAGGS GluD1 plasmid (gift from Dr. Michisuke Yuzaki) or mutant pCAGGS GluD1R526K using PEI at a ratio of 1:1.5. Following a 48-h transfection period and removal of the transfection reagent, cells were treated with different D-serine concentrations for 30 min followed by recombinant Cbln1 protein (1 μg/ml) in the presence of D-serine (or vehicle) for 4 h. Subsequently, the medium was aspirated, and the cells were rinsed twice with phosphate-buffered saline (PBS, pH 7.4) before fixation with 4% paraformaldehyde (PFA) for 15 min. After fixation, the cells were washed with PBS and incubated with 3% bovine serum albumin (BSA) for 1 h at room temperature to block nonspecific binding sites. Following blocking, the cells were treated with an anti-HA primary antibody (Cat# 2367S, Cell Signaling Technologies, Danvers, MA, USA) for 2 h at room temperature, followed by three PBS washes. Subsequently, the cells were incubated with an anti-mouse secondary antibody conjugated to Alexa Fluor488 (Cat# A21202, Thermo Fisher Scientific, Waltham, MA, USA) for 1 h, followed by three PBS washes. Finally, the coverslips were mounted on glass slides using Fluoromount-G (0100-01, SouthernBiotech, Birmingham, AL, USA), and fluorescent images were captured using a Nikon Eclipse upright microscope.

Cell binding followed by immunoblotting

Quantitative cell binding was performed with some modifications as described previously [20]. Briefly, HEK293T cells were transfected with pCAGGS GluD1. Following a 48-h transfection period and removal of the transfection reagent, cells were treated with D-serine (or vehicle) for 30 min followed by recombinant Cbln1 protein (1 μg/ml) in the presence of D-serine (or vehicle) for 4 h. Subsequently, the medium was aspirated, and the cells were rinsed thrice with PBS. The cell lysate was collected and subjected to SDS-PAGE. Membranes were probed using 1:1000; mouse monoclonal anti-his-tag (#D-291-3, MBL International Corporation) and 1:1000; guinea pig monoclonal anti-GluD1 (#MSFR102510, Frontiers Institute Co. Ltd.) antibodies.

Experimental animals

C57BL/6 and GluD1 KO male and female mice were used in this study. All studies were approved by Creighton University and Texas A&M IACUC. All efforts were made to minimize distress and discomfort in the animals. Mice were housed at 22 ± 1 °C with a 12 h light–dark cycle, with access to food and water ad libitum. Mice weighing 20–30 g–2–4 months old were used for all experiments.

Brain slice electrophysiology

Whole-cell brain slice electrophysiology was conducted to record mEPSC currents from CeA neurons, as previously described [5], with slight modifications. Briefly, mice were anesthetized with isoflurane, followed by decapitation and removal of the brain. The isolated brain was transferred to the ice-cold artificial cerebrospinal fluid (aCSF) of the composition 130 mM NaCl, 24 mM NaHCO3, 3.5 mM KCl, 1.25 mM NaH2PO4, 2.4 mM CaCl2, 2.5 mM MgCl2 and 10 mM glucose saturated with 95% O2/5% CO2. 300 μm thick sections were cut using a vibratome (Leica VT1200S; Buffalo Grove, IL, USA). These sections were incubated with either D-serine (300 μM) (or vehicle) for 15 min, followed by the addition of rCbln1 (1 μg/ml) or vehicle and incubation for 30 min. Effect of reversing the drug exposure sequence was tested by incubation with rCbln1 followed by D-serine. Incubation was performed in the presence of MK-801 (50 μM) to block NMDA receptor-mediated activity. Whole-cell patch clamp recordings were obtained from neurons in the lateral capsular region of the CeA (CeLC) in a voltage-clamp configuration with an Axopatch 200 B (Molecular Devices, Sunnyvale, CA, USA). Glass pipette (resistance of 4–6 MΩ) filled with an internal solution consisting of 110 mM Cs gluconate, 30 mM CsCl, 5 mM HEPES, 4 mM NaCl, 0.5 mM CaCl2, 2 mM MgCl2, 5 mM BAPTA, 2 mM Na2ATP, and 0.3 mM Na2GTP (pH 7.35) was used for patching. QX314 was added to the internal solution to block voltage-gated sodium channels. The aCSF solution contained 1.5 mM of CaCl2 and 1.5 mM of MgCl2. mEPSC’s were recorded at holding potential −70 mV in the presence of 0.5 μM tetrodotoxin and 100 μM picrotoxin. The signal was filtered at 5 kHz and digitized at 10 kHz using an Axon Digidata 1440A analog-to-digital board (Molecular Devices). mEPSC recordings were analyzed with MiniAnalysis software (Synaposoft, Atlanta, GA, USA) using a 5-pA amplitude threshold. The frequencies and amplitudes of the miniature currents were measured.

Immunohistochemistry

Immunohistochemical analysis of GluD1 and PKCδ expression was performed as previously described [5], with minor modifications. Mice were anesthetized with isoflurane and decapitated, their brains were removed, and brain slices (150 μm) were prepared using a vibratome (Leica VT1200S, Buffalo Grove, IL, USA). Brain slices were incubated with D-serine (300 μM) (or vehicle) for 15 min, and then Cbln1 (1 μg/ml) (or vehicle) was added, and slices were incubated for an additional 30 min. These slices were then transferred to a 4% paraformaldehyde solution for 15 min and subjected to immunohistochemistry. After washing with 0.1 M phosphate buffer, sections were incubated in blocking solution containing 10% normal goat serum (Jackson Immuno Research Laboratories Inc., West Grove, PA, Cat # 005-000-121) for 1 h at room temperature. After blocking, the sections were incubated in a cocktail of guinea pig anti-GluD1 (1:500, GluD1C-GP-Af860, Frontier Institute Co. Ltd., Sapporo, Japan), and mouse PKCδ (1:500, Cat # 10,397, BD Biosciences, San Jose, CA, USA) antibodies overnight at 4 °C. The following day, sections were washed and incubated in anti-guinea pig conjugated to Alexa Fluor 488 (1:500, A-11073, Life Technologies) or in a cocktail of goat anti-guinea pig conjugated to Alexa Fluor 488 (1:500, A-11073, Life Technologies) and goat anti-mouse conjugated to Alexa Fluor 594 (1:500, A-11032, Life Technologies) secondary antibodies for 2 h at room temperature. Sections were washed and mounted using Fluoromount-G (Southern Biotech, Birmingham, AL, USA). Confocal images were acquired using a Nikon Eclipse Ti2-E inverted confocal microscope equipped with an AX point scanner. Images of an equivalent region, 1024 × 1024 pixels, were captured using a 60 × oil immersion or 20 × objective at a 1 × zoom. CeA sections were scanned at 0.3 µm intervals along the z-axis, and an optical Sect. (18 µm thick) was taken from each tissue section. GluD1 puncta number and volume were analyzed using NIS-Element image analysis software (Nikon, NY, USA). Images from four mice per group were analyzed and plotted. Images were analyzed by a trained observer who was blinded to the treatments.

Synaptoneurosomes preparation and immunoblotting

Brain slices were prepared and incubated with drugs as described for the electrophysiology experiments. The CeA was punched with reference to the Allen Brain Atlas and processed for synaptoneurosomes. Synaptoneurosome preparation was performed as previously described [19] with slight modifications. RCeA was homogenized in a synaptoneurosome buffer with following composition: 10 mM HEPES, 1-mM EDTA, 2-mM EGTA, 0.5-mM DTT, 0.5-mM PMSF, 50 μg/ml soybean trypsin inhibitor, 0.25% of phosphatase inhibitor cocktail 2% and 3%, and 0.25% of protease inhibitor cocktail followed by 3 times sonication (FB50, Fisher Scientific, Pittsburgh, PA, USA). Samples obtained from the previous step were filtered twice using three layers of a pre-wetted 100 µm pore nylon filter (CMN-0105-D, Small Parts Inc., Logansport, IN, USA) and once through a pre-wetted 5 µm pore hydrophilic filter (CMN-0005-D, Small Parts Inc.) held in a 13 mm diameter filter holder (XX3001200, Millipore, MA, USA). The filtrate was centrifuged at 1000 × g for 10 min at 4 °C and the synaptoneurosome fraction was obtained and later resuspended in synaptoneurosome buffer containing 0.32 M sucrose and 1 mM NaHCO3. The protein concentration was estimated using the Pierce™ BCA Protein Assay Kit (Cat # 23227, Thermo Fisher Scientific). For western blotting, 15 μg of synaptoneurosomes was loaded onto 12% SDS-PAGE gels and run at 100 V for 90 min. The gels were transferred to methanol-activated PVDF membranes (GE Healthcare) at 115 V for 1 h 45 min and then blocked with 5% milk in TBST for 1 h at RT. The membranes were incubated with primary antibodies overnight at 4 °C, washed with TBST, and incubated with secondary antibodies for 1 h at RT. Primary antibodies used were: 1:1000; guinea pig monoclonal anti-GluD1 (#MSFR102510, Frontiers Institute), 1:1000; rabbit monoclonal anti-Cbln1 (#ab181379, Abcam), 1:1000; rabbit polyclonal anti-Neurexin1α (#ANR-031, Alomone Labs), 1:1000; rabbit monoclonal anti-HA (#C29F4, Cell Signaling Technology), 1:1000; rabbit monoclonal anti-β-Actin (#13E5, Cell Signaling Technology). After washing, the blots were treated with SuperSignal ™ West Pico PLUS substrate (Thermo Fisher) and developed using a ChemiDoc imaging system (Bio-Rad). The optical density was analyzed using ImageJ and normalized to that of β-actin.

Drugs for in vivo experiments

D-serine (Acros Organics Cat # 227070250) and MK-801 (Sigma Aldrich Cat # M107) were dissolved in PBS to prepare stock solutions of 100 μg/μl and 10 μg/μl, respectively, and were administered at a dose of 30 μg and 1 μg, respectively, bilaterally in the CeA region in a volume of 0.3 μl. Cbln1 (R&D Systems Cat# 6934-CB) was dissolved in PBS to prepare a stock solution of 1 μg/μl and was administered at a dose of 250 ng bilaterally in the CeA.

Stereotaxic surgery

Cannulation surgery was performed as described previously [21], with minor adjustments. The mice were anesthetized using isoflurane and positioned in a stereotaxic apparatus. Following skull exposure, a small aperture was created, and bilateral implantation of 26-gauge stainless steel guide cannulas into the CeA was performed. Stereotaxic coordinates for the procedure were set at: AP: −1.22 mm, ML: ± 2.75 mm, DV: −4.0 mm. Secure fixation of the guide cannulas onto the skull was achieved using stainless-steel screws and dental acrylic cement. Animals were assigned to the experimental groups after a post-surgery recovery period of 7–10 days. Verification of cannula placement was conducted after behavioral experiments by histological examination of fixed brain tissue using light microscopy.

CFA inflammatory pain model

Inflammation was induced by intra-plantar injection of 10 μl of complete Freund’s adjuvant (CFA; Sigma, St. Louis, MO, USA) to the right hind paw. Animals injected with 10 μl saline in the left paw served as controls.

Von frey filament test

Mechanical hypersensitivity of the mice was assessed by the Von Frey Filament test using an electronic Von Frey aesthesiometer (IITC systems). During habituation, the mice were placed in the testing chambers and conditioned to a rigid filament (from IITC systems). The test was performed by placing the mice on the perforated bottom of the test chambers with free access to their paws. The filaments were applied perpendicular to the plantar surface of the hind paw, and the paw withdrawal threshold was measured. The nocifensive response to filament application included licking, shaking, or brisk paw withdrawal. The force required for the nocifensive response was also noted. The average of three readings was taken at each time point.

Statistical analysis

All experiments adhered to protocols ensuring uniform group sizes through randomization and blinded analysis. Statistical analyses were performed using the Prism 8.0 software (GraphPad Prism software, San Diego, CA, USA). The results were graphically represented as the mean value with standard error of the mean (SEM). Pairwise comparisons between two groups were conducted using the unpaired t-test, while multiple group comparisons were performed using analysis of variance (ANOVA) followed by Bonferroni’s post-hoc test for multiple comparisons.