Neuroscience

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

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.

Honey bees use a complex form of spatial referential communication. Their waggle dance communicates to nestmates the direction, distance, and quality of a resource by encoding celestial cues, retinal optic flow, and relative food value into motion and sound within the nest. This protocol was developed to investigate the potential for social learning of this waggle dance. Using this protocol, we showed that correct waggle dancing requires social learning. Bees (Apis mellifera) that did not follow any dances before they first danced produced significantly more disordered dances, with larger waggle angle divergence errors, and encoded distance incorrectly. The former deficits improved with experience, but distance encoding was set for life. The first dances of bees that could follow other dancers had none of these impairments. Social learning, therefore, shapes honey bee signaling, as it does communication in human infants, birds, and multiple other vertebrate species. However, much remains to be learned about insects’ social learning, and this protocol will help to address knowledge gaps in the understanding of sophisticated social signal learning, particularly in understanding the molecular bases for such learning.

Key features

• It was unclear if honey bees (Apis mellifera) could improve their waggle dance by following experienced dancers before they first waggle dance.

• Honey bees perform their first waggle dances with more errors if they cannot follow experienced waggle dancers first.

• Directional and disorder errors improved over time, but distance error was maintained. Bees in experimental colonies continued to communicate longer distances than control bees.

• Dancing correctly, with less directional error and disorder, requires social learning.

• Distance encoding in the honey bee dance is largely genetic but may also include a component of cultural transmission.

The development of excessive alcohol (ethanol) and/or highly palatable food self-administration is an essential task to elucidate the neurobiological mechanisms that underlie these behaviors. Previous work has highlighted that ethanol self-administration is modulated by both the induction of aversive states (i.e., stress or frustration) and by the concurrent availability of appetitive stimuli (e.g., food). In our protocol, rats are food deprived for three days until they reach 82%–85% of their ad libitum weight. After that, rats are exposed daily for 10 days to a brief binge or control eating experience with highly sugary and palatable food (i.e., the ingestion of 11.66 and 0.97 kcal/3 min, respectively), which is followed by a two-bottle-choice test (ethanol vs. water) in their home cages for 90 min. This model induces robust binge eating, which is followed by a selective increase in ethanol self-administration. Therefore, this protocol allows to study: a) behavioral and neurobiological factors related to binge eating, b) different stages of alcohol use, and c) interactions between the latter and other addictive-like behaviors, like binge eating.

Anorexia nervosa (AN) is a psychiatric disorder mainly characterized by extreme hypophagia, severe body weight loss, hyperactivity, and hypothermia. Currently, AN has the highest mortality rate among psychiatric illnesses. Despite decades of research, there is no effective cure for AN nor is there a clear understanding of its etiology. Since a complex interaction between genetic, environmental, social, and cultural factors underlines this disorder, the development of a suitable animal model has been difficult so far. Here, we present our protocol that couples a loss-of-function mouse model to the activity-based anorexia model (ABA), which involves self-imposed starvation in response to exposure to food restriction and exercise. We provide insights into a neural circuit that drives survival in AN and, in contrast to previous protocols, propose a model that mimics the conditions that mainly promote AN in humans, such as increased incidence during adolescence, onset preceded by negative energy balance, and increased compulsive exercise. This protocol will be useful for future studies that aim to identify neuronal populations or brain circuits that promote the onset or long-term maintenance of this devastating eating disorder.

Sleep is not homogenous but contains a highly diverse microstructural composition influenced by neuromodulators. Prior methods used to measure neuromodulator levels in vivo have been limited by low time resolution or technical difficulties in achieving recordings in a freely moving setting, which is essential for natural sleep. In this protocol, we demonstrate the combination of electroencephalographic (EEG)/electromyographic (EMG) recordings with fiber photometric measurements of fluorescent biosensors for neuromodulators in freely moving mice. This allows for real-time assessment of extracellular neuromodulator levels during distinct phases of sleep with a high temporal resolution.

A fascinating question in neuroscience is how sensory stimuli evoke calcium dynamics in neurons. Caenorhabditis elegans is one of the most suitable models for optically recording high-throughput calcium spikes at single-cell resolution. However, calcium imaging in C. elegans is challenging due to the difficulties associated with immobilizing the organism. Currently, methods for immobilizing worms include entrapment in a microfluidic channel, anesthesia, or adhesion to a glass slide. We have developed a new method to immobilize worms by trapping them in sodium alginate gel. The sodium alginate solution (5%), polymerized with divalent ions, effectively immobilizes worms in the gel. This technique is especially useful for imaging neuronal calcium dynamics during olfactory stimulation. The highly porous and transparent nature of alginate gel allows the optical recording of cellular calcium oscillations in neurons when briefly exposed to odor stimulation.