Machine Learning and Confirmatory Factor Analysis Show that Buprenorphine Alters Motor and Anxiety-like Behaviors in Male, Female, and Obese C57BL/6J Mice

Neurology Chemistry Medicinal Chemistry Biochemistry Life Sciences Psychiatry Medicine
Buprenorphine Machine Learning Confirmatory Factor Analysis Motor Behavior Anxiety-like Behavior C57BL/6J Mice Obesity

In recent years, with the global increase in drug abuse, particularly opioids, scientists have become increasingly focused on the neurobehavioral effects of such substances. Among them, buprenorphine, an opioid, is widely used for medication-assisted treatment of opioid addiction. However, beyond its analgesic and addiction treatment effects, buprenorphine has also been reported to potentially contribute to the clinical management of anxiety symptoms. Although anxiety is a highly prevalent disorder in humans, it is a latent construct that cannot be directly measured, especially in animal models. Therefore, this study aims to evaluate the effects of buprenorphine on motor and anxiety-like behaviors in C57BL/6J mice by combining machine learning techniques and confirmatory factor analysis (CFA), exploring its relationship with dose, sex, and body mass.

Paper Source

This research was conducted by a team of researchers including Ohm Sharma, Michael Mykins, and Rebecca E. Bergee from The University of Tennessee. The team members are affiliated with various departments at the university, including the Neuroscience Program in Psychology, Department of Biochemistry & Cellular and Molecular Biology, and Office of Innovative Technologies. The paper was first published in the Journal of Neurophysiology on December 31, 2024, and officially released on February 10, 2025.

Research Process and Methods

Subjects and Experimental Design

The study involved 30 C57BL/6J mice, divided into three groups: normal-weight male mice (n=10), normal-weight female mice (n=10), and diet-induced obese male mice (n=10). The mice were purchased from Jackson Laboratory at 12 weeks of age and underwent a 2-3 week adaptation period upon arrival. All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Tennessee and followed the ARRIVE-2 guidelines.

Experimental Procedure

  1. Drug Administration: In the control phase of the experiment, all mice received a subcutaneous injection of 0.3 mL of 0.9% saline (vehicle control). After 14 days, the mice were administered either 1.0 mg/kg or 10 mg/kg of buprenorphine in a repeated-measures, within-subject design. One hour after injection, the mice were placed in the Elevated Zero Maze (EZM) for a 5-minute behavioral observation.

  2. Behavioral Recording and Analysis: The experiment used a high-definition camera (1080p, 30 frames per second) to record the mice’s behavior in the EZM. The videos were uploaded to cloud storage and analyzed using DeepLabCut (DLC) and Simple Behavioral Analysis (SimBA) software for automated pose estimation and behavioral analysis. DLC employed a convolutional neural network (CNN) to label and track seven body parts of the mice (nose, left ear, right ear, shoulder, center spine, left hindlimb, right hindlimb, and tail base), generating a two-dimensional digital model of the mouse’s position in the maze.

  3. Data Analysis: The study analyzed five motor behaviors: time spent in the open areas of the EZM, latency to enter the open areas, running speed, distance traveled, and the frequency of head dips (HD) and stretch-attend postures (SAP). Data analysis employed repeated-measures mixed-model analysis of variance (ANOVA) and confirmatory factor analysis (CFA) to assess the effects of buprenorphine on anxiety-like behavior in mice.

Key Findings

  1. Effects of Buprenorphine on Motor Behavior: The study found that buprenorphine significantly altered the five motor behaviors of the mice. Specifically, male mice spent significantly less time in the open areas of the EZM after buprenorphine administration, exhibited increased latency to enter the open areas, and showed significant increases in running speed and distance traveled. Female mice and obese mice also displayed similar changes in running speed and distance traveled, although the effects were less pronounced in obese mice.

  2. Confirmatory Factor Analysis Results: The CFA results indicated that the latent construct of anxiety-like behavior accounted for a statistically significant amount of variance in all five motor behaviors. Specifically, the anxiety-like behavior construct explained 28% of the variance in motor behavior, suggesting that buprenorphine significantly influenced the mice’s motor behavior through the construct of anxiety-like behavior.

Conclusions and Implications

The findings of this study support the hypothesis that buprenorphine affects motor behavior in mice by altering anxiety-like behavior. This discovery not only deepens our understanding of the neurobehavioral effects of buprenorphine but also provides new perspectives for future research on its potential applications in anxiety treatment. Additionally, the study demonstrates the strong potential of machine learning techniques in animal behavior research, particularly in automated behavioral analysis and the quantification of latent psychological constructs.

Highlights of the Study

  1. Innovative Research Methods: This study is the first to apply machine learning algorithms such as DeepLabCut and Simple Behavioral Analysis to investigate the effects of buprenorphine on motor and anxiety-like behaviors in mice, significantly enhancing the objectivity and accuracy of behavioral analysis.

  2. Multidimensional Research Design: The study examined the modulating effects of dose, sex, and body mass on buprenorphine’s impact, revealing for the first time the differential effects of buprenorphine on anxiety-like behavior under different physiological conditions.

  3. Theoretical Contributions: The findings support Darwin’s theory of the evolutionary continuity of emotional expression, indicating that anxiety-like behavior in mice shares a similar biological basis with human anxiety.

Additional Valuable Information

The study also highlights the potential clinical applications of buprenorphine in humans, particularly in the treatment of comorbid anxiety and depression. While buprenorphine is widely used for opioid addiction treatment, its role in anxiety management requires further research. Furthermore, the study suggests the long-term effects of chronic buprenorphine exposure on anxiety-like behavior in mice, providing new directions for future research.