DOI: 10.34810/data2715
title: Replication Data for: The role of Fezolinetant in fear memory consolidation

description: All behavioral sessions were divided into the following phases:
• FA – Fear acquisition
• FE1 – Fear extinction 1
• FE2 – Fear extinction 2
• FE3 – Fear extinction 3
• FE4 – Fear extinction 4
The two output files obtained from the EzTrack software after video analysis are included in the dataset. These are FreezingOutput (freezing time, frame by frame) and SummaryStats (containing key information such as file length, motion cutoff, freeze threshold, etc.).
All files are labeled as FA, FE1, FE2, FE3, or FE4, followed by the corresponding animal identification number. Due to experimental conditions, datasets for both males and females were collected in two independent batches. Accordingly, the corresponding freezing analyses were organized by batches. When the animal number is followed by “.1,” it refers to the first batch (1n) (e.g., 8.1 corresponds to animal 8 from batch 1), whereas numbers followed by “.2” refer to the second batch (e.g., 8.2 corresponds to animal 8 from batch 2).
Details for all animals, their respective experimental groups, and their freezing percentages are provided in the Excel files “1.FC fezolinetant 1n males and females” (first batch) and “1.FC fezolinetant 2n males and females” (second batch). Please download it as original file format so you can see that each session (FA, FE1, FE2, FE3, FE4) is divided in one tab of the excel file. 
For the proestrus analysis, animals were labeled using the format c1a3, where c indicates the cage and a indicates the animal number, following the specific experimental design. The analyzed freezing data and corresponding treatment for each animal are included in the Excel file “1.FC fezolinetant proestrus.”
description: METHODOLOGICAL INFORMATION

Mice
All experiments were conducted using adult male and female (8-10 weeks old) C57BL/6J mice (Charles River Laboratories). The animals were housed in groups of four to six animals under standard conditions, including a 12:12 h light/dark cycle (lights on from 8:00 am to 8:00 pm), with a controlled temperature of 22 ± 1 °C and humidity (∼40 %). Behavioral procedures and pharmacological manipulations began at 8:00 am. All procedures were approved by the Ethics Committee of the Universitat Autònoma de Barcelona (Ref: 4541) and the Generalitat de Catalunya (Ref: 12033) and were carried out in accordance with the European Communities Council Directive (2010-63-UE) and Spanish legislation (RD 53/2013).

Cued-Fear conditioning
All stages of the Cued-Fear Conditioning test – Habituation, Fear Acquisition (FA), and Fear Extinction (FE) were performed in a fear chamber (25 × 25 × 25 cm3) inside a sound-attenuating chamber (67 × 53 × 55 cm3) with a computerized Startle system (Panlab-Harvard, Barcelona, Spain). Delivery of tones and shocks was controlled by Packwin v2.0 software (Panlab-Harvard, Barcelona, Spain).
This procedure was carried out according to Florido et al. (2021, https://doi.org/10.1038/s41467-021-22911-9). Briefly, animals were habituated to the chambers for 5 min/day for two consecutive days before FA. All animals were exposed to a 5-min habituation period in the fear chamber before the onset of the first tone. During FA, all groups received 5 trials consisting of a tone as the Conditioned Stimulus (CS) (30 s, 6 kHz, 75 dB) that coterminated with a footshock which served as the Unconditioned Stimulus (US) (1 s, 0.3 mA). The intertrial interval (ITI) was 3 min, and 3 additional min followed the last CS. All animals received treatments 30 min after FA to manipulate the consolidation of memory. The first FE test was performed 24 h after FA, and repeated for 3 consecutive days. Thus, mice were exposed to 15 trials of the 30 s CS tone alone (with a 0.5 min of ITI interval. To ensure that freezing was specific tone-shock conditioning, different contexts were utilized for FA and FE. FA context consisted of a yellow light source (∼10 lx), a grid floor that dispensed the footshocks, and a solution of ethanol (EtOH) 70 % (v/v) odor that was used for cleaning between sessions. FE context consisted of a red light source (∼10 lx), a gray floor covering the bars, no background noise, and CR36—0.11 % didecyldimethylammonium chloride, and isopropyl alcohol 41 % (José Collado, Barcelona, Spain) for cleaning. Changes in the length and turns of the transportation route from the vivarium to the testing room between FA and FE.

Vaginal smears cytology
Determination of the estrous stage in females was performed by assessing vaginal smear cytology (Florido et al., 2021). Before the fear acquisition, female mice had the estrous cycle assessed for 2 consecutive cycles (approximately 8–10 days) for habituation. The vaginal lavage was performed with a 20 μl pipette loaded with 10 μl of NaCl 0.9 % (w/v) solution. The 10 μl of saline was unloaded and collected 5 consecutive times and placed on an adhesion slide (Superfrost Plus, Avantor-VWR, Pensilvanya, EUA). All vaginal smear samples were collected between 8:00 and 10:00 am. Slides were dried using a hot plate (RT2 Advanced, Thermo Fisher Scientific, Massachusetts, EUA) at 37 °C for 30 min and later stained in Cresyl Violet Acetate (C5042, Sigma-Aldrich, Spain) 0.1 % (v/v), washed twice for 1 min in distilled water and read with a 10× or 20× objective in an Eclipse Ei microscope (Zeiss, Spain). The different stages of the estrous cycle were assessed depending on the proportion of cornified epithelial cells, round nucleated epithelial cells, or leukocytes. After the cycle assessment, the female mice were immediately exposed to FA when in the Proestrus phase (maximum 2 h from vaginal lavage collection) – characterized by a high proportion (&gt;80 %) of nucleated epithelial cells and small amounts of cornified epithelial cells. Conditioned animals were housed separately from non-conditioned animals.

1. Description of methods used for collection-generation of data: 
Freezing behavior was analyzed using ezTrack (Pennington et al., 2021: doi: 10.1002/cpz1.554).

2. Methods for processing the data: 
Statistical analyses were performed using IBM SPSS Statistics 27.0, and graphs were generated using GraphPad 9.0. Trials from FA, or the mean of groups of 4 trials for FE tests, were analyzed using two-way repeated measures ANOVA. The Least Significant Difference (LSD) post-hoc test was applied to evaluate overall treatment effects and individual differences in cases of significant interactions. Fear expression was analysed by Paired T-Test when comparing the freezing time in habituation x the first five CS of each group. Unpaired T-Test was used when comparing the fear expression between groups. Results are presented as means ± SEM for 11–12 animals per group, divided into two replicates. Statistical significance was set at P ≤ 0.05. Outlier detection was conducted using Grubb’s test and removed when appropriate. The sample size was determined by using G*Power 3.1.9.7 (effect size: 0.45, α error probability: 0.05 and Power: 0.8).

3. Instrument- or software- specific information needed to interpret the data:
Excel and SPSS. 

4. Instruments, calibration and standards information:
Fear conditioning system from PanLab: LE116 experimental chamber, LE100-26 shocker. 

5. Environmental or experimental conditions:
Considering that they were performed in the animal facility of institute of neurosciences - UAB, no changes in the Environmental affects the results. 

6. Quality-assurance procedures performed on the data:
All recording cameras were regularly inspected and confirmed to be functioning properly throughout data acquisition. The freezing detection threshold was validated to accurately capture the animals’ behavioral responses, ensuring consistent and reliable quantification of freezing episodes. Statistical analyses were conducted using appropriate methods for the experimental design and data distribution, and all datasets were checked for completeness and consistency prior to analysis.
description: MS Excel, null
description: SPSS, null