This work was supported by startup funds from the University of Nevada, Reno and by NIGMS of the National Institute of Health under grant number P20 GM103650.

1. Building a Behavior Arena and Preparing Hardware to Enable Optogenetic Stimulation in the Behavior Arena
<ol>
	<li>To build a light-deprived behavior arena, construct a box with a dimension of 89 x 61 x 66 cm3 (35&#34; L x 24&#34; W x 26&#34; H) made of black colored plexiglass acrylic sheets (3 mm thick) (see Table of Materials). Materials to build such a box should be available at local hardware stores. Place this box on a table-top in the behavior room (Figure 1</s...

<ol>
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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+wiring+diagram+of+a+glomerular+olfactory+system.">The wiring diagram of a glomerular olfactory system.</a> eLife. 5, (2016).</li></ol>

The authors have nothing to disclose.

Here, we described a method that allows for the measurement of Drosophila larval behavior in response to simultaneous optogenetic activation of olfactory neurons. Previously described larval tracking methods1,8 use different odor delivery technology to activate ORNs. However, these methods cannot control for either the specificity or temporal patterns of ORN activation. Our method overcomes these deficits by using light stimuli instead of odor stimuli fo...

Olfactory information in a Drosophila larva&#39;s environment is sensed by only 21 functionally distinct ORNs, the activities of which ultimately determine larval behavior1,2,3,4. Yet, relatively little is known about the logic by which sensory information is encoded in the activities of these 21 ORNs. There is thus a need to experimentally measure the functional contributions of each larval ORN to behavior.
Although the sensory response profile of the entire repertoire of Drosophila larval ORNs has been studied in detail1,4,5, the contributions of individual ORNs to the olfactory circuit and thereby to navigational behavior remain largely unknown. Difficulties in larval behavior studies, so far, arise due to the inability to spatially and temporally activate single ORNs. A panel of odorants that specifically activate 19 of the 21 Drosophila larval ORNs was recently described1. Each odorant in the panel, at low concentrations, elicits a physiological response only from its cognate ORN. However, at higher concentrations that are normally used for conventional behavior assays, each odorant elicits physiological responses from multiple ORNs1,5,6. Further, odorants in this panel have varied volatilities that complicate interpretation of behavior studies that depend on formation of stable odor gradients7,8. Finally, naturally occurring odor stimuli have a temporal component that is difficult to replicate under laboratory conditions. It is therefore important to develop a method that can measure larval behavior while simultaneously activating individual ORNs in a spatial and temporal manner.
Here, we demonstrate a method that has advantages over previously described larval tracking assays1,8. The tracking assay described in Gershow et al. uses electronically controlled valves to maintain a stable gradient of odor in the behavior arena8. However, due to the level of complex engineering involved to build the odor stimulus setup, this method is difficult to replicate in other laboratories. Further, the issues related to using odorants to specifically activate single ORNs remain unresolved. The tracking assay described in Mathew et al. uses a simpler odor delivery system, but the resulting odor gradient is dependent on the volatility of test odorant and is unstable for long durations of the assay1. Thus, by replacing odor stimuli with light stimuli, our method has the advantages of specificity and precise temporal control of ORN activation and is not dependent on formation of odor gradients of different strengths.
Our method is easy to set up and is appropriate for researchers interested in measuring aspects of Drosophila larval navigation. This technique could be adapted to other model systems provided that the researcher is able to drive the expression of CsChrimson in their favorite system&#39;s neuron(s) of choice. CsChrimson is a red-shifted version of channel rhodopsin. It is activated at wavelengths that are invisible to the larva&#39;s phototaxis system. We are therefore able to manipulate the activity of neurons with specificity, reliability, and reproducibility9. By modifying the custom written software to account for size changes of the subjects, this method could easily be adapted for crawling larvae of other insect species.

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			<td height="40" style="height:40px;width:359px;">Video camera to capture larval movement</td>
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			<td height="33" style="height:33px;">M52 to M55 Filter Thread Adapter</td>
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			<td>59-446</td>
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			<td height="33" style="height:33px;">2&#34; Square Threaded Filter Holder for Imaging Lenses&#160;</td>
			<td style="width:296px;">Edmund Optics</td>
			<td>59-445</td>
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			<td height="33" style="height:33px;">RG-715, 2&#34; Sq. Longpass Filter</td>
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			<td>46-066</td>
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			<td height="33" style="height:33px;">Electronics for optogenetic setup</td>
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			<td>RASPBERRY-PI.org</td>
			<td>RPI2-MODB-V1.2</td>
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			<td height="33" style="height:33px;">3 Channel programmable power supply</td>
			<td>newegg.com</td>
			<td>9SIA3C62037092</td>
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			<td>amazon.com</td>
			<td>6454319</td>
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			<td height="33" style="height:33px;">630nm Quad-row LED strip lights</td>
			<td>environmentallights.com</td>
			<td>red3528-450-reel</td>
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			<td height="33" style="height:33px;width:359px;">850nm LED strips</td>
			<td>environmentallights.com</td>
			<td>wp-4000K-CC5050-60x2-kit</td>
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			<td height="33" style="height:33px;width:359px;">Software&#160;</td>
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			<td height="33" style="height:33px;width:359px;">Matlab</td>
			<td>Mathworks Inc.</td>
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			<td height="33" style="height:33px;width:359px;">Ubuntu MATE v16.04</td>
			<td>Nubuntu</td>
			<td>https://github.com/yslo/nubuntu</td>
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			<td height="33" style="height:33px;">Other items</td>
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			<td height="33" style="height:33px;">Plexiglass black acrylic</td>
			<td>Home Depot</td>
			<td style="width:208px;">MC1184848bl</td>
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			<td></td>
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			<td height="33" style="height:33px;">Nutrifly fly food</td>
			<td style="width:296px;">Genesee Scientific</td>
			<td>66-112</td>
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			<td height="33" style="height:33px;">Agarose powder</td>
			<td style="width:296px;">Genesee Scientific</td>
			<td>20-102</td>
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			<td height="33" style="height:33px;">22cm X 22cm square petri-dish</td>
			<td style="width:296px;">VWR Inc.</td>
			<td>25382-327</td>
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			<td height="33" style="height:33px;">DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
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			<td height="33" style="height:33px;">Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84097</td>
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			<td height="33" style="height:33px;">All trans-retinal</td>
			<td>Sigma-Aldrich</td>
			<td>R2500</td>
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			<td height="33" style="height:33px;">Flies</td>
			<td></td>
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			<td height="33" style="height:33px;">UAS-IVS-CsChrimson&#160;</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>55134</td>
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			<td height="33" style="height:33px;width:359px;">Orco-Gal4</td>
			<td>Bloomington Drosophila Stock Center</td>
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			<td height="33" style="height:33px;">Or42a-Gal4</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>9970</td>
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			<td height="33" style="height:33px;">Or7a-Gal4</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>23907</td>
			<td></td>
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tracking drosophila larval behavior in response to optogenetic stimulation of olfactory neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

To demonstrate the specificity of ORN activation, our method was successfully applied to determine the impact of two different ORN (ORN::7a &#38; ORN::42a) (ORNs expressing either Or7a or Or42a) activation on larval behavior (Figure 3). Consistent with recent studies that individual larval ORNs are functionally distinct1,10,13, our representative data demonstrates th...

Watch this Scientific Journal Video about Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons at JoVE.com

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

The overall goal of this methodology is to analyze navigational behavior of drosophila larva in response to simultaneously optogenetic stimulation of its olfactory neurons. This method enables the comprehensive dissection of olfactory circuit function and complements studies on how olfactory circuit function translates into behavior response. The main advantage of this technique is its specificity and temporal control by using a light instead of traditional odor gradients to activate individual olfactory neurons.To begin the experiment, build a light deprived behavior arena. Construct a box that is 89 by 61 by 66 centimeters, made of black-colored plexiglass acrylic sheets, three millimeters thick. Place the box on a tabletop in the behavior room.Next, mount a monochrome USB 3.0 CCD camera fitted with an IR Longpass 830 nanometer filter and an eight millimeter F1.4 C mount lens on the center of the ceiling of the black box. Place two infrared LED strips on the tabletop to illuminate the larvae in the dark arena. Then, obtain a 22 centimeter by 22 centimeter square aluminum plate to build the LED platform.In the center of the plate, use a metal cutter to cut a hole that is large enough to fit around the CCD camera. Cover the metal plate with red LED strip lights. Solder the LED light strip wires in series, and feed the strip wires into an optocoupler relay controlled by a Raspberry Pi 2B microprocessor.Next, mount the LED platform around the CCD camera. Then install and configure an Ubuntu MATE Raspbian Jessie Linux-based operating system on the Raspberry Pi processor before connecting the optocoupler relay to the LED strips. Attach a power supply to power the LED strips and the optocoupler.Ensure there is homogenous irradiance at various points around the CCD camera in the behavior arena. Ensure the absolute irradiance at the surface of the arena with the help of a spectrometer and determine it to be approximately 1.3 watts per meter squared throughout the surface of the arena. Maintain the flies on standard fly food at 25 degrees Celsius, 50 to 60%relative humidity in a 12-hour, 12-hour light/dark cycle.In order to express CsChrimson in a single pair of ORNs, cross the virgin females from a UAS-IVS-CsChrimson line to males from an ORX-GAL4 line. After male and female flies in a cross have been allowed to mate and lay eggs for 48 hours, transfer the adults to a fresh vial. On the surface of the food vial containing eggs, add 400 microliters of a mixture containing 400 micromolar all-trans-retinal, or ATR, dissolved in dimethyl sulphoxide, or DMSO, in 89 millimolar sucrose dissolved in distilled water.Once ATR is added to the food vials containing eggs, incubate the vials in the dark for an additional 72 hours. After incubation, extract third instar larvae from the surface of fly food by floating them using a high-density 15%sucrose solution. Using a P1000 micro-pipette, separate the larvae floating on the surface of the sucrose solution into a 1, 000 milliliter glass beaker.Wash larvae four times by exchanging 800 milliliters of fresh distilled water in the glass beaker each time. After the final wash, allow the larvae to rest for 10 minutes before subjecting them to the behavior assay. For the behavior assay, maintain a consistent temperature and humidity in the behavior room.Prepare larval crawling medium by pouring 150 milliliters of melted 1.5%agarose into a 22 centimeter by 22 centimeter square Petri dish. Allow the agarose to solidify and cool for one to two hours in the Petri dishes before using them in the behavior assay. After the agarose has solidified, transfer no more than 20 of the prepped third instar larvae to the center of the Petri dish and cover the Petri dish with its lid.Place the Petri dish in the behavior arena under the CCD camera. Turn on the 850 nanometer infrared LED light source to visualize larvae in the video. Start the CCD camera to record larval movement.Finally, move on to data processing and analysis. When ORN 7a expressing CsChrimson was stimulated by light, there was a significant decrease in run length compared to control animals. Conversely, when ORN 42a expressing CsChrimson was stimulated by light, there was a significant increase in run length compared to control animals.We found that for ORN 42a, different temporal patterns of light stimulation elicited different behavioral responses. While we have demonstrated this method for the first order olfactory receptor neurons, our method also permits a variety of future applications, such as measuring the impact of downstream neurons such as projection neurons and local neurons on larval behavior. We expect this technique to pave the way for a comprehensive dissection of larval olfactory circuit function.

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7.1K 조회수.  University of Nevada. The overall goal of this methodology is to analyze navigational behavior of drosophila larva in response to simultaneously optogenetic stimulation of its olfactory neurons. This method enables the comprehensive dissection of olfactory circuit function and complements studies on how olfactory circuit function translates into behavior response. The main advantage of this technique is its specificity and temporal control by using a light instead of traditional odor gradients to activate individu...