Mechanism of Kongsheng Zhenzhong Dan In Autism-Related Pathways: A Study Based On Network Pharmacology, Molecular Docking, And Experimental Validation

Summary

This study integrates network pharmacology, molecular docking, and molecular dynamics (MD) simulation to elucidate core active components and therapeutic targets of Kongsheng Zhenzhong Dan (KSZZD) in autism spectrum disorder (ASD), identifies key binding residues via alanine scanning, and validates effects in a valproic acid-induced ASD rat model.

Abstract

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder, and its pathogenesis is closely associated with chronic neuroinflammation. As a classic traditional Chinese medicine (TCM) formula, Kongsheng Zhenzhong Dan (KSZZD) is used for neurodevelopmental and memory disorders because of its reported neuroprotective effects, but its specific molecular mechanism in ASD remains unclear. This study integrates network pharmacology, molecular docking analysis, and MD simulation to systematically elucidate the core active components and candidate therapeutic targets of KSZZD for ASD at both the systemic and atomic levels. Subsequently, key binding residues are identified by alanine flexible scanning, and the biological effects are assessed using a valproic acid (VPA)-induced ASD rat model. Network pharmacology analysis revealed that quercetin, a key component in KSZZD, may act partly by regulating the TNF-α-mediated inflammatory signaling pathway. Molecular docking results showed that the binding energies of the core components of KSZZD to their targets were all lower than -5.0 kcal/mol, among which quercetin exhibited the strongest binding affinity to TNF (-8.52 kcal/mol). MD simulation suggested that quercetin could maintain stable binding to the active pocket of TNF-α, and alanine scanning further identified GLU104 and GLN102 as key amino acid residues for maintaining the structural stability of the complex. In vivo experiments demonstrated that quercetin intervention significantly improved locomotor and exploratory behaviors in VPA-induced ASD rats, and effectively reduced TNF-α expression levels in serum and brain tissue. These findings suggest that quercetin may contribute to the predicted effects of KSZZD, at least in part, through inhibition of the TNF-α signaling pathway.

Introduction

Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder characterized by impaired social communication, restricted interests, and repetitive stereotyped behaviors¹. The pathological mechanism of ASD is highly complex, involving the interplay of genetic variations, epigenetic regulation, and environmental factors, which ultimately leads to impaired synaptic plasticity and abnormal neural circuit connectivity^2,3. Chronic inflammation in the central nervous system (CNS) plays a central role in the pathogenesis of ASD, and interventions targeting neuroinflammation have emerged as promising therapeutic strategies.

Kongsheng Zhenzhong Dan (KSZZD), a classic TCM formula, was first recorded in Essential Prescriptions Worth a Thousand Gold for Emergencies. It consists of four medicinal herbs: Tortoise Carapace, Os Draconis, Polygalae Radix, and Acori Tatarinowii Rhizoma. KSZZD is traditionally used to tonify the heart and kidney, tranquilize the mind, and stabilize the spirit. Clinically, it is commonly used to treat intellectual developmental delay and memory impairment⁴. Modern pharmacological studies have suggested that KSZZD and its active components exert neuroprotective, anti-inflammatory, and antioxidant effects, and may improve learning, memory, and anxiety-like behaviors in neurodevelopmental disorder models⁵. Our phytochemical analysis revealed that quercetin and other flavonoids are the key active components of this formula, which can penetrate the blood-brain barrier and exhibit significant anti-inflammatory activity⁶. However, the mechanism by which these active components mediate the therapeutic effects of KSZZD in ASD remains poorly understood.

With the rapid development of computational biology, techniques such as network pharmacology, molecular docking, molecular dynamics simulation, and quantum chemistry calculation have provided powerful tools for elucidating the interactions between active components of traditional Chinese medicine and disease targets at the atomic level^7,8,9,10,11. By integrating heterogeneous data sources, network pharmacology enables the construction of a complex, interactive "component-target-pathway" network from a systems biology perspective, thereby preliminarily identifying key biological nodes for traditional Chinese medicine intervention in diseases.

On this basis, molecular docking can be further used to simulate the geometric matching and energy fitting between components and target proteins, and predict their binding modes and potential activities. Subsequently, a molecular dynamics (MD) simulation is performed to investigate the structural fluctuation and binding stability of the complex under aqueous and physiological conditions. Combined with alanine scanning, site‑directed mutagenesis simulation is applied to accurately locate the core hot‑spot residues responsible for maintaining binding affinity, and to clarify the contribution weight of the pharmacophore at the amino acid level. Finally, verification based on well‑designed pharmacological experiments can effectively reveal the molecular mechanism underlying the therapeutic effects of traditional Chinese medicine compounds on specific diseases.

Accordingly, this study first aims to screen the core components and targets of KSZZD in the treatment of ASD via network pharmacology. Subsequently, combined with molecular docking, molecular dynamics simulation, and alanine flexible scanning, the “hot‑spot residues” maintaining complex stability will be identified, and the dynamic interaction pattern between quercetin and TNF‑α will be evaluated. Finally, the computational results will be experimentally verified in a VPA‑induced ASD rat model using assessments of animal behavior, brain histopathology, and inflammatory factor expression levels. This study aims to explore potential mechanisms by which KSZZD-derived components may affect autism-related inflammatory pathways from a multi-scale perspective, providing a basis for further investigation of traditional Chinese medicine interventions in neurodevelopmental disorders.

Protocol

Network pharmacology analysis

"Autism" was used as the keyword to search the GeneCards database (https://www.genecards.org/) for autism-related targets^12,13,14. Targets retrieved from the databases were merged after duplicate entries were removed. Known targets of active components not captured by database predictions were supplemented based on literature reports¹⁵. The disease targets and potential targets of drug components were uniformly standardized to Gene Symbols using the UniProt protein database (https://www.uniprot.org/), and these two target sets were mapped to identify the potential therapeutic targets of KSZZD for autism¹⁶.

Based on the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, http://lsp.nwu.edu.cn/tcmsp.php), the potential bioactive compounds in KSZZD and their corresponding targets were screened out in accordance with the criteria of oral bioavailability (OB ≥ 0.30) and drug-likeness index (DL ≥ 0.18)¹⁷. The SMILES identifiers of the compounds were retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/), and Swiss Target Prediction (http://swisstargetprediction.ch/) was further employed to explore the potential targets not included in the TCMSP platform¹⁸. Subsequently, the HERB database (http://herb.ac.cn) was used for supplementary screening in accordance with the Lipinski's Rule of Five, with the criteria as follows: molecular weight (MW ≤ 500 Da), octanol-water partition coefficient (AlogP ≤ 5), number of hydrogen bond donors (Hdon ≤ 5), number of hydrogen bond acceptors (Hacc ≤ 10), and number of rotatable bonds (RBN ≤ 10). Then, target screening was performed using the BATMAN-TCM database (http://bionet.ncpsb.org.cn/batman-tcm/) with the following criteria: score cut-off (≥0.84), druggability score (≥0.10), and P value (≤0.05). Finally, the UniProt database (https://www.uniprot.org) was employed to convert the selected target names into standard Gene Symbols.

The overlapping targets between the drug targets and autism targets were identified using the online Draw Venn Diagram tool (http://bioinformatics.psb.ugent.be). These overlapping targets were imported into the STRING database (https://string-db.org/) to construct a protein-protein interaction (PPI) network¹⁹. The species was set to Homo sapiens, and protein-protein interactions with confidence scores below 0.40 were filtered out. The resulting network was imported into Cytoscape 3.10.0 software for visual analysis¹⁶, where drugs, compounds, and targets were represented by red diamonds, blue circles, and green triangles, respectively, and the edge weights reflected the degree centrality of the nodes. The CytoNCA plugin was then used to calculate degree centrality values, rank the core compounds, and identify the main core compounds. Bioinformatics enrichment analysis of the target genes was performed using the Metascape platform²⁰, including GO analysis (biological process, BP; molecular function, MF; cellular component, CC) and KEGG pathway analysis.

Molecular docking

The autism-related receptor proteins identified above were preprocessed before docking by repairing missing residues, optimizing the protonation state, and removing crystal water molecules to maintain receptor structural integrity. The semi-flexible docking method in the CDOCKER module²¹ was then employed. Residues within 10 Å of the co-crystal ligand were defined as the active pocket, and ligand-receptor binding was simulated within this region.

Docking results were assessed using CDOCKER Interaction Energy as the key index, with lower energy values indicating more stable predicted binding between the ligand and receptor. The reliability of the docking mode was evaluated by comparing the spatial binding conformation of the core component with that of the co-crystal ligand in the active pocket. The complex with the optimal binding energy and strongest interaction was selected as the initial conformation for the subsequent molecular dynamics simulation.

Molecular dynamics simulation

Based on the molecular docking findings, a molecular dynamics simulation was performed to explore the binding mechanism between the quercetin complex and TNF-α. This approach simulates molecular motion and interactions at the atomic level and analyzes dynamic changes in proteins, ligands, and the surrounding environment, thereby providing information on molecular conformational change, binding stability, and protein-ligand dynamics. The receptor-ligand complex was solvated using the TIP3P water model, with a buffer distance of at least 12 Å between the complex and the system boundary to ensure full solvation and reduce boundary effects. The ionic concentration was set to 0.154 M, and Na⁺ and Cl⁻ ions were added to neutralize the system charge. To improve simulation accuracy, the Amber14SB protein force field²² was adopted, which effectively describes non-bonded intermolecular interactions and binding modes and is particularly suitable for protein-ligand complex studies²³.

For initial energy minimization, the steepest descent method was run for 5000 steps to remove unreasonable contacts and high-energy conformations, using a convergence threshold of 10 kJ/mol/nm to ensure relaxation of the force field parameters. The conjugate gradient method was then run for 2,000 steps to further optimize the thermodynamic state and ensure system stability²⁴.

During equilibration, NVT ensemble simulation was first performed for 100 ps with a 2 fs timestep. The system was gradually heated to 300 K to reduce the influence of the initial structure and reach thermodynamic equilibrium. The ensemble was then switched to NPT, and an additional 100 ps equilibration was performed at a constant pressure of 1 bar to stabilize density and pressure. This stage was used to bring the system to a stable thermodynamic state before production simulation²⁵. The formal molecular dynamics simulation was performed for 20 ns, with the temperature maintained at 300 K, pressure at 1 bar, and a time step of 2 fs. The trajectory was saved every 10 ps. To ensure simulation stability and accuracy, physical parameters, including temperature, pressure, and volume, were monitored regularly to confirm that they remained within the expected ranges.

Alanine flexible scanning

Based on the stable conformation obtained from molecular dynamics simulation, alanine scanning was performed on all amino acid residues within a 3 Å radius of the ligand-binding interface. In this procedure, target residues are systematically replaced with alanine, thereby truncating the side chain while preserving the main-chain conformation and removing side-chain-mediated specific interactions. The contribution weight of each residue to binding affinity was quantified by calculating the change in binding free energy between the wild-type and mutant complexes. Unlike traditional static models, this study introduced a side-chain flexible relaxation mechanism, allowing the environment around the mutation site to undergo structural relaxation and more realistically simulate the dynamic response of the binding interface. This analysis aimed to identify candidate hotspot residues that maintain complex stability, providing an energetic fingerprint for lead compound optimization targeting autism-related proteins.

Animal experiments

Experimental animals

Healthy SPF-grade Sprague-Dawley (SD) rats (three males and three females, aged 3 months), born and reared under identical conditions, were selected. The room temperature was controlled at 18–22 °C, the relative humidity was maintained at 60%–70%, and the light cycle was 12 h: 12 h (light: dark). All animal experimental operations were approved by the Experimental Animal Ethics Committee of The First People's Hospital of Zunyi (Approval No.: LunShen (2025)-2-362).

Animal mating, pregnancy identification, and grouping

All rats were adaptively fed in an SPF environment for 1 week after purchase. One female rat and one male rat were caged together at 18:00 every afternoon. Vaginal plug examination was performed at 8:00 the next morning (12 h after caging). The presence of a vaginal plug was considered a successful mating, and the same day was designated as gestational day 0.5 (GD0.5). Pregnant rats were housed individually in separate cages. The body weight of pregnant rats was measured and recorded daily. The body weight of pregnant rats increased continuously, with an average daily gain of 2–5 g, and the total body weight could increase by about 30% before delivery. After approximately 10 days of pregnancy, a typical pear-shaped asymmetric bulge of the abdomen was visible, and hard masses of fetuses could be felt by palpation, which was distinguished from the uniform and soft abdominal circumference caused by obesity.

Model establishment and control group setting:

For the model group, two pregnant rats were randomly selected and intraperitoneally injected with VPA solution (600 mg/kg) once on gestational day 12.5 (GD12.5). VPA was administered via a single intraperitoneal injection at a dose of 600 mg/kg on gestational day 12.5. This regimen was selected based on the seminal work by Schneider and Przewłocki²⁶, who established that VPA exposure at this specific gestational time point recapitulates both the neuroanatomical and behavioral features of human ASD. This protocol has since become the standard model and has been consistently validated in recent pharmacological studies using identical parameters²⁷. For the blank control group, one pregnant rat was selected and intraperitoneally injected with an equal volume of 0.9% normal saline at the same time point. Pregnant rats delivered naturally, and the birth day of the offspring was recorded as postnatal day 0 (PND0). All offspring rats were weaned and housed separately by gender on PND21.

Offspring grouping and intervention

On PND28, 12 male pups were randomly selected from the offspring of VPA-exposed pregnant rats (6 in the model group and 6 in the quercetin intervention group), and 6 male pups were randomly selected from the offspring of normal saline-exposed pregnant rats (6 in the blank group). It was ensured that there was no significant difference in body weight among the pups of each group. Offspring born to pregnant rats treated with normal saline were assigned to the blank group, those born to pregnant rats treated with VPA to the model group, and those born to pregnant rats treated with VPA to the quercetin intervention group.

Continuous intervention was performed for 4 weeks starting from PND28. For the quercetin intervention group, quercetin suspension was intragastrically administered at a fixed time every day at a dose of 100 mg/kg/day. This dosage was selected based on the following integrated evidence: (i) a prior dose-ranging study identified 100 mg/kg as the optimal dose for alleviating anxiety-like behaviors and reducing pro-inflammatory cytokines in an LPS-induced neuroinflammation rat model²⁸; (ii) quercetin at 50 mg/kg has been shown to prevent social interaction deficits and oxidative brain damage in a prenatal VPA-induced autism rat model²⁹; and (iii) oral quercetin was recently demonstrated to reduce brain TNF-α levels and ameliorate autistic-like behaviors in a propionic acid-induced autism rat model³⁰. Collectively, these independent validations support the selection of 100 mg/kg to ensure robust engagement of the TNF-α-mediated inflammatory pathway in the present postnatal VPA-induced ASD model. For the blank and model groups, an equal volume of 0.5% CMC-Na normal saline solution was intragastrically administered every day. All animals had free access to food and water during the intervention period, and their body weights were measured every week to adjust the administration volume according to their body weights.

Open field test

After 4 weeks of intervention (approximately PND56), the open field test was performed to evaluate spontaneous activity and anxiety levels. The open field test (OFT) is a classic behavioral experiment for evaluating anxiety-related behavior in experimental animals. The measured indicators were the spontaneous movement ability of rats in an open environment and the time spent in the center of the open field. The OFT apparatus for rats was 30 cm in height, 50 cm in length, and 50 cm in width at the bottom, with white inner walls, and was artificially divided into 16 small grids, including 4 grids in the inner area and 12 grids in the outer area. The experimental site was kept quiet to avoid sound stimulation that might affect the accuracy of the experimental results. Each rat was placed in the center of the bottom of the box, and video recording and timing were carried out simultaneously. The camera field of view covered the entire open field and recorded the spontaneous movement of the rats and the number of crossings between grids. Each test lasted 5 min, after which video recording was stopped. The inner wall and bottom of the open box were wiped with 75% alcohol to prevent feces and body odor left by one animal from affecting the test results of the next animal. The operation was repeated after replacing the rats until all rats completed the test.

Pathological detection method

After fixation with 4% paraformaldehyde, brain tissue was subjected to gradient dehydration using a fully automatic dehydrator: 75% ethanol for 2 h, 85% ethanol for 1 h, 95% ethanol for 1 h, and absolute ethanol I-IV for 20 min each. The tissues were then cleared with clearing agent I for 25 min and clearing agent II for 30 min, followed by paraffin embedding. Sections with a thickness of 5 µm were dewaxed with dewaxing solution I and II for 30 min each and rehydrated with gradient ethanol. The sections were stained with hematoxylin for 5–10 min, differentiated with hydrochloric acid alcohol for 3 s, and counterstained with alkaline water to turn blue. They were then counterstained with alcohol-soluble eosin for 3 min, dehydrated with gradient ethanol, cleared, coverslipped with neutral mounting medium, and observed under a microscope.

Detection of TNF-α levels in serum and brain tissue

After the last administration, abdominal aortic blood was collected from rats and centrifuged at 3,000 r/min at 4 °C for 15 min, and the serum supernatant was collected. Meanwhile, hippocampal and cortical tissues were dissected, and precooled PBS was added at a ratio of 1:9 for mechanical homogenization. The homogenate was then centrifuged at 12,000 r/min at 4 °C for 20 min to collect the supernatant. The BCA method was used for tissue protein quantification. Following the ELISA kit instructions, the samples to be tested and biotinylated antibodies were added to the microplate in sequence. After incubation and washing, the avidin-peroxidase complex was added and incubated. Following another wash, substrate solution was added for color development, the reaction was terminated with stop solution, and absorbance was measured at 450 nm using a microplate reader. The concentrations of TNF-α in serum (pg/mL) and brain tissue (pg/mg prot) were calculated using the standard curve.

Statistical analysis

The RANDBETWEEN function in Microsoft Excel was used to randomize the rats and generate random numbers for rat allocation. All experimental data in this study were expressed as mean ± standard deviation (SD). Before statistical analysis, normality test (Shapiro-Wilk test) and homogeneity of variance test (Levene test) were first performed on the data of each group. For data that conformed to normal distribution and homogeneity of variance, one-way analysis of variance (one-way ANOVA) was used. If the difference was statistically significant, LSD method was further used for multiple comparisons. A value of *p < 0.05, **p < 0.01, and ***p < 0.001 was considered statistically significant.

Results

Analysis of the results of network pharmacology study on KSZZD in the treatment of autism

A total of 15,770 autism-related targets were retrieved from the GeneCards database. Targets with a relevance score ≥ 4.21 were selected as potential targets for autism, resulting in 1014 disease targets. The 827 targets of KSZZD were intersected with the 1,014 autism targets, and 125 common targets were obtained. Visualization was performed using the SRplot platform³¹

Discussion

A critical question is why quercetin, rather than other core components, was selected for experimental validation and whether it can represent the holistic effects of KSZZD. This selection was based on the following converging lines of evidence: (i) in a prenatal VPA-induced rat model, quercetin significantly prevented social interaction deficits and oxidative brain damage²⁹; (ii) in a propionic acid-induced autism model, oral quercetin reduced brain TNF-α levels and ameliorated autistic-like...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5% CMC-Na normal saline solution	Not specified in manuscript	Not specified	Vehicle used for blank/model groups and quercetin suspension.
0.9% normal saline	Not specified in manuscript	Not specified	Vehicle for blank-control maternal injections.
4% Paraformaldehyde in PBS	Thermo Scientific Chemicals	J61899.AK	Used for fixation of brain tissue.
75% alcohol	Not specified in manuscript	Not specified	Used to clean the open-field apparatus between animals.
Absolute ethanol	Not specified in manuscript	Not specified	Used for tissue dehydration before embedding.
Alkaline water	Not specified in manuscript	Not specified	Used to blue hematoxylin-stained sections.
Amber14SB protein force field	Not specified in manuscript	Not applicable	Protein force field used for molecular dynamics simulations.
BATMAN-TCM database	Not specified in manuscript	Not applicable	Online target-screening database: http://bionet.ncpsb.org.cn/batman-tcm/
BCA Protein Assay Kit	Thermo Scientific	23225	Used for protein quantification of tissue homogenates.
Bluing reagent	Thermo Scientific Richard-Allan Scientific	7301	Used after hematoxylin staining to blue nuclei.
CDOCKER module	Not specified in manuscript	Not applicable	Semi-flexible docking module used for molecular docking.
Clearing / dewaxing reagent	Thermo Scientific Richard-Allan Scientific	Clear-Rite 3, 6901	Can be used for clearing and deparaffinization in histology workflows.
CytoNCA plugin	Not specified in manuscript	Not applicable	Cytoscape plugin used for degree centrality analysis.
Cytoscape software	Not specified in manuscript	Version 3.10.0	Used for network visualization and protein-protein interaction analysis.
Discovery Studio software	Not specified in manuscript	Versions 2019 and 2021	Used for receptor preprocessing, docking, and alanine scanning.
Draw Venn Diagram online tool	Not specified in manuscript	Not applicable	Online tool for Venn diagrams: http://bioinformatics.psb.ugent.be
Eosin-Y (alcoholic)	Thermo Scientific Richard-Allan Scientific	7111	Used for cytoplasmic counterstaining in H&E staining.
Fully automatic dehydrator	Not specified in manuscript	Not specified	Used for gradient dehydration of fixed brain tissue.
GeneCards database	Not specified in manuscript	Not applicable	Online database used to retrieve autism-related targets: https://www.genecards.org/
GraphPad Prism software	Not specified in manuscript	Version 9.5	Used for statistical analysis and graphing.
Hematoxylin staining solution	Thermo Scientific Richard-Allan Scientific	7211	Used for nuclear staining in H&E staining.
HERB database	Not specified in manuscript	Not applicable	Online database used for supplementary compound screening: http://herb.ac.cn
Hydrochloric acid alcohol	Not specified in manuscript	Not specified	Used for differentiation during H&E staining.
Metascape platform	Not specified in manuscript	Not applicable	Online platform used for GO and KEGG enrichment analysis.
Microplate reader	Thermo Scientific	Multiskan FC, 51119000	Used for absorbance reading at 450 nm.
Microscope	Not specified in manuscript	Not specified	Used to observe H&E-stained brain sections.
Microsoft Excel software	Microsoft	Not specified	Used to randomize rats with the RANDBETWEEN function.
Mounting medium	Thermo Scientific Richard-Allan Scientific	4111	Used for coverslipping and permanent mounting of stained sections.
Open field test apparatus	Not specified in manuscript	Not specified	50 cm × 50 cm × 30 cm box used for behavioral testing.
Paraffin	Not specified in manuscript	Not specified	Used for paraffin embedding of brain tissue.
Phosphate-buffered saline (PBS), pH 7.4	Gibco	10010023	Used as pre-cooled buffer for tissue homogenization.
PLIP interaction fingerprinting	Not specified in manuscript	Not applicable	Used for protein-ligand interaction fingerprint analysis.
PubChem database	Not specified in manuscript	Not applicable	Online database used to retrieve SMILES identifiers: https://pubchem.ncbi.nlm.nih.gov/
Quercetin	Not specified in manuscript	Not specified	Administered by gavage at 100 mg/kg/day.
Rat TNF-α ELISA Kit	Invitrogen	ERA56RB	Suitable for serum and tissue homogenate TNF-α detection.
Refrigerated centrifuge	Thermo Scientific	Sorvall ST 16R, 75004383	Used for serum and tissue homogenate centrifugation.
Schrödinger Suite software	Schrödinger, LLC	Version 2024	Used to construct the molecular dynamics simulation system.
SPF-grade Sprague-Dawley rats	Not specified in manuscript	Not specified	3 male and 3 female rats used for animal model establishment.
SPSS software	Not specified in manuscript	Version 25.0	Used for statistical analysis.
SRplot platform	Not specified in manuscript	Not applicable	Online platform used for visualization: https://www.bioinformatics.com.cn/
STRING database	Not specified in manuscript	Version 12.0	Used to construct the protein-protein interaction network: https://string-db.org/
SwissTargetPrediction platform	Not specified in manuscript	Not applicable	Online platform used for target prediction: http://swisstargetprediction.ch/
TCMSP database	Not specified in manuscript	Not applicable	Online platform used for bioactive compound screening: http://lsp.nwu.edu.cn/tcmsp.php
TIP3P water model	Not specified in manuscript	Not applicable	Water model used for molecular dynamics simulation.
UniProt protein database	Not specified in manuscript	Not applicable	Used to standardize target names to Gene Symbol: https://www.uniprot.org/
Valproic acid (VPA) solution	Not specified in manuscript	Not specified	Administered intraperitoneally at 600 mg/kg on gestational day 12.5.
Video recording system/camera	Not specified in manuscript	Not specified	Used to record rat movement during the open field test.