Name: a simple neuronal mechanical injury methodology to study drosophila motor neuron degeneration
Uploaded: 2017-07-19T15:00:00
Description: Watch this Scientific Journal Video about A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration at JoVE.com

We would like to thank all members of the Keller and Magie Labs at Quinnipiac University for helpful suggestions. In particular, we would like to thank Barron L. Lincoln II for development of this injury assay within the Keller Lab. We would also like to thank Quinnipiac University College of Arts and Science Grant-In-Aid awarded to L.C. Keller.

1. Preparation of Reagents and Equipment
<ol>
	<li>Prepare 1x Dissection Buffer (70 mM NaCl, 5 mM KCl, 0.02 mM CaCl2, 20 mM MgCl2, 10 mM NaHCO3, 115 mM sucrose, 5 mM trehalose, 5 mM HEPES;&#160;pH 7.2).</li>
	<li>Prepare 1x Phosphate-Buffered Saline (PBS).</li>
	<li>Prepare 1x PBT using 1x PBS with 0.01% Triton X-100.</li>
	<li>Prepare fruit juice agar plates.14 Briefly, mix 30 g agar in 700 mL of H2O and autoclave. Dissolve 0.5 g of methyl paraben in ...

<ol>
	<li>Bier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophilia,+the+golden+bug,+emerges+as+a+tool+for+human+genetics.">Drosophilia, the golden bug, emerges as a tool for human genetics.</a> Nat. Rev. Genet. 6 (1), 9-23 (2005).</li><li>Ugur, B., Chen, K., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+tools+and+assays+for+the+study+of+human+diseases.">Drosophila tools and assays for the study of human diseases.</a> Dis Model Mech. 9 (3), 235-244 (2016).</li><li>McGurk, L., Berson, A., Bonini, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+an+in+vivo+model+for+human+neurodegenerative+disease.">Drosophila as an in vivo model for human neurodegenerative disease.</a> Genetics. 201 (2), 377-402 (2015).</li><li>Shin, J. E., Cho, Y., Beirowski, B., Milbrandt, J., Cavalli, V., DiAntonio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+leucine+zipper+kinase+is+required+for+retrograde+injury+signaling+and+axonal+regeneration.">Dual leucine zipper kinase is required for retrograde injury signaling and axonal regeneration.</a> Neuron. 74 (6), 1015-1022 (2012).</li><li>Xiong, X., Wang, X., Ewanek, R., Bhat, P., Diantonio, A., Collins, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+turnover+of+the+Wallenda/DLK+kinase+regulates+a+retrograde+response+to+axonal+injury.">Protein turnover of the Wallenda/DLK kinase regulates a retrograde response to axonal injury.</a> J Cell Biol. 191 (1), 211-223 (2010).</li><li>Xiong, X., Collins, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conditioning+lesion+protects+axons+from+degeneration+via+the+Wallenda/DLK+MAP+kinase+signaling+cascade.">A conditioning lesion protects axons from degeneration via the Wallenda/DLK MAP kinase signaling cascade.</a> J Neurosci. 32 (2), 610-615 (2012).</li><li>Mishra, B., Ghannad-Rezaie, M., Li, J., Wang, X., Hao, Y., Ye, B., Chronis, N., Collins, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+microfluidics+chips+for+live+imaging+and+study+of+injury+responses+in+Drosophila+larvae.">Using microfluidics chips for live imaging and study of injury responses in Drosophila larvae.</a> J Vis Exp. (84), e50998 (2014).</li><li>Eaton, B. A., Fetter, R. D., Davis, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynactic+is+necessary+for+synapse+stabilization.">Dynactic is necessary for synapse stabilization.</a> Neuron. 34 (5), 729-741 (2002).</li><li>Eaton, B. A., Davis, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LIM+Kinase1+controls+synaptic+stability+downstream+of+the+type+II+BMP+receptor.">LIM Kinase1 controls synaptic stability downstream of the type II BMP receptor.</a> Neuron. 47 (5), 695-708 (2005).</li><li>Pielage, J., Fetter, R. D., Davis, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+spectrin+is+essential+for+synapse+stabilization.">Presynaptic spectrin is essential for synapse stabilization.</a> Curr Biol. 15 (10), 918-928 (2005).</li><li>Pielage, J., Cheng, L., Fetter, R. D., Carlton, P. M., Sedat, J. W., Davis, G. 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+presynaptic+giant+Ankyrin+stabilizes+the+NMJ+through+regulation+or+presynaptic+microtubules+and+transsynaptic+cell+adhesion.">A presynaptic giant Ankyrin stabilizes the NMJ through regulation or presynaptic microtubules and transsynaptic cell adhesion.</a> Neuron. 58 (2), 195-209 (2008).</li><li>Massaro, C. M., Pielage, J., Davis, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+that+enhance+synapse+stability+despite+persistent+disruption+of+the+spectrin/ankryin/microtubule+cytoskeleton.">Molecular mechanisms that enhance synapse stability despite persistent disruption of the spectrin/ankryin/microtubule cytoskeleton.</a> J Cell Biol. 187 (1), 101-117 (2009).</li><li>Lincoln, B. L., Alabsi, S. H., Frendo, N., Freund, R., Keller, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+neuronal+injury+follows+a+temporal+sequence+of+cellular+events+leading+to+degeneration+at+the+neuromuscular+junction.">Drosophila neuronal injury follows a temporal sequence of cellular events leading to degeneration at the neuromuscular junction.</a> J Exp Neurosci. 9, 1-9 (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+apple+juice-agar+plates.">Drosophila apple juice-agar plates.</a> Cold Spring Harb Protoc. , (2011).</li><li>Brent, J. R., Werner, K. M., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+larval+NMJ+immunohistochemistry.">Drosophila larval NMJ immunohistochemistry.</a> J Vis Exp. (25), (2009).</li><li>Smith, R., Taylor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+and+imaging+of+active+zones+in+the+Drosophila+neuromuscular+junction.">Dissection and imaging of active zones in the Drosophila neuromuscular junction.</a> J Vis Exp. (50), (2011).</li><li>Jan, L., Jan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+to+horseradish+peroxidase+as+specific+neuronal+markers+in+Drosophila+and+in+grasshopper+embryos.">Antibodies to horseradish peroxidase as specific neuronal markers in Drosophila and in grasshopper embryos.</a> Proc. Natl. Acad. Sci. 79 (8), 2700-2704 (1982).</li><li>Lahey, T., Gorczyca, M., Jia, X., Budnik, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+tumor+suppressor+gene+dlg+is+required+for+normal+synaptic+bouton+structure.">The Drosophila tumor suppressor gene dlg is required for normal synaptic bouton structure.</a> Neuron. 13 (4), 823-835 (1994).</li></ol>

The neuronal mechanical injury described earlier and demonstrated here can be used to induce injury/stress in the segmental nerves of Drosophila larvae.4,5,6,14 This experimental technique has been employed previously to dissect the temporal sequence of events leading to neurodegeneration, as well as to examine transcriptional changes in the motor neuron cell bodies post injury.<sup cl...

Neuronal degeneration occurs during normal development and can be caused by the natural aging process, injury, stress, or disease states. Drosophila melanogaster, the common fruit fly, provides a simple and powerful model organism to study neurodegeneration due to the remarkable similarities in the molecular mechanisms that drive the degeneration of neurons. These similarities are highlighted by the fact that approximately 70% of disease-associated human genes have a Drosophila homolog.1 Additionally, numerous assays and technological tools to study human neurodegenerative diseases have been developed and utilized in Drosophila.2,3 Within Drosophila, the neuromuscular junction (NMJ) allows for analysis of both cellular and electrophysiological properties and has proven to be an important system to study neuromuscular disease due to the visible neuron-muscle connections.2 In this study, we describe an in vivo neuron injury assay in Drosophila larvae which allows for the reproducible injury of segmental nerves. This motoneuron injury results in a temporal sequence of cellular events resulting in neurodegeneration at the NMJ 24 h&#160;post injury. The ability to reproducibly injury motoneurons resulting in neurodegeneration has diverse applications such as identifying specific genes required for the degenerative process, the dissection of transcriptional responses to neuronal injury, and the analysis of protective signaling cascades.4,5,6 This method has also been used in combination with microfluidics to study neuronal degeneration and regeneration in live animals.7
We utilize an established quantitative assay to examine motor neuron degeneration at the Drosophila NMJ after mechanical injury. This assay is based on the fact that loss of presynaptic membrane and proteins precedes the disassembly of the subsynaptic reticulum (SSR) characterized by the postsynaptic muscle membrane folds.8,9,10,11,12,13 This assay allows for the quantification of &#34;synaptic footprints&#34; where the pre-synaptic neuron has lost connection to the adjacent postsynaptic muscle. The degenerative process has been shown to be progressive throughout larval development12 and cannot be accounted for by altered synapse development or sprouting.8,9,10,11,12 The advantage of using mechanical injury over preexisting mutations is that it allows for dissection of the temporal sequence of cellular events leading up to neurodegeneration at the NMJ.13

<table>
	<tbody>
		<tr>
			<td>Micro-dissecting scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #3 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11231-30</td>
			<td>Some people prefer size 3, while others prefer size 5</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td>Some people prefer size 3, while others prefer size 5</td>
		</tr>
		<tr>
			<td>CO2 Air Tank</td>
			<td>Tech Air</td>
			<td>UN 1013</td>
			<td>Various tank sizes can be purchased/</td>
		</tr>
		<tr>
			<td>CO2 Anesthetizing Apparatus</td>
			<td>Genesee Scientific</td>
			<td>59-114</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless-steel pins, size 0.1</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td></td>
		</tr>
		<tr>
			<td>SylGard 184 Silicone Elastomer, Base and Curing Agent</td>
			<td>Dow Corning</td>
			<td>3097358-1004</td>
			<td>To pour dissecting plates</td>
		</tr>
		<tr>
			<td>Bouin&#39;s Solution</td>
			<td>Sigma</td>
			<td>HT 10132-1L</td>
			<td>Antibodies should be tested for their efficiency in Bouin&#39;s and PFA</td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde in PBS</td>
			<td>Affymetrix</td>
			<td>FLY-8030-20</td>
			<td>Antibodies should be tested for their efficiency in Bouin&#39;s and PFA</td>
		</tr>
		<tr>
			<td>Dissecting Stereo MIcroscope</td>
			<td>AmScope</td>
			<td>SM-1BZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Light Source</td>
			<td>AmScope</td>
			<td>HL150-AY-220V</td>
			<td></td>
		</tr>
		<tr>
			<td>anti- nc82 antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>nc82-s</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-discs large antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>AF3</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor anti-horseradish peroxidase</td>
			<td>Jackson Immunoresearch</td>
			<td>123-545-021; 123-585-021; 123-605-021</td>
			<td>One can you Alexa Fluor&#174; 488, 594 or 647</td>
		</tr>
		<tr>
			<td>Flystuff Grape Juice Agar Premix</td>
			<td>Genesee Scientific</td>
			<td>47-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Genesee Scientific</td>
			<td>29-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-545-87</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Nalgene Utility Box</td>
			<td>Fisher Scientific</td>
			<td>03-484C</td>
			<td>Used to create humid chamber for larval recovery</td>
		</tr>
	</tbody>
</table>

a simple neuronal mechanical injury methodology to study drosophila motor neuron degeneration

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal degeneration are remarkably similar in humans and invertebrates as are the molecular mechanisms that drive these processes. The fruit fly, Drosophila melanogaster, provides a powerful yet simple genetic model organism to study the cellular complexities of neurodegenerative diseases. In fact, approximately 70% of disease-associated human genes have a Drosophila homolog and a plethora of tools and assays have been described using flies to study human neurodegenerative diseases. More specifically the neuromuscular junction (NMJ) in Drosophila has proven to be an effective system to study neuromuscular diseases because of the ability to analyze the structural connections between the neuron and the muscle. Here, we report on an in vivo motor neuron injury assay in Drosophila, which reproducibly induces neurodegeneration at the NMJ by 24 h. Using this methodology, we have described a temporal sequence of cellular events resulting in motor neuron degeneration. The injury method has diverse applications and has also been utilized to identify specific genes required for neurodegeneration and to dissect transcriptional responses to neuronal injury.

Using the procedure presented here, we have demonstrated that mechanical neuronal injury allows for temporal dissection of neurodegenerative events.14,18 The sequence of events has been previously characterized and begins with an immediate disruption of the cytoskeleton, followed by axonal trafficking defects, an accumulation of ubiquitinated proteins, and subsequent neurodegeneration 24 h post-injury. Prior to injury, WT NMJs sho...

Watch this Scientific Journal Video about A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration at JoVE.com

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

Here we describe a simple and widely accessible method to injure segmental nerves in Drosophila larvae to visualize and quantify neurodegeneration of motor neurons at the neuromuscular junction (NMJ) of third instar larvae.

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal ...

The overall goal of this procedure is to induce mechanical injury of motor neurons in Drosophila larvae. This method can help answer key questions in the field of neurodegenerative research such as the temporal sequence of cellular events leading to neurodegeneration. The main advantages of this technique are that it is inexpensive and uses equipment that most Drosophila labs already have.The implications of this technique extend towards the understanding of the molecular mechanisms that drive neurodegeneration. To begin the protocol, select 10 second or third instar larvae from a vial or bottle of Drosophila containing wandering third instar larvae that have been cultured at 25 degrees Celsius. Use forceps to carefully pick individual larvae up by forceps or a paintbrush without compressing them.Directly place the 10 larvae into a glass dish containing cold 1x PBS to remove any food debris and to decelerate larval motility. Identify third instar larvae by their anterior branch spiracles and second instar larvae by their smaller size and club-like anterior spiracles. Place each larva onto a CO2 anesthetizing apparatus.Under a dissecting microscope, carefully roll each larva onto its dorsal side to visualize the segmental nerves throughout the cuticle. It is essential to carefully roll each larva onto their dorsal side in order to visualize the segmental nerves through the cuticle before injury. Starting at the mouth hooks, position size 5 forceps approximately one-half to two-thirds of the way down the length of the larva.Once positioned, pinch approximately one-third of the ventral cuticle containing the segmental nerves with size 5 foreceps. To ensure that larval segmental nerves are injured, apply sufficient force to crush the segmental nerves but leave the cuticle intact. To determine the correct amount of force, crush 10 larvae and insure the death rate after five hours is under 50%After injury, carefully transfer the larvae to a fruit juice agar plate containing approximately 0.5 grams of yeast paste.Place each larva so its anterior end is on the yeast paste for continued feeding. Place agar plates containing yeast paste and 10 injured larvae onto the bottom of an empty 100 by 15 millimeter Petri dish. Cover the Petri dish with a moist paper towel, ensuring that the paper towel does not touch the larvae or the agar plates.Then place the Petri dish into an airtight container at room temperature. Finally, retrieve larvae for dissection after specified periods post-injury. Prior to injury, wild-type neuromuscular junctions, or NMJs, show the presynaptic active zone marker Brp in apposition to the postsynaptic marker Dlg throughout the entire NMJ.Mechanical injury of segmental nerves can induce moderate to severe neurodegeneration in which butons stained with Dlg now lack the presynaptic active zone protein Brp. After its development, this technique paved the way for researchers studying neuronal injury to explore the temporal sequence of cellular events leading to the neurodegeneration at the neuromuscular junction in Drosophila.

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Research

JoVE Journal

Neuroscience

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...