1Center for Research on Occupational and Environmental Toxicology, Oregon Health & Science University, 2Department of Behavioral Neuroscience, Oregon Health & Science University
Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.
Published December 8, 2013. Keywords: Neuroscience, Synaptic Transmission, Action Potentials, Circadian Rhythm, Excitatory Postsynaptic Potentials, Life Sciences (General), circadian rhythm, suprachiasmatic nucleus, membrane potential, patch clamp recording, fluorescent probe, intracellular calcium
1Department of Physiology and Biophysics, Boston University School of Medicine, 2Boston University Photonics Center
With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.
Published April 10, 2013. Keywords: Developmental Biology, Physiology, Biophysics, Neurobiology, Cellular Biology, Molecular Biology, Anatomy, Developmental Biology, Biomedical Engineering, Medicine, Caenorhabditis elegans, C. elegans, Microscopy, Fluorescence, Neurosciences, calcium imaging, genetically encoded calcium indicators, cameleon, GCaMP, neuronal activity, time-lapse imaging, laser ablation, optical neurophysiology, neurophysiology, neurons, animal model
1Department of Neurobiology, University of California, Los Angeles, 2Veterans Administration Greater Los Angeles Healthcare System, 3Departments of Physiology & Biophysics and Ophthalmology & Visual Sciences, Dalhousie University, 4Departments of Neurobiology and Medicine, Jules Stein Eye Institute, CURE-Digestive Diseases Research Center, David Geffen School of Medicine, University of California, Los Angeles
Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.
Published October 13, 2014. Keywords: Neuroscience, immunohistochemistry, antibody, fluo-4, calcium imaging, ganglion cells, retina, rat
1Rangos Research Center, Pediatric Gastroenterology, Hepatology, and Nutrition, Children's Hospital of Pittsburgh of UPMC, 2Department of Surgery, Tufts University Medical Center
We describe a reproducible method of preparing mouse pancreatic acinar cells from a mouse for the purpose of examining acinar cell calcium signals and cellular injury with physiologically and pathologically relevant stimuli. A method for adenoviral infection of these cells is also provided.
Published July 5, 2013. Keywords: Cancer Biology, Cellular Biology, Molecular Biology, Medicine, Biochemistry, Biomedical Engineering, Acinar Cells, Pancreatitis, Transfection, Microscopy, Confocal, Calcium Signaling, Pancreatic Acinar Cells, Pancreatitis, Calcium Signaling, Cytotoxicity, LDH Leakage, cell injury, imaging
1Department of Neurosurgery and Cellular & Molecular Physiology, Yale University School of Medicine
A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.
Published September 19, 2012. Keywords: Neuroscience, Molecular Biology, Anatomy, Physiology, subventricular zone, adult neurogenesis, gap junction, calcium imaging, neural stem cell
1Department of Integrative Neurophysiology, VU University, Amsterdam
Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.
Published October 22, 2011. Keywords: Neuroscience, calcium, imaging, mouse, network, development, cortex, multiphoton
1Department of Neurobiology, Stanford University, 2Department of Neurobiology, Stanford University School of Medicine
Calcium signals play a key role in many cellular processes including gene expression, survival and differentiation. Here we demonstrate how to perform calcium imaging using Fura-2 AM. Calcium imaging is a valuable tool to study the regulation of intracellular calcium in real time and its regulation of signaling cascades.
Published January 19, 2009. Keywords: Neuroscience, calcium imaging, calcium channels, calcium, neurons, excitable cells, time-lapse, Fura-2, Calcium indicator, intracellular calcium
1Medical Biochemistry and Molecular Biology, Saarland University, 2Experimental and Clinical Pharmacology and Toxicology, Saarland University
The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.
Published July 7, 2011. Keywords: Cell Biology, Cellular calcium homeostasis, calmodulin, complementation, endoplasmic reticulum, ER calcium leakage, gene silencing, IQ motif, mutant analysis, Sec61 complex
1Friedrich Miescher Institute for Biomedical Research, 2Max Planck Institute of Neurobiology, 3Department of Biosystems Science and Engineering, ETH Zurich
Here we describe the experimental procedures involved in two-photon imaging of mouse cortex during behavior in a virtual reality environment.
Published February 20, 2014. Keywords: Behavior, Two-photon imaging, Virtual Reality, mouse behavior, adeno-associated virus, genetically encoded calcium indicators
1Center for Integrative Genomics, University of Lausanne, 2Department of Biology, University of Konstanz
We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.
Published March 14, 2012. Keywords: Neuroscience, Drosophila, calcium imaging, antennal lobe, olfaction, neuroscience
1Department of Neurology, University of California, Los Angeles
To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .
Published March 13, 2008. Keywords: Neuroscience, 2-photon, two-photon, GFP mice, craniotomy, spine dynamics, cranial window
1Institute for Clinical Neurobiology, University of Wuerzburg, 2Department of Synapses - Circuits - Plasticity, Max Planck Institute of Neurobiology, Martinsried, 3Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians University of Munich
Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.
Published May 7, 2013. Keywords: Cellular Biology, Neurobiology, Neuroscience, Molecular Biology, Biochemistry, Biomedical Engineering, Bioengineering, Virology, Medicine, Anatomy, Physiology, Surgery, Endoplasmic Reticulum, ER, Calcium Signaling, calcium store, calcium imaging, calcium indicator, metabotropic signaling, Ca2+, neurons, cells, mouse, animal model, cell culture, targeted esterase induced dye loading, imaging
1Center for Genetic Medicine Research, Children's National Medical Center, 2Department of Integrative Systems Biology, George Washington University
The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.
Published March 24, 2014. Keywords: Biochemistry, cell injury, lysosome exocytosis, repair, calcium, imaging, total internal reflection fluorescence (TIRF) microscopy, laser ablation
1Department of Biology, College of William and Mary
Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.
Published December 23, 2012. Keywords: Developmental Biology, Neuroscience, Cellular Biology, Surgery, Anatomy, Physiology, Ophthalmology, retina, primary cell culture, dissection, confocal microscopy, calcium imaging, fluorescent in situ hybridization, FISH, Xenopus laevis, animal model
1Biological Sciences, University of Illinois at Chicago
Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals.
Published October 1, 2014. Keywords: Neuroscience, reticulospinal synapse, reticulospinal axons, presynaptic terminal, presynaptic calcium, voltage-gated calcium channels, vesicle fusion, synaptic transmission, neurotransmitter release, spinal cord, lamprey, synaptic vesicles, acute dissociation
1Department of Physiology and Biophysics, University of Miami
We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.
Published September 21, 2013. Keywords: Developmental Biology, Eukaryota, Biological Phenomena, Cell Physiological Phenomena, C. elegans, cell culture, embryonic cells
1Department of Molecular, Cellular and Developmental Biology, University of Michigan, 2Department of Biomedical Engineering, University of Michigan, 3Life Sciences Institute, University of Michigan, 4Department of Cell and Developmental Biology, University of Michigan, 5Department of Mechanical Engineering, University of Michigan
Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the 'larva chip' for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.
Published February 7, 2014. Keywords: Bioengineering, Drosophila melanogaster, Live Imaging, Microfluidics, axonal injury, axonal degeneration, calcium imaging, photoconversion, laser microsurgery
1Department of Horticulture and Landscape Architecture, Purdue University, 2Bindley Bioscience Center, Purdue University, 3Institute of Biotechnology, Jiangsu Academy of Agricultural Sciences, 4College of Environmental & Resource Science, Zhejiang University, 5Dryland Agriculture Research Centre, Shanxi Academy of Agricultural Sciences, 6Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences
Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.
Published September 2, 2014. Keywords: Plant Biology, Aequorin, Case12, abiotic stress, heavy metal stress, copper ion, calcium imaging, Arabidopsis
1Department of Neuroscience, Medical University of South Carolina
A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.
Published December 12, 2012. Keywords: Neuroscience, Molecular Biology, Cellular Biology, Anatomy, Physiology, Two-photon imaging, non-rodent, cortical maps, functional architecture, orientation pinwheel singularity, optical imaging, calcium-sensitive dye, bulk loading, single-cell electroporation
1Neurobiology and Behavior, University of California, Irvine, 2Physiology and Biophysics, University of California, Irvine
Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.
Published March 3, 2015. Keywords: Cellular Biology, Calcium, imaging, total internal reflection microscopy, algorithm, automation, fluorescence
1Department of Behavioral Physiology and Sociobiology (Zoology II) Biozentrum, University of Würzburg
Simultaneous extracellular long term recordings from two different brain neuropiles or two different anatomical tracts were established in honeybees. These recordings allow the investigation of temporal aspects of neuronal processing across different brain areas at the single neuron as well as at the ensemble level in a behaving animal.
Published July 21, 2014. Keywords: Neuroscience, honeybee brain, olfaction, extracellular long term recordings, double recordings, differential wire electrodes, single unit, multi-unit recordings
1Medical Pharmacology and Physiology, University of Missouri, 2Dalton Cardiovascular Research Center
We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.
Published November 25, 2013. Keywords: Basic Protocol, endothelial tubes, microcirculation, calcium signaling, resistance vasculature, Confocal microscopy
1Departments of Pediatrics and Neurology, University of Michigan
Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.
Published November 13, 2013. Keywords: Basic Protocol, Zebrafish, Neuromuscular Diseases, Muscular Diseases, Muscular Dystrophies, Primary Cell Culture, Immunohistochemistry (IHC), skeletal muscle, myofiber, live imaging
1Fishberg Department of Neuroscience and Friedman Brain Institute, Mt. Sinai School of Medicine, 2Phase Five Communications Inc.
We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.
Published July 15, 2012. Keywords: Neuroscience, Molecular Biology, Marine Biology, calcium imaging, intracellular recording, invertebrate, mollusc, Aplysia, Calcium Orange, facilitation, synaptic plasticity
1Section of Neurobiology, Center for Learning and Memory, The University of Texas at Austin
Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.
Published September 5, 2012. Keywords: Neuroscience, functional calcium imaging, spatiotemporal patterns of activity, dithered random-access scanning
1Section on Synaptic Pharmacology/Laboratory for Integrative Neuroscience, National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism, 2Department of Electronics Engineering, Ewha Womans University, 3Section on Transmitter Signaling/Laboratory of Molecular Physiology, National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism
This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.
Published May 25, 2011. Keywords: Neuroscience, neuronal dissociation, synaptic transmission, GABA, calcium imaging, electrophysiology, hippocampus, striatum
1Department of Neuroscience and Cell Biology, University of Texas Medical Branch
This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.
Published April 1, 2011. Keywords: Cellular Biology, Ratiometric pericam, mitochondria, calcium, apoptosis, staurosporine, live cell imaging
1Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, 2Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, 3School of Electrical Engineering, Tel-Aviv University, 4School of Physics and Astronomy, Tel-Aviv University, 5Department of Informatics, Bioengineering, Robotics and System Engineering, University of Genova
This manuscript describes a protocol to grow in vitro modular networks consisting of spatially confined, functionally inter-connected neuronal circuits. A polymeric mask is used to pattern a protein layer to promote cellular adhesion over the culturing substrate. Plated neurons grow on coated areas establishing spontaneous connections and exhibiting electrophysiological activity.
Published April 15, 2015. Keywords: Neuroscience, In vitro, patterning, PDMS stencils, SU8-2075, silicon wafer, calcium imaging, Micro Electrode Array
1Department of Pharmacology and Physiology, Rutgers New Jersey Medical School, 2Center for Taste and Feeding Behavior, Universite de Bourgogne
The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.
Published November 27, 2013. Keywords: Neuroscience, membrane potential dye, ventromedial hypothalamus, adult neurons, glucose sensing, fluorescence imaging, arcuate nucleus
1Department of Pharmacology, University of South Alabama, 2Department of Physiology, University of South Alabama
Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.
Published June 16, 2014. Keywords: Basic Protocol, signaling, ImageJ, detection, microscopy, algorithm, calcium
1Department of Pharmacology and Toxicology, University of Lausanne, 2Department of Genetics and Evolution, University of Geneva
In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.
Published December 6, 2011. Keywords: Neuroscience, Vomeronasal organ, VNO, pheromone, calcium imaging, tissue slice preparation, floating immunohistochemistry, GFP
1Department of Biological Sciences and Geology, Queensborough Community College, 2Department of Physiology & Biophysics, Stony Brook University
We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.
Published November 29, 2011. Keywords: Cellular Biology, Calcium fluxes, laser scanning microscopy, cardiomyocytes, fluorescent indicators
1The Vollum Institute, Oregon Health and Science University
Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.
Published January 19, 2012. Keywords: Neuroscience, synaptic physiology, axon terminal, synaptic ribbon, retina microcircuits, goldfish, zebrafish, brain slices, patch-clamp, membrane capacitance measurements, calcium-imaging, exocytosis, transmitter release
1Discipline of Biomedical Science, School of Medical Sciences, Sydney Medical School, University of Sydney, 2School of Biomedical Sciences and Pharmacy, University of Newcastle
Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.
Published June 13, 2013. Keywords: Neurobiology, Neuroscience, Cellular Biology, Molecular Biology, Biomedical Engineering, Anatomy, Physiology, Surgery, Vestibular, Hair cells, Epithelium, two-photon microscopy, isolated, semi-intact, electrophysiology, electroporation, microscopy, tissue, isolation, animal model
1Neuroglial Interactions in Cerebral Physiopathology, Center for Interdisciplinary Research in Biology, CNRS UMR 7241, INSERM U1050, Collège de France, 2Paris Diderot University
The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.
Published November 26, 2012. Keywords: Neuroscience, Physiology, Anatomy, Medicine, hippocampus preparation, acute brain slice, electrophysiology, patch-clamp, neurons, astrocytes, astroglial, neuroglial interactions, glutamate transporter current, potassium current, paired recordings, synaptic activity, synaptically-evoked responses
1Departments of Physiology and Neurobiology, David Geffen School of Medicine, University of California, Los Angeles
We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.
Published April 26, 2009. Keywords: Neuroscience, calcium imaging, TIRF, astrocyte, hippocampus, culture, neuroscience, brain, rat
1Department of Physiology, School of Medicine, University of Saarland, Homburg, Germany
In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.
Published August 24, 2012. Keywords: Neuroscience, Molecular Biology, Medicine, GFP, fura-red, calcium, confocal imaging, neuron, hypothalamus, brain, olfaction, mouse, slice preparation
1Department of Biomedical Engineering, Washington University in St. Louis
This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.
Published September 13, 2011. Keywords: Bioengineering, optical mapping, rabbit heart, action potential, calcium transient, voltage-sensitive dye, calcium dye
1Institute for Complex Molecular Systems & Laboratory of Macromolecular and Organic Chemistry, Eindhoven University of Technology & NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, 2NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR & IIT@NEST, Center for Nanotechnology Innovation, 3NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, 4IIT@NEST, Center for Nanotechnology Innovation
Fluorescence sensors are powerful tools in life science. Here we describe a methodology to synthesize and use dendrimer-based fluorescent sensors to measure pH in living cells and in vivo. The dendritic scaffold enhances the properties of conjugated fluorescent dyes leading to improved sensing properties.
Published September 10, 2013. Keywords: Chemistry, Investigative Techniques, Chemistry and Materials (General), dendrimer, fluorescence, sensors, pH, delivery, confocal
1Department of Biomedical Engineering, Washington University in St. Louis
This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.
Published September 13, 2011. Keywords: Bioengineering, optical mapping, action potential, calcium transient, mouse, heart
1Unit on Neural Circuits and Adaptive Behaviors, Genes Cognition and Psychosis Program, National Institute of Mental Health, 2Department of Neuroscience, Brown University - National Institutes of Health Graduate Partnership Program, 3Section on Synaptic Pharmacology, Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism, 4Champalimaud Neuroscience Programme, Champalimaud Center for the Unknown
Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.
Published January 5, 2013. Keywords: Neuroscience, Medicine, Anatomy, Neurobiology, Surgery, Cerebral Cortex, Frontal Cortex, Stereotaxic Techniques, Molecular Imaging, Neuronal Plasticity, Neurosciences, In Vivo Imaging, Two-photon Microscopy, Experience-dependent Gene Expression, Arc-GFP Mice, Cranial Window, in situ hybridization, immunohistochemistry, animal model
1Graduate Program in Neuroscience, SMBS, University at Buffalo, SUNY, 2Department of Biochemistry, SMBS, University at Buffalo, SUNY, 3Molecular and Cellular Neuroscience Department, The Scripps Research Institute, 4Department of Biochemistry and Graduate Program in Neuroscience, SMBS, University at Buffalo, SUNY
Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.
Published June 9, 2014. Keywords: Neuroscience, biophysics, ion channels, single-channel recording, NMDA receptors, gating, electrophysiology, patch-clamp, kinetic analysis
1Monell Chemical Senses Center, 2New York University College of Dentistry, 3AFB International
We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.
Published May 17, 2012. Keywords: Neuroscience, Fungiform, taste cells, cell culture, gustducin, calcium imaging, molecular biology, human fungiform papillae
1Department of Neurobiology, University of Massachusetts Medical School
The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.
Published April 26, 2012. Keywords: Neuroscience, feeding, proboscis extension, calcium imaging, Drosophila, fruit fly, GCaMP, suboesophageal ganglion (SOG), live imaging, FLIES
1Department of Natural Sciences, Assumption College
Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.
Published September 24, 2014. Keywords: Neuroscience, dorsal root gangia, DRG, chicken, in vitro, avian, laminin-1, embryonic, primary
1Institute of Neurophysiology and Cellular Biophysics and Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), University of Göttingen
We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.
Published October 30, 2014. Keywords: Neuroscience, Xenopus laevis, Anura, electroporation, single cell electroporation, sensory neurons, olfactory system, axon growth, glomerulus, olfactory bulb, olfactory map formation
1Institute for Molecular Cell Biology, Universty of Saarland
Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.
Published September 17, 2009. Keywords: Cellular Biology, cardiac myocyte, adenoviral gene transfer, fluorescent protein, confocal microscopy, calcium imaging, golgi
1Monell Chemical Senses Center
Olfactory receptor activation patterns encode odor identity, but the lack of published data identifying odorant ligands for mammalian olfactory receptors hinders the development of a comprehensive model of odor coding. This protocol describes a method to facilitate high-throughput identification of olfactory receptor ligands and quantification of receptor activation.
Published June 2, 2014. Keywords: Neuroscience, Firefly luciferase, Renilla Luciferase, Dual-Glo Luciferase Assay, olfaction, Olfactory receptor, Odorant, GPCR, High-throughput
1Institut für Biologie - Neurobiologie, Freie Universität Berlin, 2Institut für Biologie - Neurobiologie, Free University Berlin - Freie Universitaet Berlin
Bees can be conditioned in an appetitive olfactory learning paradigm (PER-conditioning). Using odors as stimuli, we established a method in which behavior is recorded while simultaneously Calcium Imaging is used to measure odor evoked activity in mushroom body neurons in vivo.
Published August 18, 2009. Keywords: Neuroscience, Calcium Imaging, Insects, Mushroom Body, PER Conditioning, Olfaction, Fura-2
1Department of Cellular and Molecular Physiology, Yale University School of Medicine
An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.
Published November 29, 2012. Keywords: Neuroscience, Medicine, Physiology, Molecular Biology, Cellular Biology, voltage-sensitive dyes, brain, imaging, dendritic spines, axons, dendrites, neurons