An Image-Free Opto-Mechanical System for Creating Virtual Environments and Imaging Neuronal Activity in Freely Moving Caenorhabditis elegans

Faumont, Serge; Rondeau, Gary; Thiele, Tod R.; Lawton, Kristy J.; McCormick, Kathryn E.; Sottile, Matthew; Griesbeck, Oliver; Heckscher, Ellie S.; Roberts, William M.; Doe, Chris Q.; Lockery, Shawn R.

doi:10.1371/journal.pone.0024666

Cited by 126 publications

(141 citation statements)

References 35 publications

(55 reference statements)

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“…1). In contrast to previous dual-objective worm trackers (18), this system operates in three dimensions. Custom real-time software uses fluorescence imaging to identify the location of the worm's brain in three dimensions and adjusts a motorized stage and piezoelectric objective mount to follow the brain as it moves and to scan through its brain volume as it crawls.…”

Section: Resultsmentioning

confidence: 99%

“…15. To facilitate higher-throughput recordings of behavior, a number of tracking microscopes (16)(17)(18) or multiworm imagers were developed (19,20) along with sophisticated behavioral analysis software and analytical tools (21,22). Motivated in part to understand these behaviors, calcium imaging systems were Significance Large-scale neural recordings in freely moving animals are important for understanding how patterns of activity across a population of neurons generates animal behavior.…”

mentioning

confidence: 99%

“…24 and and then developing rapidly (17,18,(25)(26)(27)(28)(29). One limitation of these freely moving calcium imaging systems is that they are limited to imaging only a very small subset of neurons and lack the ability to distinguish neurons that lie atop one another in the axial direction of the microscope.…”

mentioning

confidence: 99%

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Whole-brain calcium imaging with cellular resolution in freely behaving Caenorhabditis elegans

Nguyen

Shipley

Linder

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

363

355

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The ability to acquire large-scale recordings of neuronal activity in awake and unrestrained animals is needed to provide new insights into how populations of neurons generate animal behavior. We present an instrument capable of recording intracellular calcium transients from the majority of neurons in the head of a freely behaving Caenorhabditis elegans with cellular resolution while simultaneously recording the animal's position, posture, and locomotion. This instrument provides whole-brain imaging with cellular resolution in an unrestrained and behaving animal. We use spinning-disk confocal microscopy to capture 3D volumetric fluorescent images of neurons expressing the calcium indicator GCaMP6s at 6 head-volumes/s. A suite of three cameras monitor neuronal fluorescence and the animal's position and orientation. Custom software tracks the 3D position of the animal's head in real time and two feedback loops adjust a motorized stage and objective to keep the animal's head within the field of view as the animal roams freely. We observe calcium transients from up to 77 neurons for over 4 min and correlate this activity with the animal's behavior. We characterize noise in the system due to animal motion and show that, across worms, multiple neurons show significant correlations with modes of behavior corresponding to forward, backward, and turning locomotion.calcium imaging | large-scale recording | behavior | C. elegans | microscopy H ow do patterns of neural activity generate an animal's behavior? To answer this question, it is important to develop new methods for recording from large populations of neurons in animals as they move and behave freely. The collective activity of many individual neurons appears to be critical for generating behaviors including arm reach in primates (1), song production in zebrafinch (2), the choice between swimming or crawling in leech (3), and decision-making in mice during navigation (4). New methods for recording from larger populations of neurons in unrestrained animals are needed to better understand neural coding of these behaviors and neural control of behavior more generally.Calcium imaging has emerged as a promising technique for recording dynamics from populations of neurons. Calcium-sensitive proteins are used to visualize changes in intracellular calcium levels in neurons in vivo which serve as a proxy for neural activity (5). To resolve the often weak fluorescent signal of an individual neuron in a dense forest of other labeled cells requires a high magnification objective with a large numerical aperture that, consequently, can image only a small field of view. Calcium imaging has traditionally been performed on animals that are stationary from anesthetization or immobilization to avoid imaging artifacts induced by animal motion. As a result, calcium imaging studies have historically focused on small brain regions in immobile animals that exhibit little or no behavior (6).No previous neurophysiological study has attained whole-brain imaging with cellular resolution in a...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Whole-brain calcium imaging with cellular resolution in freely behaving Caenorhabditis elegans

Nguyen

Shipley

Linder

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

363

355

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“…An inverted Olympus IX81 fluorescent microscope equipped with a ×20, NA0.75 objective lens was used to measure the time course of Ca 2 + . To keep the neuron of a moving worm within the field of view of the charge-coupled device, we used the PhotoTrack system from Applied Scientific Instrumentation (ASI, Eugene, USA) combined with ASI's closedloop XY stages 42 . With the help of the PhotoTrack system, the G-CaMP2.0 fluorescence of NSM or RIM/RIC neurons in freely moving worms was recorded with an Andor Luca EM-CCD at 2 Hz.…”

Section: Molecular Biologymentioning

confidence: 99%

Dissecting a central flip-flop circuit that integrates contradictory sensory cues in C. elegans feeding regulation

et al. 2012

Nat Commun

110

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Feeding behaviour is modulated by both environmental cues and internal physiological states. Appetite is commonly boosted by the pleasant smell (or appearance) of food and destroyed by a bad taste. In reality, animals sense multiple environmental cues at the same time and it is not clear how these sensory inputs are integrated and a decision is made to regulate feeding behaviour accordingly. Here we show that feeding behaviour in Caenorhabditis elegans can be either facilitated by attractive odours or suppressed by repellents. By identifying mutants that are defective for sensory-mediated feeding regulation, we dissected a central flip-flop circuit that integrates two contradictory sensory inputs and generates bistable hormone output to regulate feeding behaviour. As feeding regulation is fundamental to animal survival, we speculate that the basic organizational logic identified here in C. elegans is likely convergent throughout different phyla.

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“…The nematode Caenorhabditis elegans is particularly ideal for optical neurophysiology owing to its small size, optical transparency, compact nervous system, and ease of genetic manipulation. Imaging systems for tracking the activity of small numbers of neurons have been effective in determining their role during nematode locomotion and navigational behaviors like chemotaxis, thermotaxis, and the escape response (1)(2)(3)(4)(5)(6).…”

mentioning

confidence: 99%

Pan-neuronal imaging in roaming Caenorhabditis elegans

Venkatachalam

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

221

216

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We present an imaging system for pan-neuronal recording in crawling Caenorhabditis elegans. A spinning disk confocal microscope, modified for automated tracking of the C. elegans head ganglia, simultaneously records the activity and position of ∼80 neurons that coexpress cytoplasmic calcium indicator GCaMP6s and nuclear localized red fluorescent protein at 10 volumes per second. We developed a behavioral analysis algorithm that maps the movements of the head ganglia to the animal's posture and locomotion. Image registration and analysis software automatically assigns an index to each nucleus and calculates the corresponding calcium signal. Neurons with highly stereotyped positions can be associated with unique indexes and subsequently identified using an atlas of the worm nervous system. To test our system, we analyzed the brainwide activity patterns of moving worms subjected to thermosensory inputs. We demonstrate that our setup is able to uncover representations of sensory input and motor output of individual neurons from brainwide dynamics. Our imaging setup and analysis pipeline should facilitate mapping circuits for sensory to motor transformation in transparent behaving animals such as C. elegans and Drosophila larva.C. elegans | Drosophila | calcium imaging | thermotaxis U nderstanding how brain dynamics creates behaviors requires quantifying the flow and transformation of sensory information to motor output in behaving animals. Optical imaging using genetically encoded calcium or voltage fluorescent probes offers a minimally invasive method to record neural activity in intact animals. The nematode Caenorhabditis elegans is particularly ideal for optical neurophysiology owing to its small size, optical transparency, compact nervous system, and ease of genetic manipulation. Imaging systems for tracking the activity of small numbers of neurons have been effective in determining their role during nematode locomotion and navigational behaviors like chemotaxis, thermotaxis, and the escape response (1-6).Recordings from large numbers of interconnected neurons are required to understand how neuronal ensembles carry out the systematic transformations of sensory input into motor patterns that build behavioral decisions.Several methods for fast 3D imaging of neural activity in a fixed imaging volume have been developed for different model organisms (7)(8)(9)(10)(11)(12)(13)(14). High-speed light sheet microscopy, light field microscopy, multifocus microscopy, and two-photon structured illumination microscopy have proved effective for rapidly recording large numbers of neurons in immobilized, intact, transparent animals like larval zebrafish and nematodes (15)(16)(17)(18)(19). However, these methods are problematic when attempting to track many neurons within the bending and moving body of a behaving animal. Panneuronal recording in moving animals poses higher demands on spatial and temporal resolution. Furthermore, extracting neuronal signals from recordings in a behaving animal requires an effective analysis pipel...

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An Image-Free Opto-Mechanical System for Creating Virtual Environments and Imaging Neuronal Activity in Freely Moving Caenorhabditis elegans

Cited by 126 publications

References 35 publications

Whole-brain calcium imaging with cellular resolution in freely behaving Caenorhabditis elegans

Whole-brain calcium imaging with cellular resolution in freely behaving Caenorhabditis elegans

Dissecting a central flip-flop circuit that integrates contradictory sensory cues in C. elegans feeding regulation

Pan-neuronal imaging in roaming Caenorhabditis elegans

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