Name: DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord (Video) | JoVE Journal
Uploaded: 2011-12-13T12:00:00.000+00:00
Duration: 9 min 14 s
Description: Learn to visualize axonal branching patterns of DRG neurons using DiI labeling in mouse embryonic spinal cord whole mounts. This method enables analysis of T- or Y-shaped bifurcation at the dorsal root entry zone.

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5 minutes

The authors wish to thank Dr. Alistair Garratt (Max Delbr&uuml;ck Center, Berlin) for helpful comments. This work was supported by the collaborative research center (SFB665) of the German Research Council (DFG).

1. Dissection procedure
Note: Experimental use of mice should follow officially approved guidelines for the care and use of laboratory animals.
<ol>
	<li>Before preparation, set up your dissecting microscope and lay out the surgical instruments needed for dissection including large and small scissors, large toothed forceps, curved forceps and four sets of Dumont No.5 dissecting forceps (two of which have inside-polished tips) (for details see the table of specific reagents and equipment). Place a sheet of filter paper in a 100-mm Sylgard-coated Petri dish. Pour cold PBS in the wells of a 12-well plate, a 100-mm petri dish and a 12-ml tube and leave on ice. Pipette 2ml of fixation buffer (4% paraformaldehyde in PBS, pH 7.4) in each well of a second 12-well plate and place on ice.</li>
	<li>After anesthetization, sacrifice the timed pregnant dam at E12.5 (detection of vaginal plug was designated as E0.5), place the mouse with its ventral side up on three sheets of paper towels and soak the abdominal area with 70% ethanol.</li>
	<li>Open the abdominopelvic cavity, isolate the bilateral uterine horns and transfer them into one of the 100-mm dishes with ice-cold PBS.</li>
	<li>Under microscopic control, make a longitudinal incision of the uterine wall with small, straight scissors and cut away each amniotic sac from the placenta.</li>
	<li>Peel away the amniotic sac from each embryo and cut the umbilical cord.</li>
	<li>Using small scissors, decapitate the embryos and put the heads or a part of them aside for the isolation of genomic DNA if genotyping should be required. Subsequently, transfer the torsi to the respective wells of the 12-well plate with cold PBS.</li>
	<li>Wet the filter paper in the Sylgard dish with a few drops of PBS and position an embryonic torso with its dorsal side up on the paper. For better stability, straighten its tail and extremities away from the body.</li>
	<li>Working under a dissecting microscope with a magnification of approximately 16x, carefully pinch the skin of the embryo above the spinal cord with two pairs of fine tipped forceps and gently tear it apart. Beginning in the middle, proceed first towards the tail and then resuming from the middle towards the anterior side.</li>
	<li>Wet the embryo from time to time with two or three drops of PBS to prevent it from drying.</li>
	<li>To prevent that DRG are teared off from the spinal cord when it is removed from the embryo detach the DRG and the spinal cord from the surrounding cartilaginous vertebral column by horizontal sliding movements with the blade of a fine forceps with inside-polished tips. Starting in the middle of the embryo's right side, work your way towards the tail and then to the anterior end. Afterwards, repeat the procedure on the other side. Be careful not to rip off the associated DRG.</li>
	<li>To detach completely the loosened spinal cord from the embryo, pick up its cervical part with fine forceps and pull it out in one piece towards its caudal end.</li>
	<li>Submerge the isolated spinal cord with attached DRG in a well of the 12-well plate filled with fixation buffer and proceed with the preparation of spinal cords from the remaining embryos.</li>
	<li>Fix spinal cords at least two hours on ice.</li>
</ol>
2. DiI-labeling of DRG neurons
<ol>
	<li>For each spinal cord to be labeled, prepare two glass needles with a micropipette puller. We use a Sutter model P-97 electrode puller (programme setting: 1. HEAT=625, VEL= 35, TIME=175; 2. HEAT=600, VEL= 25, TIME=175; 3. HEAT=630, VEL= 30, TIME=150).</li>
	<li>Under microscopic control dip the tip of each glass needle three to five times in a 5 % (w/v) solution of DiI in ethanol. Evaporation of the ethanol creates a thin layer of DiI crystals on the needle.</li>
	<li>Working under a dissecting microscope with a magnification of approximately 40x, place a spinal cord with its ventral side up on a slide. Using spring scissors, open the cord on the ventral side at its whole length by cutting through the floorplate to allow the later mounting of the preparation in an inverted open-book mode (see section C).</li>
	<li>Position the spinal cord with the dorsal side up on a slide. Then manually approach the cord with the DiI-covered tip of the glass needle under microscopic visualization and carefully pierce every second DRG on one side of the spinal cord. Almost no traces of DiI should be visible in the pierced DRG to ensure the labeling of only a small number of neurons. Take a second needle for the other side of the cord. Aiming only every second DRG avoids an overlap of labeled axonal projections from neighboring DRG which might complicate the differentiation of individual axons.</li>
	<li>Return the spinal cord to the fixation buffer and incubate in the dark for at least six hours at ambient temperature or overnight at 4 °C to give time for the dye to diffuse along the axon within the plasma membrane. Allow for a longer incubation period (up to two days at 4 °C) if the analysis of collateral growth into the spinal cord is desired.</li>
</ol>
3. Mounting and microscopic analysis
<ol>
	<li>Following dye diffusion, position a single spinal cord with its dorsal side down on a coverslip in a drop of PBS. Take care that the attached DRG are correctly oriented laterally and do not intermingle. Aspirate excess fluid and flatten out the cord in an inverted open-book mode using forceps.</li>
	<li>Mount the preparation on a microscope slide using PBS.</li>
	<li>To prevent the increase of unwanted background staining fluorescence microscopic analysis of labeled axonal projections should be carried out the day of mounting. Until then keep the slides in the dark at 4 °C.</li>
</ol>
4. Representative Results:
The spinal cord of the mouse receives afferent projections from 8 pairs of cervical, 13 pairs of thoracic, 5 pairs of lumbar and 4 pairs of sacral DRG totaling to 60 spinal ganglia. After some training a spinal cord with most DRG still attached can be isolated from an embryo in under five minutes. This procedure is suited for the isolation of spinal cords with attached DRG from E11.5 to E13.5 mouse embryos. However, best results are achieved from E12.5. Exemplary outcomes of the labeling procedure described here are shown in Figure 3. The labeling of DRG over the entire length of the spinal cord may then be used to quantify axonal branching behaviour at different vertebral levels (Fig. 4).
<img alt="Neuronal growth diagram; Bifurcation vs interstitial branching processes in neural development." src="/files/ftp_upload/3667/3667fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. Scheme depicting the two principal modes of axonal branching. (A) Branching by the activities of the growth cone that could lead to a bifurcation - as indicated here - as well as complex terminal arbors or (B) collateral formation at the axon shaft (interstitial branching). The latter mode is the dominating branching type of projecting cortical and thalamocortical axons6,7.
<img alt="Neuronal branching diagram; axon bifurcation, stem axon, DRG neuron, cutaneous pathways." src="/files/ftp_upload/3667/3667fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2. Afferent projections of DRG neurons into the embryonic spinal cord display both types of axonal branching: axons first branch at the DREZ by bifurcation (1) and from the resulting daughter branches collaterals form after a waiting period by interstitial branching (2).
<img alt="Spinal cord sections, dorsal root ganglia (DRG) diagram; neuronal morphology; histological analysis." src="/files/ftp_upload/3667/3667fig3.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
Figure 3. Visualization of single axonal trajectories of embryonic mouse DRG neurons. (A) Spinal cord with attached DRG prepared from an E12.5 mouse embryo. (Scale bar, 1 mm.) (B-D) Dorsal views of DiI-labeled DRG of wild-type mice at increasing magnification are shown. In B every second DRG is labeled by DiI. Fluorescence images are inverted, caudal is at the left and in C and D, lateral is at the bottom. In C a small number of axons is labeled and at higher magnification the presence of T-like branches can be identified in the DREZ of the spinal cord. (Scale bars, 250 μm (B), 100 μm (C) and 50 μm (D).)
<img alt="Bar chart showing spinal region data for C-turns, R-turns, and T-branches; analysis of WT and mutants." src="/files/ftp_upload/3667/3667fig4.jpg" data-altupdatedat="1787630408000" data-alt="Figure 4"> 
Figure 4. Quantification of T-shaped branches, single rostral or caudal turns in wild-type and C-type natriuretic peptide (CNP)-deficient mice at E13.5. The numbers of single axons counted are given in brackets for different trunk levels for each genotype. C- or R-turns - growth only in caudal or rostral direction, respectively.
<img alt="CNP signaling pathway diagram with wild-type and CNP-LacZ mouse embryo fluorescence images." src="/files/ftp_upload/3667/3667fig5.jpg" data-altupdatedat="1787630408000" data-alt="Figure 5"> 
Figure 5. A cGMP signaling pathway triggers sensory axon bifurcation at the DREZ of the spinal cord. (A) Scheme of the cGMP signaling pathway composed of the ligand CNP, the receptor guanylyl cyclase Npr2 and the serine/threonine kinase cGKIα in embryonic DRG neurons. Npr2 generates cGMP from GTP upon stimulation by CNP. (B) DiI tracing of single axons of DRG neurons in wild-type and CNP-deficient mice. (Scale bar, 25 μm.)

<ol><li>Honig, M. G., Hume, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dil+and+diO%3A+versatile+fluorescent+dyes+for+neuronal+labelling+and+pathway+tracing%5BTitle%5D)+AND+%22Trends.+Neurosci%22%5BJournal%5D)">Dil and diO: versatile fluorescent dyes for neuronal labelling and pathway tracing.</a> Trends. Neurosci. 12 (9), 333-333 (1989).</li>
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<li>Schmidt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+receptor+guanylyl+cyclase+Npr2+is+essential+for+sensory+axon+bifurcation+within+the+spinal+cord%5BTitle%5D)+AND+%22J.+Cell+Biol%22%5BJournal%5D)">The receptor guanylyl cyclase Npr2 is essential for sensory axon bifurcation within the spinal cord.</a> J. Cell Biol. 179 (2), 331-331 (2007).</li>
<li>Gibson, D. A., Ma, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developmental+regulation+of+axon+branching+in+the+vertebrate+nervous+system%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Developmental regulation of axon branching in the vertebrate nervous system.</a> Development. 138 (2), 183-183 (2011).</li>
<li>O'Leary, D. D., Terashima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cortical+axons+branch+to+multiple+subcortical+targets+by+interstitial+axon+budding%3A+implications+for+target+recognition+and+%26quot%3Bwaiting+periods%26quot%3B%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Cortical axons branch to multiple subcortical targets by interstitial axon budding: implications for target recognition and &quot;waiting periods&quot;.</a> Neuron. 1 (10), 901-901 (1988).</li>
<li>Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diverse+modes+of+axon+elaboration+in+the+developing+neocortex%5BTitle%5D)+AND+%22PLoS.+Biol%22%5BJournal%5D)">Diverse modes of axon elaboration in the developing neocortex.</a> PLoS. Biol. 3 (8), e272-e272 (2005).</li>
<li>Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vital+imaging+and+ultrastructural+analysis+of+individual+axon+terminals+labeled+by+iontophoretic+application+of+lipophilic+dye%5BTitle%5D)+AND+%22J.+Neurosci.+Methods%22%5BJournal%5D)">Vital imaging and ultrastructural analysis of individual axon terminals labeled by iontophoretic application of lipophilic dye.</a> J. Neurosci. Methods. 93 (1), 13-13 (1999).</li>
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<li>Zhao, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulate+axon+branching+by+the+cyclic+GMP+pathway+via+inhibition+of+glycogen+synthase+kinase+3+in+dorsal+root+ganglion+sensory+neurons%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Regulate axon branching by the cyclic GMP pathway via inhibition of glycogen synthase kinase 3 in dorsal root ganglion sensory neurons.</a> Journal of Neuroscience. 29 (5), 1350-1350 (2009).</li>
<li>Zhao, Z., Ma, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+axonal+development+by+natriuretic+peptide+hormones%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.+S.+A%22%5BJournal%5D)">Regulation of axonal development by natriuretic peptide hormones.</a> Proc. Natl. Acad. Sci. U. S. A. 106 (42), 18016-18016 (2009).</li>
<li>Schmidt, H., Rathjen, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Signalling+mechanisms+regulating+axonal+branching+in+vivo%5BTitle%5D)+AND+%22Bioessays%22%5BJournal%5D)">Signalling mechanisms regulating axonal branching in vivo.</a> Bioessays. , (2010).</li>
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The authors have nothing to disclose.

Stereotyped projection patterns comprising both types of axonal branch formation together with ease of preparation in combination with the use of fixed tissue for DiI labeling makes the embryonic spinal cord with attached DRG a favourable model to study axonal branching. The application of minute amounts of DiI using coated glass needles allows - in contrast to the bulk labeling of DRG - the visualization of small groups of DRG neurons and thereby the analysis of individual axons and their branching patterns. The describ...

<table><tbody><tr><td>Stereomicroscope Stemi DRC</td><td>Carl Zeiss, Inc.</td><td></td><td></td></tr><tr><td>Phosphate-buffered solution (PBS)</td><td>Biochrom AG</td><td>L182-50</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Merck &amp;amp; Co., Inc.</td><td>8.18715.1000</td><td></td></tr><tr><td>Standard surgical scissors</td><td>Fine Science Tools</td><td>14001-13</td><td></td></tr><tr><td>Toothed standard forceps</td><td>Fine Science Tools</td><td>11021-14</td><td></td></tr><tr><td>Extra fine iris scissors</td><td>Fine Science Tools</td><td>14088-10</td><td></td></tr><tr><td>Curved forceps</td><td>Fine Science Tools</td><td>11003-13</td><td></td></tr><tr><td>Dumont No.5 fine tips forceps</td><td>Fine Science Tools</td><td>11254-20</td><td></td></tr><tr><td>Dumont No.5 mirror finish forceps</td><td>Fine Science Tools</td><td>11252-23</td><td></td></tr><tr><td>Vannas-T&amp;#252;bingen spring scissors</td><td>Fine Science Tools</td><td>15008-08</td><td></td></tr><tr><td>Filter paper</td><td>Fisher Scientific</td><td>FB59041</td><td></td></tr><tr><td>Sylgard 184</td><td>World Precision Instruments, Inc.</td><td>SYLG184</td><td></td></tr><tr><td>100-mm Petri dishes</td><td>Greiner Bio-One</td><td>663102</td><td></td></tr><tr><td>12-ml polypropylene tube</td><td>Carl Roth GmbH</td><td>ECO3.1</td><td></td></tr><tr><td>12-well culture plate</td><td>BD Biosciences</td><td>35-3043</td><td></td></tr><tr><td>Ethanol </td><td>Merck &amp;amp; Co., Inc.</td><td>1.00983.2500</td><td></td></tr><tr><td>Flaming/Brown micropipette puller P-97</td><td>Sutter Instrument Co.</td><td></td><td></td></tr><tr><td>Borosilicate glass capillaries</td><td>Harvard Apparatus</td><td>30-0066</td><td></td></tr><tr><td>DiI (1,1&amp;#8242;-Dioctadecyl-3,3,3&amp;#8242;,3&amp;#8242;-tetramethyl&lt;strong&gt;-&lt;/strong&gt;indocarbocyanine perchlorate)</td><td>Sigma-Aldrich</td><td>468495</td><td></td></tr><tr><td>Microscope slides SuperFrost Plus</td><td>Carl Roth GmbH</td><td>H867.1</td><td></td></tr><tr><td>Glass cover slips</td><td>Carl Roth GmbH</td><td>1870.2</td><td></td></tr></tbody></table>

dii-labeling of drg neurons to study axonal branching in a whole mount preparation of mouse embryonic spinal cord

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 ...

The overall goal of this procedure is to visualize the branching patterns of individual axons of DRG neurons in a whole mount preparation of mouse embryonic spinal cord. In order to identify molecules involved in axonal branching, the projections of DRG neurons into the mouse spinal cord offer a simple, easy, accessible experimental system to study axonal branching. When ENT axons of DRG neurons reach the dorsal root entry zone of the cord, they display a stereotype pattern of T or Y shaped bifurcation.The two resulting daughter axons then proceed in rostral or cordal directions, respectively at the dorsal lateral margin of the cord. Only after a waiting period collateral sprout from these stem axons to penetrate the gray matter and project a relay neurons in specific laminate of the spinal cord where terminal arborization occurs. This video demonstrates first how to dissect the spinal cord.With its attached DRG from an embryonic day 12.5 mouse following fixation of the specimen minute mounts of dii are applied to the 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 branching of axons is an important aspect in the development of neural connectivity. The method presented here allows a comparison of branching patterns of Y type with those of mutant mice, and thereby enables the identification of molecules that regulate axonal branching.First, set up the dissecting microscope and lay out the surgical instruments needed for dissection. Place a sheet of filter paper in a 100 millimeter silk guard coated Petri dish. Next, pour cold PBS In the wells of a 12 well plate a 100 millimeter Petri dish and a 12 milliliter tube and leave on ice.Pipette two milliliters of 4%paraldehyde fixation buffer in each well of a second 12 well plate and place on ice. After isolating the bilateral uterine horns from a mouse, transfer them into the 100 millimeter dish with ice cold PBS. After decapitating the embryos transfer the toci of the embryos to the 12.Well plate wet the filter paper in the S guard dish with a few drops of PBS and position an embryonic torso with its dorsal side up on the paper for better stability. Straighten its tail and extremities away from the body working under dissecting microscope with a magnification of approximately 16 times. Carefully pinch the skin of the embryo above the spinal cord with two pairs of fine tipped forceps beginning in the middle.Gently tear the skin first towards the tail, and then resuming from the middle towards the anterior side. Wet the embryo from time to time with two or three drops of PBS to prevent it from drying to ensure that the dorsal root ganglia or DRG are not torn from the spinal cord. When it is removed, detach the DRG and the spinal cord from the surrounding cartilaginous vertebral column by starting in the middle of the embryo's right side and working towards the tail, and then to the anterior end to completely detach the loosened spinal cord from the embryo.Pick up its cervical part with fine forceps and pull it out in one piece towards its cordal.End. Submerge the isolated spinal cord with attached DRG in a well. Of the 12 well plate filled with fixation buffer, and proceed with the preparation of spinal cords from the remaining embryos.Fix the spinal cords for at least two hours on ice for each vinyl cord to be labeled, prepare two glass needles using a micro pipette puller under microscopic control. Dip the tip of each glass needle three to five times in a 5%solution of dye in ethanol to create a thin layer of dai crystals on the needle, working under a dissecting microscope with a magnification of approximately 40 times. Place a spinal cord with its ventral side up on a slide using spring scissors.Open the cord on the ventral side at its whole length by cutting through the floor plate to allow for inverted open book mounting. Position the spinal cord with the dorsal side up on the slide. Then manually approach the cord with the dai covered tip of the glass needle and carefully pierce every second DRG on one side of the spinal cord.Almost no traces of dai should be visible in the pierce DRG to ensure the labeling of only a small number of neurons using the second needle. Repeat for the other side of the cord, aiming only every second. DRG avoids an overlap of labeled axonal projections from neighboring, which might complicate the differentiation of individual axons.Return the spinal cord to the fixation buffer and incubate in the dark for at least six hours at ambient temperature or overnight at four degrees Celsius to give time for the diet to diffuse along the axon within the plasma membrane. Following diet diffusion position a single spinal cord with its dorsal side down on a cover slip in a drop of PBS aspirate excess fluid, and flatten out the cord in an inverted open book mode. Using forceps.Take care that the attached DRG are correctly oriented laterally and do not intermingle mount the preparation on a microscope slide using PBS to prevent the increase of unwanted background staining. Fluorescence microscopic analysis of the labeled axonal projections should be carried out on the day of mounting. Until then, keep the slides in the dark at four degrees Celsius.Here, dorsal views of Dai labeled DRG from wild type mice at increasing magnification are shown as described every second. DRG has been labeled by Dai. Here, a small number of axons are labeled and at a higher magnification, the presence of T like branches can be identified in the dorsal root entry zone of the spinal cord.T-shaped branches. Single rostral R or cordal C turns are quantified here at different vertebral levels in both wild type and C type natriuretic peptide CMP deficient mice. At E 13.5.The total number of neurons counted is shown in bracket and the percentage of trajectory type is shown below. In contrast to the wild type axons of DRG neurons in CMP signaling mutants turn only in one direction instead of bifurcating at the dorsal root entry zone of the spinal cord. This method helped us to identify a CGP signaling cascade that is essential for the bifurcation of sensory accident as the dorsal root entry zone.In contrast to the wild type accents of cgm, P signaling movement only turn in one direction instead of bifurcating at the dors root entry zone. These studies also revealed that co formation is not affected in CG P signaling mutants, indicating that the second type of axonal branching observed in DGen neurons is differently regulated.

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Research

JoVE Journal

Neuroscience

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...

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Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

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Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

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An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

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Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

In this report, the advantages of organotypic cultures and dissociated primary cultures of mouse-derived dorsal root ganglia are highlighted to investigate a wide range of mechanisms associated with neuron-glial interaction, neuroplasticity, neuroinflammation, and response to viral infection.

Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Presented here is the development for consistently acquiring high-quality dorsal root ganglion cryostat sections.

A Procedure for Mouse Dorsal Root Ganglion Cryosectioning

Dorsal root ganglia (DRG) contain primary sensory neuron cell bodies and relay peripheral sensory signals to the spinal cord. This protocol describes a method for mouse DRG dissection that supports standardized isolation of structurally intact ganglia, anatomical segmental identification, intuitive guidance for novice users, and reduced procedural tissue injury.

Dissection and Isolation of Mouse Dorsal Root Ganglia

Learn to visualize axonal branching patterns of DRG neurons using DiI labeling in mouse embryonic spinal cord whole mounts. This method enables analysis of T- or Y-shaped bifurcation at the dorsal root entry zone.

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

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