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1.
J Neurosci ; 44(8)2024 Feb 21.
Article in English | MEDLINE | ID: mdl-38195508

ABSTRACT

The olivo-cerebellar system plays an important role in vertebrate sensorimotor control. Here, we investigate sensory representations in the inferior olive (IO) of larval zebrafish and their spatial organization. Using single-cell labeling of genetically identified IO neurons, we find that they can be divided into at least two distinct groups based on their spatial location, dendritic morphology, and axonal projection patterns. In the same genetically targeted population, we recorded calcium activity in response to a set of visual stimuli using two-photon imaging. We found that most IO neurons showed direction-selective and binocular responses to visual stimuli and that the functional properties were spatially organized within the IO. Light-sheet functional imaging that allowed for simultaneous activity recordings at the soma and axonal level revealed tight coupling between functional properties, soma location, and axonal projection patterns of IO neurons. Taken together, our results suggest that anatomically defined classes of IO neurons correspond to distinct functional types, and that topographic connections between IO and cerebellum contribute to organization of the cerebellum into distinct functional zones.


Subject(s)
Olivary Nucleus , Zebrafish , Animals , Larva , Olivary Nucleus/physiology , Neurons/physiology , Cerebellum/physiology
2.
Science ; 366(6471): 1311-1312, 2019 12 13.
Article in English | MEDLINE | ID: mdl-31831656

Subject(s)
Odorants , Pheromones , Smell
3.
Neuron ; 102(6): 1211-1222.e3, 2019 06 19.
Article in English | MEDLINE | ID: mdl-31054873

ABSTRACT

Sensory systems must reduce the transmission of redundant information to function efficiently. One strategy is to continuously adjust the sensitivity of neurons to suppress responses to common features of the input while enhancing responses to new ones. Here we image the excitatory synaptic inputs and outputs of retinal ganglion cells to understand how such dynamic predictive coding is implemented in the analysis of spatial patterns. Synapses of bipolar cells become tuned to orientation through presynaptic inhibition, generating lateral antagonism in the orientation domain. Individual ganglion cells receive excitatory synapses tuned to different orientations, but feedforward inhibition generates a high-pass filter that only transmits the initial activation of these inputs, removing redundancy. These results demonstrate how a dynamic predictive code can be implemented by circuit motifs common to many parts of the brain.


Subject(s)
Neural Inhibition/physiology , Retinal Bipolar Cells/physiology , Retinal Ganglion Cells/physiology , Space Perception/physiology , Synapses/physiology , Vision, Ocular/physiology , Animals , Escherichia coli Proteins , Glutamic Acid/metabolism , Green Fluorescent Proteins , Larva , Optical Imaging , Orientation, Spatial , Recombinant Fusion Proteins , Retina , Retinal Bipolar Cells/metabolism , Retinal Ganglion Cells/metabolism , Synapses/metabolism , Visual Pathways , Zebrafish
4.
Theranostics ; 8(17): 4750-4764, 2018.
Article in English | MEDLINE | ID: mdl-30279735

ABSTRACT

Cardiac arrhythmias are among the most challenging human disorders to diagnose and treat due to their complex underlying pathophysiology. Suitable experimental animal models are needed to study the mechanisms causative for cardiac arrhythmogenesis. To enable in vivo analysis of cardiac cellular electrophysiology with a high spatial and temporal resolution, we generated and carefully validated two zebrafish models, one expressing an optogenetic voltage indicator (chimeric VSFP-butterfly CY) and the other a genetically encoded calcium indicator (GCaMP6f) in the heart. Methods: High-speed epifluorescence microscopy was used to image chimeric VSFP-butterfly CY and GCaMP6f in the embryonic zebrafish heart, providing information about the spatiotemporal patterning of electrical activation, action potential configuration and intracellular Ca2+ dynamics. Plotting VSFP or GCaMP6f signals on a line along the myocardial wall over time facilitated the visualization and analysis of electrical impulse propagation throughout the heart. Administration of drugs targeting the sympathetic nervous system or cardiac ion channels was used to validate sensitivity and kinetics of both zebrafish sensor lines. Using the same microscope setup, we imaged transparent juvenile casper fish expressing GCaMP6f, demonstrating the feasibility of imaging cardiac optogenetic sensors at later stages of development. Results: Isoproterenol slightly increased heart rate, diastolic Ca2+ levels and Ca2+ transient amplitudes, whereas propranolol caused a profound decrease in heart rate and Ca2+ transient parameters in VSFP-Butterfly and GCaMP6f embryonic fish. Ikr blocker E-4031 decreased heart rate and increased action potential duration in VSFP-Butterfly fish. ICa,L blocker nifedipine caused total blockade of Ca2+ transients in GCaMP6f fish and a reduced heart rate, altered ventricular action potential duration and disrupted atrial-ventricular electrical conduction in VSFP-Butterfly fish. Imaging of juvenile animals demonstrated the possibility of employing an older zebrafish model for in vivo cardiac electrophysiology studies. We observed differences in atrial and ventricular Ca2+ recovery dynamics between 3 dpf and 14 dpf casper fish, but not in Ca2+ upstroke dynamics. Conclusion: By introducing the optogenetic sensors chimeric VSFP-butterfly CY and GCaMP6f into the zebrafish we successfully generated an in vivo cellular electrophysiological readout tool for the zebrafish heart. Complementary use of both sensor lines demonstrated the ability to study heart rate, cardiac action potential configuration, spatiotemporal patterning of electrical activation and intracellular Ca2+ homeostasis in embryonic zebrafish. In addition, we demonstrated the first successful use of an optogenetic sensor to study cardiac function in older zebrafish. These models present a promising new research tool to study the underlying mechanisms of cardiac arrhythmogenesis.


Subject(s)
Anti-Arrhythmia Agents/metabolism , Biological Clocks/drug effects , Electrophysiologic Techniques, Cardiac/methods , Electrophysiological Phenomena , Heart Rate/drug effects , Optogenetics/methods , Animals , Heart/embryology , Humans , Isoproterenol/metabolism , Microscopy, Fluorescence , Piperidines/metabolism , Propranolol/metabolism , Pyridines/metabolism , Zebrafish/embryology
5.
Nat Methods ; 14(11): 1079-1082, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28967889

ABSTRACT

A long-standing objective in neuroscience has been to image distributed neuronal activity in freely behaving animals. Here we introduce NeuBtracker, a tracking microscope for simultaneous imaging of neuronal activity and behavior of freely swimming fluorescent reporter fish. We showcase the value of NeuBtracker for screening neurostimulants with respect to their combined neuronal and behavioral effects and for determining spontaneous and stimulus-induced spatiotemporal patterns of neuronal activation during naturalistic behavior.


Subject(s)
Behavior, Animal , Fishes/physiology , Animals , Microscopy/methods , Neurons/physiology , Swimming/physiology
6.
Elife ; 52016 08 18.
Article in English | MEDLINE | ID: mdl-27536944

ABSTRACT

A simple neural circuit motif in the zebrafish brain enables robust and reliable behavioral choices.


Subject(s)
Interneurons , Zebrafish , Animals , Choice Behavior
7.
Nature ; 499(7458): 295-300, 2013 Jul 18.
Article in English | MEDLINE | ID: mdl-23868258

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.


Subject(s)
Action Potentials , Calcium-Binding Proteins/chemistry , Fluorescent Dyes/chemistry , Luminescent Proteins/chemistry , Animals , Calcium/metabolism , Calcium-Binding Proteins/genetics , Cells, Cultured , Dendritic Spines/metabolism , GABAergic Neurons/metabolism , Luminescent Proteins/genetics , Mice , Molecular Imaging , Mutagenesis , Protein Engineering , Pyramidal Cells/metabolism , Pyramidal Cells/physiology , Visual Cortex/cytology , Visual Cortex/physiology
8.
Methods ; 62(3): 255-67, 2013 Aug 15.
Article in English | MEDLINE | ID: mdl-23727462

ABSTRACT

Rapidly developing imaging technologies including two-photon microscopy and genetically encoded calcium indicators have opened up new possibilities for recording neural population activity in awake, behaving animals. In the small, transparent zebrafish, it is even becoming possible to image the entire brain of a behaving animal with single-cell resolution, creating brain-wide functional maps. In this chapter, we comprehensively review past functional imaging studies in zebrafish, and the insights that they provide into the functional organization of neural circuits. We further offer a basic primer on state-of-the-art methods for in vivo calcium imaging in the zebrafish, including building a low-cost two-photon microscope and highlight possible challenges and technical considerations.


Subject(s)
Brain/physiology , Calcium/metabolism , Larva/physiology , Microscopy, Fluorescence, Multiphoton/methods , Nerve Net/physiology , Neurons/physiology , Zebrafish/physiology , Animals , Behavior, Animal/physiology , Brain/cytology , Brain Mapping/instrumentation , Brain Mapping/methods , Larva/cytology , Molecular Imaging/instrumentation , Molecular Imaging/methods , Neurons/cytology , Single-Cell Analysis/instrumentation , Single-Cell Analysis/methods
9.
Nat Methods ; 10(2): 162-70, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23314171

ABSTRACT

We describe an intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) with signal-to-noise ratio and kinetics appropriate for in vivo imaging. We engineered iGluSnFR in vitro to maximize its fluorescence change, and we validated its utility for visualizing glutamate release by neurons and astrocytes in increasingly intact neurological systems. In hippocampal culture, iGluSnFR detected single field stimulus-evoked glutamate release events. In pyramidal neurons in acute brain slices, glutamate uncaging at single spines showed that iGluSnFR responds robustly and specifically to glutamate in situ, and responses correlate with voltage changes. In mouse retina, iGluSnFR-expressing neurons showed intact light-evoked excitatory currents, and the sensor revealed tonic glutamate signaling in response to light stimuli. In worms, glutamate signals preceded and predicted postsynaptic calcium transients. In zebrafish, iGluSnFR revealed spatial organization of direction-selective synaptic activity in the optic tectum. Finally, in mouse forelimb motor cortex, iGluSnFR expression in layer V pyramidal neurons revealed task-dependent single-spine activity during running.


Subject(s)
Escherichia coli Proteins , Fluorescent Dyes , Glutamic Acid/metabolism , Green Fluorescent Proteins , Recombinant Fusion Proteins , Synaptic Transmission/physiology , Animals , Astrocytes/metabolism , Biosensing Techniques , Caenorhabditis elegans , Calcium Signaling/physiology , Escherichia coli Proteins/chemical synthesis , Excitatory Postsynaptic Potentials/physiology , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/metabolism , Green Fluorescent Proteins/chemical synthesis , Hippocampus/metabolism , Mice , Motor Cortex/metabolism , Neurons/metabolism , Photic Stimulation , Pyramidal Cells/metabolism , Recombinant Fusion Proteins/chemical synthesis , Retina/physiology , Signal-To-Noise Ratio , Zebrafish
10.
Eur J Neurosci ; 33(4): 658-67, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21299656

ABSTRACT

Vision of high temporal resolution depends on careful regulation of photoresponse kinetics, beginning with the lifetime of activated photopigment. The activity of rhodopsin is quenched by high-affinity binding of arrestin to photoexcited phosphorylated photopigment, which effectively terminates the visual transduction cascade. This regulation mechanism is well established for rod photoreceptors, yet its role for cone vision is still controversial. In this study we therefore analyzed arrestin function in the cone-dominated vision of larval zebrafish. For both rod (arrS ) and cone (arr3 ) arrestin we isolated two paralogs, each expressed in the respective subset of photoreceptors. Labeling with paralog-specific antibodies revealed subfunctionalized expression of Arr3a in M- and L-cones, and Arr3b in S- and UV-cones. The inactivation of arr3a by morpholino knockdown technology resulted in a severe delay in photoresponse recovery which, under bright light conditions, was rate-limiting. Comparison to opsin phosphorylation-deficient animals confirmed the role of cone arrestin in late cone response recovery. Arr3a activity partially overlapped with the function of the cone-specific kinase Grk7a involved in initial response recovery. Behavioral measurements further revealed Arr3a deficiency to be sufficient to reduce temporal contrast sensitivity, providing evidence for the importance of arrestin in cone vision of high temporal resolution.


Subject(s)
Arrestin/metabolism , Retinal Cone Photoreceptor Cells/physiology , Vision, Ocular/physiology , Zebrafish Proteins/metabolism , Zebrafish , Animals , Arrestin/classification , Arrestin/genetics , Electroretinography , Gene Knockdown Techniques , Larva/anatomy & histology , Larva/physiology , Protein Isoforms/genetics , Protein Isoforms/metabolism , Retinal Cone Photoreceptor Cells/cytology , Zebrafish/anatomy & histology , Zebrafish/embryology , Zebrafish/metabolism , Zebrafish Proteins/classification , Zebrafish Proteins/genetics
11.
Neurogenetics ; 12(2): 97-116, 2011 May.
Article in English | MEDLINE | ID: mdl-21267617

ABSTRACT

Over the past three decades, the zebrafish has been proven to be an excellent model to investigate the genetic control of vertebrate embryonic development, and it is now also increasingly used to study behaviour and adult physiology. Moreover, mutagenesis approaches have resulted in large collections of mutants with phenotypes that resemble human pathologies, suggesting that these lines can be used to model diseases and screen drug candidates. With the recent development of new methods for gene targeting and manipulating or monitoring gene expression, the range of genetic modifications now possible in zebrafish is increasing rapidly. Combined with the classical strengths of the zebrafish as a model organism, these advances are set to substantially expand the type of biological questions that can be addressed in this species. In this review, we outline how the potential of the zebrafish can be harvested in the context of eye development and visual function. We review recent technological advances used to study the formation of the eyes and visual areas of the brain, visual processing on the cellular, subcellular and molecular level, and the genetics of visual behaviour in vertebrates.


Subject(s)
Behavior, Animal/physiology , Vision, Ocular/genetics , Vision, Ocular/physiology , Visual Perception/genetics , Zebrafish , Adult , Animals , Genetic Techniques , Humans , Models, Animal , Models, Biological , Zebrafish/genetics , Zebrafish/physiology
12.
Brain Pathol ; 13(4): 582-97, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14655762

ABSTRACT

Until recently, it was generally accepted that the vascularization of solid tumors occurred exclusively through the sprouting and co-option from pre-existing blood vessels. Growing evidence now suggests that bone marrow-derived endothelial progenitor cells (EP) circulate in the blood and may play an important role in the formation of new blood vessels in certain tumors. Whether endothelial progenitors participate in the vascularization of brain tumors has not yet been evaluated. In this study, we examined the contribution of EP to tumor angiogenesis in a murine glioma tumor model. Donor bone marrow cells obtained from transgenic mice constitutively expressing beta-galactosidase or GFP either ubiquitously or transcriptionally regulated by an endothelial specific promotor Tie-2 were injected into lethally irradiated adult mice. After bone marrow reconstitution by donor cells, mice were implanted with syngeneic GL261 murine glioma cells. Morphological and confocal 3-dimensional analysis showed that the majority of the engrafted donor marrow cells were expressing hematopoietic and/or microglia markers, but did not appreciably contribute to the tumor vasculature. Implantation of glioma cells genetically engineered to overexpress VEGF produced highly vascularized tumors. However, the number of endothelial progenitors incorporated in the tumor vasculature did not increase. These data strongly suggest that neovascularization in the brain might fundamentally be regulated by the sprouting of pre-existing vessels and implicate that circulating endothelial progenitors do not play a significant role in this process.


Subject(s)
Glioma/physiopathology , Hematopoietic Stem Cells/physiology , Neovascularization, Pathologic/physiopathology , Animals , Antigens, CD , Antigens, Differentiation/metabolism , Blotting, Northern , Bone Marrow/physiology , CD11b Antigen/metabolism , Cell Line, Tumor , Endoglin , Glial Fibrillary Acidic Protein/metabolism , Glioma/metabolism , Glioma/veterinary , Glycoside Hydrolases/metabolism , Hematopoietic Stem Cell Transplantation/methods , Immunohistochemistry , Infections , Lac Operon , Leukocyte Common Antigens/metabolism , Lung/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microglia/metabolism , Microscopy, Confocal , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , RNA, Messenger/biosynthesis , Receptors, Cell Surface , Reverse Transcriptase Polymerase Chain Reaction/methods , Spleen/metabolism , Time Factors , Vascular Cell Adhesion Molecule-1/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , von Willebrand Factor/metabolism
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