We gratefully acknowledge the important contributions of Dr. Robert E. Buxbaum in the development of this methodology.

1. Making glass needles.
<ol>
	<li>An adjustable micro-needle puller is used to fabricate needles with a tapered tip about 4 mm in length and that are closed solid beams. As opposed to a long flexible tip, this short 4 mm length limits vibrations of the needle tip during experiments. At the proximal region of the 4 mm fiber, the needle tapers rapidly from the diameter of the glass tubing to 15 &mu;m within 1 mm, while the distal-most 1 mm of the fiber is 2.5 &mu;m in diameter. We use R-6 glass capillary tube, OD 0.9 ...

<ol>
	<li>Zheng, J., Buxbaum, R. E., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+growth+cone+adhesion+to+culture+surfaces+by+micromanipulation.">Measurements of growth cone adhesion to culture surfaces by micromanipulation.</a> J Cell Biol. 127, 2049-2060 (1994).</li><li>Chada, S., Lamoureux, P., Buxbaum, R. E., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytomechanics+of+neurite+outgrowth+from+chick+brain+neurons.">Cytomechanics of neurite outgrowth from chick brain neurons.</a> J Cell Sci. 110, 1179-1186 (1997).</li><li>Heidemann, S. R., Lamoureux, P., Buxbaum, R. E., Haynes, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Neuron in Tissue Culture. , 105-119 (1999).</li><li>Lamoureux, P., Altun-Gultekin, Z. F., Lin, C., Wagner, J. A., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac+is+required+for+growth+cone+function+but+not+neurite+assembly.">Rac is required for growth cone function but not neurite assembly.</a> J Cell Sci. 110, 635-641 (1997).</li><li>Lamoureux, P., Ruthel, G., Buxbaum, R. E., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+tension+can+specify+axonal+fate+in+hippocampal+neurons.">Mechanical tension can specify axonal fate in hippocampal neurons.</a> J Cell Biol. 159, 499-508 (2002).</li><li>Lamoureux, P., Heidemann, S. R., Martzke, N. R., Miller, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+elongation+within+and+along+the+axon.">Growth and elongation within and along the axon.</a> Dev Neurobiol. 70, 135-149 (2010).</li><li>Dennerll, T. J., Lamoureux, P., Buxbaum, R. E., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytomechanics+of+axonal+elongation+and+retraction.">The cytomechanics of axonal elongation and retraction.</a> J Cell Biol. 109, 3073-3083 (1989).</li><li>Heidemann, S. R., Kaech, S., Buxbaum, R. E., Matus, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observations+of+the+mechanical+behaviors+of+the+cytoskeleton+in+living+fibroblasts.">Direct observations of the mechanical behaviors of the cytoskeleton in living fibroblasts.</a> J Cell Biol. 145, 109-122 (1999).</li><li>Yoneda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+Exerted+by+a+Single+Cilium+of+Mytilus-Edulis+.1.">Force Exerted by a Single Cilium of Mytilus-Edulis .1.</a> Journal of Experimental Biology. 37, (1960).</li><li>Bray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+Tension+Produced+by+Nerve-Cells+in+Tissue-Culture.">Mechanical Tension Produced by Nerve-Cells in Tissue-Culture.</a> Journal of Cell Science. 37, 391-410 (1979).</li><li>Pfister, B. J., Iwata, A., Meaney, D. F., Smith, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extreme+stretch+growth+of+integrated+axons.">Extreme stretch growth of integrated axons.</a> J Neurosci. 24, 7978-7983 (2004).</li><li>Fass, J. N., Odde, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensile+force-dependent+neurite+elicitation+via+anti-beta1+integrin+antibody-coated+magnetic+beads.">Tensile force-dependent neurite elicitation via anti-beta1 integrin antibody-coated magnetic beads.</a> Biophys J. 85, 623-636 (2003).</li><li>Yang, S., Saif, M. T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabricated+Force+Sensors+and+Their+Applications+in+the+Study+of+Cell+Mechanical+Response.">Microfabricated Force Sensors and Their Applications in the Study of Cell Mechanical Response.</a> Exp Mech. 49, 135-151 (2009).</li><li>Bernal, R., Melo, F., Pullarkat, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drag+Force+as+a+Tool+to+Test+the+Active+Mechanical+Response+of+PC12+Neurites.">Drag Force as a Tool to Test the Active Mechanical Response of PC12 Neurites.</a> Biophysical Journal. 98, 515-523 (2010).</li><li>Lamoureux, P., Buxbaum, R. E., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+that+growth+cones+pull.">Direct evidence that growth cones pull.</a> Nature. 340, 159-162 (1989).</li><li>Heidemann, S. R., Lamoureux, P., Buxbaum, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+cone+behavior+and+production+of+traction+force.">Growth cone behavior and production of traction force.</a> J Cell Biol. 111, 1949-1957 (1990).</li><li>O'Toole, M., Lamoureux, P., Miller, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+physical+model+of+axonal+elongation:+force,+viscosity,+and+adhesions+govern+the+mode+of+outgrowth.">A physical model of axonal elongation: force, viscosity, and adhesions govern the mode of outgrowth.</a> Biophys J. 94, 2610-2620 (2008).</li><li>Bray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+growth+in+response+to+experimentally+applied+mechanical+tension.">Axonal growth in response to experimentally applied mechanical tension.</a> Dev Biol. 102, 379-389 (1984).</li><li>Heidemann, S. R., Lamoureux, P., Ngo, K., Reynolds, M., Buxbaum, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open-dish+incubator+for+live+cell+imaging+with+an+inverted+microscope.">Open-dish incubator for live cell imaging with an inverted microscope.</a> Biotechniques. 35, 708-708 (2003).</li></ol>

Techniques to apply and measure cellular forces have a long history9. Our method was originally motivated by the work of Dennis Bray, who used glass needles similar to ours to 'tow' neurons at a constant rate using a motorized hydraulic device10. There are many alternative means of applying forces to cells which include: stepper motors11, magnetic beads12, microfabricated beams13 and fluid flows14. The latter are similar to our approach in that the cellula...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>R-6 cap. Tube</td>
<td>&nbsp;</td>
<td>Drummond Scientific Co., Broomall, PA, USA</td>
<td>9-000-3111</td>
<td>R-6 glass OD 0.9mm, ID 0.6 mm, 8"</td>
</tr>
<tr>
<td>BB-CH puller</td>
<td>&nbsp;</td>
<td>Mecanex S.A., Geneva, Switzerland</td>
<td>BB-CH puller</td>
<td>Use Mode 4 Alt by CP=100, PP=10, SP1=1000, SP2=1000</td>
</tr>
<tr>
<td>0.001" Chromel wire</td>
<td>&nbsp;</td>
<td>Omega Engineering, Stamford, CT, USA</td>
<td>SPCH-001-50</td>
<td>unsheathed, themocouple wire, 50ft spool now called Chromega</td>
</tr>
<tr>
<td>0.003" Constatan wire</td>
<td>&nbsp;</td>
<td>Omega Engineering, Stamford, CT, USA</td>
<td>SPCI-003-50</td>
<td>unsheathed, themocouple wire, 50 ft spool</td>
</tr>
<tr>
<td>fine forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools, USA</td>
<td>91150-20</td>
<td>Dumont Inox #5</td>
</tr>
<tr>
<td>universal microscope boom stand</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>76135 or 90430</td>
<td>most brands or types of boom stand will work for this use</td>
</tr>
<tr>
<td>mechanical micromanipulator</td>
<td>&nbsp;</td>
<td>Narishige </td>
<td>M-152</td>
<td>three-axis direct-drive coarse micromanipulator</td>
</tr>
<tr>
<td>hydraulic micromanipulator</td>
<td>&nbsp;</td>
<td>Narishige </td>
<td>MO-203</td>
<td>now available as MMO-203, three movable axis type</td>
</tr>
<tr>
<td>needle holder</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>11520145</td>
<td>set of 3</td>
</tr>
<tr>
<td>single instrument holder</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>11520142</td>
<td>&nbsp;</td>
<tr>
<td>double instrument holder</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>11520143</td>
<td>&nbsp;</td>
<tr>
<td>mechanical micromanipulator</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>39430001</td>
<td>post mount,1 prob holder, RH Model 430001</td>
</tr>
<tr>
<td>joystick mech. micromanipulator</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>11520137</td>
<td>&nbsp;</td>
<tr>
<td>Leica DM IRB</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>&nbsp;</td>
<td>inverted microscope</td>
</tr>
<tr>
<td>Vibraplane isolation table</td>
<td>&nbsp;</td>
<td>Kinetic System, Boston, MA, USA</td>
<td>1200 series</td>
<td>ours is model 1201-02-12</td>
</tr>
<tr>
<td>Ringcubator</td>
<td>&nbsp;</td>
<td>self manufactured see reference 19</td>
<td>&nbsp;</td>
<td>reference 19, requires updated controller listed below</td>
</tr>
<tr>
<td>programable temperature controller</td>
<td>&nbsp;</td>
<td>Instrumart.com</td>
<td>Fuji Electric PXR3</td>
<td>replaces the retired PXV3 temperature controller</td>
</tr>
<tr>
<td>Nikon Diaphot TMD</td>
<td>&nbsp;</td>
<td>Nikon Instruments, Inc.</td>
<td>&nbsp;</td>
<td>inverted microscope, circa 1980</td>
</tr>
<tr>
<td>Nikon SMZ-10 binocular dissecting </td>
<td>&nbsp;</td>
<td>Nikon Instruments, Inc.</td>
<td>&nbsp;</td>
<td>other dissecting microscopes will work</td>
</tr>		</tbody>
		</table>
		</div>

mechanical manipulation of neurons to control axonal development

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the 10-1000 &mu;dyne range1,2. Force measurements are obtained through observation of the Hookean bending of the glass needles, which are calibrated by a direct and empirical method3. Equipment requirements and procedures for fabricating, calibrating, treating, and using the needles on cells are fully described. The force regimes previously used and different cell types to which these techniques have been applied demonstrate the flexibility of the methodology and are given as examples for future investigation4-6. The technical advantages are the continuous 'visualization' of the forces produced by the manipulations and the ability to directly intervene in a variety of cellular events. These include direct stimulation and regulation of axonal growth and retraction7; as well as detachment and mechanical measurements on any type of cultured cell8.

Watch this Scientific Journal Video about Mechanical Manipulation of Neurons to Control Axonal Development at JoVE.com

Mechanical Manipulation of Neurons to Control Axonal Development

Application and direct measurements of forces on neurons in the 2-1000 microdyne range are achieved with high precision using calibrated glass needles. This methodology can be used to control and measure several aspects of axonal development, including axonal initiation, axonal tension, velocity of axonal elongation, and force vectors.

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the ...

mechanical-manipulation-of-neurons-to-control-axonal-development

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Neuroscience

10.4K vues.  Michigan State University, East Lansing. Mesures d'application directe et des forces sur les neurones dans la gamme 2000-1000 Microdyne sont atteints avec une grande précision en utilisant des aiguilles de verre calibré. Cette méthodologie peut être utilisée pour contrôler et mesurer plusieurs aspects du développement axonal, y compris l'initiation axonale, la tension axonale, la vitesse d'élongation des axones, et les vecteurs de la force.

Vidéo: Mechanical Manipulation of Neurons to Control Axonal Development

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

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.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

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&#8217;s disease (AD) and Huntington&#8217;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 &#956;m/sec. This system can be used to investigate mechanisms of disrupted axonal function in AD or HD, as well as other degenerative disorders.

Axonal transport of BDNF, a neurotrophic factor, is critical for the survival and function of several neuronal populations. Some degenerative disorders are marked by disruption of axonal structure and function. We demonstrated the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons.

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle (Mercynorrhina torquata)

The rise of radio-enabled digital electronic devices has prompted the use of small wireless neuromuscular recorders and stimulators for studying in-flight insect behavior. This technology enables the development of an insect-machine hybrid system using a living insect platform described in this protocol. Moreover, this protocol presents the system configuration and free flight experimental procedures for evaluating the function of the flight muscles in an untethered insect. For demonstration, we targeted the third axillary sclerite (3Ax) muscle to control and achieve left or right turning of a flying beetle. A thin silver wire electrode was implanted on the 3Ax muscle on each side of the beetle. These were connected to the outputs of a wireless backpack (i.e., a neuromuscular electrical stimulator) mounted on the pronotum of the beetle. The muscle was stimulated in free flight by alternating the stimulation side (left or right) or varying the stimulation frequency. The beetle turned to the ipsilateral side when the muscle was stimulated and exhibited a graded response to an increasing frequency. The implantation process and volume calibration of the 3 dimensional motion capture camera system need to be carried out with care to avoid damaging the muscle and losing track of the marker, respectively. This method is highly beneficial to study insect flight, as it helps to reveal the functions of the flight muscle of interest in free flight.

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle Mercynorrhina torquata

This protocol describes the process of constructing an insect-machine hybrid system and carrying out wireless electrical stimulation of the flight muscles required to control the turning motion of a flying insect.

The rise of radio-enabled digital electronic devices has prompted the use of small wireless neuromuscular recorders and stimulators for studying in-flight ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

Visualizing Axonal Growth Cone Collapse and Early Amyloid &#946; Effects in Cultured Mouse Neurons

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the brains of AD patients, these do not improve memory functions. Since A&#946; aggregates in the brain before the appearance of memory impairments, targeting A&#946; may be inefficient for treating AD patients who already exhibit memory deficits. Therefore, downstream signaling due to A&#946; deposition should be blocked before AD development. A&#946; induces axonal degeneration, leading to the disruption of neuronal networks and memory impairments. Although there are many studies on the mechanisms of A&#946; toxicity, the source of A&#946; toxicity remains unknown. To help identify the source, we propose a novel protocol that uses microscopy, gene transfection, and live cell imaging to investigate early changes caused by A&#946; in axonal growth cones of cultured neurons. This protocol revealed that A&#946; induced clathrin-mediated endocytosis in axonal growth cones followed by growth cone collapse, demonstrating that inhibition of endocytosis prevents A&#946; toxicity. This protocol will be useful in studying the early effects of A&#946; and may lead to more efficient and preventative AD treatment.

Visualizing Axonal Growth Cone Collapse and Early Amyloid &amp;#946; Effects in Cultured Mouse Neurons

Here a protocol to investigate the early effects of amyloid-&#946; (A&#946;) in the brain is presented. This shows that A&#946; induces clathrin-mediated endocytosis and collapse of axonal growth cones. The protocol is useful in studying early effects of A&#946; on axonal growth cones and may facilitate prevention of Alzheimer's disease.

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the ...