Neuroscience

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.

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.

Pavlovian fear conditioning is a widely used procedure to assess learning and memory processes that has also been extensively used as a model of post-traumatic stress disorder (PTSD). Freezing, the absence of movement except for respiratory-related movements, is commonly used as a measure of fear response in non-human animals. However, this measure of fear responses can be affected by a different baseline of locomotor activity between groups and/or conditions. Moreover, fear conditioning procedures are usually restricted to a single conditioned stimulus (e.g., a tone cue, the context, etc.) and thus do not depict the complexity of real-life situations where traumatic memories are composed of a complex set of stimuli associated with the same aversive event. To overcome this issue, we use a conditioned lick suppression paradigm where water-deprived mice are presented with a single conditioned stimulus (CS, a tone cue or the context) previously paired with an unconditioned stimulus (US, a foot shock) while consuming water. We use the ratio of number of licks before and during the CS presentation as a fear measure, thereby neutralizing the potential effect of locomotor activity in fear responses. We further implemented the conditioned lick suppression ratio to assess the effect of cue competition using a compound of contextual and tone cue conditioned stimuli that were extinguished separately. This paradigm should prove useful in assessing potential therapeutics and/or behavioral therapies in PTSD, while neutralizing potential confounding effects between locomotor activity and fear responses on one side, and by considering potential cue-competition effects on the other side.

Graphical abstract

Schematic representation of the compound context-cue condition lick suppression procedure. Illustration reproduced from Bouchekioua et al. (2022).

Rodent spatial navigation is a key model system for studying mammalian cognition and its neural mechanisms. Of particular interest is how animals memorize the structure of their environments and compute multi-step routes to a goal. Previous work on multi-step spatial reasoning has generally involved placing rodents at the start of a maze until they learn to navigate to a reward without making wrong turns. It thus remains poorly understood how animals rapidly learn about the structure of naturalistic open environments with goals and obstacles. Here we present an assay in which mice spontaneously memorize two-step routes in an environment with a shelter and an obstacle. We allow the mice to explore this environment for 20 min, and then we remove the obstacle. We then present auditory threat stimuli, causing the mouse to escape to the shelter. Finally, we record each escape route and measure whether it targets the shelter directly (a ‘homing-vector’ escape) or instead targets the location where the obstacle edge was formerly located (an ‘edge-vector’ escape). Since the obstacle is no longer there, these obstacle-edge-directed escape routes provide evidence that the mouse has memorized a subgoal location,i.e., a waypoint targeted in order to efficiently get to the shelter in the presence of an obstacle. By taking advantage of instinctive escape responses, this assay probes a multi-step spatial memory that is learned in a single session without pretraining. The subgoal learning phenomenon it generates can be useful not only for researchers working on navigation and instinctive behavior, but also for neuroscientists studying the neural basis of multi-step spatial reasoning.

The ability to adapt one's behavior in response to changing circumstances, or cognitive flexibility, is often altered in neuropsychiatric and neurodevelopmental conditions. In rodents, cognitive flexibility is frequently assessed using associative learning paradigms with a reversal component. The majority of existing protocols rely on unrestrictive exploration with no discouragement of wrong responses and are often influenced by spatial cues, at least during the test's learning phase. Here, we present a rewarded contingency discrimination learning test that minimizes the task's spatial component and contains an element that actively discourages pure exploratory responses. The method described herein is a manual version that can be performed using home-made equipment, but the test setup is amenable to automatization and can be adapted to address more complex cognitive demands, including conditional associative learning, attentional set formation, and attention shifting.

The novel object recognition (NOR) task is a behavioral test commonly used to evaluate episodic-like declarative memory and it relies on the innate tendency of rodents to explore novelty. Here we present a maze used to evaluate NOR memory in mice that reduces the time of the assay while improving reliability of the measurements by increasing the exploratory behavior. This memory test, being performed in a two-arms maze, is suitable for several strains of mice (including inbreed and outbreed) and does not require extended training sessions allowing an accurate temporal assessment of memory formation. This particular maze increases the mouse exploration time and reduces variability compared to other arenas used before to assess NOR. As both long- and short-term NOR memory can be easily and accurately quantified using this paradigm, this improved methodology can be easily applied to study pharmacological, genetic or age-related modulation of cognitive function.

Recording neural activity in unrestricted animals is necessary to unravel the neural basis of ethological behaviors. Recently, Neuropixels probes have made important strides in improving yield and lowering noise, but have limited use cases in freely moving animals. Although there are a number of studies demonstrating the use of these probes in headfixed mice, there are not established protocols for the use and reuse of them in a freely moving mouse. We therefore designed a novel device (the AMIE) that maximizes the potential value of these powerful probes. Here, we provide the technical drawings for the AMIE and detail its preparation, implantation, and explantation. With our approach, researchers can record hundreds of neurons during freely moving behavior across weeks of experiments, and then recycle valuable probes for future use.

The Morris water maze (MWM) is one of the most commonly used tests for assessing spatial learning and memory in mice. While the MWM is highly amenable to testing the effects of memory modifying drugs, most studies do not consider the timing or duration of drug exposure when conducting the MWM assay; factors that can strongly influence the effect of the drug on different stages of memory and interfere with data interpretation. Herein we describe a MWM protocol which offers the advantage of distinguishing the impact of a fast acting intraperitoneally (IP) injected drug on the different stages of spatial memory: acquisition, consolidation, and retrieval. Mice initially undergo habituation to both the MWM apparatus and IP injection procedure over the course of three days. For assessing the effect of a drug on memory acquisition, mice are injected with the drug prior to training sessions over four consecutive days, where mice learn to find an escape platform in a circular water tank using distal spatial cues. To determine the effect of the drug on memory consolidation, mice are injected with the drug immediately after each training session. For testing the effect of a drug on memory retrieval, mice receive mock IP injections on each training day and the drug is IP injected only once, prior to a probe trial, where mice attempt to locate the platform following its removal from the tank. This protocol provides a simple strategy for distinguishing the effect(s) of a CNS acting drug on the different stages of memory.

The Displaced Object Recognition (DOR) task, sometimes called the Novel Object Location task, assesses spatial recognition memory without navigational demands, explicit instruction, or the need for multiple days of training. This memory task has two phases. First, the subject is familiarized to an open arena with two objects and is allowed to explore the objects. Following a delay period, the subject returns to the arena, but one of the previous objects has been moved to a new location. Greater exploration of the displaced object is used as the index of memory for the previous object location. An advantage of the DOR task is that subjects can be tested without explicit training, since this task exploits the natural tendency to be more interested in something novel. The spontaneous aspect of this task allows for the testing of animals as well as human populations that are unable to follow verbal instructions, such as babies. Therefore, this powerful test of recognition memory can be administered similarly for many species, including rats and humans, allowing for better translatability.