Neuroscience

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.

Feeding behavior is a complex experience that involves not only sensory (i.e., visual, odor, taste, or texture) but also affective or emotional aspects (i.e., pleasure, palatability, or hedonic value) of foods. As such, behavioral tests that assess the hedonic impact of foods are necessary to fully understand the factors involved in ingestive behavior. In this protocol, we use the taste reactivity (TR) test to characterize the hedonic responses of rats to flavors paired with either lithium chloride–induced nausea or internal pain produced by hypertonic NaCl, two treatments that reduce voluntary consumption. This application of the TR test demonstrates how emetic and non-emetic (somatic pain in particular) treatments produce dissociable patterns of hedonic reactions to fluids: only emetic treatments result in the production of aversive orofacial responses, reflecting conditioned nausea, whereas somatic pain produces immobility, reflecting conditioned fear. Other methods, such as the microstructural analysis of licking behavior, do not reliably distinguish conditioned nausea and fear, a key advantage of the more selective TR procedure. This protocol also contains guidance for adaptation to other species and designs.

In neuroscience, it is fundamental to understand how sensory stimuli are translated into neural activity at the entry point of sensory systems. In the olfactory system, odorants inhaled into the nasal cavity are detected by ~1,000 types of odorant receptors (ORs) that are expressed by olfactory sensory neurons (OSNs). Since each OSN expresses only one type of odorant receptor, the odor-evoked responses reflect the interaction between odorants and the expressed OR. The responses of OSN somata are often measured by calcium imaging and electrophysiological techniques; however, previous techniques require tissue dissection or cell dissociation, rendering it difficult to investigate physiological responses. Here, we describe a protocol that allows us to observe odor-evoked responses of individual OSN somata in the mouse olfactory epithelium in vivo. Two-photon excitation through the thinned skull enables highly-sensitive calcium imaging using a genetically encoded calcium indicator, GCaMP. Recording of odor-evoked responses in OSN somata in freely breathing mice will be fundamental to understanding how odor information is processed at the periphery and higher circuits in the brain.

Olfactory behavior is among the most fundamental animal behaviors both in the wild and in the laboratory. To elucidate the neural mechanisms underlying olfactory behavior, it is critical to measure neural responses to odorant concentration changes resembling those that animals actually sense during olfactory behavior. However, reproducing the dynamically changing olfactory stimuli to an animal during such measurements of neural activity is technically challenging. Here, we describe technical details and protocols for odor stimulation during calcium imaging of the sensory neurons of the nematode Caenorhabditis elegans. In this system, the neuronal activity of C. elegans is measured using ratiometric calcium imaging during exposure to quantitatively controlled olfactory stimuli over time. Temporal changes in odor concentrations around the animal are precisely controlled according to a predesigned temporal odor gradient to reproduce a realistic odor concentration change during olfactory behavior in a behavioral arena. By monitoring neural activity in response to the realistic olfactory stimulus, it is possible to elucidate the mechanisms by which olfactory input is processed by neural activities and reflected in behavioral output.

Classical fear conditioning typically involves pairing a discrete cue with a foot shock. Quantifying behavioral freezing to the learned cue is a crucial assay for neuroscience studies focused on learning and memory. Many paradigms utilize discrete stimuli such as tones; however, given mice are odor-driven animals and the wide variety of odorants commercially available, using odors as conditioned stimuli presents advantages for studies involving learning. Here, we describe detailed procedures for assembling systems for presenting discrete odor cues during single-day fear conditioning and subsequent analysis of freezing behavior to assess learning.