DOI: 10.20350/digitalcsic/17761
title: SNAP-47 mediates somatic oxytocin dynamics in hypothalamic neurons [Dataset]

description: This repository includes the required data to recreate the graphs presented in the paper"SNAP-47 mediates somatic oxytocin dynamics in hypothalamic neurons", Aznar-Escolano et al., 2026 Comms Biol [Acepted] .xlsx file with the data for recreating the main results of the article
description: Methodological information: Animals: Immunohistochemistry and cultured neurons were obtained from either in house-bred wild-type BL6/C57 mice or OT-tdTomato mice, generated by crossing OT-Cre animals43 (Jackson Laboratories; strain ID 024234) with the Ai9 (RCL-tdT) reporter line from Jackson Laboratories (strain ID 007909) to identify OT-positive neurons 43. We have complied with all relevant ethical regulations for animal use. All experiments were performed according to Spanish and European Union regulations regarding animal research (2010/63/EU), and the experimental procedures were approved by the Bioethical Committee at the Instituto de Neurociencias and the Consejo Superior de Investigaciones Científicas (CSIC). Animals were housed in ventilated cages in a standard pathogen-free facility, with free access to food and water on a 12 h light/dark cycle. -- Hypothalamic cultured neurons: Dissociated hypothalamic cultures were prepared from either newborn wild type C57BL/6 or OTtdTomato mice. Briefly, hypothalami were isolated and dissociated in a digestion solution containing papain for 18 minutes at 37 ºC. Papain inactivation solution containing trypsin inhibitor type III Ovomucoid was then applied. Tissue was mechanically disaggregated through a glass pipette and cells were plated on poly-D-lysine coated glass coverslips placed in 12 mm wells at a density of 75,000 cells per cover slips. Cells were grown in Neurobasal medium (Invitrogen) supplemented with B-27 (Thermo Fisher) and Glutamax (Invitrogen). Half of the culture medium was replaced every 2-3 days. Neurons were infected with an adeno-associated virus (AAV) expressing a specific shRNA to block SNAP-47 expression (Jurado et al., 2013) and GFP as a reporter. Cultures were infected 7-10 days prior experiments. -- Cytotoxicity assay: Primary hypothalamic neurons at 10-14 DIV were incubated in Tyrode’s basal solution (mM: 125 NaCl; 2 KCl; 30 glucose; 25 HEPES; 2 CaCl2; 2 MgCl2; pH 7.4), 70 % EtOH, 50 mM KCl Tyrode’s solution (mM: 77 NaCl; 50 KCl; 30 glucose; 25 HEPES; 2 CaCl2; 2 MgCl2; pH 7.4), or 100 mM KCl Tyrode’s solution (mM: 27 NaCl; 100 KCl; 30 glucose; 25 HEPES; 2 CaCl2; 2 MgCl2; pH 7.4) for 1 or 10 minutes. A LIVE/DEAD® Cell Assay (Thermo Fisher) was then performed by incubating the cells for 15 minutes at 20-25°C. Cells were subsequently stained with DAPI (Sigma Aldrich) for cell quantification, fixed in PFA, and mounted on coverslips. At least 5 images per condition from two different cultures were acquired using a Leica SPEII confocal microscope. Image analysis was conducted with the open-source software Fiji (National Institutes of Health, USA). -- Immunohistochemistry: Brains were dissected from young adult (2-3-months-old) C57BL/6 mice fixed in PFA 4 % and embedded in agarose 4 % for processing in a vibratome (Leica VT 1000S) to obtain 50 µm thick slices. Immunohistochemistry for OT and AVP was performed using mouse anti-OT and anti-AVP primary antibodies, originally developed in Dr. H. Gainer´s laboratory77 and kindly provided by Dr. A. H. Veenema (Michigan State University). Additional antibodies used included: rabbit anti-SNAP-47 (Synaptic Systems, Ref: 111 403, IHC and ICC 1:100), rabbit anti-SNAP-25 (Synaptic Systems, Ref: 111 008, IHC and ICC: 1:1000), mouse anti-GFAP (Millipore, Ref: MAB360, ICC: 1:1000), and mouse anti--tubulin (Abcam, Ref: Ab7291, ICC: 1:5000). Cultured cells were incubated for 1 hour in a blocking solution containing 0.5% PBS azide, 99.5% sodium azide, 0.1% Triton X-100, and 2% Normal Goat Serum. Tissue samples were incubated for 1 hour in a similar blocking solution, with 0.5% PBS azide, 0.3% Triton X-100, and 2% Normal Goat Serum. Primary antibodies were incubated overnight at 4°C. Secondary Alexa antibodies (anti-rabbit Alexa 488 and anti-rabbit Alexa 594; Invitrogen) were added for 2 hours at room temperature. Samples were then incubated with DAPI and mounted on coverslips with Mowiol (Merk). Imaging was performed using a Leica SPEII confocal microscope with oil immersion objectives at 20x, 40x, and 63x magnifications. -- Chasing of OT-containing compartments in cultured neurons: Hypothalamic primary neuronal cultures at 10-14 DIV were incubated in tempered Tyrode's solution (mM: 125 NaCl; 2 KCl; 30 glucose; 25 HEPES; 2 CaCl2; 2 MgCl2; pH 7.4) for 3 minutes on a shaker. Cells were then incubated with either tempered Tyrode’s basal or 100 mM KCl Tyrode (mM: 27 NaCl; 100 KCl; 30 glucose; 25 HEPES; 2 CaCl2; 2 MgCl2; pH 7.4) for 1 or 10 minutes, or with chelated calcium 100 mM KCl Tyrode (mM: 125 NaCl; 2 KCl; 30 glucose; 25 HEPES; 0.5 EGTA; 2 MgCl2; pH 7.4) for 1 minute at 37ºC. To arrest calcium-dependent endocytosis, cells were placed on ice and rinsed with calcium-free Tyrode’s solution. Primary antibodies (anti-OT, anti-SNAP-47) were then added for 2 hours on ice. After antibody incubation, plates were rinsed and fixed in 4% PFA at 4ºC for 10 minutes. Cells were incubated with secondary antibodies (anti-Rabbit Alexa 488; anti-Rabbit Alexa 594; Invitrogen) prepared in 1X PBS for 2 hours at room temperature on a shaker. Images were acquired using a confocal laser Olympus Fluoview FV300 (Olympus) with a 100x oil immersion objective (A.N = 1.45) and FluoView 5.0 software. Image analysis was performed with the open-source software Fiji (National Institutes of Health, USA), using a custom macro and plugins “UCSD Control” and JACoP78. Results were analyzed with GraphPad Prism 8. -- Colocalization analysis: Images were opened using the "UCSD Control" plugin in Fiji. Each image was separated into different Z planes, and a 3D projection was constructed from the stacks. OT-containing compartments from the 3D projection were outlined and saved in the ROI manager for colocalization analysis using the JACoP plugin. Colocalization parameters for individual OT compartments in each Z stack were obtained using an automatic threshold. The colocalization analysis produced two parameters: Pearson´s and Manders´ coefficients79. Pearson's coefficient measures the linear relationship between the intensity levels from two channels, with values ranging from -1 (perfect inverse correlation) to +1 (perfect correlation), with 0 indicating no correlation78. Two Pearson's coefficients can be derived: Pearson_A, which measures the correlation within the entire volume of the analyzed signal, and Pearson_B, which refers to the interaction within the region of colocalization (Fig. 2e, f). Manders' overlap coefficient measures the degree of co-occurrence between two channels, independent of the linearity of the relationship. Manders' overlap coefficient values range from 0 (no overlap) to +1 (total overlap). Two Manders' coefficients can be calculated: Manders_A, which refers to the correlation of channel 1 (red) with channel 2 (green), and Manders_B, which measures the co-occurrence of channel 2 (green) with channel 1 (red) (Fig. 2g, h). Pearson's and Manders' coefficients are not expressed as percentage values. Although both parameters provide information about the colocalization of pixels between two channels, Manders' and Pearson's coefficients exhibit notable differences. Manders' overlap coefficient is independent of object size and channel intensity, while Pearson's correlation coefficient compares channels pixel by pixel and requires that both objects be of similar size to provide accurate comparisons. Since the OT-containing compartments were smaller than the SNAP-47 staining, Manders' overlap coefficient was considered more reliable than Pearson's coefficient. Additionally, a random data set was generated as an internal control to demonstrate that the observed colocalization was not occurring by chance. The random data set was generated from the real stack that exhibited the highest Manders' overlap value for each individual OT-containing compartment, using the "UCSD Control" and JACoP plugins in Fiji. Random OT-containing compartments were created by selecting a real OT-containing compartment with a size closest to the mean OT-containing compartment size and placing it at random x and y positions within the cell body, excluding the nucleus and extracellular locations. The control picture was generated by overlapping the image containing the randomly generated OT-containing compartments with experimental SNAP-47 data, followed by colocalization analysis. Basal and stimulation conditions used in these simulations mirror the treatments applied in the chasing experiments. -- Viral vectors: A specific shRNA targeting the untranslated (UTR) region of the mouse SNAP-47 (SNAP-47 shRNA: ATAGCAATAGAATCAGCAGAGC) was cloned downstream of the human polyubiquitin promoter-C. shRNA effectiveness was tested in cultured neurons as described in Jurado et al.33 (see Supplementary Fig. S3). The effectiveness of the shRNA was also evaluated in hypothalamic slices from injected animals using immunohistochemistry to detect endogenous SNAP-47. SNAP-47 shRNA was subcloned into a floxed AAV vector containing GFP as a reporter. -- In vivo injections : OT-Cre mice (1-2-months-old) were prepared for stereotactic injection using standard procedures approved by the Bioethical Committee at the Instituto de Neurociencias and the Consejo Superior de Investigaciones Científicas (CSIC). Briefly, animals were anesthetized with isoflurane and administered analgesic (Buprenex; 0.1 mg kg−1, s.c.). For knockdown of SNAP-47 in the PVN, an AAV-SNAP-47KD-GFP was injected bilaterally into the PVN using the following coordinates from bregma: AP: ± 0 mm, ML: ± 0.25 mm, DV: – 4.75 mm. A virus containing just GFP was used as a control. Pipette was left in the ROI for 5 min to allow the diffusion of the AAVs (200 nl) into the brain. The scalp was then sealed and the animals were left to recover in a warm chamber. Electrophysiological recordings and behavioral assays were conducted 3-5 weeks after the injections. -- Electrophysiology: Acute hypothalamic slices from 2-3-months-old mice (male and females) were obtained in a Leica 1200S vibratome. The recording chamber was perfused with cold ACSF gassed with 5% CO2 / 95% O2. Whole-cell voltage-clamp recordings were obtained with patch recording pipettes (5–7 MΩ) filled with a solution containing (mM): 115 CsMeSO3, 20 CsCl, 10 HEPES, 2.5 MgCl2, 4 Na2-ATP, 0.4 Na-GTP, 10 sodium phosphocreatine, 0.6 EGTA, pH 7.25, osmolarity 290 mOsm. Recordings were performed using transmitted light illumination under a microscope equipped with a 40X water immersion objective. Neurons were held either at -70 mV to record spontaneous excitatory postsynaptic currents (sEPSC) or at 0 mV to record spontaneous inhibitory postsynaptic currents (sIPSCs). Access resistance was monitored throughout the experiments, cells with changes above 20 % were discarded. Recordings were analyzed using Clampfit 10 (Molecular Devices). Data acquisition was performed with a Multiclamp 700B amplifier, a 1550B digitizer and pClamp 10 software (Molecular Devices). Data were acquired at a 20 kHz sampling rate and were filtered at 10 kHz. -- Social behavior: Social behavior was assayed by a three chamber test following standard procedures45,46. In brief, the test was conducted in a cage measuring 60 × 40 × 22 cm, featuring dividing walls with openings that allowed access into each chamber. The test mouse was placed in the middle chamber and allowed to explore for 10 minutes. Following this habituation period, an unfamiliar subject of the same sex (mouse 1, M1) was placed in one of the side chambers within a small, wire cage that permitted contact. In the sociability session, the test mouse had a choice of spending time in either the empty chamber (E) or the chamber occupied by M1. At the end of the sociability session, each mouse was tested in a second 10-min session to evaluate social preference for a new subject (M2) during the social novelty session. Control mice commonly spent more time exploring the novel animal (M2)45,46. Exploration time was quantified offline from continuous video recordings using BORIS and SMART video-tracking software (PanLab S.L.). -- Statistics and reproducibility: All of the statistical details of experiments can be found in the figure legends which include the statistical tests used, exact value of n, exclusion of any data, and what n represents (cells, slices, mice). Statistical analyses were conducted using GraphPad Prism 8 data analysis software. Results in dispersion plots are presented as the mean ± standard error of the mean (SEM). In violin plots, thick black bar represents the 25 % - 75 % interquartile (IQR) range; thin bars indicate the range within 1.5 x IQR; white dot indicates the median. P values &lt; 0.05 were considered statistically significant. Statistical tests used for each specific data set are indicated in the corresponding figure legends. The sample size was determined based on values used in previous experiments and informed by earlier studies in the field using the same methods and measured comparable outcomes. Male and female animals were indistinctively used and data was pooled for the analysis. No sex differences were observed.