Calcium Imaging of Neuronal Activity under Gradually Changing Odor Stimulation in Caenorhabditis elegans

[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. ms 2

olfactory stimuli are still largely unknown.
The nematode Caenorhabditis elegans has been widely used to study olfactory behavior. C. elegans shows attractive and repulsive behaviors to odor stimuli, and has a strong advantage for neural studies because of its simple nervous system with complete connectome information (White et al., 1986;Bargmann et al., 1993;Bargmann, 2006). C. elegans is also useful for calcium imaging of neural activity because of its transparent body and the availability of various cell-specific promoters to express genetically encoded calcium indicators (GECIs) (Kerr, 2006). Furthermore, recent technical advances have enabled the application of calcium imaging to freely moving C. elegans by using automated tracking of the animal using a motorized stage (Faumont et al., 2011;Kawano et al., 2011;Piggott et al., 2011;Zheng et al., 2012;Tanimoto et al., 2016). This method allows for the simultaneous analysis of behavioral output and neural activity, such as motor neuron or command interneuron activity. However, during calcium imaging, it has remained difficult to present a realistic gradual odorant concentration change. A widely used microfluidic flow channel delivers only up-step or down-step odorant stimuli to a physically restricted animal in a small chamber (Chronis et al., 2007). Systems that deliver gradually changing concentrations of water-soluble chemicals/odorants were recently developed (Albrecht and Bargmann, 2011;Larsch et al., 2013;Itskovits et al., 2018), but these odor delivery methods use only the aqueous phase, which differs from the widely used air phase olfactory paradigm (Bargmann, 2006).
In addition, such microfluidic channels are not compatible with auto-tracking microscope systems for calcium imaging of freely moving C. elegans at high magnification. Therefore, it has been technically difficult to simultaneously analyze neural activity and behavior in response to naturalistic olfactory stimuli in freely moving C. elegans.
Here we describe the technical details and protocols of our original calcium imaging microscope system integrated with a specialized odor stimulation device. In this system, temporal changes in odor concentration around the animal are precisely controlled according to predesigned temporal odor gradients to reproduce olfactory stimuli during olfactory behavior. These temporal gradients are designed based on measured odor concentration changes sensed by the animal during actual olfactory navigation behavior in a classic Petri dish experiment (Yamazoe-Umemoto et al., 2015 and 2018). This odor stimulation device can also be combined with an auto-tracking motorized stage to apply the realistic graded odorant stimuli to freely moving C. elegans. By reproducing realistic odor concentration changes during the measurement of neural activity and behavior, it was possible to elucidate the neural mechanisms that underlie the dynamic aspects of olfactory processing by the nervous system, such as temporal differentiation and integration (Tanimoto et al., 2017). Our protocol for the repulsive odor 2-nonanone described below should be applicable to other odorants.   The ImagEM electron-multiplying charge-coupled device (EM-CCD) camera is used to acquire fluorescent images sensitively (Hamamatsu Photonics). IR, infrared light.

Odor delivery system
Although 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  system is used with the auto-tracking system, the odor flow from the tube also continuously stimulates the animal since the animal is tracked in the center of the view field ( Figure 1).
Temporal changes in the 2-nonanone concentration in the odor flow are controlled by two HV-SSP01 syringe pumps (HawkVision, Japan), each holding two 25 ml Gastight syringes (Hamilton) (Figure 2). One pump is used to infuse the 2-nonanone gas, while the other infuses air, and the infused gas and air are mixed through a custom-made microchannel (see Figure 2 and Equipment step 3). The two pumps are programmed to deliver a constant 8 ml/min gas flow in total from the tube end with varying combinations of pump speed to cause the temporal changes in 2-nonanone concentration. For example, when the pump speed for 2-nonanone is changed from 2 ml/min to 5 ml/min, for air it is changed from 6 ml/min to 3 ml/min during the same period. With the 8 ml/min odor flow, the animal's entire body is exposed to an essentially uniform odor flow without significant stagnation, diffusion, or turbulence. We found that the 8 ml/min air flow rate did not affect the animal's behavior, although stronger flow rates caused aversive responses of the animal. Flow rates slower than 8 ml/min reduce the temporal precision of the temporal odor gradient when measured with a semiconductor odor sensor (see Equipment steps 5-6). The experimental room, particularly around the microscope, must be kept windless during the experiment. The behavior of the odor flow can be visualized by filling fog produced by Wizard Stick (Zero Toys, USA) instead of the odor gas (see Tanimoto et

Custom-made microchannel for odor delivery
We fabricated a custom microchannel for odor delivery. Because 2-nonanone, the odorant we used here, is highly adsorptive, we chose glass, stainless steel, and PFA Teflon to fabricate the microchannel since they are less adsorptive to 2-nonanone. Other kinds of plastics or silicones are highly adsorptive to 2-nonanone, and we avoided using them for the channel.       Figures 8A, 8B). This agar plate is especially useful for the auto-tracking experiment because the dish has no brim that can interfere with the objective lens and the bamboo skewer. Also, by filling the dish with 10 ml NGM agar, we can reduce the meniscus and increase the flatness of the agar's surface to ensure stable focusing during tracking. In these conditions, ~100 synchronized young adult animals will be grown with a few eggs laid on the plate per animal at the beginning of the experiment.
Note: Because some transgenic strains lay fewer eggs than wild-type animals, the number of such transgenic animals transferred to the new plate can be increased to obtain ~100 animals per plate. 3. Bring the medium and dishes on the bench and keep the medium on a warming magnetic stirrer to retain medium-low viscosity. 4. Quickly pour 10 ml of the medium into the dish using a 5-ml Pipetman P5000 (Gilson) before the pre-warmed dish cools to room temperature. Prepare one agar plate for one trial planned on the experimental day. Be careful not to create any bumps or dents on the agar surface that may disturb the imaging. 5. Cover four plates on the bench with a lid of a 34 cm × 24 cm plastic case 2321E (Sanplatec) to avoid dust and excessive dryness ( Figure 8A). 6. After 15 min, move the agar plates to the 34 cm × 24 cm plastic case. Close its lid and store it at room temperature ( Figure 8B).