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  • 8/6/2019 Op to Genetic Manipulation of Neural Activity in Freely Moving Caenorhabditis Elegans

    1/8

    2011

    NatureAmerica,Inc.Allrightsreserved.

    Articles

    nAture methods | ADVANCE ONLINE PUBLICATION |

    W p a p a y apab a- vy w paa pa y v Caenorhabditis elegans. A a

    p a a wxp ap-2 ap p

    yp. ia p wa aayz wp a v a, apy a a a a a a v a a w a apppa

    wav a b avy. Ba a a a w a apy v a, ypa a p (~50 a p ) pv

    paa (~30 m). t aay, fxbya y y, w p p aay w , -ay a ay a av b pb w pv .

    Researchers in systems neuroscience aim to understand how neural

    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.

    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

    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

    op apa a avy yv Caenorhabditis elegansAndrew M Leifer1,4, Christopher Fang-Yen1,2,4, Marc Gershow1, Mark J Alkema3 & Aravinthan D T Samuel1

    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

    animals, requiring a more sophisticated instrument.Here we describe an optogenetic illumination system that allows

    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

    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.

    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

    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.

    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).

    Received 23 August 2010; Accepted 16 decembeR 2010; published online 16 JAnuARy 2011; doi:10.1038/nmeth.1554

    http://www.nature.com/doifinder/10.1038/nmeth.1554http://www.nature.com/doifinder/10.1038/nmeth.1554
  • 8/6/2019 Op to Genetic Manipulation of Neural Activity in Freely Moving Caenorhabditis Elegans

    2/8

    2011

    NatureAmerica,Inc.Allrightsreserved.

    2 | ADVANCE ONLINE PUBLICATION | nAture methods

    Articles

    To accelerate real-time image analysis of worm posture, we

    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.

    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

    are identified as local maxima of boundary curvature (the head is

    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.

    For our current system, the total latency between image acquisi-tion and DMD illumination is 20 ms: image exposure, 2 ms; data

    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

    working at ~50 frames per second (FPS)

    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

    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,

    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.

    Time(s)

    +5 5+2.5 0 2.5

    Normalized curvature

    Fractional distance along centerline (head = 0; tail = 1)

    0 0.25 10.50 0.75

    Normalizedcurvature

    Time (s)

    Time (s)

    6

    0

    6

    4

    2

    2

    4

    8

    12

    10

    6 0 64224 8 1210

    6 0 64224 8 1210

    Bending dynamics at 0.10 along centerline

    Bending dynamics at 0.75 along centerline

    a b+10

    10

    +5

    5

    0

    Body-wall muscle cells

    +10

    10

    +5

    5

    0

    Normalizedcurvature

    DMD

    Custom computervision software

    Laser532 nm or 473 nm

    High-speed

    camera

    Worm x-ymotorized stage

    Objective

    Red lighta

    Dichroic

    mirror

    b

    c d

    Acquire image Threshold

    Identify head, tail,

    boundary and centerline

    Define coordinate

    system

    Illuminate

    selected target

    HSN

    Bendingwavespeed

    (bodylengths1)

    2

    1

    0

    Illumination

    outside boundaryIlluminationinside boundary

    Time (s)

    0 2

    6

    3

    0

    1

    2

    4

    5

    Fractional distance along centerline (head = 0; tail = 1)

    Numberofegg-layingevents

    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

    Body-wall muscle cells

    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-