Neuroscience

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).

Drosophila larvae exhibit rolling motor behavior as an escape response to avoid predators and painful stimuli. We introduce an accessible method for applying optogenetics to study the motor circuits driving rolling behavior. For this, we simultaneously implement the Gal4-UAS and LexA-Aop binary systems to express two distinct optogenetic channels, GtACR and Chrimson, in motor neuron (MN) subsets and rolling command neurons (Goro), respectively. Upon exposure to white LED light, Chrimson permits the influx of positive ions into Goro neurons, leading to depolarization, whereas GtACR mediates chloride influx into MNs, resulting in hyperpolarization. This method allows researchers to selectively activate certain neurons while simultaneously inhibiting others within a circuit of interest, offering a unique advantage over current optogenetic approaches, which often utilize a single type of optogenetic actuator. Here, we provide a detailed protocol for the dual silencing-activation approach using GtACR and Chrimson optogenetic channels and present a robust methodological framework for investigating the neuromuscular basis of rolling in larvae. Our cost-effective and scalable approach utilizes readily accessible equipment and can be applied to study other locomotor behaviors in Drosophila larvae, thereby enhancing our understanding of the neural circuit mechanisms underlying sensorimotor transformation.

Behavioral neuroscience requires precise and unbiased methods for animal behavior assessment to elucidate complex brain–behavior interactions. Traditional manual scoring methods are often labor-intensive and can be prone to error, necessitating advances in automated techniques. Recent innovations in computer vision have led to both marker- and markerless-based tracking systems. In this protocol, we outline the procedures required for utilizing Augmented Reality University of Cordoba (ArUco) markers, a marker-based tracking approach, to automate the assessment and scoring of rodent engagement during an established intracortical microstimulation-based nose-poking go/no-go task. In short, this protocol involves detailed instructions for building a suitable behavioral chamber, installing and configuring all required software packages, constructing and attaching an ArUco marker pattern to a rat, running the behavioral software to track marker positions, and analyzing the engagement data for determining optimal task durations. These methods provide a robust framework for real-time behavioral analysis without the need for extensive training data or high-end computational resources. The main advantages of this protocol include its computational efficiency, ease of implementation, and adaptability to various experimental setups, making it an accessible tool for laboratories with diverse resources. Overall, this approach streamlines the process of behavioral scoring, enhancing both the scalability and reproducibility of behavioral neuroscience research. All resources, including software, 3D models, and example data, are freely available at https://github.com/tomcatsmith19/ArucoDetection.

Long-lasting memories are a core aspect of an animal’s life. Such memories are characterized by unique molecular mechanisms and often unique circuitry, neither of which are completely understood in vivo. The deep knowledge of the identity and connectivity of neurons of the fruit fly Drosophila melanogaster, as well as the sophisticated genetic tools that allow in vivo perturbations and physiology monitoring, make it a remarkably useful organism in which to investigate the molecular mechanisms of long-term memories. In this protocol, we focus on habituation, a non-associative form of learning, and describe a reliable, semi-automated technique to induce and assess long-term olfactory habituation (LTH) in Drosophila using the olfactory arena, thus providing a method aligned with recent technological progress in behavioral measurement. Prior work has shown that LTH is induced by a 4-day exposure to an odorant and is characterized by a long-lasting (> 24 h) reduction in behavioral response to the exposed odorant, measured using a manual and skill-intensive Y-maze assay. Here, we present a semi-automated protocol for obtaining quantifiable measures of LTH, at the level of detail required for other investigators in the field. Unlike previously described methods, the protocol presented here provides quantitative and detailed behavioral measurements obtained by video recording that can be shared with the scientific community and allows sophisticated forms of offline analysis. We suggest that this procedure has the potential to advance our understanding of molecular and circuit mechanisms of olfactory habituation, its control via neuromodulation, and its interactions with other forms of memory.

C. elegans is a well-established nematode model organism, with 83% of its genes conserved in humans with translation potential. C. elegans is translucent, with clearly defined cellular organization, and robustly identifiable under a microscope, being an excellent model for studying feeding behavior. Its neuromuscular pharyngeal pump undergoes a pumping motion that can be quantified to study feeding behavior at specific treatment conditions and in genetically modified worms. Understanding the evolutionarily conserved feeding behaviors and regulatory signals is vital, as unhealthy eating habits increase the risk of associated diseases. The current protocol was developed to identify and study evolutionary conserved signals regulating feeding behavior. The protocol described here is very robust in calculating the pumping rate (pumping per minute) as it directly counts the pharyngeal pumping for 30 s. This protocol uses basic laboratory instrumentation, such as a stereomicroscope with an attached camera and a computer with a video program that can be used to count manually. The advantages of studying C. elegans feeding include understanding the genetic basis of feeding regulation, dysregulation of feeding behavior in a disease model, the influence of toxic or environmental substances in feeding behavior, and modulation of feeding behavior by pharmacological agents.

Physiological changes during awake immobility–related brain states remain one of the great unexplored behavioral states. Controlling periods of awake immobility is challenging because restraining the animal is stressful and is accompanied by altered physiological states. Here, we describe the ThermoMaze, a behavioral paradigm that allows for the collection of large amounts of physiological data while the animal rests at distinct experimenter-determined locations. We found that the paradigm generated long periods of immobility and did not alter the brain temperature. We combined the ThermoMaze with electrophysiology recordings in the CA1 region of the hippocampus and found a location-specific distribution of sharp-wave ripple events. We describe the construction of the ThermoMaze with the intention that it helps enable large-scale data recordings on immobility-related brain states.

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.

Sleep is an essential behavior that is still poorly understood. Sleep abnormalities accompany a variety of psychiatric and neurological disorders, and sleep can serve as a modifiable behavior in the treatment of these disorders. Zebrafish (Danio rerio) has proven to be a powerful model organism to study sleep and the interplay between sleep and these disorders due to the high conservation of the neuro-modulatory mechanisms that control sleep and wake states between zebrafish and humans. The zebrafish is a diurnal vertebrate with a relatively simple nervous system compared to mammalian models, exhibiting conservation of sleep ontogeny across different life stages. Zebrafish larvae are an established high-throughput model to assess sleep phenotypes and the biological underpinnings of sleep disturbances. To date, sleep measurement in juvenile and adult zebrafish has not been performed in a standardized and reproducible manner because of the relatively low-throughput nature in relation to their larval counterparts. This has left a gap in understanding sleep across later stages of life that are relevant to many psychiatric and neurodegenerative disorders. Several research groups have used homemade systems to address this gap. Here, we report employing commercially available equipment to track activity and sleep/wake patterns in juvenile and adult zebrafish. The equipment allows researchers to perform automated behavior assays in an isolated environment with light/dark and temperature control for multiple days. We first explain the experimental procedure to track the sleep and activity of adult zebrafish and then validate the protocol by measuring the effects of melatonin and DMSO administration.

Many studies on mosquito biology rely on laboratory-reared colonies, emphasizing the need for standardized protocols to investigate critical aspects such as disease biology, mosquito behavior, and vector control methods. While much knowledge is derived from anthropophilic species from genera like Anopheles, Aedes, and Culex, there is a growing interest in studying mosquitoes that feed on non-human hosts. This interest stems from the desire to gain a deeper understanding of the evolution of diverse host range use and host specificity. However, there is currently a limited number of comprehensive protocols for studying such species. Considering this gap, we present a protocol for rearing Uranotaenia lowii, a mosquito species specialized in feeding on anuran amphibians by eavesdropping on host-emitted sound cues. Additionally, we provide instructions for successfully shipping live specimens to promote research on this species and similar ones. This protocol helps fill the current gap in comprehensive guidelines for rearing and maintaining colonies of anuran host–biting mosquitoes. It serves as a valuable resource for researchers seeking to establish colonies of mosquito species from the Uranotaeniini tribe. Ultimately, this protocol may facilitate research on the evolutionary ecology of Culicidae, as this family has recently been proposed to have originated from a frog-feeding ancestor.