The main functions of the Analysis Module are to automatically detect behaviors and analyze the results of experiments. The Analysis Module imports videos and accompanying keypoint tracking data and smooths the tracking data. It can accommodate previously recorded videos since keypoint tracking models can be trained posthoc. Users can track any keypoints they choose. We used DeepLabCut (DLC) (Mathis et al., 2018) for keypoint tracking. DLC produces a list of comma-separated values that contains framewise estimates of the X-Y coordinates for designated body parts as well as a likelihood statistic for each estimated point. The Analysis Module applies a threshold based on DLC likelihood and performs a Hampel transformation (Hampel, 1974) to remove outliers. Then, a local regression smoothing method (LOWESS) is applied to the data (Cleveland, 1981), and sub-threshold tracking values are estimated using surrounding data and spline interpolation (Figure 2A). The module then performs a feature expansion step, calculating additional keypoint positions based on the originally tracked set (Supplementary file 1). We designed a set of postural and kinematic metrics that are calculated automatically for each keypoint (Supplementary file 2). These metrics serve as the inputs for BehaviorDEPOT’s heuristics.

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We created a set of heuristics to detect several human-defined behaviors including freezing, rearing, escape, locomotion, and novel object investigation. From the graphical interface of the Analysis Module, users can select a heuristic and indicate if they want to analyze behaviors during particular time windows or within regions of interest (ROI) and whether they plan to do batched analyses. The Analysis Module also includes spatial functions for analyzing the open field test, elevated plus maze, T-maze and three chamber assays. By allowing users to perform integrated spatial-temporal-behavioral analyses, BehaviorDEPOT’s utility extends beyond existing open-source behavioral classification software, which typically just report which behaviors occur in each video frame. Later in the manuscript, we describe how users can create their own heuristics and incorporate them into the graphical interface.

The Analysis Module exports all relevant data in a set of MATLAB structures (Supplementary file 3) so that users can easily perform additional analyses if needs arise (Figure 2A). For instance, users may want to align the data to neural signals, a process we discuss in upcoming sections. A structure called ‘Tracking’ stores raw and smoothed keypoint tracking data. ‘Params’ stores the parameters of video recordings, smoothing functions, heuristics used, and arena metrics (e.g. ROI size and location). ‘Metrics’ stores kinematic and postural statistics calculated from keypoint positions (Supplementary file 2). ‘Behavior’ stores bout-wise and vectorized representations of identified behaviors across the entire video, or with respect to user-defined spatiotemporal filters.

The Analysis Module also generates a series of graphical data representations (Supplementary file 3). For instance, trajectory maps show when an animal was in a particular location and where behaviors occurred. Bout maps indicate when behaviors occurred and for how long. These visual representations help users understand behavioral phenotypes in great spatiotemporal detail and can inform further custom analyses using the data structures that the Analysis Module generates. In the following sections, we describe the development and validation of BehaviorDEPOT’s heuristics and demonstrate its utility in numerous behavioral assays.

Freezing behavior, defined as the absence of movement except for respiration (Grossen and Kelley, 1972; Fanselow and Bolles, 1979; Fanselow, 1984), is widely studied as a proxy for both learned and innate fear (Anagnostaras et al., 1999; Anagnostaras et al., 2010; Perusini and Fanselow, 2015). In many laboratory assays, rodents freeze in response to perceived environmental threats including conditioned cues, predator odor, or looming disks that mimic a predator approaching from above (Perusini and Fanselow, 2015). Despite the heavy study of freezing behavior, most labs still score freezing manually or using expensive commercially available software programs that often fail when animals have tethered headmounts. Here, we describe the development and validation of the BehaviorDEPOT freezing detection heuristic and demonstrate its accurate performance across a range of experimental setups, including in mice wearing head-mounts for optogenetics and Miniscopes.

The BehaviorDEPOT freezing detection heuristic combines a convolution-based smoothing operation with a low-pass velocity filter and then applies a minimum duration threshold to identify periods of freezing. To develop the freezing heuristic, we began by training a deep neural network in DLC on videos recorded with a high resolution and high frame rate camera (Chameleon3 USB3, FLIR) at 50 frames per second (fps). The network tracked 8 points on the body and BehaviorDEPOT smoothed the raw tracking data (Figure 2B and C). An expert rater annotated freezing in three randomly selected videos (27,000 total frames). This served as a reference set for heuristic development. We reasoned that freezing could be reliably detected based on periods of low keypoint velocity. After exploring the predictive value of different keypoints using the Data Exploration Module (described below), we determined that thresholding the velocity of a midpoint on the back and the angular velocity of the head produced the most accurate freezing detection heuristic (Figure 2D). We smoothed the heuristic output by applying a sliding window to produce a convolved freezing vector in which each value represented the number of freezing frames visible when the window is centered at a given frame. We then applied an adjustable count threshold to convert the convolved freezing vector into the final binary freezing vector (Figure 2E).

To validate heuristic performance, we manually annotated a different, randomly selected set of videos that were never referenced while setting the parameters (Supplementary file 4). These videos were recorded in several different behavioral chambers under varied lighting conditions. BehaviorDEPOT freezing detection was highly consistent with human annotations (Figure 2F). Accuracy of the freezing heuristic was estimated based on precision, recall, F1 score, and specificity. Precision and recall quantify the positive predictive value against the tendency to produce false positive or false negative errors, respectively. The F1 score, the harmonic mean of the precision and recall, is useful as a summary statistic of overall performance. Specificity quantifies the ability to accurately label true negative values and helps ensure that the heuristic is capturing data from only a single annotated behavior. Our heuristic had very low error rates (Figure 2F).

We also assessed how good the DLC tracking needed to be for the freezing heuristic to work well. We trained ten different DLC networks with mean tracking errors ranging from 1 to 8 pixels. We used each network to analyze a set of six videos and then used BehaviorDEPOT to automatically detect freezing in each video. A linear regression analysis revealed that tracking error had a significant effect on precision and on the F1 score without affecting recall or specificity (Figure 2—figure supplement 1; Supplementary file 5), with tracking errors <4 pixels producing the highest F1 scores in our analysis.

To ensure that our heuristic would generalize to other camera types and DLC networks, we trained a second network based on videos recorded with a standard webcam at 30fps. On the webcam, lower sensor quality and lower frame rate produces more blur in the recorded images, so we tracked 4 body parts that are easy to see (nose, ears, tail base; Figure 2G). When compared to human annotations (Supplementary file 4), the webcam videos also scored highly for precision, recall, F1 score, and specificity (Figure 2G), indicating that our freezing heuristic can indeed generalize across camera types and DLC networks.

Since the same rules may not necessarily generalize to all settings, we developed a second freezing heuristic that uses a changepoint function to find frames at which the mean velocity changes most significantly and then separates frames into groups that minimize the sum of the residual error from the local mean. Binarized freezing vectors are then processed using a convolution algorithm and minimum duration threshold (Figure 2E). This heuristic was also highly accurate, performing similarly to the velocity-based heuristic on videos recorded with a webcam (Figure 2—figure supplement 2). Users can tune the heuristic by adjusting a minimum residual threshold for the changepoint function. We termed this the ‘jitter’ heuristic since the minimum residual threshold will be determined by the pixel error in keypoint position estimates. In other words, the threshold will be determined by how much frame-to-frame ‘jitter’ there is in DLC’s estimate of the keypoint location. Keypoint tracking ‘jitter’ may arise as a function of video resolution, framerate, and number of frames used to train a keypoint tracking model. As such, the ‘jitter’ heuristic may accommodate a wider range of video qualities and keypoint tracking models. Also, in videos recorded from the side, the velocity-based freezing heuristic may be slightly affected by distortions in velocity calculations caused by the angle (e.g. when the mouse is moving towards/away from the camera).

As a companion to the heuristic for freezing, we also developed the ‘Experiment Module’, a MATLAB app that allows users to design and run fear conditioning experiments. This extends the utility of BehaviorDEPOT by providing a fully open-source software pipeline that takes users from data collection to data analysis. The Experiment Module controls commercially available shockers, lasers for optogenetics, and sound generators via a set of Arduinos (Figure 1—figure supplement 1). Users can download the ‘Fear Conditioning Experiment Designer’ app from our Github repository and install it with a single button click. From a graphical interface, users can design experimental protocols for contextual or cued fear conditioning, with or without optogenetics. All experimental parameters (e.g. timestamps for laser, tones, shocks) are saved in MATLAB structures that can be referenced by the BehaviorDEPOT analysis pipeline (Figure 1).

In commercial freezing detection software, algorithms often fail when a rodent is wearing a patch cord for routine optogenetics experiments. To ensure that BehaviorDEPOT’s freezing heuristic maintains high levels of accuracy under these conditions, we tested its performance in an optogenetics experiment. mPFC plays a well-established role in fear memory retrieval (Corcoran and Quirk, 2007; LeDoux, 2000), extinction (Giustino and Maren, 2015; Sierra-Mercado et al., 2011), and generalization (Giustino and Maren, 2015; Xu and Südhof, 2013; Pollack et al., 2018). While silencing mPFC subregions can promote fear memory generalization in remote memory (Xu and Südhof, 2013; Frankland et al., 2004), less is known about its role in recent memory generalization. We used an adeno-associated virus (AAV) to express the soma-targeted neuronal silencer stGtACR2 (Mahn et al., 2018) bilaterally in the mPFC and implanted optogenetic cannula directly above the AAV injection sites (Figure 3A, Figure 3—figure supplement 1). We performed contextual fear conditioning (CFC) in context A. The next day, we measured freezing levels in the conditioned context (context A) as well as a novel context (context B) that was never paired with shocks. During these retrieval sessions, 2-min laser-on periods were separated by two minute laser-off intervals (Figure 3B).

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We first tested the accuracy of the freezing heuristic for animals wearing tethered head mounts. We trained an optogenetics-specific DLC network that tracks 9 points on the animal, including the fiber-optic cannula (Figure 3C). Our rationale for creating a separate keypoint tracking network was twofold. First, while you can train one ‘master’ DLC network that can track mice in many different arenas, we find that DLC tracking errors are lowest when you have dedicated networks for particular camera heights, arenas, and types of head-mounted hardware. DLC networks are easy to train and to make it even easier for new users, we provide links to download our DLC models in our GitHub repository. Second, this was another opportunity to test how well the BehaviorDEPOT freezing heuristic generalizes to a different DLC network with a different number of keypoints. For a randomly selected set of videos with different floors and lighting conditions, we compared heuristic performance to expert human raters (Supplementary file 4). Even with the patch cord attached, the freezing heuristic had excellent scores for precision, recall, F1 and specificity (Figure 3D).

Having confirmed the accuracy of the freezing heuristic, we then used BehaviorDEPOT to quantify freezing behavior and compared the results to the annotations of an expert human rater. As expected, fear conditioned mice readily froze following shocks during CFC, while non-shocked controls did not (Figure 3E). During retrieval sessions, silencing mPFC in previously shocked animals significantly enhanced freezing in the novel context but did not affect freezing in the fear conditioned context (Figure 3F and G). mPFC silencing thereby produced a significant decrease in the discrimination index in fear conditioned mice (Figure 3H), indicating that mPFC plays a key role in the specificity of recent fear memories. In all analyses, BehaviorDEPOT freezing estimates were comparable to a highly trained human rater (Figure 3F–H). By maintaining performance levels even in videos with visual distractors like a patch cord, the BehaviorDEPOT freezing heuristic overcomes a major point of failure in commercially available freezing detection software.

As new open-source tools for neurophysiology become available, more labs are performing simultaneous neurophysiological and behavioral recordings. Miniature head-mounted microscopes now allow us to image the activity of hundreds of neurons simultaneously in freely moving animals (Ghosh et al., 2011; Cai et al., 2016; Shuman et al., 2020). These miniscopes pair with genetically encoded Ca²⁺ indicators (Dana et al., 2019) that can be targeted to specific neuronal populations and GRIN lenses (Barretto et al., 2009) that can be targeted anywhere in the brain. With these tools in hand, we can discover how the encoding of complex cognitive and emotional behaviors maps onto specific cell types across the brain. By recording the activity of hundreds of neurons simultaneously, we can also study the population codes that produce complex behaviors (Jercog et al., 2021; Stout and Griffin, 2020). To do so, however, we need improved open-source methods that allow us to quantify freely moving behaviors with reference to salient environmental stimuli and to align these detailed behavioral measurements with neurophysiological recordings.

Here, we demonstrate the utility of BehaviorDEPOT for aligning behavioral measurements with Ca²⁺ signals during a platform-mediated avoidance (PMA) task that has temporally and spatially salient features. PMA is an mPFC-dependent task in which a fear conditioned tone prompts mice to navigate to a safety platform that protects them from receiving a footshock (Bravo-Rivera et al., 2015; Diehl et al., 2020). We recorded the activity of hundreds of mPFC neurons in freely behaving animals using head-mounted microendoscopes (UCLA Miniscopes Cai et al., 2016; Shuman et al., 2020) while simultaneously recording behavior using a new open-source USB camera, the UCLA MiniCAM.

Together with BehaviorDEPOT and the UCLA Miniscopes, the MiniCAM provides a fully open-source data acquisition and analysis pipeline for in vivo Ca²⁺ imaging during freely moving behavior. The MiniCAM is an open-source behavioral imaging platform that natively integrates and synchronizes with open-source UCLA Miniscope hardware and software (Figure 4A). It is composed of an M12 optical lens mount, a custom printed circuit board housing a CMOS image senor and supporting electronics, an LED illumination ring, and a 3D printed case. The MiniCAM is powered and communicates over a single coaxial cable that can be up to 15 m long. The coaxial cable connects to a Miniscope data acquisition board (DAQ) which then connects over USB3 to a host computer. A range of commercial M12 lenses can be used to select the view angle of the camera system. The image sensor used is a 5MP CMOS image sensor (MT9P031I12STM-DP, ON Semiconductor) with 2592x1944 pixel resolution and a full resolution frame rate of approximately 14FPS. For this application, the MiniCAM’s pixels were binned and cropped to achieve 1024x768 pixels at approximately 50FPS. The optional LED illumination ring uses 16 adjustable red LEDs (LTST-C190KRKT, Lite-On Inc, 639 nm peak wavelength) for illumination in dark environments (Figure 4A). We trained a separate DLC network for videos of animals wearing Miniscopes recorded with MiniCAMs. Our network tracked 8 keypoints on the body (ears, nose, midback, hips, tailbase, and tail) and the Miniscope itself (Figure 4B). For these videos, BehaviorDEPOT freezing ratings were highly consistent with expert human annotations (Figure 4C).

Figure 4

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We used BehaviorDEPOT to analyze behavior during PMA so we could align it to the underlying Ca²⁺ activity. In this task, animals are placed in a fear conditioning chamber in which an acrylic safety platform occupies 25% of the electrified grid floor. Three baseline tones are followed by nine tones that co-terminate with a mild foot shock. The following day, we measure avoidance and freezing behaviors during six unreinforced tones (Figure 4D). The Analysis Module automatically produces trajectory maps that make it quick and easy to assess the spatiotemporal characteristics of rodent behavior. In our representative example, the color-coded trajectory and freezing locations (denoted as black squares) converge on the platform at the end of the session, indicating the mouse indeed learned to avoid shocks by entering the platform (Figure 4E). We used BehaviorDEPOT to produce summary data, showing that mice readily learned the cue-avoidance association during training (Figure 4F) and remembered it the next day (Figure 4G).

During a retrieval session, we recorded Ca²⁺ activity using a UCLA Miniscope and behavior using a UCLA MiniCAM (Figure 4H). Using MIN1PIPE (Lu et al., 2018), we extracted and processed neural signals from 513 mPFC neurons across 3 mice. We then determined whether individual neurons encoded specific behaviors that we had quantified using BehaviorDEPOT (Figure 4I). We computed a receiver operating characteristic (ROC) curve that measures a neuron’s stimulus detection strength over a range of thresholds (Figure 4J). We identified numerous neurons that were modulated by freezing and avoidance on the safety platform. These neurons were organized in a salt and pepper manner in mPFC (Figure 4K). Nearly half of all neurons that exhibited task relevant behavior and were specific for either freezing or threat avoidance, or their combination (Figure 4L). These experiments demonstrate that the BehaviorDEPOT heuristic for detecting freezing is robust across a wide variety of experimental setups with different camera types, keypoint tracking networks, arenas and headmounts. Further, this suite of open-source hardware and software will enable a broader user base to combine Ca²⁺ imaging with high resolution behavioral analysis.

BehaviorDEPOT also supports behavioral analyses beyond the realm of conditioned fear. In a number of commonly used assays, behaviors are defined based on animal location or the intersection of location and specific movements (e.g. head turns in a choice zone, described below). Such assays, including elevated plus maze (EPM), open field test (OFT), novel object exploration (NOE), object location memory, social preference/memory, decision-making assays (T-maze), and working memory assays (Y-mazes), examinenumerous cognitive and emotional processes including anxiety, memory, and decision-making and are published in tens of thousands of studies each year. To extend the utility of BehaviorDEPOT to this broad user base, the Analysis Module includes functions that quantify time spent in user defined ROIs. We demonstrate the utility of these functions in four representative assays: EPM, OFT, NOE, and a T-maze. However, the same functions could be used to analyze numerous assays including Y-mazes, real-time or conditioned place preference, as well as social preference and social memory tests.

EPM and OFT are used to measure anxiety-related behaviors in the laboratory (La-Vu et al., 2020), but these assays are often scored manually or with expensive software such as Ethovision (Noldus et al., 2001). In the EPM, rodents navigate through an elevated (~1 foot off the ground) plus-shaped maze consisting of two open arms without walls and two enclosed arms with walls. Animals that spend more time in the closed arms are interpreted to have higher anxiety (La-Vu et al., 2020). In the OFT, rodents are placed in an empty arena without a ceiling for 10–15 minutes. Experimenters measure the fraction of time spent in the perimeter of the box vs. the center. Increased time in the perimeter zone is interpreted as higher anxiety. OFT is also commonly used to measure locomotion in rodents. We trained a DLC network to track animals in an EPM or OFT. We used the BehaviorDEPOT functions to indicate the closed arms, open arms, and center zone for EPM (Figure 5A) and the center zone for OFT (Figure 5B). BehaviorDEPOT uses the midback keypoint to track the animal as it traverses the zones, reporting time spent in each zone. BehaviorDEPOT annotations were highly consistent with an expert human rater (Figure 5D and E ).

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NOE assays can be used to assess exploration and novelty preference (Ennaceur and Delacour, 1988; Leger et al., 2013; Vogel-Ciernia and Wood, 2014; Zeidler et al., 2020). An object is placed in the center of an open field and investigation time is measured. NOE is typically defined as instances when the animal’s head is within 2 cm of the object and oriented towards the object (Leger et al., 2013; Vogel-Ciernia and Wood, 2014). After importing DLC tracking data, BehaviorDEPOT allows users to draw an ROI at a set radius around the object (Figure 5C). The NOE heuristic uses a combination of head angle and distance with respect to the ROI to label ‘investigation’ frames. BehaviorDEPOT quantified NOE at levels that were highly consistent with expert human raters (Figure 5F).

Decision-making is a widely studied area of neuroscience. Many labs study contingency-based, cost-benefit decision-making in mazes that involve choice points. For example, effort-based decision-making involves weighing whether it is worth exerting greater effort for a more valuable outcome. A common rodent assay for effort-based decision-making is the barrier T-maze task in which animals choose whether to climb over a barrier for a large reward (high effort, high value reward; HE/HVR) vs. taking an unimpeded path for a smaller reward (low effort, low value reward; LE/LVR) (Bailey et al., 2016). Well trained animals typically prefer HE/HVR choices, taking a direct route over the barrier with stereotyped trajectories. However, when reward or effort contingencies are adjusted, animals demonstrate vicarious trial and error (VTE), thought to be a marker of deliberative (rather than habitual) decision-making. During VTE trials, animals may pause, look back-and-forth, or reverse an initial choice (Redish, 2016). Several groups have identified neural signatures of VTE in hippocampus, striatum, and prefrontal cortex suggesting that animals may be playing out potential choices (i.e. vicariously) based on an internal model of maze contingencies (Redish, 2016). Simple, flexible, and automated analysis tools for detecting VTE and aligning this behavior with neural data would significantly enhance our understanding of deliberative decision-making.

We used BehaviorDEPOT to automatically detect VTE in a barrier T-maze task and to report the ultimate choice animals made during each effort-based decision-making trial. First, we trained a new DLC network to track mice as they navigated the T-maze. In one version of the task, mice decided whether to turn left to collect a small reward or to turn right and climb a wire mesh barrier for a large reward. We then changed the effort contingency by adding a second barrier to equalize effort across choices. We used the BehaviorDEPOT Analysis Module to perform automated analysis of the task. We defined 8 regions of interest (Approach, Choice, Left Effort, Left Reward, Left Food Cup, Right Effort, Right Reward, and Right Food Cup, Figure 6A). We then used the stored tracking data to automatically detect trials, which we defined as the first frame when the animal entered the approach zone until the first frame when the animal entered the reward zone, and to report the outcome of each trial (left choice or right choice) (Figure 6B–D).

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To develop a VTE heuristic, we used the head angle data stored in the BehaviorDEPOT ‘Metrics’ data structure. For each trial, we analyzed the head angles when the mouse was in the choice zone and used these values to determine the number of head turns per trial. Manually annotated VTE trials tended to have 1 or more head turns, while non-VTE trials tended to have 0 head turns, so we defined VTE trials as having 1 or more head turns in the choice zone (Figure 6E). We then used our BehaviorDEPOT VTE heuristic to detect the fraction of trials with VTE in T-maze sessions with 1 vs 2 barriers, finding a significant increase in the occurrence of VTE trials when a second barrier was added. Importantly, the BehaviorDEPOT performance was highly consistent with a human rater (Figure 6F).

Together, these analyses showcase the many functions of the Analysis Module and highlight the utility of the repository of postural and kinematic statistics that is automatically generated in BehaviorDEPOT. By calculating and storing information including velocity, angular velocity, head angle, and acceleration in a framewise manner, BehaviorDEPOT allows users to design automated analysis pipelines for a wide range of commonly studied cognitive tasks. Indeed, BehaviorDEPOT can be tailored to meet individual needs. In the following sections, we describe additional modules that help users develop custom heuristics and integrate them into the Analysis Module.

To broaden the utility of BehaviorDEPOT, we created four additional modules that guide users through the process of developing their own heuristics. The Inter-Rater, Data Exploration, Optimization and Validation modules help researchers identify and validate the combinations of metrics that track best with their behaviors of interest. Reference annotations can come from human raters or other software.

A major hurdle in developing classifiers or heuristics using supervised approaches is settling on a ground truth definition of the behavior of interest. The Inter-Rater Module compares annotations from multiple human raters and identifies points of overlap and disagreement (Figure 7A). In response to the initial output of the Inter-Rater Module, human raters can discuss the points of disagreement and modify their manual ratings until they converge maximally. The resulting ‘ground truth’ definitions of behavior can be used to benchmark the performance of newly developed heuristics or to optimize existing ones.

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Here, we demonstrate the outputs of the Inter-Rater module using freezing and novel object exploration as examples. This module imports multiple human annotations and users can select a reference dataset (e.g. the most highly trained expert rater, Figure 7B1 and C1). The module compares each set of annotations to the reference, scoring the annotations frame-by-frame as true positive, true negative, false positive, or false negative for each rater. These values are first used to calculate percent overlap and percent error between all raters. Precision, recall, specificity, and F1 score are calculated and reported for each rater relative to the chosen reference (Figure 7B2 and C2). Additionally, visualizations of frame-by-frame percent agreement (Figure 7B3 and C3) and user-by-user disagreement (Figure 7B4 and C4) are automatically generated to assist identifying areas of conflict between users. The automated features of the Inter-Rater Module make it fast and easy to perform iterative comparisons of manual annotations, interleaved with human updates, until a satisfactory level of agreement is achieved. The Inter-Rater Module can function as an independent unit and can thus support heuristic development with any application.

For users who want to develop new heuristics, the Data Exploration Module helps to identify combinations of keypoint metrics that have high predictive value for behaviors of interest. Users can choose between two different exploration modes: broad or focused. In focused mode, researchers use their intuition about behaviors to select the metrics to examine. The user chooses two metrics at a time and the Data Exploration Module compares values between frames where behavior is present or absent and provides summary data. A generalized linear model (GLM) also estimates the likelihood that the behavior is present in a frame across a range of threshold values for the two selected metrics (Figure 8A), allowing users to optimize parameters in combination. Focused mode is fast and easy to use and users can iteratively test as many combinations of metrics as they like.

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In broad mode, the module uses all available keypoint metrics to generate a GLM that can predict behavior as well as a rank-order list of metrics based on their predictive weights. Poorly predictive metrics are removed from the model if their weight is sufficiently small. Users also have the option to manually remove individual metrics from the model. Once suitable metrics and thresholds have been identified using either mode, users can plug any number and combination of metrics into a heuristics template script that we provide and incorporate their new heuristics into the Analysis Module. Detailed instructions for integrating new heuristics are available in our GitHub repository.

We used focused mode to create our freezing heuristic. First, the Data Exploration Module imports the ‘Metrics’ and ‘Behavior’ data structures we produced using the Analysis Module along with reference annotations for the same videos. We used the annotations of an expert human rater for reference. In focused mode, users iteratively select combinations of two keypoint metrics (e.g. head velocity, tail angular velocity, etc.) and a behavior label from the human annotations file (e.g. ‘Freezing’). We reasoned that low velocity of certain bodyparts would correlate with freezing and thus examined the predictive value of several keypoint velocities. The module creates two data distributions: one containing video frames labeled with the chosen behavior and a second containing the remaining video frames. The larger set is randomly downsampled to ensure that each distribution contains equal numbers of frames and then a series of analyses quantify how reliably chosen metrics align with the behavior of interest. Boxplots (Figure 8B) and histograms (Figure 8C) identify features that reliably segregate with frames containing a behavior of interest. Indeed, we discovered that the linear velocity of the back and angular velocity of the head tracked particularly well with freezing (Figure 8B and C). The GLM revealed the combinations of threshold values that best predicted freezing (Figure 8D).

In general, we found that metrics that are well-suited for behavior detection contrast with metrics on frames that do not contain the behavior and have a low standard deviation within the behavior set. Distributions of useful metrics also tend to differ substantially from the total set of frames, especially when compared to frames that do not contain the behavior. The GLM predictions are useful for determining which of the selected metrics best predict the behavior and whether they enhance the predictive value when combined. This module will broaden the utility of BehaviorDEPOT, allowing researchers to tailor its automated functions to fit their needs.

A major advantage of using heuristics to detect behaviors is that parameters can be quickly and easily tuned to optimize behavioral detection in out-of-sample data. Some users of the Optimization Module may have just developed a new heuristic in the Data Exploration Module. Others may want to optimize an existing heuristic for their experimental setup since camera position, framerate and resolution and lighting conditions may influence behavior detection thresholds. To use the module, researchers provide reference annotations and the outputs from the Analysis Module (‘Params’, ‘Behavior’, and ‘Metrics’). By sweeping through the parameter space for chosen metrics, the Optimization Module identifies the set of thresholds that maximize detection accuracy (Figure 9A). Through the graphical interface, users can then update the heuristic threshold values and save the settings for future use. Commercially available freezing classifiers also allow users to adjust the thresholds for freezing detection to ensure that accurate classification can be achieved in a variety of experimental conditions. However, their algorithms are hidden and there is no way to precisely measure error rates. Our Optimization Module adds a level of rigor to the optimization step by reporting heuristic performance as F1 score, precision, recall, and specificity values.

Figure 9

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We used the Optimization Module to tune our freezing heuristic for videos recorded in other laboratories. Two research groups provided videos of rats and mice, respectively, recorded using their own acquisition hardware and software. For each dataset, we trained keypoint tracking networks (Figures 9B1 and 2) and manually annotated freezing in a subset of the videos. We then analyzed behavior using the Analysis Module with the default settings of our freezing heuristic. Using the ‘Parameters’, ‘Behavior’, and ‘Metrics’ files produced by the Analysis Module, the Optimization Module iteratively swept through a range of threshold values for the four metrics that define the freezing heuristic: back velocity, head angular velocity (Figures 9C1 and 2), window width, and count threshold in the convolution algorithm and reported performance for different combinations of values (Figures 9D1 and 2).

The Validation Module can assess a heuristic’s predictive quality by comparing automated behavioral detection to human annotations. The user must first generate a reference set of annotations (either manual or otherwise) and analyze the video using the heuristic of interest in the Analysis Module. In the validation module, the user is prompted to indicate which heuristic to evaluate and to select a directory containing behavior videos and the output files from the Analysis Module (Metrics, Behavior, etc). For each video, the module will categorize each frame as true positive, false positive, true negative, or false negative, using the human data as a reference. Precision, recall, specificity, and F1 score are then calculated and visualized for each video. These statistics are also reported for the total video set by concatenating all data and recalculating performance (Figure 9E). We validated the performance of the BehaviorDEPOT freezing heuristic on the rat (Figure 9F) and mouse (Figure 9G) videos acquired in external laboratories which we had optimized using the Optimization Module. In both cases, the optimal combination of thresholds for each video set (Figure 9C and D) achieved high F1 scores (>0.9), indicating that our chosen features for freezing detection are robust across a range of cameras, keypoint tracking networks, experimental conditions, and rodent species.

Finally, we benchmarked BehaviorDEPOT’s performance against JAABA (Kabra et al., 2013). Like BehaviorDEPOT, JAABA is geared toward a non-coding audience and its classifiers are based on an intuitive understanding of what defines behaviors. JAABA uses supervised machine learning to detect behaviors and has been widely adopted by the Drosophila community with great success. However, fewer studies have used JAABA to study rodents, suggesting there may be some challenges associated with using JAABA for rodent behavioral assays. The rodent studies that used JAABA typically examined social behaviors or gross locomotion, usually in an open field or homecage (Sangiamo et al., 2020; Neunuebel et al., 2015; Phillips et al., 2019; Nomoto and Lima, 2015; van den Boom et al., 2017).

As the basis for its classifiers, JAABA uses a large feature set that it calculates based on animal poses. JAABA is built around trackers like MoTr (Ohayon et al., 2013) and Ctrax (Branson et al., 2009), which model animal position and orientation by fitting ellipses. We used MoTr and Ctrax to track animals in fear conditioning videos we had recorded previously. The segmentation algorithms performed poorly when rodents were in fear conditioning chambers which have high-contrast bars on the floor but could fit ellipses to mice in an open field, in which the mouse is small relative to the environment and runs against a clean white background (Figure 10A–B). Poor tracking in visually complex environments such as a fear conditioning chamber may explain, at least in part, why relatively few rodent studies have employed JAABA.

Figure 10

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To circumvent these issues, we fit ellipses to the mice based on DLC keypoints (Figure 10C). Briefly, we used the nose-to-tail distance to calculate the long axis and the hip-to-hip distance to calculate the short axis and used a MATLAB function to generate well-fit ellipses for each video frame. We then imported the ellipse features (animal position (centroid), semi-axis lengths, and orientation) into JAABA. We first trained a freezing classifier using JAABA. Even when we gave JAABA more training data than we used to develop BehaviorDEPOT heuristics (6 videos vs. 3 videos), the BehaviorDEPOT heuristic for freezing had significantly higher recall and F1 scores when tested on a separate set of videos (Figure 10D). We also trained a VTE classifier with JAABA. When we tested its performance on a separate set of videos, JAABA could not distinguish VTE vs. non-VTE trials. It labeled every trial as containing VTE (Figure 10E), suggesting a well-fit ellipse is not sufficient to detect these fine angular head movements.

Another strength of BehaviorDEPOT relative to JAABA is that BehaviorDEPOT can quantify behaviors with reference to spatial or temporal cues of interest. JAABA does allow users to draw spatial ROIs but uses this information as the basis of behavioral classification in ways that cannot be detected or controlled by the user. These direct comparisons highlight the need for new behavioral analysis software targeted towards a noncoding audience, particularly in the rodent domain. BehaviorDEPOT can now fill this role.

Female and male C57B1/6 J mice (JAX Stock No. 000664) aged 10–16 weeks were group housed (2–5 per cage) and kept on a 12 hr light cycle. Following behavior conditioning, animals were individually housed until the memory retrieval sessions. All animal procedures followed animal care guidelines approved by the University of California, Los Angeles Chancellor’s Animal Research Committee.

Mice were handled for 5 days preceding the behavioral testing procedure. The conditioning chamber consisted of an 18cm x 18cm x 30 cm cage with a grid floor wired to a scrambled shock generator (Lafayette Instruments) surrounded by a custom-built acoustic chamber. The chamber was scented with 50% Windex. Mice were placed in the chamber and then after a 2-min baseline period, received five 0.75mA footshocks spaced 1 min apart. Mice were removed 1 min after the last shock. Non-shocked control animals freely explored the conditioning chamber but never received any shocks. The following day, mice were returned to the conditioning chamber and a novel context (different lighting and metal floor, scented with 1% acetic acid), separated by a 1 hr interval. Context presentation order on day 2 was counterbalanced across mice.

PMA used the fear conditioning chamber described above, except 25% of the floor was covered with a thin acrylic platform (3.5x4 × 0.5 inches). During training, mice were presented with three baseline 30 s 4 kHz tones (CS), followed by nine presentations of the CS that co-terminated with a 2 s footshock (0.13mA). The following day, mice were presented with six CS in the absence of shocks.

AAV1-syn-jGCaMP7f.WPRE (ItemID: 104488-AAV1) was purchased from Addgene and diluted to a working titer of 8.5x10¹² GC/ml. AAV1-CamKIIa-stGtACR2-FusionRed (ItemID: 105669-AAV1) was purchased from Addgene and diluted to a working titer of 9.5x10¹¹ GC/ml.

Adult wildtype C57/Bl6 mice were anesthetized with isoflurane and secured to a stereotaxic frame (Kopf, 963). Mice were placed on a heating blanket and artificial tears kept their eyes moist throughout the surgery. After exposing the skull, we drilled a burr hole above mPFC in both hemispheres (AP +1.8, ML +/-0.3 from bregma). A Hamilton syringe containing AAV1-CaMKIIa-stGtACR2-WPRE was lowered into the burr hole and 400 nL of AAV was pressure injected into each site (DV –2.25 mm and –2.50 mm from bregma) at 100 nL/min using a microinjector (WPI, UMP3T-1). The syringe was left in place for 10 min to ensure the AAV did not spill out of the target region. After injecting the AAV, chronic fiber-optic cannula (0.37NA, length = 2 mm, diameter = 200 um) were implanted bilaterally above the injection site and secured to the skull with Metabond (Parkell, S371, S396, S398). After recovery, animals were housed in a regular 12 hr light/dark cycle with food and water ad libitum. Carprofen (5 mg/kg) was administered both during surgery and for 2 days after surgery together with amoxicillin (0.25 mg/mL) in the drinking water for 7 days after surgery.

Animals were habituated to the patch-cord for 3 days in advance of optogenetic stimulation. A patch-cord was connected to the fiber-optic cannula and animals were allowed to explore a clean cage for 5 min. On the testing day, optical stimulation through the fiber-optic connector was administered by delivering light through a patch-cord connected to a 473 nm laser (SLOC, BL473T8-100FC). Stimulation was delivered continuously with 2.5 mW power at the fiber tip.

For Miniscope recordings, all mice underwent two stereotaxic surgeries (Cai et al., 2016). First, adult WT mice were anesthetized with isoflurane and secured to a stereotaxic frame (Kopf, 963). Mice were placed on a heating blanket and artificial tears kept their eyes moist throughout the surgery. After exposing the skull, a burr hole was drilled above PL in the left hemisphere (+1.85, –0.4, –2.3 mm from bregma). A Hamilton syringe containing AAV1-Syn-jGCaMP7f-WPRE was lowered into the burr hole and 400 nL of AAV was pressure injected using a microinjector (WPI, UMP3T-1). The syringe was left in place for 10 min to ensure the AAV did not spill out of the target region and then the skin was sutured. After recovery, animals were housed in a regular 12 hr light/dark cycle with food and water ad libitum. Carprofen (5 mg/kg) was administered both during surgery and for 2 days after surgery together with amoxicillin (0.25 mg/mL) for 7 days after surgery. Two weeks later, mice underwent a GRIN lens implantation surgery. After anesthetizing the animals with isoflurane (1–3%) and securing them to the stereotaxic frame, the cortical tissue above the targeted implant site was carefully aspirated using 27-gauge and 30-gauge blunt needles. Buffered ACSF was constantly applied throughout the aspiration to prevent tissue desiccation. The aspiration ceased after full termination of bleeding, at which point a GRIN lens (1 mm diameter, 4 mm length, Inscopix 1050–002176) was stereotaxically lowered to the targeted implant site (–2.0 mm dorsoventral from skull surface relative to bregma). Cyanoacrylate glue was used to affix the lens to the skull. Then, dental cement sealed and covered the exposed skull, and Kwik-Sil covered the exposed GRIN lens. Carprofen (5 mg/kg) and dexamethasone (0.2 mg/kg) were administered during surgery and for 7 days after surgery together with amoxicillin (0.25 mg/mL) in the drinking water. Two weeks after implantation, animals were anesthetized again with isoflurane (1–3%) and a Miniscope attached to an aluminum baseplate was placed on top of the GRIN lens. After searching the field of view for in-focus cells, the baseplate was cemented into place, and the Miniscope was detached from the baseplate. A plastic cap was locked into the baseplate to protect the implant from debris.

Mice were handled and habituated to the weight of the microscope for 4 days before behavioral acquisition. On the recording day, a V4 Miniscope was secured to the baseplate with a set screw and the mice were allowed to acclimate in their home cage for 5 min. Imaging through the Miniscope took place throughout the entire PMA training (~30 min) and retrieval session the following day. Behavior was simultaneously recorded with a UCLA MiniCAM.

Frames in which animals were freezing and/or on the safety platform were determined using BehaviorDEPOT. Cell footprints and Ca²⁺ fluorescence timeseries were extracted from Miniscope recordings using MIN1PIPE. We identified 513 neurons across three mice. Custom MATLAB software was used to align data from the behavior camera and the Miniscope camera. To identify neurons that were active during freezing or when the animal was on the safety platform, we plotted receiver operating characteristic curves (ROC) for individual neurons and measured the area under the curve (AUC). ROCs plot the true positive rate (true positive/(true positive +false negative)) against the false positive rate (false positive/(false positive +true negative)) over a range of probability thresholds. Neurons with high AUC values therefore predict the behavioral variable of interest with a high true positive rate and low false positive rates over a large range of thresholds. To determine if a neuron significantly encoded a particular behavioral event, we generated a null distribution of AUCs by circularly shuffling event timing and recalculating the AUC over 1000 permutations. Neurons were considered significantly activated during a behavior if their AUC was greater than 97.5% of AUCs in the null distributions and significantly suppressed if their AUC value was in the lowest 2.5% of all AUCs in the null distribution.

Mice were transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. Brains were dissected, post-fixed in 4% PFA for 12–24 hr and placed in 30% sucrose for 24–48 hr. They were then embedded in Optimum Cutting Temperature (OCT, Tissue Tek) and stored at –80 °C until sectioning. 60 µm floating sections were collected into PBS. Sections were washed 3x10 min in PBS and then blocked in 0.3% PBST containing 10% normal donkey serum (Jackson Immunoresearch, 17-000-121) for 2 hr. Sections were then stained with rabbit anti-RFP (Rockland 600-41-379 at 1:2000) in 0.3% PBST containing 3% donkey serum overnight at 4 °C. The following day, sections were washed 3x5 min in PBS and then stained with secondary antibody (Jackson Immunoresearch Cy3 donkey anti-rabbit IgG(H+L) 711-165-152, 1:1000) in 0.3% PBST containing 5% donkey serum for 2 hr at room temperature. Sections were then washed 5 min with PBS, 15 min with PBS +DAPI (Thermofisher Scientific, D1306, 1:4000), and then 5 min with PBS. Sections were mounted on glass slides using FluoroMount-G (ThermoFisher, 00-4958-02) and then imaged at 10 x with a Leica slide scanning microscope (VT1200S).

For the open field test, a 50 cm x 50 cm arena with 38 cm high walls was used. The total area of the arena was 2500 cm² and the center was determined to be one fourth of this, 625 cm² or a 25 cm x 25 cm square. Mice were placed into the room 20 min prior to test for habituation. At the beginning of the test, mice were placed in the arena facing the wall and allowed to explore for 10 min. Arena was cleaned with 70% ethanol between animals. The total time spent in the center was determined using the OFT heuristic in BehaviorDEPOT and a 2-min epoch was compared to human annotations.

The EPM consisted of a cross-shaped platform 38 cm off the ground with four arms, two that are enclosed by walls 20 cm in height. Mice were placed into the room 20 min prior to test for habituation. For the test, mice were placed in the center of the platform (5 cm x 5 cm) facing a closed arm and allowed to explore for 8 min. The EPM was cleaned between animals with 70% ethanol. The number of entries into the open arms and the time spent in the open arms were determined using the EPM heuristic in BehaviorDEPOT and compared to human annotations for six full-length videos.

The NOE used the same arena as OF and mice were placed into the room 20 min before testing took place. The objects used were an empty isoflurane bottle (6.5 cm x 6.5 cm x 10 cm) and an ice pack (6 cm x 6.5 cm x 15 cm). At the beginning of each session, the object, cleaned with 70% ethanol, was placed into the center of the arena. The mouse was placed facing a wall into the arena and allowed to explore for 10 min. The arena and objects were cleaned between animals. The time spent exploring the novel object was defined by a human annotator as when the mouse’s head was oriented toward the object with the nose within 2 cm of the object. If the mouse was on top of the object, this was not included. These human annotations were compared to the output of BehaviorDEPOT using the NOE heuristic.

For T-maze experiments, mice were food deprived to ~85% of the ad libitum initial weight (3–4 days) and habituated to handling and maze exploration. Reward pellets were chopped Reese’s peanut butter chips (~0.01 g each). One arm of the maze was designated as high value reward (‘HVR’, 3 pellets), the other low value reward (‘LVR’, 1 pellet). Mice were trained to perform effort-reward decision-making via a sequential process. Once mice learned to choose the HVR arm (>80%), a 10 cm wire-mesh barrier was inserted to block that arm. Training was complete when mice achieved >70% success high effort/HVR choices on 2 consecutive days. Forced trials were used to encourage sampling of the barrier arm during training. Once mice achieved stable performance, a second barrier was inserted in the LVR arm to equalize effort between choices.

Behavioral videos were acquired using one of the following three setups:

1. 50fps using a Chameleon3 3.2 megapixel monochrome USB camera fitted with a Sony 1/1.8 sensor (FLIR systems, CM3-U3-31S4M-CS) and a 1/1.8 lens with a 4.0–13 mm variable focal length (Tamron, M118VM413IRCS). We recorded 8-bit videos with a 75% M-JPEG compression.

2. 30fps using a ELP 2.8–12 mm Lens Varifocal Mini Box 1.3 megapixel USB Camera.

3. 50fps using a UCLA MiniCAM 5 megapixel CMOS sensor (MT9P031I12STM-DP, ON Semiconductor)

Two-minute samples of each video recording were manually annotated by 1–3 highly trained individuals for freezing behavior using QuickTime7, FIJI (FFMPEG plugin), or MATLAB. One-minute intervals were chosen from the beginning and end of the video recordings to capture diverse behaviors. In some cases, the entire video was annotated. Freezing was defined as the absence of movement except for respiration. Novel object exploration was defined as when the mouse’s head was oriented toward the object with the nose within 2 cm of the object. If the mouse was on top of the object this was not included. Time in zones of the EPM and OFT was defined by when the middle of the back crossed the threshold between zones. VTE was defined as when the animal was in the choice zone and swept its head back and forth across ~180°. Annotations were iteratively refined after discussion between users until they converged maximally and then each heuristic was developed based on the annotations of one human expert.

To develop heuristics, videos were randomly assigned to heuristic development and test sets. Dividing up the dataset by video rather than by frame ensures that highly correlated temporally adjacent frames are not sorted into training and test sets, which can cause overestimation of accuracy. Since the videos in the test set were separate from those used to develop the heuristics, our validation data reflects the accuracy levels users can expect from BehaviorDEPOT. Heuristic performance was assessed using precision (true positive frames / true positive frames + false positive frames), recall (true positive frames / true positive frames + false negative frames), F1 score (2*(precision*recall / precision + recall)) and specificity (true negative frames / true negative frames + false positive frames).

Heuristic parameters for freezing, NOE, and VTE were chosen based on human definitions of these behaviors and examined in a test dataset of videos. For freezing, we explored linear and angular velocity metrics for various keypoints, finding that angular velocity of the head and linear velocity of a back point tracked best with freezing. Common errors in our heuristics were identified as short sequences of frames at the beginning or end of a behavior bout. This may reflect failures in human detection. Other common errors were sequences of false positive or false negative frames that were shorter than a typical behavior bout. We included the convolution algorithm to correct these short error sequences, finding empirically that window widths that are the length of the smallest bout of ‘real’ behavior and count thresholds approximately one third of the window width yielded the best results.

Statistical analyses were performed in MATLAB or GraphPad Prism.

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

We thank A Klein and N Gogolla for contributing mouse behavioral videos for analysis with BehaviorDEPOT. We thank A Adhikari and P Schuette for helpful advice in designing a freezing heuristic. We thank J Reichl for assistance with DLC network training. We thank C Klune for helpful suggestions on the manuscript. This work was funded by NIH K01MH116264 (L.A.D.), Whitehall Foundation Research Grant (L.A.D), Klingenstein-Simons Foundation Grant (L.A.D.), NARSAD Young Investigator Award (L.A.D.), NIH T32MH073526 (B.J.), ARCS Pre-doctoral Fellowship (C.J.G.), NSF Graduate Research Fellowship (C.M.G) and NIH K08MH116125 (S.A.W.).

All of the animals were handed according to the approved institutional animal care and use committee protocols of the University of California, Los Angeles. The protocol was approved by the University of California, Los Angeles Chancellor's Animal Research Committee (ARC-2019-012-AM-004).

© 2022, Gabriel et al.

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