神经科学

Social behavior is highly dynamic and context-dependent, yet many commonly used rodent social assays rely on short testing periods and simplified measures of proximity or investigation. Here, we present a detailed protocol for constructing and using the immersive social interaction assay (ISIA), a behavioral paradigm designed to capture prolonged, voluntary social interactions in freely moving mice. The ISIA apparatus consists of two modified standard rodent home-cage chambers connected by a 3D-printed tube with an adjustable inner diameter. This design enables flexible experimental control: a removable restrictor can confine a head-bar-implanted focal mouse to one chamber while permitting a freely moving conspecific to traverse the full apparatus, enabling assessment of social motivation. Animals can be recorded over extended time windows with familiar or novel conspecifics, in their home territory or in novel environments, and under varying thermal or enrichment conditions, while expressing a broad repertoire of affiliative and aggressive behaviors, including huddling and aggression. The apparatus can be readily integrated with machine learning–based tracking and behavioral classification pipelines for high-throughput analysis. This protocol describes step-by-step construction, setup, and experimental implementation of the ISIA, from apparatus construction and head-bar implantation to video-based behavioral analysis, with the goal of facilitating broader adoption of ethologically relevant behavioral assays in neuroscience research.

Cognitive flexibility is a process that involves dynamically adapting behavior to obtain a desired series of outcomes during continuously changing stimulus-response-reward associations. Attentional set-shifting is a multi-modal decision-making paradigm that tests cognitive flexibility, which can be useful for investigating neural circuitry that is disrupted in multiple neuropsychiatric disorders, including schizophrenia, Alzheimer’s disease, depression, and addiction. The canonical human attentional set-shifting paradigm for measuring cognitive flexibility is the Wisconsin Card Sorting Test, in which subjects sort cards based on rules that change periodically and adapt their behavior by utilizing feedback in the form of correct and incorrect outcomes. To transfer these tests to rodent models, previous techniques involved attentional set-shifting paradigms in free-roaming test chambers, in which animals associated cues with reward locations. The protocol presented here involves an analogous attentional set-shifting paradigm, in which water-restricted mice are head-restrained on a platform and must keep track of periodically switching stimulus-response-outcome associations. The mice must learn to associate odor or whisker vibration cues with a binary directional lick response that triggers water delivery. The mice are trained to respond by licking one of two spouts, in which the correct decision is dependent on the current stimulus rule. This protocol allows for behaviorally measuring cognitive flexibility alongside neural activity by pairing the head-restrained paradigm with 2-photon calcium imaging, optogenetics, and extracellular and intracellular physiology.

The advent of geroscience engendered the development of approaches to quantify the aging process and estimate biological age on an individual level. Recognizing that declines observed in aging are not only physical but also social led us to develop a mouse Social Frailty Index (mSFI) designed to quantify age-related impairments of social functioning in mice. The mSFI consists of seven behavioral assays that measure essential facets of social behavioral functioning in mice: social communication, social interaction, and social functional ability. The assays that comprise the mSFI are all minimally disruptive, relatively simple to execute, and optimized for compatibility with longitudinal studies utilizing experimental interventions relevant to geroscience. The mSFI is conducted over AM and PM sessions spanning a maximum of 3.5 days, using materials common to most animal facilities. The data for all assays is obtained observationally, manually recorded, and entered into predefined template sheets that automate the computation of the mSFI. We have demonstrated the validity and applicability of the mSFI across multiple laboratory sites and experiments. This index has proven to discriminate between differential trajectories of biological aging driven by sex, progeria, or social stress-relevant contexts. The mSFI represents a novel index to quantify trajectories of biological aging in mice, and its application may help elucidate the social dimensions of the aging process.

Pupil size is a non-invasive and highly sensitive technique used to measure changes in pupil diameter. It not only responds to light but also reflects inner cognitive processes (e.g., attention and emotion perception). Recently, it has been introduced to the traditional cognitive neuroscience field as a useful tool to objectively and sensitively capture the current cognitive state and its temporal dynamics. Importantly, this index is automatic and requires no explicit reports, thus it could be used to investigate the rarely explored realm of implicit cognitive processing. Here, we describe a comprehensive protocol that records pupil responses during the passive viewing of emotional biological motion (BM). Our results reliably reveal the multi-level implicit processing mechanism of BM emotion, as indicated by the fine-grained emotion processing in intact BM and the rapid but rather coarse emotion processing in local BM. Moreover, the emotion modulation effects observed in intact BM are indicative of individual autistic tendencies. We believe this protocol could be adapted to unveil the automatic processing of emotions and other attributes in social signals and further assist the early detection of social-cognitive disorders (e.g., autism).

Alzheimer's disease (AD) poses a global health threat, progressively robbing patients of their memory and cognitive abilities. While it is recognized that meaningful social contact can alleviate the symptoms of dementia in AD patients, the precise mechanisms by which social stimulation mitigates AD symptoms remain poorly understood. We found that social interaction with novel mice, also known as novel social, simulated meaningful socializing. Therefore, we developed the multiple novel social (MNS) stimulation paradigm to train AD model mice and found that MNS effectively alleviated cognitive deficits in AD mice. This discovery not only opens up a new avenue for investigating the relationship between social stimulation and Alzheimer's disease but also lays the groundwork for delving into the underlying mechanisms, thereby providing crucial theoretical support for developing novel strategies to treat Alzheimer's disease.

Measuring autonomic parameters like heart rate in behaving mice is not only a standard procedure in cardiovascular research but is applied in many other interdisciplinary research fields. With an electrocardiogram (ECG), the heart rate can be measured by deriving the electrical potential between subcutaneously implanted wires across the chest. This is an inexpensive and easy-to-implement technique and particularly suited for repeated recordings of up to eight weeks. This protocol describes a step-by-step guide for manufacturing the needed equipment, performing the surgical procedure of electrode implantation, and processing of acquired data, yielding accurate and reliable detection of heartbeats and calculation of heart rate (HR). We provide MATLAB graphical user interface (GUI)–based tools to extract and start processing the acquired data without a lot of coding knowledge. Finally, based on an example of a data set acquired in the context of defensive reactions, we discuss the potential and pitfalls in analyzing HR data.

Key features

• Next to surgical steps, the protocol provides a detailed description of manufacturing custom-made ECG connectors and a shielded, light-weight patch cable.

• Suitable for recordings in which signal quality is challenged by ambient noise or noise from other recording devices.

• Described for 2-channel differential recording but easily expandable to record from more channels.

• Includes a summary of potential analysis methods and a discussion on the interpretation of HR dynamics in the case study of fear states.

Visual learning in animals is a remarkable cognitive ability that plays a crucial role in their survival and adaptation. Therefore, the ability to learn is highly conserved among animals. Despite lacking a centralized nervous system like vertebrates, invertebrates have demonstrated remarkable learning abilities. Here, we describe a simple behavioral assay that allows the analysis of visual associative learning in individually traceable freely walking adult fruit flies. The setup is based on the simple and widely used behavioral assay to study orientation behavior in flies. A single wing-clipped fly that has been starved for 21 h is placed on a platform where two unreachable opposite visual sets are displayed. This visual learning protocol was initially developed to study the cognitive ability of fruit flies to process numerical information. Through the application of the protocol, flies are able to associate a specific visual set with an appetitive reward. This association is revealed 2 h later during the testing session where we observed a change in their preference upon learning (i.e., change in their spontaneous preference). Moreover, this protocol could potentially be used to associate any other visual object/property to the reward, expanding the opportunities of studying visual learning in freely walking fruit flies at individual level.

Graphical overview

In this protocol, we describe for the first time a judgment bias paradigm to phenotype the way zebrafish assess ambiguous stimuli. We have developed and validated a protocol for a judgment bias test based on a Go/No-go task, and performed using a half radial maze. After a habituation phase, fish are trained to discriminate between two reference arms [positive (P) and negative (N)]. For this purpose, they experience a positive event (food reward in P), when presented with a specific location/color cue, and a negative event (chasing with net in N), when presented with a different location/color cue. Acquisition of the discrimination learning between P and N is revealed by the latencies to enter the experimental arms of the behavioral maze being significantly lower for the P arm than for the N arm. Once zebrafish are able to discriminate between P and N arms, their latency to enter other maze arms spatially located between P and N [(Near Positive (NP), Ambiguous (A) = half-way between P and N, and Near Negative (NN)] is analyzed. Latencies (L) to enter NP, A and NN maze arms are interpreted as indicating the individual expectancy to experience a reward/punishment on each of them. A judgment bias score (JBS) is calculated from the latencies to enter the P, N, and A arms for each fish [JBS = (L_A–L_P)*100/(L_N–L_P)], based on which fish can be classified into an optimistic/pessimistic axis. A JBS below 50 indicates that fish perceive the ambiguous stimulus as a positive one (optimistic bias), while JBS above 50 indicates that fish perceive the ambiguous stimulus as a negative one (pessimistic bias). However, for classification criteria, it could be advantageous to use the method of selecting extreme phenotypes (e.g., upper and lower quartiles of the JBS), since JBS in zebrafish falls into a bimodal distribution (unpublished data). Therefore, this protocol provides a unique, inexpensive, and effective alternative to other methods of measuring affective states in zebrafish that might be of great interest to a broad target audience and have a large number of applications.

Graphic abstract:

Flow chart of the judgment bias protocol in zebrafish.

Understanding the neural basis of reward processing is a major concern, as it holds the key to alleviating symptoms of addiction and poor mental health. However, this goal seems difficult to attain as long as research on reward processing cannot easily be compared across species and reward types, due to methodological differences and the presence of confounding factors. We recently developed an experimental paradigm that allows monitoring anticipatory and consummatory responses to matched social (touch) and nonsocial (food) rewards in adult humans. The following protocol describes in detail the materials and the paradigm, which measures reward wanting and liking with a real effort task and subjective ratings. It can also be used in combination with facial electromyography (EMG), brain imaging (e.g., fMRI), and pharmacological interventions. It is our firm belief that the field will profit greatly from more research being conducted on reward processing using this and similarly controlled paradigms, which allow for cross-species comparison.

Space and time are both essential features of episodic memory. However, while spatial tasks have been used effectively to study the behavioral relevance of place cells, the behavioral paradigms utilized for the study of time cells have not used time duration as a variable that animals need to be aware of to solve the task. In order to evaluate how time flow is coded into memory, time duration needs to be a variable that animals use to solve the behavioral task. This protocol describes a novel behavioral paradigm, the time duration discrimination (TDD) task, which is designed to directly investigate the neurological mechanisms that underlie temporal processing. During the TDD task, rats navigate around a Figure-8 Maze, which contains a rectangular track with a central arm and a delay box at the end of the central arm. While confined to the delay box, rats experience a 10- or 20-second time delay, during which a tone will play for the duration of the 10- or 20-second delay. When the delay box opens, the rat will choose whether to turn left or right out of the delay box and receive a reward for the correct choice (e.g., 10 seconds = left turn; 20 seconds = right turn). By directly manipulating elapsed time, we can better explore the behavioral relevance of hippocampal time cells and whether the time-dependent activity seen in physiological recordings of hippocampal neurons reflects a neuronal representation of time flow that can be used by the animal for learning and storing memories.

Graphic abstract:

Elapsed time duration discrimination in rats