Op to Genetic Manipulation of Neural Activity in Freely Moving Caenorhabditis Elegans

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<ul><li><p>8/6/2019 Op to Genetic Manipulation of Neural Activity in Freely Moving Caenorhabditis Elegans</p><p> 1/8</p><p>2011</p><p>NatureAmerica,Inc.Allrightsreserved.</p><p>Articles</p><p>nAture methods | ADVANCE ONLINE PUBLICATION | </p><p>W p a p a y apab a- vy w paa pa y v Caenorhabditis elegans. A a</p><p>p a a wxp ap-2 ap p</p><p> yp. ia p wa aayz wp a v a, apy a a a a a a v a a w a apppa</p><p>wav a b avy. Ba a a a w a apy v a, ypa a p (~50 a p ) pv </p><p>paa (~30 m). t aay, fxbya y y, w p p aay w , -ay a ay a av b pb w pv .</p><p>Researchers in systems neuroscience aim to understand how neural</p><p>dynamics create behavior. Optogenetics has accelerated progress inthis area by making it possible to stimulate or inhibit neurons thatexpress light-activated proteins, for example, channelrhodopsin-2(ChR2) and halorhodopsin (also known as Halo/NpHR), by illu-minating them17. The nematode C. elegans is particularly ame-nable to optogenetics owing to its optical transparency, compactnervous system and ease of genetic manipulation811.</p><p>The ability to deliver light to one cell with spatial selectivity isessential for targeted optogenetic perturbation in the many casesin C. elegans in which genetic methods do not provide adequatespecificity. In the worm motor circuit, for example, single neuron-specific promoters are not available to drive expression of light-activated proteins in only one or a few neurons of the ventral</p><p>nerve cord (VNC). Optogenetics has been applied to the mech-anosensory circuit in C. elegans, but only through simultaneousstimulation of all touch receptor neurons, because promoters spe-cific to each neuron are unavailable1. Researchers can use laserkilling to study the contribution of single touch receptor neuronsto overall behavior by removing neurons, but it is often prefer-able to work with intact circuits1214. Recently, a digital micro-mirror device (DMD) has been used to deliver light with high</p><p>op apa a avy yv Caenorhabditis elegansAndrew M Leifer1,4, Christopher Fang-Yen1,2,4, Marc Gershow1, Mark J Alkema3 &amp; Aravinthan D T Samuel1</p><p>spatial selectivity in immobilized C. elegans10 and immobilizedDanio rerio zebrafish15; each element of a DMD may be inde-pendently controlled to deliver light to a corresponding pixel ofa microscopes field of view. In many cases, however, the normaloperation of neural circuits can be studied only in freely behaving</p><p>animals, requiring a more sophisticated instrument.Here we describe an optogenetic illumination system that allows</p><p>perturbations of neural activity with high spatial and temporalresolution in an unrestrained worm, enabling us to control loco-motion and behavior in real time (Colbert) in C. elegans. In theColbert system, a video camera follows a worm under dark-fieldillumination, and a motorized stage keeps the worm centered inthe cameras field of view. Machine-vision algorithms estimatethe coordinates of targeted cells within the worm body and gen-erate an illumination pattern that is projected onto the worm bya DMD with laser light, and the cycle repeats itself for the nextframe. Because the worm is a moving target, the faster an imagecan be captured and translated into DMD directives, the more</p><p>accurately an individual cell can be targeted. The Colbert systemcarries out all of these functions in ~20 ms, providing a spatialresolution of ~30 m in optogenetic control for freely swimmingC. elegans. We analyzed the motor circuit and mechanosensorycircuit of unrestrained worms, demonstrating the performanceof the Colbert system, a new tool that enhances the flexibility andpower of optogenetic approaches in C. elegans.</p><p>resultsexpa pTo stimulate neurons using ChR2 or inhibit neurons using Halo/NpHR, we used a 473-nm or 532-nm wavelength diode-pumpedsolid state (DPSS) laser, respectively (Fig. 1a). Either laser was</p><p>incident onto a DMD with 1,024 768 elements. Laser light wasreflected onto the specimen only when an individual micromirrorwas turned to the on position. We illuminated the specimenunder dark-field illumination by red light to avoid excitingChR2 or Halo/NpHR. Filter cubes reflected the wavelengths foroptogenetic illumination from the DMD onto the sample, whilepassing longer wavelengths for dark-field illumination to acamera. A motorized stage kept the specimen in the field of view.</p><p>1Department of Physics and Center for Brain Science, Harvard University, Cambridge, Massachusetts, USA. 2Department of Bioengineering, University of Pennsylvania,Philadelphia, Pennsylvania, USA. 3Department of Neurobiology, University of Massachusetts Medical School, Worcester, Massachusetts, USA. 4These authors contributedequally to this work. Correspondence should be addressed to C.F.-Y. (fangyen@seas.upenn.edu) or A.S. (samuel@physics.harvard.edu).</p><p>Received 23 August 2010; Accepted 16 decembeR 2010; published online 16 JAnuARy 2011; doi:10.1038/nmeth.1554</p>http://www.nature.com/doifinder/10.1038/nmeth.1554http://www.nature.com/doifinder/10.1038/nmeth.1554</li><li><p>8/6/2019 Op to Genetic Manipulation of Neural Activity in Freely Moving Caenorhabditis Elegans</p><p> 2/8</p><p>2011</p><p>NatureAmerica,Inc.Allrightsreserved.</p><p>2 | ADVANCE ONLINE PUBLICATION | nAture methods</p><p>Articles</p><p>To accelerate real-time image analysis of worm posture, we</p><p>developed the MindControl software package using the open-source OpenCV computer vision library16. With the graphical userinterface (GUI), the user can dynamically target specific regions offreely moving worms. The MindControl software and documenta-tion are available as Supplementary Software.</p><p>The MindControl software carries out a sequence of image ana-lysis operations on each frame received from the camera (Fig. 1b).An image is captured by the computer, filtered and thresholded.Next, the boundary of the worm is calculated, and head and tail</p><p>are identified as local maxima of boundary curvature (the head is</p><p>blunt and the tail is sharp). The worm centerline is calculated andthe body is divided into 100 evenly spaced segments. These seg-ments define a worm coordinate system invariant to worm postureor orientation, within which the user may define target positions.The software maps the position of targets onto the coordinates ofthe real image and finally sends the appropriate pattern to the DMDfor illumination.</p><p>For our current system, the total latency between image acquisi-tion and DMD illumination is 20 ms: image exposure, 2 ms; data</p><p>transfer to computer, 3 ms; image analysis,10 ms; and data transfer to DMD, 5 ms.Given the size and speed of a swimmingworm at 10 magnification, our system</p><p>working at ~50 frames per second (FPS)</p><p>F | High-resolution optogenetic controlo reely moving C. elegans. (a) An individualworm swims or crawls on a motorized stageunder red dark-ield illumination. A high-speed camera images the worm. Customsotware instructs a DMD to relect laser lightonto targeted cells. (b) Images are acquiredand processed at ~50 FPS. Each 1,024 768</p><p>pixel image is thresholded and the wormboundary is ound. Head and tail are locatedas maxima o boundary curvature (red arrows).Centerline is calculated rom the midpoint oline segments connecting dorsal and ventralboundaries (blue bar) and is resampled tocontain 100 equally spaced points. The wormis partitioned into segments by indingvectors (green arrows) rom centerline toboundary, and selecting one that is mostperpendicular to the centerline (orangearrow). Targets deined in worm coordinatesare transormed into image coordinates andsent to the DMD or illumination (green bar).() Schematic o body-wall muscles. Anterior,</p><p>to let; dorsal, to top. Bending wave speedo swimming worm expressing Halo/NpHRin its body-wall muscles subjected to greenlight (10 mW mm2) outside or inside theworm boundary (n = 5 worms, representativetrace). () Schematic o HSN. A swimmingworm expressing ChR2 in HSN was subjected toblue light (5 mW mm2). Histogram, positionat which egg-laying occurred when a narrowstripe o light was slowly scanned along theworms centerline (n = 13 worms). Once anegg was laid, the worm was discarded.</p><p>Time(s)</p><p>+5 5+2.5 0 2.5</p><p>Normalized curvature</p><p>Fractional distance along centerline (head = 0; tail = 1)</p><p>0 0.25 10.50 0.75</p><p>Normalizedcurvature</p><p>Time (s)</p><p>Time (s)</p><p>6</p><p>0</p><p>6</p><p>4</p><p>2</p><p>2</p><p>4</p><p>8</p><p>12</p><p>10</p><p>6 0 64224 8 1210</p><p>6 0 64224 8 1210</p><p>Bending dynamics at 0.10 along centerline</p><p>Bending dynamics at 0.75 along centerline</p><p>a b+10</p><p>10</p><p>+5</p><p>5</p><p>0</p><p>Body-wall muscle cells</p><p>+10</p><p>10</p><p>+5</p><p>5</p><p>0</p><p>Normalizedcurvature</p><p>DMD</p><p>Custom computervision software</p><p>Laser532 nm or 473 nm</p><p>High-speed</p><p>camera</p><p>Worm x-ymotorized stage</p><p>Objective</p><p>Red lighta</p><p>Dichroic</p><p>mirror</p><p>b</p><p>c d</p><p>Acquire image Threshold</p><p>Identify head, tail,</p><p>boundary and centerline</p><p>Define coordinate</p><p>system</p><p>Illuminate</p><p>selected target</p><p>HSN</p><p>Bendingwavespeed</p><p>(bodylengths1)</p><p>2</p><p>1</p><p>0</p><p>Illumination</p><p>outside boundaryIlluminationinside boundary</p><p>Time (s)</p><p>0 2</p><p>6</p><p>3</p><p>0</p><p>1</p><p>2</p><p>4</p><p>5</p><p>Fractional distance along centerline (head = 0; tail = 1)</p><p>Numberofegg-layingevents</p><p>4 6 8 10 12 14 0 0.2 0.4 0.6 0.8 10.1 0.3 0.5 0.7 0.9</p><p>Body-wall muscle cells</p><p>F 2 | Optogenetic inactivation omuscle cells. (a) Kymograph o time-varyingbody curvature along the centerline o aPmyo3Halo/NpHRCFPtransgenic worm.Between 0 s and 4 s, the worm was stimulatedwith green light (10 mW mm2) in a regionspanning the worm diameter and between 0.38and 0.6 o the ractional distance along thecenterline. (b) For the kymograph in a, time-varying curvature at two points along the wormcenterline, both anterior (top) and posterior(bottom) to the illuminated region.</p></li><li><p>8/6/2019 Op to Genetic Manipulation of Neural Activity in Freely Moving Caenorhabditis Elegans</p><p> 3/8</p><p>2011</p><p>NatureAmerica,Inc.Allrightsreserved.</p><p>nAture methods | ADVANCE ONLINE PUBLICATION | 3</p><p>Articles</p><p>delivers optogenetic illumination with aspatial resolution of ~30 m, not far fromthe spatial resolution limit imposed bythe pixel density of the DMD (~5 m at10 magnification).</p><p>spaa a yFirst, we confirmed that illumination is restricted to the targetedarea. We examined a transgenic worm expressing Halo/NpHRCFPin all body-wall muscles. Whole-animal illumination of transgenic</p><p>Pmyo-3Halo/NpHR worms causes all muscles to relax6. Weplaced individual swimming worms in the Colbert system and usedgreen light (532 nm, 10 mW mm2) to alternately illuminate theentire region outside and inside the worm boundary (Fig. 1c andSupplementary Video 1). Illuminating the entire region outsidethe worm boundary had no effect as bending waves propagatedfrom head to tail at normal speed. Illuminating the entire regioninside the worm boundary, however, arrested locomotion as thebody relaxed and the speed of bending waves dropped to zero.</p><p>To quantify the spatial resolution of the Colbert system, wemeasured its targeting accuracy in evoking egg-laying eventsby stimulating the HSN motor neurons. We used transgenicworms expressing ChR2 under the egl-6promoter, which drives</p><p>expression in the bilaterally symmetric HSN neurons (HSNL andHSNR) as well as glia-like cells in the worms head17. Optogeneticstimulation of the HSN neurons, which innervate the vulvalmusculature, evokes egg-laying behavior (L. Emtage andN. Ringstad, personal communication).</p><p>The two HSN neurons lie on top of one another when theworm is viewed laterally, so our system targets both neurons.We projected a thin stripe of blue l ight (473 nm, 5 mW mm2)on the body of swimming Pegl-6ChR2 transgenic worms. Thelong axis of the stripe was orthogonal to the worm centerline andspanned its diameter. The stripe width corresponded to 2% of theanterior-posterior length of the worm centerline (that is, ~20 mof the ~1-mm-long young adult worm). We used narrow stripes</p><p>so that our illumination would be less probable to stimulate HSNwhen illuminating its process. We slowly moved the illuminationstripe along the centerline of swimming worms while recordingegg-laying events. Of 14 worms studied, we observed 13 egg-</p><p>laying events, eight in which the stripe started at the head andfive in which the stripe started at the tail. Egg-laying frequencysharply peaked when the center of the stripe coincided with thecenterline coordinate of the HSN cell bodies, or 49.6% of thetotal distance from the anterior to the posterior of the body with3.2% s.d. (Fig. 1d and Supplementary Video 2). The width ofthis distribution suggests that the Colbert system provides at least~30 m of spatial resolution.</p><p>op apa In C. elegans, forward movement is driven by motor neurons inthe VNC, which coordinate the activity of 95 body wall musclecells along the dorsal and ventral sides of the VNC 18. The circuit</p><p>for worm locomotion is poorly understood in comparison to thatof other undulatory animals such as the leech and lamprey1921.Because this circuit probably operates normally only duringnormal movement, technology such as the Colbert system isnecessary to dissect cellular activity in unrestrained animals .</p><p>We used the Colbert system to suppress muscle activity in aregion of the body in myo-3Halo/NpHRCFP transgenicworms (Fig. 2 and Supplementary Video 3). This perturbationof undulatory dynamics can be shown graphically using a red-blue color map to represent the curvature of the body centerlinein nondimensional units (that is, the curvature calculated at eachpoint along the centerline, , multiplied by worm length, L) as afunction of time and fractional distance along the centerline,</p><p>Time (s)</p><p>6 0 64224 8</p><p>+20</p><p>20</p><p>+10</p><p>10</p><p>0</p><p>+20</p><p>20</p><p>+10</p><p>10</p><p>Bending dynamics at 0.10 along centerline</p><p>Bending dynamics at 0.75 along centerline</p><p>Time (s)</p><p>6 0 64224 8</p><p>0</p><p>Time (s)</p><p>4 1 64023 21 53</p><p>Time (s)</p><p>4 1 64023 21 53</p><p>Ventral cord illumination</p><p>Dorsal cord illumination</p><p>b</p><p>d</p><p>W</p><p>avespeed</p><p>(bo</p><p>dylengths1) 0.5</p><p>0.4</p><p>0</p><p>0.3</p><p>0.2</p><p>0.1</p><p>Wavespeed</p><p>(bodylengths1) 0.5</p><p>0.4</p><p>0</p><p>0.3</p><p>0.2</p><p>0.1</p><p>Normalizedcurvature</p><p>Normalizedcurvature</p><p>3.0 s 2.6 2.2</p><p>1.8 1.4 1.0</p><p>0.6 0.2 0.2</p><p>0.7 1.1 1.5</p><p>1.9 2.3 2.7</p><p>Time(s</p><p>)</p><p>+10 10+5 0 5</p><p>Normalized curvature</p><p>Fractional distance along centerline (head = 0; tail = 1)</p><p>0 0.25 10.50 0.75</p><p>5</p><p>0</p><p>2</p><p>4</p><p>DB motor neuronsVB motor neurons</p><p>3</p><p>1</p><p>5</p><p>2</p><p>4</p><p>3</p><p>1</p><p>6</p><p>a</p><p>c</p><p>F 3 | Inhibition o motor neurons.(a) Schematic o cholinergic DB and VB motorneurons. Anterior, to let; dorsal, to top.Kymograph o time-varying body curvaturealong the centerline o a Punc-17Halo/NpHRCFPtransgenic worm illuminated bya stripe o green light (10 mW mm2) alongits VNC between t = 0 s and 1.6 s. In the dorsal-</p><p>ventral direction, the stripe width was equalto 50% o the worm diameter and centered onthe ventral boundary. In the anterior-posteriordirection, the stripe length was between 0.14and 0.28 o the ractional distance alongthe body. (b) For the kymograph in a, time-varying curvature at two points along the wormcenterline, both anterior (top) and posterior(bottom) to the illuminated region. () Videosequence o worm illuminated by a long stripeo green light (10 mW mm2) spanning the VNCbetween t = 0 s and 1.8 s. Scale bar, ~100 m.() Bending wave speed o a swimming wormilluminated by a long stripe o green light(10 mW mm2) lasting 1.8 s and spanning</p><p>the VNC (top) and dorsal nerve cord (bottom).</p></li><li><p>8/6/2019 Op to Genetic Manipulation of Neural Activity in Freely Moving Caenorhabditis Elegans...</p></li></ul>