Name: characterizing the composition of molecular motors on moving axonal cargo using "cargo mapping" analysis
Uploaded: 2014-10-30T13:00:00
Description: Watch this Scientific Journal Video about Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis at JoVE.com

We thank Ge Yang, Gaudenz Danuser, Khuloud Jaqaman, and Daniel Whisler for assistance with the adaptation and development of the software to quantitate cargo mapping analyses, and Emily Niederst for help in making the microfluidic devices. This work was supported in part by NIH-NIA grant AG032180 to L.S.B.G., and the Howard Hughes Medical Institute. L.S. was supported in part by a NIH Bioinformatics Training Grant T32 GM008806, S.E.E. was supported by a Damon Runyon Cancer Research Foundation Fellowship, NIH Neuroplasticity Training Grant AG000216, and by a grant from The Ellison Medical Foundation New Scholar in Aging Award, G.E.C was supported by an NIH/NCATS&#160;1 TL1 award TR001114 and by the Achievement Rewards for College Scientists foundation.

All experiments were conducted following approved protocols and according to institutional guidelines for the humane care of research animals. Neonate mice were euthanized by decapitation.
1. Preparation of Microfluidic Devices for Cell Culture
<ol>
	<li>Prepare polydimethyl siloxane (PDMS) microfluidic devices for growth of hippocampal neurons as described by Harris and colleagues17-19. Below are some modifications that were adapted to the cargo mapping protocol. 
	NOTE: Mic...

<ol>
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The protocol presented here enables the correlation of directionality of movement of individual fluorescent microtubule-based moving cargo particles with the relative type and amount of associated motor proteins in live neurons. Previously, the total motor composition of axonal vesicular cargoes was assayed on heterogeneous populations of biochemically purified vesicles and organelles9,15. However, characterizing motor composition for a single type of cargo inside cells has been chal...

Intracellular transport is critical in all cell types for the delivery of proteins, membranes, organelles, and signaling molecules to various cellular domains1. Neurons are highly specialized cells with long, polarized projections that critically depend on intracellular transport of essential cargoes for their long-distance delivery to various axonal microdomains. This transport is mediated by kinesins and dyneins &#8212; two large families of molecular motor proteins &#8212; that bind to cargoes and track along polarized microtubules in anterograde and retrograde directions, respectively. While retrograde movement is mainly mediated by dynein, movement in the anterograde direction is facilitated by a large, functionally diverse family of kinesin motors. Consequently, anterograde transport of axonal cargoes could be mediated by various family members of the kinesin superfamily1-5. Though some cargoes move persistently in either direction, most cargoes move bidirectionally and reverse frequently on their way to their final destinations1,5-13. Furthermore, it has been shown that motors of opposing directionality associate simultaneously to cargoes, raising the question as to how regulated movement of cargoes is coordinated by opposite-polarity motors5-7. Together, transport of axonal cargoes is a concerted process that is regulated by the composition of motors and their specific biochemical activities, which in turn are dependent on various adaptors and regulatory binding partners14.
To faithfully describe the mechanism of axonal transport for a specific cargo and to uncover the underlying regulation of that transport, it is paramount to determine the composition of motor proteins and their regulatory binding partners associated with individual cargoes during their live transport. Other methods, for example biochemical approaches, provide estimates of average motor compositions on purified heterogeneous vesicle populations, but these estimates do not reveal the type or amounts of motors associated to single moving vesicles. Also, reconstitution of vesicle transport along preassembled microtubules in vitro enabled measuring the amount of one type of motor on a single vesicle level15. However, these experiments did not directly correlate the amount of motors with the transport characteristics of those vesicles, and measured transport in the absence of cellular regulatory factors.
A protocol is presented here, which determines the motor composition (type and relative amount of motors) of individual moving vesicles from immunofluorescence (IF) data measuring endogenously expressed motor proteins, and correlates these parameters to the live transport of the exact same vesicles in neurons16. This method entails precise mapping of IF-to-live cargo movement data. This is accomplished by growing hippocampal mouse neurons in microfluidic devices following established protocols17-19. These devices allow for the identification and correlation (&#8220;mapping&#8221;) of axons and single moving cargoes in fixed and live light microscopy modalities (Figure 1). Cultured neurons are transfected with fluorescently labeled cargo proteins whose transport is imaged at high spatial and temporal resolution to obtain detailed movement information that is plotted in kymographs. During the course of imaging, neurons are fixed with paraformaldehyde, and subsequently stained with antibodies against endogenous motor proteins. Fixed cargo and motor images are superimposed onto live movement kymographs to &#8220;map&#8221; (colocalize) them to the live cargo movement trajectories16. To correlate the live movement of cargoes with the association of motor proteins, colocalization is analyzed using a custom made MATLAB software package called &#8220;Motor Colocalization&#8221;16,20. Fluorescently labeled cargoes and motors generate diffraction-limited punctate features that can partially overlap. To resolve the position of overlapping puncta, the software first automatically fits Gaussian functions to each point spread function, representing individual fluorescent puncta, to determine their precise X-Y sub-pixel position coordinates and intensity amplitudes21-23. The positions of motors and cargoes are subsequently compared to each other to determine colocalization16,20. Therefore, this method more precisely assigns colocalization between fluorescent puncta as compared to other methods24.
The strength of this method is the ability to assess the colocalization of motors with individual cargo in fixed cells, for which live movement trajectories (e.g., the direction in which they were moving at the time of fixation) have been recorded. With this method, kinesins and dyneins were found to associate simultaneously to vesicles that carry the normal prion protein (PrPC-cellular), a neuronally enriched cargo that moves bidirectionally or remains stationary in axons16. This analysis allowed the formulation of a working model for the regulation of PrPC vesicle movement in which anterograde (kinesin) and retrograde (dynein) motors coordinate their activities in order to move the vesicles in either direction or to remain stationary while associated to the cargo. Another strength of this method is its potential broad applicability for characterizing colocalization/association of many fluorescently labeled cargoes that move in virtually any cell type, with any other protein(s) of interest. Thus, live/fixed correlation could potentially allow for the detection of transient protein-cargo interactions, as many individual fluorescently labeled moving particles can be analyzed over a desired period of time. Given the broad applicability and the type of questions that this method can address, this protocol will be of interest to a wide audience of cell biologists including those studying trafficking and transport in neurons or in other cell types.

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			<td>P5899-20mg</td>
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			<td height="21" style="height:21px;">D-MEM (Dulbecco&#8217;s Modified Eagle Medium) High Glucose, w/ L-Glutamine, w/o Sodium Pyruvate (1X)</td>
			<td>Life Technologies</td>
			<td>11965092</td>
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			<td>24020117</td>
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			<td height="21" style="height:21px;">DPBS (Dulbecco&#8217;s Phosphate Buffered Saline, no Magnesium, no Calcium (1X)</td>
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			<td>14190250</td>
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			<td height="21" style="height:21px;">Penicillin/Streptomycin (100X)</td>
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			<td>15140122</td>
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			<td height="21" style="height:21px;">Corning cellgro Water for Cell Culture&#160;</td>
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			<td height="21" style="height:21px;">Lipofectamine 2000</td>
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			<td height="21" style="height:21px;">Formaldehyde Solution 16% EM Grade</td>
			<td>Fisher Scientific</td>
			<td>50980487</td>
			<td>Caution: Harmful by inhalation, in contact with skin and if swallowed. Irritating to eyes, respiratory system and skin. Dispose according to official regulations.</td>
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			<td height="21" style="height:21px;">Sucrose</td>
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			<td>BP2818-100</td>
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			<td height="21" style="height:21px;">ProLong Gold antifade reagent</td>
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			<td height="21" style="height:21px;">Cover Glass 1 1/2. 24X40mm</td>
			<td>Corning</td>
			<td>2980-244</td>
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			<td height="21" style="height:21px;">Axis microfluidic device, 450 &#181;m</td>
			<td>Millipore</td>
			<td>AX450</td>
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			<td height="21" style="height:21px;">Adobe Photoshop</td>
			<td>Adobe</td>
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			<td height="21" style="height:21px;">60 mm cell culture dish</td>
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			<td height="21" style="height:21px;">150 mm cell culture dish</td>
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			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibodies Used:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Kinesin light chain, V-17</td>
			<td>Santa Cruz&#160;</td>
			<td>sc-13362</td>
			<td>specificity verified using KLC1-/- neurons (Ref. 16 ), recommended dilution 1:100.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti Dynein Heavy Chain 1, R-325</td>
			<td>Santa Cruz&#160;</td>
			<td>sc-9115</td>
			<td>specificity verified using shRNA against DYN1HC1 (Ref. 16), recommended dilution 1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 568 Donkey Anti-Rabbit IgG Antibody</td>
			<td>Life Technologies</td>
			<td>A10042</td>
			<td>&#160;recommended dilution 1:200.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 647 Donkey Anti-Rabbit IgG (H+L) Antibody</td>
			<td>Life Technologies</td>
			<td>A31573</td>
			<td>&#160;recommended dilution 1:200.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 568 Donkey Anti-Goat IgG (H+L) Antibody IgG Antibody</td>
			<td>Life Technologies</td>
			<td>A11057</td>
			<td>&#160;recommended dilution 1:200.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 647 Donkey Anti-Goat IgG (H+L) Antibody</td>
			<td>Life Technologies</td>
			<td>A21447</td>
			<td>&#160;recommended dilution 1:200.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plugins and Macros</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ</td>
			<td></td>
			<td></td>
			<td><a href="http://imagej.nih.gov/ij/index.html">&#160;http://imagej.nih.gov/ij/index.html.&#160;</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ Kymoraph Plugin</td>
			<td></td>
			<td></td>
			<td><a href="http://www.embl.de/eamnet/html/body_kymograph.html">http://www.embl.de/eamnet/html/body_kymograph.html.</a></td>
		</tr>
	</tbody>
</table>

characterizing the composition of molecular motors on moving axonal cargo using "cargo mapping" analysis

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative analysis of motor/cargo associations at the single vesicle level. The goal of this protocol is to use quantitative fluorescence microscopy to correlate (&#8220;map&#8221;) the position and directionality of movement of live cargo to the composition and relative amounts of motors associated with the same cargo. &#8220;Cargo mapping&#8221; consists of live imaging of fluorescently labeled cargoes moving in axons cultured on microfluidic devices, followed by chemical fixation during recording of live movement, and subsequent immunofluorescence (IF) staining of the exact same axonal regions with antibodies against motors. Colocalization between cargoes and their associated motors is assessed by assigning sub-pixel position coordinates to motor and cargo channels, by fitting Gaussian functions to the diffraction-limited point spread functions representing individual fluorescent point sources. Fixed cargo and motor images are subsequently superimposed to plots of cargo movement, to &#8220;map&#8221; them to their tracked trajectories. The strength of this protocol is the combination of live and IF data to record both the transport of vesicular cargoes in live cells and to determine the motors associated to these exact same vesicles. This technique overcomes previous challenges that use biochemical methods to determine the average motor composition of purified heterogeneous bulk vesicle populations, as these methods do not reveal compositions on single moving cargoes. Furthermore, this protocol can be adapted for the analysis of other transport and/or trafficking pathways in other cell types to correlate the movement of individual intracellular structures with their protein composition. Limitations of this protocol are the relatively low throughput due to low transfection efficiencies of cultured primary neurons and a limited field of view available for high-resolution imaging. Future applications could include methods to increase the number of neurons expressing fluorescently labeled cargoes.

Figure 1 shows an overview of the microfluidic device used to grow hippocampal neurons (Figure 1A, B). Neurons are plated in reservoir 1. The size of the microchannels prevents the diffusion of cell bodies (soma) into the axonal compartment while the length of the channels prevents dendritic projections from crossing all the way to the axonal compartment. After ~2-3 days in culture, neurons start extending their axons across microchannels into the axonal compartment (Figure 1B, C...

Watch this Scientific Journal Video about Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis at JoVE.com

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis

Intracellular transport of cargoes, such as vesicles or organelles, is carried out by molecular motor proteins that track on polarized microtubules. This protocol describes the correlation of the directionality of transport of individual cargo particles moving inside neurons, to the relative amount and type of associated motor proteins.

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative ...

characterizing-composition-molecular-motors-on-moving-axonal-cargo

Research

JoVE Journal

Neuroscience

Brain Imaging Investigation of the Impairing Effect of Emotion on Cognition

Emotions can impact cognition by exerting both enhancing (e.g., better memory for emotional events) and impairing (e.g., increased emotional distractibility) effects (reviewed in 1). Complementing our recent protocol 2 describing a method that allows investigation of the neural correlates of the memory-enhancing effect of emotion (see also 1, 3-5), here we present a protocol that allows investigation of the neural correlates of the detrimental impact of emotion on cognition. The main feature of this method is that it allows identification of reciprocal modulations between activity in a ventral neural system, involved in 'hot' emotion processing (HotEmo system), and a dorsal system, involved in higher-level 'cold' cognitive/executive processing (ColdEx system), which are linked to cognitive performance and to individual variations in behavior (reviewed in 1). Since its initial introduction 6, this design has proven particularly versatile and influential in the elucidation of various aspects concerning the neural correlates of the detrimental impact of emotional distraction on cognition, with a focus on working memory (WM), and of coping with such distraction 7,11, in both healthy 8-11 and clinical participants 12-14.

We present a protocol that allows investigation of the neural mechanisms mediating the detrimental impact of emotion on cognition, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants.

Emotions can impact cognition by exerting both enhancing (e.g., better memory for emotional events) and impairing (e.g., increased emotional ...

High-density EEG Recordings of the Freely Moving Mice using Polyimide-based Microelectrode

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a noninvasive brain monitoring tool in cognitive neuroscience as well as a diagnostic tool for epilepsy and sleep disorders in neurology. However, the underlying mechanism of EEG rhythm generation is still under the veil. Recently introduced polyimide-based microelectrode (PBM-array) for high resolution mouse EEG1 is one of the trials to answer the neurophysiological questions on EEG signals based on a rich genetic resource that the mouse model contains for the analysis of complex EEG generation process. This application of nanofabricated PBM-array to mouse skull is an efficient tool for collecting large-scale brain activity of transgenic mice and accommodates to identify the neural correlates to certain EEG rhythms in conjunction with behavior. However its ultra-thin thickness and bifurcated structure cause a trouble in handling and implantation of PBM-array. In the presented video, the preparation and surgery steps for the implantation of PBM-array on a mouse skull are described step by step. Handling and surgery tips to help researchers succeed in implantation are also provided.

In this article, we described the surgery procedure and handling tips for implantation of ultra-thin polyimide-based microelectrode array (PBM-array) on the mouse skull for acquisition of high-density encephalography (EEG) in a mouse model.

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a ...

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. 
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options.  We seek to define how neural immune ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

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

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

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

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

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

Screening of Axonal Degeneration in Carpal Tunnel Syndrome Using Ultrasonography and Nerve Conduction Studies

Axonal degeneration, indicative of surgical decompression, may coexist in carpal tunnel syndrome (CTS) as the disease progresses. However, the current diagnostic and severity gradation system cannot clearly indicate its coexistence, resulting in&#160;confusion of appropriate treatment prescription. There are also constraints in conventional methods for&#160;differentiation as well. This study aims at introducing an innovative, efficient, and quick screening protocol to differentiate axonal degeneration associated with CTS, using ultrasound and nerve conduction studies (NCS). It starts by using NCS to perform orthodromic stimulation at the wrist, to obtain the sensory conduction of the median and the ulnar nerves respectively. Meanwhile, the motor conduction&#160;of the median nerve is collected by stimulating the palm, wrist, and elbow, followed by the stimulation of the ulnar nerve at the wrist,&#160;below and above the elbow. Then, an ultrasound assessment is performed, using a linear array transducer, with cross-sectional area (CSA) and perimeter (P) at the wrist and at the one-third distal forearm calipered. Ratios (R-CSA, R-P) and changes from wrist to one-third distal forearm (&#916;CSA and &#916;P) are calculated according to a standard format. Potential axonal degeneration coexisting in CTS will be screened according to the criteria of NCS and cut-off values of ultrasound measurements established in a previous study. In terms of its noninvasiveness, low cost, convenience, and efficiency, it is easy to apply ultrasound complimentarily in clinical practice to prescreen patients with potential coexisting axonal degeneration. Nevertheless, the ultrasonographic imaging cannot directly reflect axonal degeneration. It still relies on conventional but invasive methods&#160;such as electromyography (EMG) and biopsy&#160;for confirmation if needed.

Here we present a protocol using nerve conduction studies and ultrasound to screen potential axonal degeneration associated with carpal tunnel syndrome. The criteria for differentiation are established. Compared to conventional approaches, this method is noninvasive, convenient, and efficient, with an overall satisfactory accuracy, sensitivity, and specificity.

Axonal degeneration, indicative of surgical decompression, may coexist in carpal tunnel syndrome (CTS) as the disease progresses. However, the current ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...