The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is subject to chemical and mechanical signals, and the mechanisms by which it senses and responds to mechanical signals are poorly understood. Elucidating the role of forces in cell maturation will enable the design of scaffolds that can promote cell adhesion and cytoskeletal coupling to the substrate, and therefore improve the capacity of different neuronal types to regenerate after injury.
Here, we describe a method to apply simultaneous force spectroscopy measurements during laser induced cell lesion. We measure tension release in the partially lesioned axon by simultaneous interferometric tracking of an optically trapped probe adhered to the membrane of the axon. Our experimental protocol detects the tension release with piconewton sensitivity, and the dynamic of the tension release at millisecond time resolution. Therefore, it offers a high-resolution method to study how the mechanical coupling between cells and substrates can be modulated by pharmacological treatment and/or by distinct mechanical properties of the substrate.
24 Related JoVE Articles!
Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method
Institutions: Cedars Sinai Medical Center, UCLA, Fourth Military Medical University, David Geffen School of Medicine, UCLA, Fourth Military Medical Univeristy.
In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is very poor 1
. The failure of axon regeneration is a combined result of several factors including the hostile glial cell environment, inhibitory myelin related molecules and decreased intrinsic neuron regenerative capacity 2
. Astrocytes are the most predominant glial cell type in central nervous system and play important role in axon functions under physiology and pathology conditions 3
. Contrast to the homologous oligodendrocytes, astrocytes are a heterogeneous cell population composed by different astrocyte subpopulations with diverse morphologies and gene expression 4
. The functional significance of this heterogeneity, such as their influences on axon growth, is largely unknown.
To study the glial cell, especially the function of astrocyte heterogeneity in neuron behavior, we established a new method by co-culturing high purified dorsal root ganglia neurons with glial cells obtained from the rat cortex. By this technique, we were able to directly compare neuron adhesion and axon growth on different astrocytes subpopulations under the same condition.
In this report, we give the detailed protocol of this method for astrocytes isolation and culture, dorsal root ganglia neurons isolation and purification, and the co-culture of DRG neurons with astrocytes. This method could also be extended to other brain regions to study cellular or regional specific interaction between neurons and glial cells.
Neuroscience, Issue 43, Dorsal root ganglia, glial cell, heterogeneity, co-culture, regeneration, axon growth
Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons
Institutions: Johns Hopkins University School of Medicine, Johns Hopkins University School of Medicine.
It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2
. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3
. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4
. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro
. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8
. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro
model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9
Neuroscience, Issue 66, Physiology, Developmental Biology, cell culture, axon regeneration, axon growth, dorsal root ganglion, spinal cord injury
Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement
Institutions: University of Maryland.
The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2
or record extracellular neuronal activity during a movement.3,4
While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo
movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo
movement thus allowing substantial insight into the role of the neuron in sensorimotor function.
Neuroscience, Issue 50, neurophysiology, sensory neuron, motor control, proprioception, neurotransmitter, sensorimotor integration, rat
Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons
Institutions: University of California, San Diego, Shanghai Jiao Tong University, University of California, San Diego, VA San Diego Healthcare System.
BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are increasingly viewed as an early feature of neurodegenerative diseases, including Alzheimer’s disease (AD) and Huntington’s disease (HD). As yet unclear is the mechanism(s) by which axonal injury is induced. We reported the development of a novel technique to produce biologically active, monobiotinylated BDNF (mBtBDNF) that can be used to trace axonal transport of BDNF. Quantum dot-labeled BDNF (QD-BDNF) was produced by conjugating quantum dot 655 to mBtBDNF. A microfluidic device was used to isolate axons from neuron cell bodies. Addition of QD-BDNF to the axonal compartment allowed live imaging of BDNF transport in axons. We demonstrated that QD-BDNF moved essentially exclusively retrogradely, with very few pauses, at a moving velocity of around 1.06 μm/sec. This system can be used to investigate mechanisms of disrupted axonal function in AD or HD, as well as other degenerative disorders.
Neuroscience, Issue 91, live imaging, brain-derived neurotrophic factor (BDNF), quantum dot, trafficking, axonal retrograde transport, microfluidic chamber
Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays
Institutions: Purdue University.
The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1
and survival factors2
located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3
. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4
. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6
Here, we describe and demonstrate the in vitro
method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth.
SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7
) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro
assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo
. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8
Neuroscience, Issue 58, chicken, dissection, morphogen, NT-3, neurite outgrowth, spinal cord, statoacoustic ganglion, Wnt5a
Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates
Institutions: University of California Riverside, University of California Riverside, University of California Riverside.
Close to two decades of research has established that astrocytes in situ
and in vivo
express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+
indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+
events using confocal microscopy. In essence, a “calcium roadmap” is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.
Neuroscience, Issue 85, astrocyte, plasticity, mGluRs, neuronal Firing, electrophysiology, Gq GPCRs, Bolus-loading, calcium, microdomains, acute slices, Hippocampus, mouse
Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons
Institutions: VU University Amsterdam.
The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive functions including decision making, sensory-guided behavior or memory. To understand how such intricate neuronal networks perform such tasks, a crucial step is to determine the function (or electrical activity) of individual cell types within the network, preferentially when the animal is performing a relevant cognitive task. Additionally, it is equally important to determine the anatomical structure of the network and the morphological architecture of the individual neurons to allow reverse engineering the cortical network. Technical breakthroughs available today allow recording cellular activity in awake, behaving animals with the valuable option of post hoc
identifying the recorded neurons. Here, we demonstrate the juxtasomal biocytin labeling technique, which involves recording action potential spiking in the extracellular (or loose-patch) configuration using conventional patch pipettes. The juxtasomal recording configuration is relatively stable and applicable across behavioral conditions, including anesthetized, sedated, awake head-fixed, and even in the freely moving animal. Thus, this method allows linking cell-type specific action potential spiking during animal behavior to reconstruction of the individual neurons and ultimately, the entire cortical microcircuit. In this video manuscript, we show how individual neurons in the juxtasomal configuration can be labeled with biocytin in the urethane-anaesthetized rat for post hoc
identification and morphological reconstruction.
Bioengineering, Issue 84, biocytin, juxtasomal, morphology, physiology, action potential, structure-function, histology, reconstruction, neurons, electrophysiological recordings
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (http://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo
Institutions: University of Göttingen.
The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo
labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis
. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo
electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo
time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.
Neuroscience, Issue 92, Xenopus laevis, Anura, electroporation, single cell electroporation, sensory neurons, olfactory system, axon growth, glomerulus, olfactory bulb, olfactory map formation
The Swimmeret System of Crayfish: A Practical Guide for the Dissection of the Nerve Cord and Extracellular Recordings of the Motor Pattern
Institutions: University of Cologne.
Here we demonstrate the dissection of the crayfish abdominal nerve cord. The preparation comprises the last two thoracic ganglia (T4, T5) and the chain of abdominal ganglia (A1 to A6). This chain of ganglia includes the part of the central nervous system (CNS) that drives coordinated locomotion of the pleopods (swimmerets): the swimmeret system. It is known for over five decades that in crayfish each swimmeret is driven by its own independent pattern generating kernel that generates rhythmic alternating activity 1-3
. The motor neurons innervating the musculature of each swimmeret comprise two anatomically and functionally distinct populations 4
. One is responsible for the retraction (power stroke, PS) of the swimmeret. The other drives the protraction (return stroke, RS) of the swimmeret. Motor neurons of the swimmeret system are able to produce spontaneously a fictive motor pattern, which is identical to the pattern recorded in vivo 1
The aim of this report is to introduce an interesting and convenient model system for studying rhythm generating networks and coordination of independent microcircuits for students’ practical laboratory courses. The protocol provided includes step-by-step instructions for the dissection of the crayfish’s abdominal nerve cord, pinning of the isolated chain of ganglia, desheathing the ganglia and recording the swimmerets fictive motor pattern extracellularly from the isolated nervous system.
Additionally, we can monitor the activity of swimmeret neurons recorded intracellularly from dendrites. Here we also describe briefly these techniques and provide some examples. Furthermore, the morphology of swimmeret neurons can be assessed using various staining techniques. Here we provide examples of intracellular (by iontophoresis) dye filled neurons and backfills of pools of swimmeret motor neurons. In our lab we use this preparation to study basic functions of fictive locomotion, the effect of sensory feedback on the activity of the CNS, and coordination between microcircuits on a cellular level.
Neurobiology, Issue 93, crustacean, dissection, extracellular recording, fictive locomotion, motor neurons, locomotion
Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays
Institutions: University Medical Center Utrecht.
Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1
. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2
. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4
. These soluble factors can be studied in vitro
by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro
. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or βIII tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro
studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.
Neuroscience, Issue 61, Axon guidance, collagen matrix, development, dissection, dopamine, medium spiny neuron, rat tail collagen, striatum, striatonigral, mesostriatal
Neural Explant Cultures from Xenopus laevis
Institutions: Harvard Medical School.
The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1
. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis
growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4
) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7
. In this method8
, eggs are collected and fertilized in vitro
, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis
growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.
Neuroscience, Issue 68, Cellular Biology, Anatomy, Physiology, Growth cone, neural explant, Xenopus laevis, live cell imaging, cytoskeletal dynamics, cell culture
Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration
Institutions: Max Planck Institute of Experimental Medicine, Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB).
Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro
and in vivo
analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo
system owing to its predominant postnatal development. We also present an in vivo
electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.
Neuroscience, Issue 85, axons, dendrites, neuronal migration, cerebellum, cultured neurons, transfection, in vivo electroporation
Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
Institutions: University of Maine.
Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.
Basic Protocol, Issue 82, Microscopy, Super-resolution imaging, Multicolor, single molecule, FPALM, Localization microscopy, fluorescent proteins
Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises
Institutions: University of Kentucky, University of Toronto.
The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+
on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.
Neuroscience, Issue 47, Invertebrate, Crayfish, neurophysiology, muscle, anatomy, electrophysiology
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography
Institutions: Baylor College of Medicine, Baylor College of Medicine, University of California at San Diego, Baylor College of Medicine.
Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the structure of neurites is critical to understanding how these processes move materials and signals that support synaptic communication. Electron microscopy (EM) has been traditionally used to assess the ultrastructural features within neurites; however, the exposure to organic solvent during dehydration and resin embedding can distort structures. An important unmet goal is the formulation of procedures that allow for structural evaluations not impacted by such artifacts.
Here, we have established a detailed and reproducible protocol for growing and flash-freezing whole neurites of different primary neurons on electron microscopy grids followed by their examination with cryo-electron tomography (cryo-ET). This technique allows for 3-D visualization of frozen, hydrated neurites at nanometer resolution, facilitating assessment of their morphological differences. Our protocol yields an unprecedented view of dorsal root ganglion (DRG) neurites, and a visualization of hippocampal neurites in their near-native state. As such, these methods create a foundation for future studies on neurites of both normal neurons and those impacted by neurological disorders.
Neuroscience, Issue 84, Neurons, Cryo-electron Microscopy, Electron Microscope Tomography, Brain, rat, primary neuron culture, morphological assay
Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers
Institutions: University of Maryland, University of Maryland.
Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200 nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125
I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125
I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.
Bioengineering, Issue 80, Antigens, Enzymes, Biological Therapy, bioengineering (general), Pharmaceutical Preparations, Macromolecular Substances, Therapeutics, Digestive System and Oral Physiological Phenomena, Biological Phenomena, Cell Physiological Phenomena, drug delivery systems, targeted nanocarriers, transcellular transport, epithelial cells, tight junctions, transepithelial electrical resistance, endocytosis, transcytosis, radioisotope tracing, immunostaining
Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors
Institutions: University College London.
Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA
Rs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials.
During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAA
Rs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other.
To elucidate the underlying molecular mechanisms, a novel in vitro
co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAA
R subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAA
R subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro
model system can be used to reproduce, at least in part, the in vivo
conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAA
Rs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.
Neuroscience, Issue 93, Developmental neuroscience, synaptogenesis, synaptic inhibition, co-culture, stable cell lines, GABAergic, medium spiny neurons, HEK 293 cell line
Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays
Institutions: Technical University of Berlin, Oregon Health & Science University.
Whereas cation transport by the electrogenic membrane transporter Na+
-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+
-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+
transport by Na+
- or gastric H+
-ATPase in single cells. Using Xenopus
oocytes as expression system, we determine the amount of Rb+
) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+
uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g.
to quantitatively determine the 3Na+
transport stoichiometry of the Na+
-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+
-ATPase. In principle, the assay is not limited to K+
-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.
Biochemistry, Issue 72, Chemistry, Biophysics, Bioengineering, Physiology, Molecular Biology, electrochemical processes, physical chemistry, spectrophotometry (application), spectroscopic chemical analysis (application), life sciences, temperature effects (biological, animal and plant), Life Sciences (General), Na+,K+-ATPase, H+,K+-ATPase, Cation Uptake, P-type ATPases, Atomic Absorption Spectrophotometry (AAS), Two-Electrode Voltage-Clamp, Xenopus Oocytes, Rb+ Flux, Transversely Heated Graphite Atomizer (THGA) Furnace, electrophysiology, animal model
Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles
Institutions: Heart Research Center Goettingen, University Medical Center Goettingen, German Center for Cardiovascular Research (DZHK) partner site Goettingen, University of Maryland School of Medicine.
In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+
release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.
Bioengineering, Issue 92, cardiac myocyte, atria, ventricle, heart, primary cell isolation, fluorescence microscopy, membrane tubule, transverse-axial tubule system, image analysis, image processing, T-tubule, collagenase
The Preparation of Electrohydrodynamic Bridges from Polar Dielectric Liquids
Institutions: Wetsus - Centre of Excellence for Sustainable Water Technology, IRCAM GmbH, Graz University of Technology.
Horizontal and vertical liquid bridges are simple and powerful tools for exploring the interaction of high intensity electric fields (8-20 kV/cm) and polar dielectric liquids. These bridges are unique from capillary bridges in that they exhibit extensibility beyond a few millimeters, have complex bi-directional mass transfer patterns, and emit non-Planck infrared radiation. A number of common solvents can form such bridges as well as low conductivity solutions and colloidal suspensions. The macroscopic behavior is governed by electrohydrodynamics and provides a means of studying fluid flow phenomena without the presence of rigid walls. Prior to the onset of a liquid bridge several important phenomena can be observed including advancing meniscus height (electrowetting), bulk fluid circulation (the Sumoto effect), and the ejection of charged droplets (electrospray). The interaction between surface, polarization, and displacement forces can be directly examined by varying applied voltage and bridge length. The electric field, assisted by gravity, stabilizes the liquid bridge against Rayleigh-Plateau instabilities. Construction of basic apparatus for both vertical and horizontal orientation along with operational examples, including thermographic images, for three liquids (e.g.
, water, DMSO, and glycerol) is presented.
Physics, Issue 91, floating water bridge, polar dielectric liquids, liquid bridge, electrohydrodynamics, thermography, dielectrophoresis, electrowetting, Sumoto effect, Armstrong effect
Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales
Institutions: Rutgers University, Rutgers University.
Neuronal morphology plays a significant role in determining how neurons function and communicate1-3
. Specifically, it affects the ability of neurons to receive inputs from other cells2
and contributes to the propagation of action potentials4,5
. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7
. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8
, and mental retardation11
. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1
Neuroscience, Issue 45, Sholl Analysis, Neurite, Morphology, Computer-assisted, Tracing
Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures
Institutions: Dana Farber Cancer Institute, Harvard Medical School.
Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.
Neuroscience, Issue 20, campenot cultures, cell culture, dorsal root ganglia (DRG) neurons, neuronal culture