A System for Tracking the Dynamics of Social Preference Behavior in Small Rodents

Summary

Described here is a novel automated experimental system that offers an alternative to the three-chamber test and also solves several caveats. This system supplies multiple behavioral parameters that enable rigorous analysis of small rodent behavioral dynamics during the social preference and social novelty preference tests.

Abstract

Exploring the neurobiological mechanisms of social behavior requires behavioral tests that can be applied to animal models in an unbiased and observer-independent manner. Since the beginning of the millennium, the three-chamber test has been widely used as a standard paradigm to evaluate sociability (social preference) and social novelty preference in small rodents. However, this test suffers from multiple limitations, including its dependence on spatial navigation and negligence of behavioral dynamics. Presented and validated here is a novel experimental system that offers an alternative to the three-chamber test, while also solving some of its caveats. The system requires a simple and affordable experimental apparatus and publicly available open-source analysis system, which automatically measures and analyzes multiple behavioral parameters at individual and population levels. It allows detailed analysis of the behavioral dynamics of small rodents during any social discrimination test. We demonstrate the efficiency of the system in analyzing the dynamics of social behavior during the social preference and social novelty preference tests as performed by adult male mice and rats. Moreover, we validate the ability of the system to reveal modified dynamics of social behavior in rodents following manipulations such as whisker trimming. Thus, the system allows for rigorous investigation of social behavior and dynamics in small rodent models and supports more accurate comparisons between strains, conditions, and treatments.

Introduction

Revealing the biological mechanisms underlying neurodevelopmental disorders (NDDs) is one of the main challenges in the field of neuroscience¹. Addressing this challenge requires behavioral paradigms and experimental systems that typify the behavior of rodents in a standard and unbiased manner. An influential study published more than a decade ago by Moy and colleagues² presented the three-chamber test. Since then, this test has been widely used to investigate social behavior in rodent models of NDDs. This test evaluates two innate tendencies of rodents: 1) to stay in the proximity of a social stimulus over an object (sociability, also termed social preference [SP]), and 2) to prefer the proximity of a novel social stimulus over a familiar one (social novelty preference [SNP])^3,4. Several subsequent studies suggested methods of automated analysis of the three-chamber test using computerized methods^5,6.

This test still suffers from several caveats. First, it principally examines social place preference rather than the motivation of the subject to directly interact with a social stimulus, although some groups also measure olfactory investigation (sniffing) time, either manually⁷ or using commercial computerized systems^8,9,10. Second, the three-chamber test is mostly used to measure the total time spent by the subject in each chamber, and it neglects behavioral dynamics. Finally, it relies on only one aspect of the social behavior, which is the time spent by the subject in each chamber (or sniffing time, if measured).

Here we present a novel and affordable experimental system that is an alternative to the three-chamber apparatus. It also allows performance of the same behavioral tests while solving the abovementioned caveats. The presented behavioral system automatically and directly measures the investigative behavior of a rodent towards two stimuli. Additionally, it analyzes the behavioral dynamics in an observer-independent manner. Moreover, this system measures multiple behavioral parameters and analyzes these at both individual and population levels; thus, it supports a rigorous analysis of social behavior and its dynamics during each test. Furthermore, random repositioning of the chambers in opposite corners of the arena during the various test stages neutralizes any effects of spatial memory or preference. This system may also be used for other discrimination tests, such as sex discrimination. The custom apparatus is easy to produce, and the analysis system is publicly accessible as an open-source code, thereby allowing its use in any laboratory. We demonstrate the ability of this system to measure multiple parameters of social behavior in rodent strains with distinct fur colors during the social preference and social novelty preference tests. We also validate the ability of the system to reveal modified dynamics of social behavior in rodents following manipulations, such as whisker trimming.

TrackRodent software: three algorithms were written in MATLAB (2014a-2019a) to track the experimental subject and its interactions with the stimuli. All algorithms were deposited in GitHub, found at <https://github.com/shainetser/TrackRodent>. The main aim of all four algorithms is to track the contours of the subject's body to detect any direct contact with the stimuli areas.

Body-based algorithm: this algorithm has three versions that track the contours of an unwired dark mouse on a white background (BlackMouseBodyBased), a white mouse on a dark background (WhiteMouseBodyBased), or a white rat on a dark background (WhiteRatBodyBased). The graphical user interface (GUI) of the software requires that the experimenter chooses an experiment using either mice or rats and then selects the correct code. For each version of the algorithm, there are two optional codes: one that presents the tracking process on the screen while it performs the analysis, and one that does not (hence, it runs faster and is termed "fast"). For example, the names of the relevant codes for the BlackMouseBodyBased algorithm are: "BlackMouseBodyBased23_7_14" and "BlackMouseBodyBased23_7_14_Fast". All algorithms ending with "fast" do not show the tracking online, and users must directly save the data to the results file (.mat file). All body-based algorithms require setting a single threshold ("low threshold" in the software's GUI) to detect the body of the subject.

Head-directionality based algorithm: the second algorithm, which is available only for black mice, is based on the body-based algorithm, in addition to determining head directionality. This algorithm detects the interactions of subject's head with the "stimuli" areas, thus avoiding false positives that can arise from random contacts of the subject' with these areas. For this algorithm, two detection thresholds of mouse body contours are defined: high threshold, which includes the brighter tail of black mice, and low threshold, which includes the body without tail. Thereafter, the algorithm fits an ellipsoid to the detected boundaries using the lower threshold and defines the location of the mouse head and tail (with no distinction between the two). The final discrimination between the tail and head is based on the boundaries defined by the higher threshold.

Wired animal algorithm: the third algorithm aims to minimize artifacts resulting from cables (i.e., electrical wire or optical fiber) connected to the animal, allowing analysis of the animal's behavior while connected to a cable. This algorithm has codes only for black mice and white rats. The code for rats requires the experimenter to define both low and high thresholds, while the mouse code requires only a low threshold.

Protocol

All methods described have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Haifa. 1. Experimental Set-up Arena Construct the experimental arena for mice (Figure 1A,D) by placing a white or black (depending on the animal's color) plexiglass box (37 cm x 22 cm x 35 cm) in the middle of an acoustic chamber (60 cm x 65 cm x 80 cm, made of 2 cm thick wood coated …

Representative Results

Using the system for the social preference test in C57BL/6J mice Figure 1 shows three versions of the experimental set-up. The first version (Figure 1A-C) is designed for mice with dark fur colors, such as C57BL/6J mice. The second (Figure 1D-F) is planned for mice with bright fur colors, such as BALB/c or ICR (CD-1) mice. The third is larger (…

Discussion

The experimental system described here, which was designed as an alternative to the three-chamber apparatus^2,5, allows performance of the same tests while solving some of its limitations. The use of triangular chambers, which are located in two opposite corners of the rectangular arena limits the subject-stimulus interaction area to a well-defined plane, thus enabling precise automated analysis of investigation behavior. One advantage is the use of the analysis s…

Materials

Flea3 1.3 MP Mono USB3 Vision	FLIR (formerly PointGrey)	FL3-U3-13Y3M-C	Monochromatic Camera
FlyCap 2.0	FLIR (formerly PointGrey)	FlyCapture 2.13.3.61X64	Video recording software
Home 5 minute Epoxy glue	Devocon	20845	For gluing the metal mesh to the Plexiglas stimuli chambers
Matlab 2014-2019	MathWorks	R2014a – R2019a	Programming environment
Plexiglas boards (6 mm thickBlack or white)	Melina (1990) LTD, Israel	NaN	For arena and stimuli chambers construction
Red led strips (60 leds per meter) connected to a 12V power supply	2012topdeal eBay supplier	NaN	For illumination of the acoustic chamber