Abstract
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.
Highlights
[Background] Olfaction is an important modality that almost all animal species use for survival and reproduction, and olfactory behaviors, such as pheromonal responses, innate/learned fear responses, and olfactory navigation, have been intensively studied (Bargmann, 2006; Jacobs, 2012; Isosaka et al, 2015; Yabuki et al, 2016)
To elucidate the neural mechanisms underlying olfactory behavior, it is crucial to measure neural responses to gradually changing olfactory stimuli similar to what animals sense during the behavior
Gradual concentration change because it is difficult to: (1) measure the odor concentration sensed by an animal during olfactory behavior; and (2) quantitatively control the odor stimuli presented to awake behaving animals to reproduce the measured realistic gradual concentration changes
Summary
This odor delivery system was initially developed as a part of an auto-tracking system for freely moving C. elegans, we later used it for multiple immobilized animals (Tanimoto et al., 2017; Yamazaki et al, 2019). Vaporizing tank ( see Yamazoe-Umemoto et al, 2018) A custom-made 50-L acrylic tank is used for vaporizing and storing the 2-nonanone gas (FIS, catalog number: DT-T1) (Figure 5) The lid of this vaporizing tank is equipped with a small metal block with a groove, a heater with a temperature controller, and a fan. 5. Semiconductor odor sensor The temporal concentration change of 2-nonanone is measured at the end of the tube by the semiconductor odor sensor (the same type of sensor used in a gas chromatograph SGVA-N2, FIS; Yamazoe-Umemoto et al, 2018) before and after the imaging experiments each day (Figure 6A). The detailed calibration and measurement procedures are shown in steps B, E, F, I in the Procedure section (Figure 6C)
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