navigational behavior of drosophila larvae in response to optogenetic stimulation of olfactory sensory neurons

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

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 1 to 2 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.

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Neuroscience

Neurophysiology

Stimulation and Modulation Techniques

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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 universal serial bus (USB) 3.0 charge-coupled device (CCD) camera fitted with an infrared (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 light-emitting diode (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 study 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% relative humidity (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).
	<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 expression. 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. 
		&#8203;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.&#8203;
	<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 min. 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>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Video camera to capture larval movement</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CCD Camera</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&#34; Square Threaded Filter Holder for Imaging Lenses</td>
			<td>Edmund Optics</td>
			<td>59-445</td>
			<td></td>
		</tr>
		<tr>
			<td>RG-715, 2&#34; Sq. Longpass Filter</td>
			<td>Edmund Optics</td>
			<td>46-066</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronics for optogenetic setup</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>Software</td>
			<td></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>Other items</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>Fly food and other reagents</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>Flies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UAS-IVS-CsChrimson</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>55134</td>
			<td></td>
		</tr>
		<tr>
			<td>Orco-Gal4</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>26818</td>
			<td></td>
		</tr>
		<tr>
			<td>Or42a-Gal4</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>9970</td>
			<td></td>
		</tr>
		<tr>
			<td>Or7a-Gal4</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>23907</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

<img alt="Figure 1" src="/files/ftp_upload/22764/22764_Figure1.jpg" /> 
Figure 1: Behavior arena and larval crawling medium. (A) Front view of the black-box behavior arena. The open door of the arena reveals a CCD camera suspended from the ceiling of the box. (B) Bottom view of a metal platform containing red LED light strips used for optogenetic stimulations is mounted around the CCD camera. (C) Top view of the larval crawling mediu...

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

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

Take control and transgenic larvae of the fruit fly Drosophila.
The transgenic larvae express a light-sensitive ion channel in the olfactory receptor neurons or ORNs. These neurons are part of the olfactory circuit, which detects odor stimuli and transmits signals to the brain, guiding larval movement in response to the stimuli.
Place the larvae on a dish with solidified agarose inside a behavior arena, and cover the dish.
Expose the larvae to a wavelength of light that stimulates the ion channels and record larval navigational behavior.
The light activates all-trans-retinal, a chromophore, which induces the opening of ion channels, known as optogenetic stimulation, and allows cation influx.
The influx generates action potentials that transmit signals along the olfactory circuit, impacting larval navigational behavior.
Transgenic larvae with light-sensitive ion channels exhibit a change in the distance traveled in a straight line before changing direction, known as run length, compared to the control larvae.

12 次观看. 果蝇幼虫对嗅觉感觉神经元光遗传学刺激的响应的导航行为

视频: 果蝇幼虫对嗅觉感觉神经元光遗传学刺激的响应的导航行为 - 实验

果蝇中 Rho1 介导的肌动球蛋白收缩性的光遗传学抑制

Optogenetic Inhibition of Rho1-Mediated Actomyosin Contractility Coupled with Measurement of Epithelial Tension in Drosophila Embryos

Contractile forces generated by actin and non-muscle myosin II ("actomyosin contractility") are critical for morphological changes of cells and tissues at multiple length scales, such as cell division, cell migration, epithelial folding, and branching morphogenesis. An in-depth understanding of the role of actomyosin contractility in morphogenesis requires approaches that allow the rapid inactivation of actomyosin, which is difficult to achieve using conventional genetic or pharmacological approaches. The presented protocol demonstrates the use of a CRY2-CIBN based optogenetic dimerization system, Opto-Rho1DN, to inhibit actomyosin contractility in Drosophila embryos with precise temporal and spatial controls. In this system, CRY2 is fused to the dominant negative form of Rho1 (Rho1DN), whereas CIBN is anchored to the plasma membrane. Blue light-mediated dimerization of CRY2 and CIBN results in rapid translocation of Rho1DN from the cytoplasm to the plasma membrane, where it inactivates actomyosin by inhibiting endogenous Rho1. In addition, this article presents a detailed protocol for coupling Opto-Rho1DN-mediated inactivation of actomyosin with laser ablation to investigate the role of actomyosin in generating epithelial tension during Drosophila ventral furrow formation. This protocol can be applied to many other morphological processes that involve actomyosin contractility in Drosophila embryos with minimal modifications. Overall, this optogenetic tool is a powerful approach to dissect the function of actomyosin contractility in controlling tissue mechanics during dynamic tissue remodeling.

作者聚焦: Rho1 介导的肌动球蛋白收缩力的光遗传学抑制与果蝇胚胎上皮张力的测量相结合  

Actomyosin contractility plays an important role in cell and tissue morphogenesis. However, it is challenging to manipulate actomyosin contractility in vivo acutely. This protocol describes an optogenetic system that rapidly inhibits Rho1-mediated actomyosin contractility in Drosophila embryos, revealing the immediate loss of epithelial tension after the inactivation of actomyosin in vivo.

Rho1介导的肌动肌蛋白收缩力的光遗传学抑制与 果蝇 胚胎上皮张力的测量

Contractile forces generated by actin and non-muscle myosin II ("actomyosin contractility") are critical for morphological changes of cells and tissues at ...

科研

JoVE Journal

发育生物学

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Optogenetic 在行为小鼠海马θ振荡中的作用

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

神经科学

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model to study animal avoidance behavior. This protocol introduces a light-spot assay to investigate larval phototactic behavior. The experimental set-up includes two main parts: a visual stimulation system that generates the light spot, and an infrared light-based imaging system that records the process of larval light avoidance. This assay allows tracking of the behavior of larva before entering, during encountering, and after leaving the light spot. Details of larval movement including deceleration, pause, head casting, and turning can be captured and analyzed using this method.

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

This protocol introduces a light-spot assay to investigate Drosophila larval phototactic behavior. In this assay, a light spot is generated as light stimulation, and the process of larval light avoidance is recorded by an infrared light-based imaging system.

光斑分析分析果蝇拉瓦尔光轴

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model ...

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

成績單

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