Neuroscience

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.

In vivo brain imaging, using a combination of genetically encoded Ca²⁺ indicators and gradient refractive index (GRIN) lens, is a transformative technology that has become an increasingly potent research tool over the last decade. It allows direct visualisation of the dynamic cellular activity of deep brain neurons and glia in conscious animals and avoids the effect of anaesthesia on the network. This technique provides a step change in brain imaging where fibre photometry combines the whole ensemble of cellular activity, and multiphoton microscopy is limited to imaging superficial brain structures either under anaesthesia or in head-restrained conditions. We have refined the intravital imaging technique to image deep brain nuclei in the ventral medulla oblongata, one of the most difficult brain structures to image due to the movement of brainstem structures outside the cranial cavity during free behaviour (head and neck movement), whose targeting requires GRIN lens insertion through the cerebellum—a key structure for balance and movement. Our protocol refines the implantation method of GRIN lenses, giving the best possible approach to image deep extracranial brainstem structures in awake rodents with improved cell rejection/acceptance criteria during analysis. We have recently reported this method for imaging the activity of retrotrapezoid nucleus and raphe neurons to outline their chemosensitive characteristics. This revised method paves the way to image challenging brainstem structures to investigate their role in complex behaviours such as breathing, circulation, sleep, digestion, and swallowing, and could be extended to image and study the role of cerebellum in balance, movement, motor learning, and beyond.

Key features

• We developed a protocol that allows imaging from brainstem neurons and glia in freely behaving rodents.

• Our refined method of GRIN lenses implantation and cell sorting approach gives the highest number of cells with the least postoperative complications.

• The revised deep brainstem imaging method paves way to understand complex behaviours such as cardiorespiratory regulation, sleep, swallowing, and digestion.

• Our protocol can be implemented to image cerebellar structures to understand their role in key functions such as balance, movement, motor learning, and more.

Graphical overview

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.

Habituation, the process by which animals learn to ignore insignificant stimuli, facilitates engagement with salient features of the environment. However, neural mechanisms underlying habituation also allow responses to familiar stimuli to be reinstated when such stimuli become potentially significant. Thus, the habituated state must allow a mechanism for habituation override. The remarkably precise knowledge of cell identity, connectivity, and information coding in Drosophila sensory circuits, as well as the availability of tools to genetically target these cells, makes Drosophila a valuable and important organism for analysis of habituation and habituation-override mechanisms. Studies of olfactory and gustatory habituation in Drosophila suggest that potentiation of GABAergic neurons underlies certain timescales of habituation and have specified some elements of a gustatory habituation-override pathway. More detailed understanding of gustatory habituation and habituation-override mechanisms will benefit from access to robust behavioral assays for (a) the proboscis extension reflex (PER) elicited by a sweet stimulus, (b) exposure paradigms that result in PER habituation, and, most critically, (c) manipulations that result in PER-habituation override. Here, we describe simple protocols for persistent sucrose exposure of tarsal hairs that lead to habituation of proboscis extension and for presentation of a novel appetitive stimuli that reinstate robust PER to habituated flies. This detailed protocol of gustatory habituation provides (a) a simple method to induce habituation by continuous exposure of the flies to sucrose for 10 min without leading to ingestion and (b) a novel method to override habituation by presenting yeast to the proboscis.

Key features

• A protocol for stimulation of Drosophila’s taste (sugar) sensory neurons that induces gustatory habituation without satiation due to ingestion.

• A chemical (yeast) stimulation protocol that rapidly induces habituation override/dishabituation in sugar-habituated Drosophila.

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

Graphical overview of conditional learning protocol. Forty-eight hours before conditioning, the wings of the flies are clipped, and individual flies are left to recover in a fresh food vial. Twenty-one hours before the conditional learning starts, flies are transferred to a starvation vial containing wet paper. The training session consists in placing a drop of sugar next to the place with the lower number of objects (numerosity) and a drop of water next to the larger numerosity. The fly is placed in the arena and left to freely walk for three minutes. Once the session is finished, the fly is placed back in their empty vial for 2 h until the testing session starts.

An emerging body of behavioural studies indicates that regular swimming in cold water has positive effects on mental health and wellbeing, such as reducing fatigue, improving mood, and lessening depressive symptoms. Moreover, some studies reported immediate effects of cold-water immersion (CWI) on elevating mood and increasing a positive emotional state. However, the neural mechanisms underlying these effects remain largely unknown. The lack of studies using neuroimaging techniques to investigate how a whole-body CWI affects neural processes has partly resulted from the lack of a tested experimental protocol. Previous protocols administered tonic limb cooling (1–10 °C) while recording functional magnetic resonance (fMRI) signals. However, using very low water temperature constitutes points of contrast to painful experiences that are different from what we experience after a whole-body head-out CWI. In our protocol, healthy adults unhabituated to cold water were scanned twice: immediately before (pre-CWI) and after (post-CWI) immersion in cold water (water temperature 20 °C) for 5 min. We recorded cardiac and ventilatory responses to CWI and assessed self-reported changes in positive and negative affects. Our protocol showed reliable changes in brain connectivity after a short exposure to cold water, thus enabling its use as a tested experimental framework in future studies.

Graphical overview

Living organisms possess the ability to respond to environmental cues and adapt their behaviors and physiologies for survival. Eusocial insects, such as ants, bees, wasps, and termites, have evolved advanced sociality: living together in colonies where individuals innately develop into reproductive and non-reproductive castes. These castes exhibit remarkably distinct behaviors and physiologies that support their specialized roles in the colony. Among ant species, Harpegnathos saltator females stand out with their highly plastic caste phenotypes that can be easily manipulated in a laboratory environment. In this protocol, we provide detailed instructions on how to generate H. saltator ant colonies, define castes based on behavioral and physiological phenotypes, and experimentally induce caste switches, including the transition from a non-reproductive worker to a reproductive gamergate and vice versa (known as reversion). The unusual features of H. saltator make it a valuable tool to investigate cellular and molecular mechanisms underlying phenotypic plasticity in eusocial organisms.

Key features

• H. saltator is one of few ant species showing remarkable caste plasticity with striking phenotypic changes, being a useful subject for studying behavioral plasticity.

• Caste switches in H. saltator can be easily manipulated in a controlled laboratory environment by controlling the presence of reproductive females in a colony.

• The relatively large size of H. saltator females allows researchers to dissect various tissues of interest and conduct detailed phenotypic analyses.