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 1A).</li>
	<li>Mount a monochrome USB 3.0 CCD camera fitted with an IR long-pass 830 nm filter and an 8 mm F1.4 C-mount lens (see Table of Materials) to the center of the ceiling of the black box. Place two infra-red LED strips (see Table of Materials) on the table-top in order to illuminate the larvae in the dark arena (Figure 1A).</li>
	<li>To build the LED platform, obtain a 22 cm &#215; 22 cm square Aluminum plate (preferably spray painted with a matte black finish to eliminate any reflections). In the center of the plate, using a metal cutter, cut a hole that is large enough to fit around the CCD camera.</li>
	<li>Cover the metal plate with red LED strip lights (see Table of Materials). Solder LED light strip wires in series and feed the strip wires into an optocoupler relay controlled by a Raspberry Pi 2B microprocessor (see Table of Materials) (Figure 1B and 2).</li>
	<li>Install and configure Ubuntu Mate/Raspian Jesse/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 (Figure 2) (see Table of Materials). Mount the LED platform around the CCD camera (Figure 1B). 
	NOTE: Ubuntu Mate v16.04 operating system is freely available. A set of simple Python based commands can be easily adapted to program patterns of LED light stimuli (see file of syntax).</li>
	<li>Ensure homogenous irradiance at various points in the behavior arena. Measure the absolute irradiance at the surface of the arena with the help of a Spectrometer and determine it to be ~1.3 W/m2 throughout the surface of the arena. 
	NOTE: At this intensity, no significant changes were observed in temperature over the course of the experiments. Another study10 used a higher irradiance of ~1.9 W/m2 and observed no changes in temperature over the course of the experiments.</li>
</ol>
2. Preparation of Drosophila Larvae for Behavior Analyses
<ol>
	<li>Maintain the flies on standard fly food (see Table of Materials) at 25 &#176;C, 50-60% RH, and a 12 h/12 h light/dark cycle.</li>
	<li>In order to express CsChrimson in a single pair of ORNs, cross virgin females from a UAS-IVS-CsChrimson line to males from an OrX-Gal4 line (&#39;X&#39; corresponds to one of 21 larval odor receptor (Or) genes that are uniquely expressed in each of 21 pairs of ORNs)9,11.
	<ol>
		<li>Alternately, in order to express CsChrimson in all 21 larval ORNs, cross virgin females from a UAS-IVS-CsChrimson line to males from an Orco-Gal4 line (&#39;Orco&#39; is the co-receptor that is expressed in all 21 ORNs).</li>
		<li>Use UAS-IVS-CsChrimson line by itself as a control in these experiments. 
		NOTE: The fly stocks listed here are all available at the Bloomington Drosophila Stock Center (see Table of Materials).</li>
	</ol>
	</li>
	<li>Once male and female flies in a cross are allowed to mate and lay eggs for 48 h, transfer adults to a fresh vial.
	<ol>
		<li>To the surface of the food vial containing eggs, add 400 &#181;L of a mixture containing 400 &#181;M all-trans retinal (ATR) dissolved in Dimethyl sulfoxide (DMSO) and 89 mM sucrose dissolved in distilled water. 
		NOTE: The small amount of sucrose promotes larval feeding of the ATR solution. ATR is a cofactor required for upregulating of CsChrimson expression9,10. ATR is light sensitive.</li>
		<li>Once ATR is added to the food vials containing eggs, incubate the vials in the dark for an additional 72 h. 
		NOTE: While this study and the above two studies have not observed effects on larval behavior due to ATR feeding, it is recommended controlling for the effects of ATR feeding by subjecting test lines to the same amount of the above mixture that does not contain ATR.</li>
	</ol>
	</li>
	<li>Extract third-instar larvae (~120 h after egg laying) from the surface of fly food by floating them using a high density (15%) sucrose solution. Using a P1000 micropipette separate the larvae floating on the surface of the sucrose solution into a 1000 mL glass beaker.</li>
	<li>Wash larvae 3&#8211;4 times by exchanging 800 mL fresh distilled water in the glass beaker each time. Allow larvae to rest for 10 min before subjecting them to the behavior assay.</li>
</ol>
3. Behavior Assay
<ol>
	<li>Maintain consistent temperature (between 22&#8211;25 &#176;C) and humidity (between 50 and 60% RH) in the behavior room.</li>
	<li>Prepare larval crawling medium by pouring 150 mL of melted agarose (1.5%) into a 22 cm x 22 cm square Petri dish. One Petri dish is poured for each trial of the assay, 8&#8211;10 trials per experiment. Allow agarose to solidify and cool for 1&#8211;2 h in the Petri dishes before using them in the behavior assay.
	<ol>
		<li>Transfer no more than 20 of the prepped third-instar larvae to the center of Petri dish (Figure 1C). Cover the Petri dish with its lid. Place the Petri dish in the behavior arena under the CCD camera. 
		NOTE: Depending on the experiment, behavior assays are typically carried out for 3&#8211;5 min. If an odorant is used in the assay to provide guidance cues, it has been observed that the resulting odor gradient remains stable for approximately 5 min1. Longer assay times are not recommended. Deleterious effects on larvae due to dehydration or from prolonged 630 nm red-light exposure have not been observed within these time points.</li>
	</ol>
	</li>
	<li>Turn ON the 850 nm infra-red LED light source to visualize larvae in the video. Start the CCD camera to record larval movement.</li>
	<li>Using the software associated with the Raspberry Pi processor, program the procedure to administer appropriate patterns of red light stimulation. 
	NOTE: A set of simple Python based commands can be easily adapted to program patterns of LED light stimuli (see file of syntax).</li>
</ol>
4. Data Processing and Analysis
<ol>
	<li>Import the recorded video of each trial into any available programming software like Matlab.</li>
	<li>Obtain XY coordinates of every larva in a movie as a function of time. Based on limits of the tracking software, 15&#8211;20 third-instar larvae can be tracked in a single movie1,8. 
	NOTE: A set of simple Matlab codes (&#39;Tracklarva&#39;) that can be easily adapted to suit appropriate conditions (see file of syntax) is provided. This program combines all trials in an experiment and outputs the XY coordinates of every larva for the entire duration of the assay (see code syntax below). Alternately, one could use several open-source based programs that are freely available for researchers. E.g. JAABA (http://jaaba.sourceforge.net/)12.</li>
	<li>Use the generated XY coordinates to plot larval trajectories and to further analyze larval locomotion.
	<ol>
		<li>For behavior analyses, use navigational statistics such as speed, path curvature, heading angle, to segment individual larval trajectories into alternating sequences of runs and turns.</li>
		<li>Runs are defined as continuous periods of forward movement. Turns separate successive runs. Turns are flagged when changes to trajectory orientation angles were &#62;45&#176; (see file of syntax).</li>
	</ol>
	</li>
	<li>Calculate average values for run speed, run length, run direction, and other parameters as required. 
	NOTE: A set of simple syntax to extract &#39;runs&#39; and &#39;stops&#39; from larval trajectories is provided (see file of syntax). Simple Matlab or Excel based functions can be applied to the extracted data to calculate values for &#39;run speed&#39;, &#39;run length&#39; etc.</li>
	<li>Represent data for each behavioral metric as mean &#177; SEM.</li>
</ol>

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A., Kwon, J. Y., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+odor+coding+in+the+Drosophila+larva.">The molecular basis of odor coding in the Drosophila larva.</a> Neuron. 46, 445-456 (2005).</li><li>Kreher, S. A., Mathew, D., Kim, J., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translation+of+sensory+input+into+behavioral+output+via+an+olfactory+system.">Translation of sensory input into behavioral output via an olfactory system.</a> Neuron. 59, 110-124 (2008).</li><li>Hallem, E. A., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coding+of+odors+by+a+receptor+repertoire.">Coding of odors by a receptor repertoire.</a> Cell. 125, 143-160 (2006).</li><li>Monte, P., 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=Characterization+of+the+larval+olfactory+response+in+Drosophila+and+its+genetic+basis.">Characterization of the larval olfactory response in Drosophila and its genetic basis.</a> Behav Genet. 19, 267-283 (1989).</li><li>Gershow, M., 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=Controlling+airborne+cues+to+study+small+animal+navigation.">Controlling airborne cues to study small animal navigation.</a> Nature methods. 9, 290-296 (2012).</li><li>Klapoetke, N. 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H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Kabra, M., Robie, A. 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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.

<table><tbody><tr><td>&lt;strong&gt;Video camera to capture larval movement&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CCD Camera&amp;nbsp;</td><td>Edmund Optics</td><td>106215</td><td></td></tr><tr><td>M52 to M55 Filter Thread Adapter</td><td>Edmund Optics</td><td>59-446</td><td></td></tr><tr><td>2&quot; Square Threaded Filter Holder for Imaging Lenses&amp;nbsp;</td><td>Edmund Optics</td><td>59-445</td><td></td></tr><tr><td>RG-715, 2&quot; Sq. Longpass Filter</td><td>Edmund Optics</td><td>46-066</td><td></td></tr><tr><td>&lt;strong&gt;Electronics for optogenetic setup&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Raspberry Pi 2B</td><td>RASPBERRY-PI.org</td><td>RPI2-MODB-V1.2</td><td></td></tr><tr><td>3 Channel programmable power supply</td><td>newegg.com</td><td>9SIA3C62037092</td><td></td></tr><tr><td>8 Channel optocoupler relay</td><td>amazon.com</td><td>6454319</td><td></td></tr><tr><td>630nm Quad-row LED strip lights</td><td>environmentallights.com</td><td>red3528-450-reel</td><td></td></tr><tr><td>850nm LED strips</td><td>environmentallights.com</td><td>wp-4000K-CC5050-60x2-kit</td><td></td></tr><tr><td>&lt;strong&gt;Software&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Matlab</td><td>Mathworks Inc.</td><td></td><td></td></tr><tr><td>Ubuntu MATE v16.04</td><td>Nubuntu</td><td>https://github.com/yslo/nubuntu</td><td></td></tr><tr><td>&lt;strong&gt;Other items&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Plexiglass black acrylic</td><td>Home Depot</td><td>MC1184848bl</td><td></td></tr><tr><td>&lt;strong&gt;Fly food and other reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Nutrifly fly food</td><td>Genesee Scientific</td><td>66-112</td><td></td></tr><tr><td>Agarose powder</td><td>Genesee Scientific</td><td>20-102</td><td></td></tr><tr><td>22cm X 22cm square petri-dish</td><td>VWR Inc.</td><td>25382-327</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>84097</td><td></td></tr><tr><td>All trans-retinal</td><td>Sigma-Aldrich</td><td>R2500</td><td></td></tr><tr><td>&lt;strong&gt;Flies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;UAS-IVS-CsChrimson&amp;nbsp;&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>55134</td><td></td></tr><tr><td>&lt;em&gt;Orco-Gal4&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>26818</td><td></td></tr><tr><td>&lt;em&gt;Or42a-Gal4&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>9970</td><td></td></tr><tr><td>&lt;em&gt;Or7a-Gal4&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>23907</td><td></td></tr></tbody></table>

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...

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

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 ...

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7.2K Views.  University of Nevada. 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.

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

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Behavioral Assays for Optogenetic Manipulation of Neural Circuits in Drosophila melanogaster

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study introduces easy-to-implement setups for applying optogenetic methods in Drosophila melanogaster. Two optogenetic tools, CsChrimson, a red-light-activated cation channel, and GtACR2, a blue-light-activated anion channel, were employed in four experimental approaches. Three of these approaches involve single-fly experiments: (1) a blue-light optogenetic thermotactic positional preference assay targeting temperature-sensitive heating cells, (2) a red-light optogenetic positional preference assay activating bitter sensing neurons, and (3) a proboscis extension response assay activating the sweet-sensing neurons. The fourth approach (4) is a fly maze setup to assess avoidance behaviors using multiple flies. The ability to manipulate neural activity temporally and spatially offers powerful insights into sensory processing and decision-making, underscoring the potential of optogenetics to advance our knowledge of neural function. These methods provide an accessible and robust framework for future research in neuroscience to enhance the understanding of specific neural pathways and their behavioral outcomes.

This paper presents methods for optogenetic manipulation in Drosophila melanogaster, utilizing CsChrimson and GtACR2 to activate and silence specific neurons. Four experiments are described to utilize optogenetics to explore thermotactic and gustatory behaviors, providing insights into the underlying neural mechanisms governing these processes.

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study ...

Navigational Behavior of Drosophila Larvae in Response to Optogenetic Stimulation of Olfactory Sensory Neurons

Source: Clark, D. A., et al. <a href="https://app.jove.com/v/57353">Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons.</a> J. Vis. Exp. (2018).
This video demonstrates a study of the navigational behavior of Drosophila larvae in response to optogenetic stimulation of olfactory sensory neurons (ORNs). Transgenic Drosophila larvae expressing light-sensitive ion channels in the ORNs are placed on agarose within a behavior arena. Optogenetic stimulation with light opens the channels, causing a cation influx that triggers the transmission of the signal to the brain, and the navigation behavior is recorded.

1. Building a Behavior Arena and Preparing Hardware to Enable Optogenetic Stimulation in the Behavior Arena

	To build a light-deprived behavior arena, ...

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

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Chapters in this video

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

Appetitive Associative Olfactory Learning in Drosophila Larvae

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Behavioral Assays for Optogenetic Manipulation of Neural Circuits in Drosophila melanogaster

Navigational Behavior of Drosophila Larvae in Response to Optogenetic Stimulation of Olfactory Sensory Neurons