Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light
TL;DR: A two-photon technique for brain-wide calcium imaging in Caenorhabditis elegans, using wide-field temporal focusing (WF-TeFo) and a nuclear-localized, genetically encoded calcium indicator, NLS-GCaMP5K, that permits unambiguous discrimination of individual neurons within the densely packed head ganglia of C. elegans.
Abstract: Recent efforts in neuroscience research have been aimed at obtaining detailed anatomical neuronal wiring maps as well as information on how neurons in these networks engage in dynamic activities. Although the entire connectivity map of the nervous system of Caenorhabditis elegans has been known for more than 25 years, this knowledge has not been sufficient to predict all functional connections underlying behavior. To approach this goal, we developed a two-photon technique for brain-wide calcium imaging in C. elegans, using wide-field temporal focusing (WF-TeFo). Pivotal to our results was the use of a nuclear-localized, genetically encoded calcium indicator, NLS-GCaMP5K, that permits unambiguous discrimination of individual neurons within the densely packed head ganglia of C. elegans. We demonstrate near-simultaneous recording of activity of up to 70% of all head neurons. In combination with a lab-on-a-chip device for stimulus delivery, this method provides an enabling platform for establishing functional maps of neuronal networks.
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TL;DR: This work demonstrates simultaneous functional imaging of neuronal activity at single-neuron resolution in an entire Caenorhabditis elegans and in larval zebrafish brain with high-speed volumetric calcium imaging.
Abstract: High-speed, large-scale three-dimensional (3D) imaging of neuronal activity poses a major challenge in neuroscience. Here we demonstrate simultaneous functional imaging of neuronal activity at single-neuron resolution in an entire Caenorhabditis elegans and in larval zebrafish brain. Our technique captures the dynamics of spiking neurons in volumes of ∼700 μm × 700 μm × 200 μm at 20 Hz. Its simplicity makes it an attractive tool for high-speed volumetric calcium imaging.
714 citations
TL;DR: As a new paradigm for neuroscience, neural network models have the potential to incorporate knowledge acquired with single-neuron approaches to help us understand how emergent functional states generate behaviour, cognition and mental disease.
Abstract: For over a century, the neuron doctrine--which states that the neuron is the structural and functional unit of the nervous system--has provided a conceptual foundation for neuroscience This viewpoint reflects its origins in a time when the use of single-neuron anatomical and physiological techniques was prominent However, newer multineuronal recording methods have revealed that ensembles of neurons, rather than individual cells, can form physiological units and generate emergent functional properties and states As a new paradigm for neuroscience, neural network models have the potential to incorporate knowledge acquired with single-neuron approaches to help us understand how emergent functional states generate behaviour, cognition and mental disease
579 citations
TL;DR: A new 3D microscopy technique that allows volumetric imaging of living samples at ultra-high speeds: Swept, confocally-aligned planar excitation (SCAPE) microscopy, demonstrated by imaging spontaneous neuronal firing in the intact brain of awake behaving mice, as well as freely moving transgenic Drosophila larvae.
Abstract: We report a new 3D microscopy technique that allows volumetric imaging of living samples at ultra-high speeds: Swept, confocally-aligned planar excitation (SCAPE) microscopy. While confocal and two-photon microscopy have revolutionized biomedical research, current implementations are costly, complex and limited in their ability to image 3D volumes at high speeds. Light-sheet microscopy techniques using two-objective, orthogonal illumination and detection require a highly constrained sample geometry, and either physical sample translation or complex synchronization of illumination and detection planes. In contrast, SCAPE microscopy acquires images using an angled, swept light-sheet in a single-objective, en-face geometry. Unique confocal descanning and image rotation optics map this moving plane onto a stationary high-speed camera, permitting completely translationless 3D imaging of intact samples at rates exceeding 20 volumes per second. We demonstrate SCAPE microscopy by imaging spontaneous neuronal firing in the intact brain of awake behaving mice, as well as freely moving transgenic Drosophila larvae.
513 citations
TL;DR: This work elucidate the key developments and define a simple set of underlying principles governing LSFM, which aim to clarify the decisions to be made for those who wish to develop and use bespoke light-sheet systems and to assist in identifying the best approaches to apply this powerful technique to myriad biological questions.
Abstract: This Review introduces the fundamental considerations for building a light sheet microscope, describes the pros and cons associated with available implementations, and offers practical advice for users.
484 citations
TL;DR: It is shown that the coordination of neuronal activity patterns into global brain dynamics underlies the high-level organization of behavior and serves as a robust scaffold for action selection in response to sensory input.
427 citations
Cites background from "Brain-wide 3D imaging of neuronal a..."
...…cyclical dynamics continuously represent action sequences and decisions d Internal representation of behavior persists when de- coupled from its execution d Brain dynamics provide a robust scaffold for sensory-driven action selection Kato et al., 2015, Cell 163, 656–669 October 22, 2015 ª2015…...
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References
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TL;DR: The structure and connectivity of the nervous system of the nematode Caenorhabditis elegans has been deduced from reconstructions of electron micrographs of serial sections as discussed by the authors.
Abstract: The structure and connectivity of the nervous system of the nematode Caenorhabditis elegans has been deduced from reconstructions of electron micrographs of serial sections. The hermaphrodite nervous system has a total complement of 302 neurons, which are arranged in an essentially invariant structure. Neurons with similar morphologies and connectivities have been grouped together into classes; there are 118 such classes. Neurons have simple morphologies with few, if any, branches. Processes from neurons run in defined positions within bundles of parallel processes, synaptic connections being made en passant. Process bundles are arranged longitudinally and circumferentially and are often adjacent to ridges of hypodermis. Neurons are generally highly locally connected, making synaptic connections with many of their neighbours. Muscle cells have arms that run out to process bundles containing motoneuron axons. Here they receive their synaptic input in defined regions along the surface of the bundles, where motoneuron axons reside. Most of the morphologically identifiable synaptic connections in a typical animal are described. These consist of about 5000 chemical synapses, 2000 neuromuscular junctions and 600 gap junctions.
5,491 citations
TL;DR: A family of ultrasensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies and mice in vivo are developed and provide new windows into the organization and dynamics of neural circuits over multiple spatial and temporal scales.
Abstract: Fluorescent calcium sensors are widely used to image neural activity. Using structure-based mutagenesis and neuron-based screening, we developed a family of ultrasensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies and mice in vivo. In layer 2/3 pyramidal neurons of the mouse visual cortex, GCaMP6 reliably detected single action potentials in neuronal somata and orientation-tuned synaptic calcium transients in individual dendritic spines. The orientation tuning of structurally persistent spines was largely stable over timescales of weeks. Orientation tuning averaged across spine populations predicted the tuning of their parent cell. Although the somata of GABAergic neurons showed little orientation tuning, their dendrites included highly tuned dendritic segments (5-40-µm long). GCaMP6 sensors thus provide new windows into the organization and dynamics of neural circuits over multiple spatial and temporal scales.
5,365 citations
"Brain-wide 3D imaging of neuronal a..." refers methods in this paper
...While the GCaMPs have been used since 2001 for detecting Ca2+ activity in the roundworm (Nakai et al., 2001; Chen et al., 2013), Caenorhabditis elegans, Schrödel et al. (2013) developed a sculpted light technique for recording activity of the majority of head ganglion neurons in response to…...
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...GENERAL COMMENTARY published: 03 April 2014 doi: 10.3389/fncir.2014.00031 Brain-wide imaging of neurons in action...
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TL;DR: In this article, the authors studied functional brain organization in healthy adults using resting state functional connectivity MRI and proposed two novel brain wide graphs, one of 264 putative functional areas, the other a modification of voxelwise networks that eliminates potentially artificial short-distance relationships.
3,517 citations
"Brain-wide 3D imaging of neuronal a..." refers background in this paper
...In order to understand how complex properties like response to stimulus, cognition, and emotion are derived from simple physical processes like an action potential, the algorithms of neural circuitry need to be described (Power et al., 2011)....
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TL;DR: The importance of ionic movements in excitable tissues has been emphasized by a number of recent experiments which are consistent with the theory that nervous conduction depends on a specific increase in permeability which allows sodium ions to move from the more concentrated solution outside a nerve fibre to the more dilute solution inside it.
Abstract: The importance of ionic movements in excitable tissues has been emphasized by a number of recent experiments. On the one hand, there is the finding that the nervous impulse is associated with an inflow of sodium and an outflow of potassiuim (e.g. Rothenberg, 1950; Keynes & Lewis, 1951). On the other, there are experiments which show that the rate of rise and amplitude of the action potential are determined by the concentration of sodium in the external medium (e.g. Hodgkin & Katz, 1949 a; Huxley & Stiimpffi, 1951). Both groups of experiments are consistent with the theory that nervous conduction depends on a specific increase in permeability which allows sodium ions to move from the more concentrated solution outside a nerve fibre to the more dilute solution inside it. This movement of charge makes the inside of the fibre positive and provides a satisfactory explanation for the rising phase of the spike. Repolarization during the falling phase probably depends on an outflow of potassium ions and may be accelerated by a process which increases the potassium permeability after the action potential has reached its crest (Hodgkin, Huxley & Katz, 1949).
1,569 citations
"Brain-wide 3D imaging of neuronal a..." refers background in this paper
...The physical substrates comprising the brain, including neurophysiology, ion gradients, neurotransmitter release, and the molecules involved in action potential firing have been studied since Hodgkin and Huxley developed the squid giant axon model in 1952 Hodgkin et al. (1952)....
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TL;DR: This work developed digital scanned laser light sheet fluorescence microscopy and recorded nuclei localization and movement in entire wild-type and mutant zebrafish embryos over the first 24 hours of development to derive a model of germ layer formation and show that the mesendoderm forms from one-third of the embryo's cells in a single event.
Abstract: A long-standing goal of biology is to map the behavior of all cells during vertebrate embryogenesis. We developed digital scanned laser light sheet fluorescence microscopy and recorded nuclei localization and movement in entire wild-type and mutant zebrafish embryos over the first 24 hours of development. Multiview in vivo imaging at 1.5 billion voxels per minute provides “digital embryos,” that is, comprehensive databases of cell positions, divisions, and migratory tracks. Our analysis of global cell division patterns reveals a maternally defined initial morphodynamic symmetry break, which identifies the embryonic body axis. We further derive a model of germ layer formation and show that the mesendoderm forms from one-third of the embryo's cells in a single event. Our digital embryos, with 55 million nucleus entries, are provided as a resource.
1,446 citations