This work was funded by a Stanford Dean's Fellowship (SEJdV), a National Institutes of Health Director's Pioneer Award (TRC DP0035350), a McKnight Foundation Scholar's Award (TRC) and R01 EY022638 (TRC).

1. Generate Channelrhodopsin Flies
<ol>
 <li>Cross UAS-ChR2 flies with the Gal4 driver of your choosing, we use G105-Gal4, which is expressed in Foma-1 neurons in the optic lobe.</li>
 <li>To eliminate the possibility of a visual response to the blue light stimulation, both fly lines are in a w+norpA background.</li>
 <li>End result: w+norpA;G105-Gal4/UAS-ChR2 +</li>
 <li>After adult flies eclose, put selected females on fresh food, supplemented with 10 &mu;M all-trans-retinal (a co-factor required for ChR2) and protected from light, for 3 days before performing the behavioral assay.</li>
</ol>
2. Make 10 &mu;M All-trans-retinal Enhanced Food
<ol>
 <li>Dissolve 100 mg of all-trans-retinal in 17.6 ml of 95% ethanol to make 20 mM retinal. Keep all-trans-retinal protected from light at all times.</li>
 <li>Melt standard cornmeal fly food in microwave, and let cool until warm to touch.</li>
 <li>Mix 50 &mu;l of 20 mM all-trans-retinal into vials of 10 ml of fly food.</li>
 <li>Let vials cool and keep protected from light.</li>
</ol>
3. Equipment
<ol>
 <li>Pipet tips: Standard 1,000 &mu;l pipet tips are cut near the tip, creating a pore diameter of ~2.25 mm.</li>
 <li>Platform (see Figure 1).
 <ol>
 <li>A Delrin base, 17 cm X 25 cm, was constructed with threaded holes at each corner to fit &frac14;" NPT coolant hose connectors.</li>
 <li>A vertical holder, made from Delrin, is attached to the center of the base. The overall dimensions are 25 mm X 40 mm X 65 mm (width X depth X height). A 10 mm wide groove runs the length of the holder, with a thumb screw at the bottom. A platform is attached to the top of the holder, 25 mm X 40 mm X 10 mm, with a 3.5 mm diameter hole aligned with the groove in the holder.</li>
 </ol>
 </li>
 <li>LED arrays (see Figure 1).
 <ol>
 <li>Four arms of coolant hose, ~18 cm long, are affixed to the platform base using the coolant hose connector. Coolant hose is used only as structural support and is not used for cooling purposes.</li>
 <li>Properly spaced grooves are cut into the final piece of coolant hosing of each arm to affix a heat sink to the end of each arm.</li>
 <li>A blue LED Rebel Tri-Stars is mounted to each heat sink using pre-cut thermal adhesive tape. A Carclo 18 &deg; Tri-lens is affixed to each Tri-Star.</li>
 <li>LED Tri-Stars are wired to the BuckPuck DC drivers and a power supply as specified. We have arranged our set-up with each BuckPuck powering two Tri-Stars in series.</li>
 <li>Illumination of all four LED Tri-Stars at 700 mA yielded an irradiance of 713 W/m2 on our platform.</li>
 </ol>
 </li>
 <li>Camera: The camera is mounted on a small tripod and focused on the top of the platform.</li>
</ol>
4. Behavioral Assay
<ol>
 <li>Briefly anaesthetize flies on ice.</li>
 <li>Place individual flies in pipet tips, using tape to close both ends of the tip.</li>
 <li>After the flies have awoken and are actively exploring the pipet tip, remove the tape and place a pipet in the groove in vertical holder. The thumbscrew is used to secure the pipet tip in place and to close the bottom of the tip.</li>
 <li>As the fly explores the pipet tip (typically 30 - 60 sec), start the camera recording just before the fly emerges from the tip onto the platform.</li>
 <li>After the fly has emerged onto the platform, wait 1-2 seco, and turn on the blue LEDs. Use a timer to manually measure the time until the fly initiates flight.</li>
</ol>

<ol>
	<li>Venken, K., Simpson, J., Bellen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulations+of+genes+and+cells+in+the+nervous+system+of+the+fruit+fly.">Genetic manipulations of genes and cells in the nervous system of the fruit fly.</a> Neuron. 72, 202-230 (2011).</li><li>Gordon, M., Scott, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+control+in+a+Drosophila+taste+circuit.">Motor control in a Drosophila taste circuit.</a> Neuron. 61, 373-384 (2009).</li><li>Suh, G. S. B., 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=Light+activation+of+an+innate+olfactory+avoidance+response+in+Drosophila.">Light activation of an innate olfactory avoidance response in Drosophila.</a> Current Biology. 17, 905-908 (2007).</li><li>Zhang, W., Ge, W., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+toolbox+for+light+control+of+Drosophila+behaviors+through+Channelrhodopsin+2-mediated+photoactivation+of+targeted+neurons.">A toolbox for light control of Drosophila behaviors through Channelrhodopsin 2-mediated photoactivation of targeted neurons.</a> European Journal of Neuroscience. 26, 2405-2416 (2007).</li><li>Nagel, G., 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=Channelrhodopsin-2,+a+directly+light-gated+cation-selective+membrane+channel.">Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.</a> Proceedings of the National Academy of Science. 100, 13940-13945 (2003).</li><li>de Vries, S., Clandinin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loom-sensitive+neurons+link+computation+to+action+in+the+Drosophila+visual+system.">Loom-sensitive neurons link computation to action in the Drosophila visual system.</a> Current Biology. 22, 353-362 (2012).</li><li>Card, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escape+behaviors+in+insects.">Escape behaviors in insects.</a> Current Opinion in Neurobiology. 22, 1-7 (2012).</li><li>Card, G., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visually+mediated+motor+planning+in+the+escape+response+of+Drosophila.">Visually mediated motor planning in the escape response of Drosophila.</a> Current Biology. 18, 1300-1307 (2008).</li><li>Fotowat, H., Fayyazuddin, A., Bellen, H. J., Gabbiani, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+neuronal+pathway+for+visually+guided+escape+in+Drosophila+melanogaster.">A novel neuronal pathway for visually guided escape in Drosophila melanogaster.</a> Journal of Neurophysiology. 102, 875-885 (2009).</li><li>Card, G., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+trade-offs+in+the+flight+initiation+of+Drosophila+melanogaster.">Performance trade-offs in the flight initiation of Drosophila melanogaster.</a> The Journal of Experimental Biology. 211, 341-353 (2008).</li><li>Hammond, S., O'Shea, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escape+flight+initiation+in+the+fly.">Escape flight initiation in the fly.</a> Journal of Comparative Phsyiology A. 193, 471-476 (2007).</li><li>Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+mutants+of+Drosophila+isolated+by+countercurrent+distribution.">Behavioral mutants of Drosophila isolated by countercurrent distribution.</a> PNAS. 58, 1112-1119 (1967).</li><li>Bloomquist, B., 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=Isolation+of+a+putative+phospholipase+C+gene+of+Drosophila,+norpA,+and+its+role+in+phototransduction.">Isolation of a putative phospholipase C gene of Drosophila, norpA, and its role in phototransduction.</a> Cell. 54, 723-733 (1988).</li><li>Gohl, D., 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=A+versatile+in+vivo+system+for+directed+dissection+of+gene+expression+patterns.">A versatile in vivo system for directed dissection of gene expression patterns.</a> Nature Methods. 8, 231-237 (2011).</li><li>Zhang, F., 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=Red-shifted+optogenetic+excitation:+a+tool+for+fast+neural+control+derived+from+Volvox+cateri.">Red-shifted optogenetic excitation: a tool for fast neural control derived from Volvox cateri.</a> Nature Neuroscience. 11, 631-633 (2008).</li></ol>

No conflicts of interest declared.

We have demonstrated optogenetic stimulation of escape behaviors by bathing freely walking flies in bright blue light. This approach can be easily adapted to examine other behaviors in freely walking flies, and can be scaled to larger platforms by simply tiling the LED arrays we used over a larger area. Using either the inexpensive camera we describe, or other available camera systems, the user can tailor the frame rate and spatial resolution of the images acquired to suit the behavior of interest. Additionally, o...

A growing arsenal of genetically encoded tools have been developed to manipulate neural activity in specific cells in Drosophila melanogaster1. These tools enable the noninvasive activation or silencing of specific neurons in the intact and freely moving animal. Among these, Channelrhodopsin2 (ChR2), a light-activated cation channel, offers key advantages, since it can be temporally controlled and quickly induced. When neurons that express ChR2 are exposed to bright blue (470 nm) light they rapidly depolarize and exhibit elevated firing rates3-5. Such targeted activation of specific neurons in freely moving animals has revealed the sufficiency of particular neurons for behaviors such as CO2 avoidance3, proboscis extension2,4, and giant-fiber mediated startle responses4. However, as the intense light sources necessary to stimulate ChR2 also stimulate photoreceptors, applying optogenetic techniques to the visual system has been limited. By combining an optogenetic approach with a mutation that impairs phototransduction, we have demonstrated that activation of a specific cluster of neurons in the fly's optic lobe can drive the escape behavior used to avoid collision6.
	Most, if not all, visual animals exhibit an escape behavior to avoid collisions with oncoming objects. Walking or stationary flies, when presented with a looming collision, take-off into flight, away from the oncoming collision7-9. These take-offs are characterized by raised wings prior to take-off and an unstable flight trajectory10,11. This response is distinct from the giant-fiber mediated startle response, jumps that are not preceded by raised wings, and usually result in a free-falling tumble4,9 . Having identified a specific cluster of loom sensitive neurons in the optic lobe, Foma-1 neurons, that are uniquely tuned to encode approaching objects, we sought to probe their involvement in the fly's loom escape behavior. Here we demonstrate the use of optogenetics to selectively activate these neurons and elicit the fly's escape behavior.
	We use the Gal4-UAS transcriptional activator system to drive the expression of ChR2 in the Foma-1 neurons. ChR2 requires the cofactor all-trans-retinal and as this is found in low levels in the Drosophila central nervous system it must be supplemented in the flies' diet.3,4 As bright light is used to activate ChR2 and flies exhibit strong phototactic behaviors12, we sought to eliminate the possibility of a visual response to the stimulus. To do this, we used animals that were homozygous mutant for a null allele of the norpA gene, which encodes a critical component of the phototransduction cascade, phospholipase C-&beta;. Photoreceptors in such mutant flies are unable to respond to light13. To test the optogenetic stimulation of the escape response, we need to isolate a single fly and bathe it in bright blue light. To do this, we place individual flies in pipet tips. One pipet tip is placed in a custom holder, such that the fly will geotactically walk up the tip and out onto a rectangular platform. The fly is able to freely walk around on the top of this platform. The platform is surrounded by four blue LED arrays, each containing 3 LEDs, focused on the top of the platform. After the fly is on the platform, the LEDs are illuminated, and the fly's response is recorded using a high-speed camera6.

<table border="1">
	 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>All-trans Retinal</td>
 <td>Advance Scientific &amp; Chemical Inc</td>
 <td>R3041</td>
 <td></td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Heat Sink 9.2 C/W</td>
 <td>Luxeonstar</td>
 <td>LPD30-30B</td>
 <td>30 mm square X 30 mm high</td>
 </tr>
 <tr>
 <td>Carclo 18 &deg; Tri-Lens</td>
 <td>Luxeonstar</td>
 <td>10507</td>
 <td></td>
 </tr>
 <tr>
 <td>Blue Rebel LED on Tri-Star Base</td>
 <td>Luxeonstar</td>
 <td>MR-B0030-20T </td>
 <td>470 nm, 174 lm @ 700 mA.</td>
 </tr>
 <tr>
 <td>700 mA BuckPuck DC Driver</td>
 <td>Luxeonstar</td>
 <td>3021-D-E-700</td>
 <td></td>
 </tr>
 <tr>
 <td>Wiring Harness for BuckPuck Driver</td>
 <td>Luxeonstar</td>
 <td>3021-HE</td>
 <td></td>
 </tr>
 <tr>
 <td>Pre-cut thermal adhesive tape</td>
 <td>Luxeonstar</td>
 <td>LXT-S-12</td>
 <td>20 mm Hex Base</td>
 </tr>
 <tr>
 <td>Snap-Loc Coolant Hose, &frac14;" ID</td>
 <td>McMaster-Carr</td>
 <td>5307K49</td>
 <td></td>
 </tr>
 <tr>
 <td>Snap-Loc Coolant Hose Connector</td>
 <td>McMaster-Carr</td>
 <td>5307K39</td>
 <td>&frac14;" NPT Male</td>
 </tr>
 <tr>
 <td>Laboratory Grade Switching Mode Programmable DC Power Supply</td>
 <td>BK Precision</td>
 <td>1698</td>
 <td></td>
 </tr>
 <tr>
 <td>Exilim camera</td>
 <td>Casio</td>
 <td>EX-FH20</td>
 <td></td>
 </tr>
	 </tbody>
 </table>

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.

Blind flies expressing either ChR2 or the G105 driver alone show a low rate of take-off following their illumination with bright blue light. Blind flies exhibited the same rate of take-off regardless of illumination (Figure 2), suggesting that these take-offs were spontaneous rather than due to the illumination with blue light. When the ChR2 is expressed in the Foma1 neurons, however, illumination with blue light elicits the escape response. Over 50% of the flies tested took off within 1 sec of illuminat...

Watch this Scientific Journal Video about Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster at JoVE.com

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.

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 ...

optogenetic-stimulation-of-escape-behavior-in-drosophila-melanogaster

Research

JoVE Journal

Neuroscience

17.3K Views.  Stanford University. 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.

Video: Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

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 ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Quantification of Drosophila Grooming Behavior

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...

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.

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