Head-fixed Mice Research Articles

Meso-Py: Dual Brain Cortical Calcium Imaging in Mice during Head-Fixed Social Stimulus Presentation.

We present a cost-effective, compact foot-print, and open-source Raspberry Pi-based widefield imaging system. The compact nature allows the system to be used for close-proximity dual-brain cortical mesoscale functional-imaging to simultaneously observe activity in two head-fixed animals in a staged social touch-like interaction. We provide all schematics, code, and protocols for a rail system where head-fixed mice are brought together to a distance where the macrovibrissae of each mouse make contact. Cortical neuronal functional signals (GCaMP6s; genetically encoded Ca2+ sensor) were recorded from both mice simultaneously before, during, and after the social contact period. When the mice were together, we observed bouts of mutual whisking and cross-mouse correlated cortical activity across the cortex. Correlations were not observed in trial-shuffled mouse pairs, suggesting that correlated activity was specific to individual interactions. Whisking-related cortical signals were observed during the period where mice were together (closest contact). The effects of social stimulus presentation extend outside of regions associated with mutual touch and have global synchronizing effects on cortical activity.

Full field-of-view virtual reality goggles for mice

Visual virtual reality (VR) systems for head-fixed mice offer advantages over real-world studies for investigating the neural circuitry underlying behavior. However, current VR approaches do not fully cover the visual field of view of mice, do not stereoscopically illuminate the binocular zone, and leave the lab frame visible. To overcome these limitations, we developed iMRSIV (Miniature Rodent Stereo Illumination VR)-VR goggles for mice. Our system is compact, separately illuminates each eye for stereo vision, and provides each eye with an ∼180° field of view, thus excluding the lab frame while accommodating saccades. Mice using iMRSIV while navigating engaged in virtual behaviors more quickly than in a current monitor-based system and displayed freezing and fleeing reactions to overhead looming stimulation. Using iMRSIV with two-photon functional imaging, we found large populations of hippocampal place cells during virtual navigation, global remapping during environment changes, and unique responses of place cell ensembles to overhead looming stimulation.

Mesotrode chronic simultaneous mesoscale cortical imaging and subcortical or peripheral nerve spiking activity recording in mice.

Brain function originates from hierarchical spatial-temporal neural dynamics distributed across cortical and subcortical networks. However, techniques available to assess large-scale brain network activity with single-neuron resolution in behaving animals remain limited. Here, we present Mesotrode that integrates chronic wide-field mesoscale cortical imaging and compact multi-site cortical/subcortical cellular electrophysiology in head-fixed mice that undergo self-initiated running or orofacial movements. Specifically, we harnessed the flexibility of chronic multi-site tetrode recordings to monitor single-neuron activity in multiple subcortical structures while simultaneously imaging the mesoscale activity of the entire dorsal cortex. A mesoscale spike-triggered averaging procedure allowed the identification of cortical activity motifs preferentially associated with single-neuron spiking. Using this approach, we were able to characterize chronic single-neuron-related functional connectivity maps for up to 60 days post-implantation. Neurons recorded from distinct subcortical structures display diverse but segregated cortical maps, suggesting that neurons of different origins participate in distinct cortico-subcortical pathways. We extended the capability of Mesotrode by implanting the micro-electrode at the facial motor nerve and found that facial nerve spiking is functionally associated with the PTA, RSP, and M2 network, and optogenetic inhibition of the PTA area significantly reduced the facial movement of the mice. These findings demonstrate that Mesotrode can be used to sample different combinations of cortico-subcortical networks over prolonged periods, generating multimodal and multi-scale network activity from a single implant, offering new insights into the neural mechanisms underlying specific behaviors.

Mesotrode allows chronic simultaneous mesoscale cortical imaging and subcortical or peripheral nerve spiking activity recording in mice

Brain function originates from hierarchical spatial-temporal neural dynamics distributed across cortical and subcortical networks. However, techniques available to assess large-scale brain network activity with single-neuron resolution in behaving animals remain limited. Here, we present Mesotrode that integrates chronic wide-field mesoscale cortical imaging and compact multi-site cortical/subcortical cellular electrophysiology in head-fixed mice that undergo self-initiated running or orofacial movements. Specifically, we harnessed the flexibility of chronic multi-site tetrode recordings to monitor single-neuron activity in multiple subcortical structures while simultaneously imaging the mesoscale activity of the entire dorsal cortex. A mesoscale spike-triggered averaging procedure allowed the identification of cortical activity motifs preferentially associated with single-neuron spiking. Using this approach, we were able to characterize chronic single-neuron-related functional connectivity maps for up to 60 days post-implantation. Neurons recorded from distinct subcortical structures display diverse but segregated cortical maps, suggesting that neurons of different origins participate in distinct cortico-subcortical pathways. We extended the capability of Mesotrode by implanting the micro-electrode at the facial motor nerve and found that facial nerve spiking is functionally associated with the PTA, RSP, and M2 network, and optogenetic inhibition of the PTA area significantly reduced the facial movement of the mice. These findings demonstrate that Mesotrode can be used to sample different combinations of cortico-subcortical networks over prolonged periods, generating multimodal and multi-scale network activity from a single implant, offering new insights into the neural mechanisms underlying specific behaviors.

Abstract 12907: Aging Drives Cerebrovascular Network Remodeling and Functional Changes in the Mouse Brain

Introduction: Cerebrovascular dysfunction has been implicated in age-related cognitive decline and dementia, but the underlying vascular mechanisms are not well understood. An improved understanding of the nature of normal cerebrovascular aging is needed to help to establish the role that vascular dysfunction might play in cognitive decline and dementia. Methods: Here, we asked how normal aging differentially impacts the vascular structure and function in different brain areas in mice. We investigated structural changes in aged cerebrovascular networks and pericytes utilizing serial two-photon tomography (STPT). To further investigate potential remodeling of different vascular compartments and pericyte subtypes, we utilized tissue clearing, 3D immunolabeling, and light sheet fluorescence microscopy (LSFM) imaging. We also assessed how healthy aging impacts brain hemodynamics in response to voluntary locomotion and whisker stimulation in awake, head-fixed mice using wide field optical signal imaging and two-photon imaging. We tested mice of both sexes at 2-month, 18-month (early aging), and 24-month (late aging) of age. Results: Whole-brain vascular tracing using STPT showed an overall ~10% decrease in vascular length and branching density, and LSFM imaging with 3D immunolabeling further revealed increased arteriole tortuosity in aged brains. We also uncovered a selective vascular and pericyte loss in deep cortical layers, basal forebrain regions, and the hippocampal network. This may contribute to their regional vulnerabilities in neurodegenerative disorders. Moreover, our in vivo imaging in awake, head-fixed mice identified delayed neurovascular coupling response and inefficient oxygen delivery in aged brains. Conclusions: Our study reveals aging-related brain-wide changes in vascular and mural cell types that can explain vulnerability and resilience of different brain areas in normal aging. Moreover, we identified an age-related decrease in brain oxygenation and delayed neurovascular coupling responses which can be linked with cognitive impairment in aged brains. These aging-related changes will serve as a common factor to understand many neurodegenerative disorders and cognition decline in the elderly population.

Awake responses suggest inefficient dense coding in the mouse retina.

The structure and function of the vertebrate retina have been extensively studied across species with an isolated, ex vivo preparation. Retinal function in vivo, however, remains elusive, especially in awake animals. Here, we performed single-unit extracellular recordings in the optic tract of head-fixed mice to compare the output of awake, anesthetized, and ex vivo retinas. While the visual response properties were overall similar across conditions, we found that awake retinal output had in general (1) faster kinetics with less variability in the response latencies; (2) a larger dynamic range; and (3) higher firing activity, by ~20 Hz on average, for both baseline and visually evoked responses. Our modeling analyses further showed that such awake response patterns convey comparable total information but less efficiently, and allow for a linear population decoder to perform significantly better than the anesthetized or ex vivo responses. These results highlight distinct retinal behavior in awake states, in particular suggesting that the retina employs dense coding in vivo, rather than sparse efficient coding as has been often assumed from ex vivo studies.

RPM: An open-source Rotation Platform for open- and closed-loop vestibular stimulation in head-fixed Mice

Head fixation allows the recording and presentation of controlled stimuli and is used to study neural processes underlying spatial navigation. However, it disrupts the head direction system because of the lack of vestibular stimulation. To overcome this limitation, we developed a novel rotation platform which can be driven by the experimenter (open-loop) or by animal movement (closed-loop). The platform is modular, affordable, easy to build and open source. Additional modules presented here include cameras for monitoring eye movements, visual virtual reality, and a micro-manipulator for positioning various probes for recording or optical interference. We demonstrate the utility of the platform by recording eye movements and showing the robust activation of head-direction cells. This novel experimental apparatus combines the advantages of head fixation and intact vestibular activity in the horizontal plane. The open-loop mode can be used to study e.g., vestibular sensory representation and processing, while the closed-loop mode allows animals to navigate in rotational space, providing a better substrate for 2-D navigation in virtual environments. The full build documentation is maintained at https://ranczlab.github.io/RPM/.

Mega-scale movie-fields in the mouse visuo-hippocampal network.

Natural visual experience involves a continuous series of related images while the subject is immobile. How does the cortico-hippocampal circuit process a visual episode? The hippocampus is crucial for episodic memory, but most rodent single unit studies require spatial exploration or active engagement. Hence, we investigated neural responses to a silent movie (Allen Brain Observatory) in head-fixed mice without any task or locomotion demands, or rewards. Surprisingly, a third (33%, 3379/10263) of hippocampal -dentate gyrus, CA3, CA1 and subiculum- neurons showed movie-selectivity, with elevated firing in specific movie sub-segments, termed movie-fields, similar to the vast majority of thalamo-cortical (LGN, V1, AM-PM) neurons (97%, 6554/6785). Movie-tuning remained intact in immobile or spontaneously running mice. Visual neurons had >5 movie-fields per cell, but only ~2 in hippocampus. The movie-field durations in all brain regions spanned an unprecedented 1000-fold range: from 0.02s to 20s, termed mega-scale coding. Yet, the total duration of all the movie-fields of a cell was comparable across neurons and brain regions. The hippocampal responses thus showed greater continuous-sequence encoding than visual areas, as evidenced by fewer and broader movie-fields than in visual areas. Consistently, repeated presentation of the movie images in a fixed, but scrambled sequence virtually abolished hippocampal but not visual-cortical selectivity. The preference for continuous, compared to scrambled sequence was eight-fold greater in hippocampal than visual areas, further supporting episodic-sequence encoding. Movies could thus provide a unified way to probe neural mechanisms of episodic information processing and memory, even in immobile subjects, across brain regions, and species.

Mega-scale movie-fields in the mouse visuo-hippocampal network

Natural visual experience involves a continuous series of related images while the subject is immobile. How does the cortico-hippocampal circuit process a visual episode? The hippocampus is crucial for episodic memory, but most rodent single unit studies require spatial exploration or active engagement. Hence, we investigated neural responses to a silent movie (Allen Brain Observatory) in head-fixed mice without any task or locomotion demands, or rewards. Surprisingly, a third (33%, 3379/10263) of hippocampal –dentate gyrus, CA3, CA1 and subiculum– neurons showed movie-selectivity, with elevated firing in specific movie sub-segments, termed movie-fields, similar to the vast majority of thalamo-cortical (LGN, V1, AM-PM) neurons (97%, 6554/6785). Movie-tuning remained intact in immobile or spontaneously running mice. Visual neurons had >5 movie-fields per cell, but only ~2 in hippocampus. The movie-field durations in all brain regions spanned an unprecedented 1000-fold range: from 0.02s to 20s, termed mega-scale coding. Yet, the total duration of all the movie-fields of a cell was comparable across neurons and brain regions. The hippocampal responses thus showed greater continuous-sequence encoding than visual areas, as evidenced by fewer and broader movie-fields than in visual areas. Consistently, repeated presentation of the movie images in a fixed, but scrambled sequence virtually abolished hippocampal but not visual-cortical selectivity. The preference for continuous, compared to scrambled sequence was eight-fold greater in hippocampal than visual areas, further supporting episodic-sequence encoding. Movies could thus provide a unified way to probe neural mechanisms of episodic information processing and memory, even in immobile subjects, across brain regions, and species.

Non-consummatory behavior signals predict aversion-resistant alcohol drinking in head-fixed mice

A key facet of alcohol use disorder is continuing to drink alcohol despite negative consequences (so called “aversion-resistant drinking”). In this study, we sought to assess the degree to which head-fixed mice exhibit aversion-resistant drinking and to leverage behavioral analysis techniques available in head-fixture to relate non-consummatory behaviors to aversion-resistant drinking. We assessed aversion-resistant drinking in head-fixed female and male C57BL/6 J mice. We adulterated 20% (v/v) alcohol with varying concentrations of the bitter tastant quinine to measure the degree to which mice would continue to drink despite this aversive stimulus. We recorded high-resolution video of the mice during head-fixed drinking, tracked body parts with machine vision tools, and analyzed body movements in relation to consumption. Female and male head-fixed mice exhibited heterogenous levels of aversion-resistant drinking. Additionally, non-consummatory behaviors, such as paw movement and snout movement, were related to the intensity of aversion-resistant drinking. These studies demonstrate that head-fixed mice exhibit aversion-resistant drinking and that non-consummatory behaviors can be used to assess perceived aversiveness in this paradigm. Furthermore, these studies lay the groundwork for future experiments that will utilize advanced electrophysiological techniques to record from large populations of neurons during aversion-resistant drinking to understand the neurocomputational processes that drive this clinically relevant behavior.This article is part of the Special Issue on “PFC circuit function in psychiatric disease and relevant models”.

Stable sound decoding despite modulated sound representation in the auditory cortex

The activity of neurons in the auditory cortex is driven by both sounds and non-sensory context. To investigate the neuronal correlates of non-sensory context, we trained head-fixed mice to perform a two-alternative-choice auditory task in which either reward or stimulus expectation (prior) was manipulated in blocks. Using two-photon calcium imaging to record populations of single neurons in the auditory cortex, we found that both stimulus and reward expectation modulated the activity of these neurons. A linear decoder trained on this population activity could decode stimuli as well or better than predicted by the animal's performance. Interestingly, the optimal decoder was stable even in the face of variable sensory representations. Neither the context nor the mouse's choice could be reliably decoded from the recorded neural activity. Our findings suggest that, in spite of modulation of auditory cortical activity by task priors, the auditory cortex does not represent sufficient information about these priors to exploit them optimally. Thus, the combination of rapidly changing sensory information with more slowly varying task information required for decisions in this task might be represented in brain regions other than the auditory cortex.

Haploinsufficiency of Shank3 in Mice Selectively Impairs Target Odor Recognition in Novel Background Odors.

Individuals with mutations in a single copy of the SHANK3 gene present with social interaction deficits. Although social behavior in mice depends on olfaction, mice with mutations in a single copy of the Shank3 gene do not have olfactory deficits in simple odor identification tasks (Drapeau et al., 2018). Here, we tested olfaction in mice with mutations in a single copy of the Shank3 gene (Peça et al., 2011) using a complex odor task and imaging in awake mice. Average glomerular responses in the olfactory bulb of Shank3B +/- were correlated with WT mice. However, there was increased trial-to-trial variability in the odor responses for Shank3B +/- mice. Simulations demonstrated that this increased variability could affect odor detection in novel environments. To test whether performance was affected by the increased variability, we tested target odor recognition in the presence of novel background odors using a recently developed task (Li et al., 2023). Head-fixed mice were trained to detect target odors in the presence of known background odors. Performance was tested using catch trials where the known background odors were replaced by novel background odors. We compared the performance of eight Shank3B +/- mice (five males, three females) on this task with six WT mice (three males, three females). Performance for known background odors and learning rates were similar between Shank3B +/- and WT mice. However, when tested with novel background odors, the performance of Shank3B +/- mice dropped to almost chance levels. Thus, haploinsufficiency of the Shank3 gene causes a specific deficit in odor detection in novel environments. Our results are discussed in the context of other Shank3 mouse models and have implications for understanding olfactory function in neurodevelopmental disorders.SIGNIFICANCE STATEMENT People and mice with mutations in a single copy in the synaptic gene Shank3 show features seen in autism spectrum disorders, including social interaction deficits. Although mice social behavior uses olfaction, mice with mutations in a single copy of Shank3 have so far not shown olfactory deficits when tested using simple tasks. Here, we used a recently developed task to show that these mice could identify odors in the presence of known background odors as well as wild-type mice. However, their performance fell below that of wild-type mice when challenged with novel background odors. This deficit was also previously reported in the Cntnap2 mouse model of autism, suggesting that odor detection in novel backgrounds is a general deficit across mouse models of autism.

Brain-machine interface learning is facilitated by specific patterning of distributed cortical feedback.

Neuroprosthetics offer great hope for motor-impaired patients. One obstacle is that fine motor control requires near-instantaneous, rich somatosensory feedback. Such distributed feedback may be recreated in a brain-machine interface using distributed artificial stimulation across the cortical surface. Here, we hypothesized that neuronal stimulation must be contiguous in its spatiotemporal dynamics to be efficiently integrated by sensorimotor circuits. Using a closed-loop brain-machine interface, we trained head-fixed mice to control a virtual cursor by modulating the activity of motor cortex neurons. We provided artificial feedback in real time with distributed optogenetic stimulation patterns in the primary somatosensory cortex. Mice developed a specific motor strategy and succeeded to learn the task only when the optogenetic feedback pattern was spatially and temporally contiguous while it moved across the topography of the somatosensory cortex. These results reveal spatiotemporal properties of the sensorimotor cortical integration that set constraints on the design of neuroprosthetics.

A Novel Head-Fixed Assay for Social Touch in Mice Uncovers Aversive Responses in Two Autism Models.

Social touch, an important aspect of social interaction and communication, is essential to kinship across animal species. How animals experience and respond to social touch has not been thoroughly investigated, in part because of the lack of appropriate assays. Previous studies that examined social touch in freely moving rodents lacked the necessary temporal and spatial control over individual touch interactions. We designed a novel head-fixed assay for social touch in mice, in which the experimenter has complete control to elicit highly stereotyped bouts of social touch between two animals. The user determines the number, duration, context, and type of social touch interactions, while monitoring an array of complex behavioral responses with high resolution cameras. We focused on social touch to the face because of its high translational relevance to humans. We validated this assay in two different models of autism spectrum disorder (ASD), the Fmr1 knock-out (KO) model of Fragile X syndrome (FXS) and maternal immune activation (MIA) mice. We observed higher rates of avoidance running, hyperarousal, and aversive facial expressions (AFEs) to social touch than to object touch, in both ASD models compared with controls. Fmr1 KO mice showed more AFEs to mice of the same sex but whether they were stranger or familiar mice mattered less. Because this new social touch assay for head-fixed mice can be used to record neural activity during repeated bouts of social touch it could be used to uncover underlying circuit differences.SIGNIFICANCE STATEMENT Social touch is important for communication in animals and humans. However, it has not been extensively studied and current assays to measure animals' responses to social touch have limitations. We present a novel head-fixed assay to quantify how mice respond to social facial touch with another mouse. We validated this assay in autism mouse models since autistic individuals exhibit differences in social interaction and touch sensitivity. We find that mouse models of autism exhibit more avoidance, hyperarousal, and aversive facial expressions (AFEs) to social touch compared with controls. Thus, this novel assay can be used to investigate behavioral responses to social touch and the underlying brain mechanisms in rodent models of neurodevelopmental conditions, and to evaluate therapeutic responses in preclinical studies.

Multiple long-range projections convey position information to the agranular retrosplenial cortex

Neuronal signals encoding the animal's position widely modulate neocortical processing. While these signals are assumed to depend on hippocampal output, their origin has not been investigated directly. Here, we asked which brain region sends position information to the retrosplenial cortex (RSC), a key circuit for memory and navigation. We comprehensively characterized the long-range inputs to agranular RSC using two-photon axonal imaging in head-fixed mice performing a spatial task in darkness. Surprisingly, most long-range pathways convey position information, but with notable differences. Axons from the secondary motor and posterior parietal cortex transmit the most position information. By contrast, axons from the anterior cingulate and orbitofrontal cortex and thalamus convey substantially less position information. Axons from the primary and secondary visual cortex contribute negligibly. This demonstrates that the hippocampus is not the only source of position information. Instead, the RSC is a hub in a distributed brain network that shares position information.

Saccade-responsive visual cortical neurons do not exhibit distinct visual response properties.

Rapid saccadic eye movements are used by animals to sample different parts of the visual scene. Previous work has investigated neural correlates of these saccades in visual cortical areas such as V1, however how saccade-responsive neurons are distributed across visual areas, cell types, and cortical layers has remained unknown. Through analyzing 818 one-hour experimental sessions from the Allen Brain Observatory, we present a large-scale analysis of saccadic behaviors in head-fixed mice and their neural correlates. We find that saccade-responsive neurons are present across visual cortex, but their distribution varies considerably by transgenically-defined cell type, cortical area, and cortical layer. We also find that saccade-responsive neurons do not exhibit distinct visual response properties from the broader neural population, suggesting the saccadic responses of these neurons are likely not predominantly visually-driven. These results provide insight into the roles played by different cell types within a broader, distributed network of sensory and motor interactions.Significance StatementWhile the existence of saccade-responsive neurons in visual cortex has been previously established, how these responses vary by transgenically-defined cell type and whether these neurons differentially process visual stimuli is unknown. In addition to identifying distinct neural cell types more likely to robustly respond to saccades, our results indicate these saccade-responsive neurons do not exhibit distinct visual responses from their non-saccade-responsive counterparts.

An open-source platform for head-fixed operant and consummatory behavior.

Head-fixed behavioral experiments in rodents permit unparalleled experimental control, precise measurement of behavior, and concurrent modulation and measurement of neural activity. Here, we present OHRBETS (Open-Source Head-fixed Rodent Behavioral Experimental Training System; pronounced 'Orbitz'), a low-cost, open-source platform of hardware and software to flexibly pursue the neural basis of a variety of motivated behaviors. Head-fixed mice tested with OHRBETS displayed operant conditioning for caloric reward that replicates core behavioral phenotypes observed during freely moving conditions. OHRBETS also permits optogenetic intracranial self-stimulation under positive or negative operant conditioning procedures and real-time place preference behavior, like that observed in freely moving assays. In a multi-spout brief-access consumption task, mice displayed licking as a function of concentration of sucrose, quinine, and sodium chloride, with licking modulated by homeostatic or circadian influences. Finally, to highlight the functionality of OHRBETS, we measured mesolimbic dopamine signals during the multi-spout brief-access task that display strong correlations with relative solution value and magnitude of consumption. All designs, programs, and instructions are provided freely online. This customizable platform enables replicable operant and consummatory behaviors and can be incorporated with methods to perturb and record neural dynamics in vivo.

The Nonclassic Psychedelic Ibogaine Disrupts Cognitive Maps

BackgroundThe ability of psychedelic compounds to profoundly alter mental function has been long known, but the underlying changes in cellular-level information encoding remain poorly understood. MethodsWe used two-photon microscopy to record from the retrosplenial cortex in head-fixed mice running on a treadmill before and after injection of the nonclassic psychedelic ibogaine (40 mg/kg intraperitoneally). ResultsWe found that the cognitive map, formed by the representation of position encoded by ensembles of individual neurons in the retrosplenial cortex, was destabilized by ibogaine when mice had to infer position between tactile landmarks. This corresponded with increased neural activity rates, loss of correlation structure, and increased responses to cues. Ibogaine had surprisingly little effect on the size-frequency distribution of network activity events, suggesting that signal propagation within the retrosplenial cortex was largely unaffected. ConclusionsTaken together, these data support proposals that compounds with psychedelic properties disrupt representations that are important for constraining neocortical activity, thereby increasing the entropy of neural signaling. Furthermore, the loss of expected position encoding between landmarks recapitulated effects of hippocampal impairment, suggesting that disruption of cognitive maps or other hippocampal processing may be a contributing mechanism of discoordinated neocortical activity in psychedelic states.

Classification of multiple emotional states from facial expressions in head-fixed mice using a deep learning-based image analysis.

Facial expressions are widely recognized as universal indicators of underlying internal states in most species of animals, thereby presenting as a non-invasive measure for assessing physical and mental conditions. Despite the advancement of artificial intelligence-assisted tools for automated analysis of voluminous facial expression data in human subjects, the corresponding tools for mice still remain limited so far. Considering that mice are the most prevalent model animals for studying human health and diseases, a comprehensive characterization of emotion-dependent patterns of facial expressions in mice could extend our knowledge on the basis of emotions and the related disorders. Here, we present a framework for the development of a deep learning-powered tool for classifying facial expressions in head-fixed mouse. We demonstrate that our machine vision was capable of accurately classifying three different emotional states from lateral facial images in head-fixed mouse. Moreover, we objectively determined how our classifier characterized the differences among the facial images through the use of an interpretation technique called Gradient-weighted Class Activation Mapping. Importantly, our machine vision presumably discerned the data by leveraging multiple facial features. Our approach is likely to facilitate the non-invasive decoding of a variety of emotions from facial images in head-fixed mice.

An open-source behavior controller for associative learning and memory (B-CALM).

Associative learning and memory, i.e., learning and remembering the associations between environmental stimuli, self-generated actions, and outcomes such as rewards or punishments, are critical for the well-being of animals. Hence, the neural mechanisms underlying these processes are extensively studied using behavioral tasks in laboratory animals. Traditionally, these tasks have been controlled using commercial hardware and software, which limits scalability and accessibility due to their cost. More recently, due to the revolution in microcontrollers or microcomputers, several general-purpose and open-source solutions have been advanced for controlling neuroscientific behavioral tasks. While these solutions have great strength due to their flexibility and general-purpose nature, for the same reasons, they suffer from some disadvantages including the need for considerable programming expertise, limited online visualization, or slower than optimal response latencies for any specific task. Here, to mitigate these concerns, we present an open-source behavior controller for associative learning and memory (B-CALM). B-CALM provides an integrated suite that can control a host of associative learning and memory behaviors. As proof of principle for its applicability, we show data from head-fixed mice learning Pavlovian conditioning, operant conditioning, discrimination learning, as well as a timing task and a choice task. These can be run directly from a user-friendly graphical user interface (GUI) written in MATLAB that controls many independently running Arduino Mega microcontrollers in parallel (one per behavior box). In sum, B-CALM will enable researchers to execute a wide variety of associative learning and memory tasks in a scalable, accurate, and user-friendly manner.