Name: visualization of motor axon navigation and quantification of axon arborization in mouse embryos using light sheet fluorescence microscopy
Uploaded: 2018-05-11T15:00:00
Description: Watch this Scientific Journal Video about Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy at JoVE.com

LSFM experiments and data analysis were performed in part using the advanced optical microscopes of the Division of Instrument Service in Academia Sinica and with the assistance of Ms. Shu-Chen Shen. We thank Ms. Sue-Ping Lee from the Imaging Core Facility of IMB for considerable technical assistance with Imaris image analysis. The IMB&#39;s Scientific English Editing Core reviewed the manuscript. This work is funded by Academia Sinica Career Development Award (CDA-107-L05), MoST (104-2311-B-001-030-MY3), and NHRI (NHRI-EX106-10315NC).

All of the live animals were kept in a specific pathogen free (SPF) animal facility, approved and overseen by IACUC of Academia Sinica.
1. Fixation
<ol>
	<li>Collect embryos of embryonic day 12.5 (E12.5) then decapitate and eviscerate them.</li>
	<li>Fix the embryos individually in 24-well plates with 1 mL/well of freshly prepared 4% paraformaldehyde in 1x phosphate buffer saline (PBS) overnight. Incubate at 4 &#176;C on a shaker.</li>
	<li>Wash the fixed embryos at least 3x, each for 5-10 m...

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Several steps in the protocol may be subjected to changes under certain circumstances. For example, the duration of fixation depends on the age of the embryos, varying from 2 h to 1 or more days using freshly-made paraformaldehyde. Since fixation is carried out prior to whole mount immunostaining, for antibodies that are sensitive to protein crosslinking, methanol can be used as an alternative fixative agent. For a high signal-to-noise ratio upon staining, it is necessary to optimize the detergent concentration (between ...

Spinal MNs are the part of the central nervous system but innervate peripheral muscles to control movement. In the developing spinal cord, MN progenitors (pMNs) are established according to the signals emanating from the notochord and adjacent somites. All differentiated post-mitotic MNs are then generated from pMNs, eventually giving rise to a series of MN subtypes along the rostrocaudal axis of the spinal cord1,2. Spinal MNs are topographically and anatomically well organized. Their morphological arrangement correlates with the position of their respective target in the periphery3. Upon reaching their muscle targets, axons receive cellular and exogenous neurotrophic factors that induce them to extend and branch further into muscles. Innervation and branching defects may contribute to the failure to form neuromuscular junctions (NMJ). For example, glial-derived neurotrophic factors (GDNF)-induced Pea3 is indispensable for axon arborization into cutaneous maximus (CM) and latissimus dorsi (LD) muscles4,5. In addition, DINE knockout mouse embryos show defective arborization of phrenic nerves in the diaphragm, causing respiratory failure and mortality immediately after birth6,7. Therefore, this last step of MN maturation (i.e., axonal projection and branching) is critical to ensure communication between neurons and target cells.
To view arborization patterns, researchers normally conduct confocal imaging or two-photon fluorescence light microscopy of sectioned or whole-mount samples8,9,10. Both of these microscopy techniques generate acceptable resolution and depth penetration. Two-photon fluorescence light microscopy involves excitation of fluorophores by simultaneous absorption of two lower-energy photons11. Since two-photon excitation uses near-infrared radiation, the decreased excitation frequency contributes to reduced scattering and better tissue penetration up to 1 mm in tissue, thus allowing imaging with greater depth. Confocal microscopy removes by filters the out-of-focus signals and only collects light within the focal plane12. With this approach, images of samples from different focal planes can be combined to produce a three-dimensional (3D) image via a Z-stack function. Nevertheless, signal intensity is reduced as most of the light is blocked and high numerical apertures obscure the depth-of-field. More importantly, both techniques contribute to the severe photodamage and phototoxicity since the whole specimen receives illumination even when only one plane is imaged at a given time.
To circumvent these shortcomings, LSFM has become a favored alternative, with the advantages of being fast, light-efficient, and less phototoxic13,14. In addition, LSFM allows multi-view imaging. This approach is particularly suited to visualizing motor axons and their dispersing terminals as they spread through 3D space. LSFM outperforms the other two options because samples are mounted on a stage that allows rotation around a vertical axis and movements along the x, y, and z axes. This set-up not only allows for a minimally blocked view of the sample but also the choice of a desirable illumination path, a shortcoming of two-photon and confocal microscopy, both of which require mounting of samples on a flat slide. Therefore, LSFM is the most suitable tool for 3D imaging of axon arborization and for quantification of motor nerve terminals in mouse embryos.

<table>
	<tbody>
		<tr>
			<td>Hb9::GFP</td>
			<td>The Jackson labortory</td>
			<td>005029</td>
			<td>Collect embryos of embryonic day 13.5 (E13.5)</td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde (PFA)</td>
			<td></td>
			<td></td>
			<td>For 200ml: Add 20ml 10X PBS, 8g PFA in ddH2O. Adjust pH to 7.4 with NaOH (10N). Filter sterilize and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Phosphate buffer saline 10X (PBS 10X)</td>
			<td></td>
			<td></td>
			<td>For 1L: Add 80 g NaCl, 2 g KCl,14.4g Na2HPO4, 2.4 g KH2PO4 and top up with ddH2O. Autoclave and store at RT.</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ThermoFisher</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheep polyclonal anti-GFP</td>
			<td>AbD Serotec</td>
			<td>4745-1051</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey anti-sheep</td>
			<td>Invitrogen</td>
			<td>A-11015</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>RapiClear 1.47 clearing reagent</td>
			<td>SunJin Lab</td>
			<td>RC147001</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5ml micro tube</td>
			<td>Sarstedt</td>
			<td>72.690.001</td>
			<td></td>
		</tr>
		<tr>
			<td>24 wells plate</td>
			<td>ThermoFisher</td>
			<td>142475</td>
			<td></td>
		</tr>
		<tr>
			<td>5 SA Tweezer</td>
			<td>ideal-tek</td>
			<td>3480641</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors striaght sharp/sharp</td>
			<td>Aesculap</td>
			<td>BC110R</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgery Scalpels, single use</td>
			<td>Aesculap</td>
			<td>BA365</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Nikon</td>
			<td>SMZ800</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>TKS</td>
			<td>RS-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Lightsheet Z.1 microscope</td>
			<td>Carl Zeiss Microscopy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris 8.4.0 image analysis software</td>
			<td>Bitplane, Zurich, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B6.Cg-Tg(Hlxb9-GFP)1Tmj/J (Hb9::GFP mice)</td>
			<td>The Jackson Laboratory</td>
			<td>005029</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of motor axon navigation and quantification of axon arborization in mouse embryos using light sheet fluorescence microscopy

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

LSFM provides detailed 3D visualization of MN trajectory and axon arborization in mouse embryos. Under bright field, tissues appear completely transparent after being immersed in the commercial clearing reagent. No shrinkage or swelling of the sample was noticed after up to one week of storage in clearing reagent before imaging. Under&#160;fluorescence channel, motor neurons are labeled with transgenically expressed GFP&#160;(Movie 1). In some instance, details of the axo...

Watch this Scientific Journal Video about Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy at JoVE.com

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Here, we describe a protocol for visualizing motor neuron projection and axon arborization in transgenic Hb9::GFP mouse embryos. After immunostaining for motor neurons, we used light sheet fluorescence microscopy to image embryos for subsequent quantitative analysis. This protocol is applicable to other neuron navigation processes in the central nervous system.

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in ...

The overall goal of this protocol is to allow qualitative and quantitative assessment of motor neuron axon arborization patterns using light sheet microscope and image analysis software on mouse embryos. This method can help to understand motor neuron development in coordination with complex movement. The main advantage of this technique is that you can observe and image axon arborization patterns of motor neurons from various angles and quantitatively analyze each individual nerve.Demonstrating the procedure will be Ya-Ping Yen and Ee Shan Liau, two graduate students from my laboratory. To begin this procedure, fix the embryos individually in a 24-well plate with one milliliter of freshly prepared 4%paraformaldehyde in PBS per well at four degrees Celsius on a shaker overnight. The next day, wash the fixed embryos at least three times, each for five to 10 minutes, with one milliliter of 1X PBS and incubate them overnight at four degrees Celsius.Next, permeabilize each embryo in one milliliter of 0.5%PBST overnight at four degrees Celsius on a shaker. Block each sample with one milliliter of 10%FBS prepared in 1X PBS overnight at 4 degrees Celsius on a shaker. Subsequently, incubate each embryo with one milliliter of anti-GFP primary antibody at 4 degrees Celsius for 72 hours with constant shaking.After the primary antibody incubation, wash each embryo with one milliliter of 0.5%PBST over the course of one to two days at four degrees Celsius on a shaker. Afterward, apply one milliliter of secondary antibody and incubate overnight in the dark at four degrees Celsius with constant shaking. The following day, wash each embryo with one milliliter of 0.5%PBST at least three times at four degrees Celsius on a shaker over the course of two to three days.Then, incubate each embryo in a commercial clearing reagent with a refractive index of 1.49 nd in a 1.5 milliliter tube protected from light at room temperature overnight. Set up 5X/0.1 illumination optics and 5X/0.16 detection optics. Assemble the sample holder and sample capillary.Prepare the sample chamber by filling it with clearing solution. To mount the embryo, prepare a P200 pipette tip by cutting away the upper part so that it fits the diameter of the capillary and remove the pointed part for the sample attachment. Then, melt the blunted end of the pipette tip using a small flame.Extinguish the flame and quickly attach the embryo vertically onto the melted end. Fit the upper part of the pipette tip with the sample capillary and put the prepared sample chamber into the microscope. Using the imaging software, select Locate Capillary under the Locate tab and adjust the X, Y, and Z axes.Next, select Locate Sample and zoom to 0.6X to focus on the embryo. Allow the chamber buffer to equilibrate with the embryo for some time to clear off any debris or bubbles. For image acquisition under the Acquisition tab, define the lightpath parameters such as Detective Objectives, Laser Blocking Filter, Beam Splitter, Cameras, and Lasers.Check the Pivot Scan check box for shadow reduction. Then, define the acquisition settings, Bit Bepth to 16-bit, Zoom to the range between 0.36 and 0.7X, single site illumination or dual site illumination. Click Continuous and set the Laser Intensity, Exposure Time, Laser Power, and Light Sheet Position.Subsequently, press Stop to end image acquisition. It is important to ensure that the transparent sample is positioned within the shortest lightpath to allow detailed acquisition of images of fine arborized structures on individual motor nerves. For multi-dimensional acquisition, define the Z-stack by moving the z position for the first and last image.Click Optimal to set the slice number. Then, click Start Experiment to acquire the selected Z-stack. When it is done, save the image in czi format.To quantify axon arborization, open the image file in the imagining analysis software. Adjust the image color, brightness, and contrast using the Display Adjustment window to detect filaments based on local intensity contrast. Next, click on the Add New Filaments icon and select the Autopath Algorithm in the dropdown menu.Select the region of interest, then define the starting and seed points by assigning the largest and thinnest diameter measurements which can be measured using the Slice mode. Assign a starting point at the edge of the region of interest. To achieve manual addition or removal of starting points, first change the pointer mode, navigate select, and then shift and right-click at the points of interest.Next, select manual thresholds for seed points to ensure that all of the visible arborization is marked. Then, manually add or remove seed points. Check the box Remove Disconnected Segments and remove seed points around starting points.Subsequently, adjust the threshold for background subtraction and finish the process. It is necessary to remove background artifacts and confirm the detector path reflect true axon arborization for accurate quantification. At the end, choose the desired style and color.Under the Statistics tab, select Detailed and use filament number, dendrite terminal points to quantify the motor nerve terminals as an indicator of motor axon arborization. Then, export the image of the reconstructed axon as a tiff file. LSFM provides detailed 3D visualization of motor axon arborization in mouse embryos.Motor neurons are subjected to the above-described protocol and labeled with transgenically-expressed GFP. To image axon arborization in greater detail and to perform quantification, magnification can be adjusted so that every finely arborized structure can be revealed. Finally, axon arborization pattern of individual forelimb nerves can be reconstructed using image analysis software.The autopath algorithm in the software traces filament and quantifies the total number of axon terminals. After watching this video, you should have a good understanding of how to prepare samples and image motor neuron axons of mouse embryos using light sheet fluorescense microscope. Also, you will know how to quantitatively assess the arborization pattern of each individual motor nerves by MRS.

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Research

JoVE Journal

Neuroscience

Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is very poor 1. The failure of axon regeneration is a combined result of several factors including the hostile glial cell environment, inhibitory myelin related molecules and decreased intrinsic neuron regenerative capacity 2. Astrocytes are the most predominant glial cell type in central nervous system and play important role in axon functions under physiology and pathology conditions 3. Contrast to the homologous oligodendrocytes, astrocytes are a heterogeneous cell population composed by different astrocyte subpopulations with diverse morphologies and gene expression 4. The functional significance of this heterogeneity, such as their influences on axon growth, is largely unknown.
To study the glial cell, especially the function of astrocyte heterogeneity in neuron behavior, we established a new method by co-culturing high purified dorsal root ganglia neurons with glial cells obtained from the rat cortex. By this technique, we were able to directly compare neuron adhesion and axon growth on different astrocytes subpopulations under the same condition.
In this report, we give the detailed protocol of this method for astrocytes isolation and culture, dorsal root ganglia neurons isolation and purification, and the co-culture of DRG neurons with astrocytes. This method could also be extended to other brain regions to study cellular or regional specific interaction between neurons and glial cells.

In this protocol, we described a new method to study the influence of glial cell heterogeneity on axon growth with an in vitro co-culture system. Rat cortical glial cells were cultured to confluence and cocultured with highly purified rat dorsal root ganglia neurons. Different glial cell influence on neurons adhesion and axon growth was compared directly in the same culture. This method provides a new way to directly study the glial cell heterogeneity influence on neuron adhesion and axon growth.

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

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. ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) axons form branches and synapses in specific layers of the cerebral cortex. Despite the obvious spatial correlation between axon branching and synapse formation, the causal relationship between them is poorly understood. To address this issue, we recently developed a method for simultaneous imaging of branching and synapse formation of individual TC axons in organotypic cocultures.
This protocol describes a method which consists of a combination of an organotypic coculture and electroporation. Organotypic cocultures of the thalamus and cerebral cortex facilitate gene manipulation and observation of axonal processes, preserving characteristic structures such as laminar configuration. Two distinct plasmids encoding DsRed and EGFP-tagged synaptophysin (SYP-EGFP) were co-transfected into a small number of thalamic neurons by an electroporation technique. This method allowed us to visualize individual axonal morphologies of TC neurons and their presynaptic sites simultaneously. The method also enabled long-term observation which revealed the causal relationship between axon branching and synapse formation.

This protocol describes a method for simultaneous imaging of thalamocortical axon branching and synapse formation in organotypic cocultures of the thalamus and cerebral cortex. Individual thalamocortical axons and their presynaptic terminals are visualized by a single cell electroporation technique with DsRed and GFP-tagged synaptophysin.

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) ...

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

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

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

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.

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...