The authors thank Dr. James Bamburg and members of his laboratory for collaboration in these studies. This work was funded by NIH grants HD19950, EY07133 and by grants from the Minnesota Medical Foundation.

For rhodamine-actin labeling, neurons are cultured on glass coverslips placed in the bottom of 35 mm plastic dishes, or on coverslips glued into "video" dishes.
1. Preparation of Coverslips or "Video" Dishes
<ol>
<li>Many neuronal types are poorly adhesive to in vitro substrates. Glass coverslips, which are required for this procedure, may not allow sufficient neuron-substrate adhesion unless they are adequately cleaned and prepared. A detailed procedure for acid washing and cleanin...

<ol>
	<li>Lowery, L. A., Van Vactor, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+trip+of+the+tip:+Understanding+the+growth+cone+machinery.">The trip of the tip: Understanding the growth cone machinery.</a> Nat Rev Mol Cell Biol. 10, 332-343 (2009).</li><li>Letourneau, P., Squire, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+Pathfinding:+Extracellular+Matrix+Role.">Axonal Pathfinding: Extracellular Matrix Role.</a> Encyclopedia of Neuroscience. , 1139-1145 (2008).</li><li>Kalil, K., Dent, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Touch+and+go:+Guidance+cues+signal+to+the+growth+cone+cytoskeleton.">Touch and go: Guidance cues signal to the growth cone cytoskeleton.</a> Curr Opin Neurobio. 15, 521-526 (2005).</li><li>Dent, E. W., Gertler, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+dynamics+and+transport+in+growth+cone+motility+and+axon+guidance.">Cytoskeletal dynamics and transport in growth cone motility and axon guidance.</a> Neuron. 40, 209-227 (2003).</li><li>Pak, C. W., Flynn, K. C., Bamburg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin-binding+proteins+take+the+reins+in+growth+cones.">Actin-binding proteins take the reins in growth cones.</a> Nat Rev Neurosci. 9, 136-147 (2008).</li><li>Guan, K. L., Rao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+mechanisms+mediating+neuronal+responses+to+guidance+cues.">Signaling mechanisms mediating neuronal responses to guidance cues.</a> Nat Rev Neurosci. 4, 941-956 (2003).</li><li>Gallo, G., Letourneau, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+growth+cone+actin+filaments+by+guidance+cues.">Regulation of growth cone actin filaments by guidance cues.</a> J. Neurobiology. 58, 92-102 (2004).</li><li>Fass, J., Gehler, S., Sarmiere, P., Letourneau, P., Bamburg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulating+filopodial+dynamics+through+actin-depolymerizing+factor/cofilin.">Regulating filopodial dynamics through actin-depolymerizing factor/cofilin.</a> Anat Sci Inter. 79, 173-183 (2004).</li><li>Bernstein, B. W., Bamburg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ADF/cofilin:+A+functional+node+in+cell+biology.">ADF/cofilin: A functional node in cell biology.</a> Trends Cell Biol. 20, 187-195 (2010).</li><li>Marsick, B. M., Flynn, K. C., Santiago, M., Bamburg, J. R., Letourneau, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+ADF/cofilin+mediates+attractive+growth+cone+turning+toward+nerve+growth+factor+and+netrin-1.">Activation of ADF/cofilin mediates attractive growth cone turning toward nerve growth factor and netrin-1.</a> Dev Neurobiol. 70, 565-588 (2010).</li><li>Symons, M. H., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+actin+polymerization+in+live+and+permeabilized+fibroblasts.">Control of actin polymerization in live and permeabilized fibroblasts.</a> J Cell Biol. 114, 503-513 (1991).</li><li>Chan, A. Y., Raft, S., Bailly, M., Wyckoff, J. B., Segall, J. E., Condeelis, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGF+stimulates+an+increase+in+actin+nucleation+and+filament+number+at+the+leading+edge+of+the+lamellipod+in+mammary+adenocarcinoma+cells.">EGF stimulates an increase in actin nucleation and filament number at the leading edge of the lamellipod in mammary adenocarcinoma cells.</a> J Cell Sci. 111, 199-211 (1998).</li></ol>

The methods presented here allow temporal and spatial resolution of cellular components that are involved in the dynamic remodeling of the actin cytoskeleton at the leading margin of migrating growth cones. The action of attractant molecules, like NGF or netrin, to rapidly stimulate actin filament polymerization is revealed as a local increase in actin barbed ends, created by actin filament severing by activated ADF/cofilin10, as shown in Figures 1 and 2. The method permits localization of other proteins invol...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>18x18 mm coverslip</td>
<td>&nbsp;</td>
<td>Gold Seal</td>
<td>3305</td>
<td>&nbsp;</td>
<tr>
<td>35mm Petri dishes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>351008</td>
<td>&nbsp;</td>
<tr>
<td>Aquarium cement</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>DAP 100% silicone aquarium sealant</td>
<td>Any hardware store</td>
</tr>
<tr>
<td>Poly-D-lysine (mw &gt; 300,000)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1024</td>
<td>&nbsp;</td>
<tr>
<td>Natural mouse laminin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>23017-015</td>
<td>&nbsp;</td>
<tr>
<td>L1 CAM</td>
<td>&nbsp;</td>
<td>R &amp; D systems</td>
<td>777-NC</td>
<td>&nbsp;</td>
<tr>
<td>Alexa-fluor 350 phalloidin</td>
<td>&nbsp;</td>
<td>Invitrogen (Molecular Probes)</td>
<td>A22281</td>
<td>&nbsp;</td>
<tr>
<td>Rhodamine non-muscle actin</td>
<td>&nbsp;</td>
<td>Cytoskeleton, Inc.</td>
<td>APHR-A</td>
<td>&nbsp;</td>
<tr>
<td>F12 Culture medium</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>21700-075</td>
<td>&nbsp;</td>
<tr>
<td>B27</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>Slowfade </td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>536937</td>
<td>&nbsp;</td>		</tbody>
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labeling f-actin barbed ends with rhodamine-actin in permeabilized neuronal growth cones

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally expressed molecular guidance cues that bind growth cone receptors and regulate the dynamics and organization of the growth cone cytoskeleton3-6. The main target of these navigational signals is the actin filament meshwork that fills the growth cone periphery and that drives growth cone motility through continual actin polymerization and dynamic remodeling7. Positive or attractive guidance cues induce growth cone turning by stimulating actin filament (F-actin) polymerization in the region of the growth cone periphery that is nearer the source of the attractant cue. This actin polymerization drives local growth cone protrusion, adhesion of the leading margin and axonal elongation toward the attractant.
Actin filament polymerization depends on the availability of sufficient actin monomer and on polymerization nuclei or actin filament barbed ends for the addition of monomer. Actin monomer is abundantly available in chick retinal and dorsal root ganglion (DRG) growth cones. Consequently, polymerization increases rapidly when free F-actin barbed ends become available for monomer addition. This occurs in chick DRG and retinal growth cones via the local activation of the F-actin severing protein actin depolymerizing factor (ADF/cofilin) in the growth cone region closer to an attractant8-10. This heightened ADF/cofilin activity severs actin filaments to create new F-actin barbed ends for polymerization. The following method demonstrates this mechanism. Total content of F-actin is visualized by staining with fluorescent phalloidin. F-actin barbed ends are visualized by the incorporation of rhodamine-actin within growth cones that are permeabilized with the procedure described in the following, which is adapted from previous studies of other motile cells11, 12. When rhodamine-actin is added at a concentration above the critical concentration for actin monomer addition to barbed ends, rhodamine-actin assembles onto free barbed ends. If the attractive cue is presented in a gradient, such as being released from a micropipette positioned to one side of a growth cone, the incorporation of rhodamine-actin onto F-actin barbed ends will be greater in the growth cone side toward the micropipette10.
Growth cones are small and delicate cell structures. The procedures of permeabilization, rhodamine-actin incorporation, fixation and fluorescence visualization are all carefully done and can be conducted on the stage of an inverted microscope. These methods can be applied to studying local actin polymerization in migrating neurons, other primary tissue cells or cell lines.

Watch this Scientific Journal Video about Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones at JoVE.com

Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones

A method to visualize and quantify F-actin barbed ends in neuronal growth cones is described. After culturing neurons on glass coverslips, cells are permeabilized with a saponin-containing solution. Then, a short incubation with the saponin buffer containing rhodamine-actin incorporates fluorescent actin onto free actin barbed ends.

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally ...

labeling-f-actin-barbed-ends-with-rhodamine-actin-permeabilized

Research

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Neuroscience

14.7K ビュー.  University of Minnesota. 神経成長円錐におけるF -アクチン有刺鉄線両端を視覚化し、定量化する方法が記載されている。ガラス製カバースリップ上で培養ニューロン後、細胞をサポニン含有溶液に透過処理されています。その後、ローダミン-アクチンを含むサポニンバッファと短いインキュベーションは無料アクチン有刺鉄線端上に蛍光アクチンを内蔵しています。

ビデオ: Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones

A method to visualize and quantify F-actin barbed ends in neuronal growth cones is described. After culturing neurons on glass coverslips, cells are permeabilized with a saponin-containing solution. Then, a short incubation with the saponin buffer containing rhodamine-actin incorporates fluorescent actin onto free actin barbed...

quantification of filamentous actin (f-actin) puncta in rat cortical neurons

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

actin co-sedimentation assay; for the analysis of protein binding to f-actin

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules.  In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind...

in vitro polymerization of f-actin on early endosomes

In Vitro Polymerization of F-actin on Early Endosomes

Early endosome functions depend on F-actin polymerization. Here, we describe a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to biochemical and genetic...

In Vitro Polymerization of F-actin on Early Endosomes

measuring protein binding to f-actin by co-sedimentation

Measuring Protein Binding to F-actin by Co-sedimentation

This protocol describes a method to test the ability of a protein to co-sediment with filamentous actin (F-actin) and, if binding is observed, to measure the affinity of the...

live cell imaging of f-actin dynamics via fluorescent speckle microscopy (fsm)

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

analyses of actin dynamics, clutch coupling and traction force for growth cone advance

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance.

neuronal cell cultures from aplysia for high-resolution imaging of growth cones

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell...

three-dimensional super resolution microscopy of f-actin filaments by interferometric photoactivated localization microscopy (ipalm)

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical...

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

reconstitution of actin-based motility with commercially available proteins

Reconstitution of Actin-Based Motility with Commercially Available Proteins

This protocol describes how to produce actin comets on the surfaces of beads using commercially available protein ingredients. Such systems mimic the protrusive structures found in cells, and can be used to examine physiological mechanisms of force production in a simplified...

study of the actin cytoskeleton in live endothelial cells expressing gfp-actin

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory...

monitoring actin disassembly with time-lapse microscopy

Monitoring Actin Disassembly with Time-lapse Microscopy

imaging intranuclear actin rods in live heat stressed drosophila embryos

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The goal of this protocol is to inject Rhodamine-conjugated globular actin into Drosophila embryos and image intranuclear actin rod assembly following heat...

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

imaging of podocytic proteins nephrin, actin, and podocin with expansion microscopy

Imaging of Podocytic Proteins Nephrin, Actin, and Podocin with Expansion Microscopy

The presented method enables visualization of fluorescently labeled cellular proteins with expansion microscopy leading to a resolution of 70 nm on a conventional...

reconstituting and characterizing actin-microtubule composites with tunable motor-driven dynamics and mechanics

Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and Mechanics

This paper presents protocols for engineering and characterizing tunable three-dimensional composite networks of co-entangled actin filaments and microtubules. Composites undergo active restructuring and ballistic motion, driven by myosin II and kinesin motors, and are tuned by the relative concentrations of actin, microtubules, motor proteins, and passive crosslinkers.

the gliding actin filament assay: an in vitro motility assay to study translocation of actin filaments on immobilized myosins

In this video, we perform an in vitro motility assay to analyze the actin-myosin interactions. The gliding movement of actin filaments on immobilized myosin molecules is visualized using fluorescence...

The Gliding Actin Filament Assay: An In Vitro Motility Assay to Study Translocation of Actin Filaments on Immobilized Myosins

aip1p dynamics are altered by the r256h mutation in actin

Aip1p Dynamics Are Altered by the R256H Mutation in Actin

Disease-causing mutations in actin can alter cytoskeletal function. Cytoskeletal dynamics are quantified through imaging of fluorescently tagged proteins using total internal fluorescence microscopy. As an example, the cytoskeletal protein, Aip1p, has altered localization and movement in cells expressing the mutant actin isoform,...

probing myosin ensemble mechanics in actin filament bundles using optical tweezers

Probing Myosin Ensemble Mechanics in Actin Filament Bundles Using Optical Tweezers

Formation of actomyosin bundles in vitro and measuring myosin ensemble force generation using optical tweezers is presented and...

using plustiptracker software to measure microtubule dynamics in xenopus laevis growth cones

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Load 8 square coverslips onto a rack, and thoroughly wash them with approximately 2 to 5 milliliters of 200-proof ethanol, followed by 2 to 5 milliliters of distilled water. Then, dry the coverslips completely using a filtered air-line or...

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Recording Large-scale Neuronal Ensembles with Silicon Probes in the Anesthetized Rat

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of behaviors from the spiking activity of its neurons. One of the most effective methods to monitor spiking activity from a large number of neurons in multiple local neuronal circuits simultaneously is by using silicon electrode arrays1-3.
Action potentials produce large transmembrane voltage changes in the vicinity of cell somata. These output signals can be measured by placing a conductor in close proximity of a neuron. If there are many active (spiking) neurons in the vicinity of the tip, the electrode records combined signal from all of them, where contribution of a single neuron is weighted by its 'electrical distance'. Silicon probes are ideal recording electrodes to monitor multiple neurons because of a large number of recording sites (+64) and a small volume. Furthermore, multiple sites can be arranged over a distance of millimeters, thus allowing for the simultaneous recordings of neuronal activity in the various cortical layers or in multiple cortical columns (Fig. 1). Importantly, the geometrically precise distribution of the recording sites also allows for the determination of the spatial relationship of the isolated single neurons4. Here, we describe an acute, large-scale neuronal recording from the left and right forelimb somatosensory cortex simultaneously in an anesthetized rat with silicon probes (Fig. 2).

Extracellular recordings of neuronal activity using silicon probes in the anesthetized rat will be described. This technique allows information to be obtained across multiple brain areas from more than 100 neurons simultaneously. It provides information with single cell resolution about neuronal ensembles dynamics in multiple local circuits.

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

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.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

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 &#8220;calcium roadmap&#8221; 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.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

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 ...

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...