Optogenetic dissection of a behavioural module in the vertebrate spinal cord. Nature

Helen Wills Neuroscience Institute and Department of Molecular and Cell Biology, University of California in Berkeley, Berkeley, California 94720, USA.
Nature (Impact Factor: 41.46). 09/2009; 461(7262):407-10. DOI: 10.1038/nature08323
Source: PubMed


Locomotion relies on neural networks called central pattern generators (CPGs) that generate periodic motor commands for rhythmic movements. In vertebrates, the excitatory synaptic drive for inducing the spinal CPG can originate from either supraspinal glutamatergic inputs or from within the spinal cord. Here we identify a spinal input to the CPG that drives spontaneous locomotion using a combination of intersectional gene expression and optogenetics in zebrafish larvae. The photo-stimulation of one specific cell type was sufficient to induce a symmetrical tail beating sequence that mimics spontaneous slow forward swimming. This neuron is the Kolmer-Agduhr cell, which extends cilia into the central cerebrospinal-fluid-containing canal of the spinal cord and has an ipsilateral ascending axon that terminates in a series of consecutive segments. Genetically silencing Kolmer-Agduhr cells reduced the frequency of spontaneous free swimming, indicating that activity of Kolmer-Agduhr cells provides necessary tone for spontaneous forward swimming. Kolmer-Agduhr cells have been known for over 75 years, but their function has been mysterious. Our results reveal that during early development in zebrafish these cells provide a positive drive to the spinal CPG for spontaneous locomotion.

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    • "On the other hand, single-and two-photon un-caging stimulation has been important to demonstrate the Ca 2+ -dependent release of gliotransmitters by astrocytes in acute brain slices (Fellin et al., 2004; Gordon et al., 2009; Liu et al., 2004; Perea and Araque, 2007) as well as permitting neuronal activation and inhibition in freely moving animals (Aravanis et al., 2007; Gradinaru et al., 2009; Wyart et al., 2009). In conjunction with spatiotemporally resolved photo-stimulation techniques, these photo-sensible tools represent the most promising alternative to electrical stimulation devices to control the activity of specific types of brain cells in time and space with sufficient precision. "
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    ABSTRACT: Present neurobiological concepts regarding superior cognitive functions are based on synaptic neurotransmission and neuronal plasticity. However, the diversity and complexity of neuro-cortical connections, circuits, maps and their relationships with memory, learning and other superior cognitive functions are not fully explained by the present neurobiological paradigms. Recent discoveries concerning the ability of neuronal cells to perceive and process very-weak electromagnetic information suggest a possible role of bio-photons generated as consequence of neuronal and astroglial metabolisms in a wide diversity of cognitive representations. Moreover, the finding that human brain has magnetite nanoparticles opens new possibilities about the role of these nano-crystals in information processing and memory. In the present chapter, a previously advanced model (Neuron-Astroglial Communication in Short-Term Memory: Bio-Electric, Bio-Magnetic and Bio-Photonic Signals?) is developed. Based in the ability of neurons and astrocytes to generate bio-photons as consequence of their metabolic activities, I propose the generation of innumerable bio-phonic-mediated nano-holograms, which are produced and modulated by magnetite nano-crystals associated to neuronal and astroglial membranes in the cerebral neocortex. Specifically, it is suggested that bio-photons generated by neuronal and astroglial cells may produce multichannel holographic pictures through their interaction with single domain and/or superparamagnetic magnetite nanoparticles, explaining retrieval of short-term and long-term memories as well as other neuro- cognitive representations such as the ―images‖ generated in dreams. Bio-chemic, bio- electric, bio-magnetic and bio-photonic activities in the cerebral cortex are not independent bio-physical phenomena, suggesting that interactions among these signals may contribute to information exchange and processing in the neocortex. This hypothesis proposes that the interactions among bio-chemic, bio-electric, bio-magnetic and bio- photonic activities in neurons and astrocytes in the human cerebral neocortex are not epiphenomena of the cerebral activity but they play important roles in cognitive functions, providing new perspectives for better understand complex cognitive functions.
    Horizons in Neuroscience Research. Volume 20, Edited by Andres Costa and Eugenio Villalba, 07/2015: chapter Holographic Memory: Magnetite Nano-Devices for Bio-Photonic Representations in the Human Brain Neocortex: pages 1-40; Nova Science Publishers., ISBN: 978-1-63482-817-8
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    • "We tested immunoreactivity pattern for both rabbit and rat anti-glycine antibodies, and we obtained similar patterns of staining. The polyclonal rabbit anti-GABA antibody (Sigma, code A2052) has previously been used in many studies with zebrafish (Higashijima et al., 2004; Mueller et al., 2006; Wyart et al., 2009) and specific subpopulations of neurons were labeled with this antibody. In Higashijima et al. (2004), double staining with immunohistochemistry with rabbit anti- GABA antibody (Sigma) and in situ hybridization with GAD probes (GAD65 and GAD67) performed and almost complete overlap of both staining was observed. "
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    ABSTRACT: Glycine is a major inhibitory neurotransmitter in the central nervous system of vertebrates. Here, we report the initial development of glycine-immunoreactive (Gly-ir) neurons and fibers in zebrafish. The earliest Gly-ir cells were found in the hindbrain and rostral spinal cord by 20 hours post-fertilization (hpf). Gly-ir cells in rhombomeres 5 and 6 that also expressed glycine transporter 2 (glyt2) mRNA were highly stereotyped; they were bilaterally located and their axons ran across the midline and gradually turned caudally, joining the medial longitudinal fascicles in the spinal cord by 24 hpf. Gly-ir neurons in rhombomere 5 were uniquely identified, since there was one per hemisegment, whereas the number of Gly-ir neurons in rhombomere 6 were variable from one to three per hemisegment. Labeling of these neurons by single-cell electroporation and tracing them until the larval stage revealed that they became MiD2cm and MiD3cm respectively. The retrograde labeling of reticulo-spinal neurons in Tg(glyt2:gfp) larva, which express GFP in Gly-ir cells, and a genetic mosaic analysis with glyt2:GFP DNA construct also supported this notion. Gly-ir cells were also distributed widely in the anterior brain by 27 hpf, whereas glyt2 was hardly expressed. Double staining with anti-glycine and anti-GABA antibodies demonstrated distinct distributions of Gly-ir and GABA-ir cells, as well as the presence of doubly immunoreactive cells in the brain and placodes. These results provide evidence of identifiable glycinergic (Gly-ir/glyt2-positive) neurons in vertebrate embryos, and they can be used in further studies of the neurons' development and function at the single-cell level. © 2013 Wiley Periodicals, Inc. Develop Neurobiol, 2013.
    Developmental Neurobiology 06/2014; 74(6). DOI:10.1002/dneu.22158 · 3.37 Impact Factor
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    • "The lateral processes of type 1 cells may also provide synaptic input to neurons in the gray matter on their way to the spinal cord margin, but there is no information on which neurons would be the targets. In the zebrafish, an optical activation of the central canal cells can trigger an activation of the locomotor network (Wyart et al., 2009), testifying to the close links to the locomotor system. Type 1 cells receive at rest spontaneous synaptic input with both IPSPs and EPSPs. "
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    ABSTRACT: Cerebrospinal fluid-contacting (CSF-c) cells are found in all vertebrates but their function remains elusive. In the lamprey spinal cord they surround the central canal and some have processes passing the gray matter to the lateral edge of the flattened spinal cord. Stimulation of CSF-c cells at the central canal elicits GABAergic IPSPs in intraspinal stretch receptor neurons (edge cells). Here, we characterize laterally projecting CSF-c cells according to their morphology, phenotype, and neuronal properties by using immunohistochemistry, retrograde tracing, calcium imaging and whole-cell recordings. We identify two types of CSF-c cells. Type 1 cells have a bulb-like ending that protrudes into the central canal and a lateral process that ramifies ventrolaterally and laterally with a dense plexus surrounding the mechanosensitive dendrites of the edge cells. Most type 1 cells fire spontaneous action potentials that are abolished by tetrodotoxin, and all display spontaneous EPSPs and IPSPs that remain in the presence of tetrodotoxin. GABA and somatostatin are co-localized in type 1 cells and they express both GABA and glutamate receptors. Type 2 cells, on the other hand, have a flat ending protruding into the central canal and a laterally projecting process that only ramifies at the lateral edge. These cells show immunoreactivity to taurine but they do not express GABA or somatostatin nor do they have any active neuronal properties. Type 2 cells might be a form of glia. Type 1 CSF-c cells are neurons and may play a modulatory role by influencing edge cells and thus the locomotor-related sensory feedback. J. Comp. Neurol., 2014. © 2014 Wiley Periodicals, Inc.
    The Journal of Comparative Neurology 06/2014; 522(8). DOI:10.1002/cne.23542 · 3.23 Impact Factor
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