Name: utilizing combined methodologies to define the role of plasma membrane delivery during axon branching and neuronal morphogenesis
Uploaded: 2016-03-16T14:00:00
Description: Watch this Scientific Journal Video about Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis at JoVE.com

This work was supported by the National Institutes of Health: RO1-GM108970 (SLG) and F31-NS087837 (CW).

Statement of research ethics: All experiments involving animals detailed herein are subject to the rules and regulations of the UNC Committee on Animal Care and to NIH standards for the care and use of laboratory animals.
1. Preparation and Plating of Dissociated Cortical Neurons
<ol>
	<li>Euthanize timed-pregnant females by CO2 inhalation followed by cervical dislocation. Remove whole brains from the skulls of embryonic day 15.5 (E15.5) mice and microdissect cort...

<ol>
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Axon branching is a fundamental neurodevelopmental process and underpins the vast neuroconnectivity of the mammalian nervous system. Understanding the mechanisms involved in localized plasma membrane expansion is integral to our understanding of both normal and pathological neurodevelopment. The use of a multipronged approach incorporating both population level and single cell level methodologies enhances reproducibility and increases spatial and temporal resolution without compromising population level analysis. At the ...

Recent estimates suggest that the human brain contains 1011 neurons with 1014 synaptic connections1, highlighting the importance of axon branching in vivo. Extracellular axon guidance cues such as netrin-1 guide axons to appropriate synaptic partners and stimulate axonal branching, thereby increasing synaptic capacity2-5. Netrin-1-dependent axonal arborization involves substantial plasma membrane expansion6, which we hypothesized requires delivery of additional membrane components via SNARE complex dependent exocytic vesicle fusion7. 
Investigating the role of SNARE-mediated exocytosis in netrin-1 dependent axon branching is complicated by several factors. First, the heterogeneity of cortical neurons increases the sample size required to identify significant effects, complicating single cell techniques like imaging. Second, although biochemical techniques permit observation of changes that occur at the population level, they lack the temporal and spatial resolution necessary to localize plasma membrane expansion to the axon in the time frame of axon branching. Lastly, although axon branches form over hours, the cellular changes that contribute to axonal extension may begin within minutes and occur on the order of seconds, thus extending the temporal scope for experimental consideration. 
We outline a multi-technique approach that addresses these diverse temporal and spatial scales of exocytosis and axon branching, and thus enhances our understanding of the fundamental cellular mechanisms. Utilizing these approaches provides evidence that supports a critical role for SNARE-mediated exocytosis in axon branching.

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			<td height="40" style="height:40px;width:179px;">6-well tissue culture treated plates</td>
			<td style="width:99px;">Olympus Plastics</td>
			<td style="width:76px;">25-105</td>
			<td style="width:187px;"></td>
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			<td height="141" style="height:141px;width:179px;">glass coverslips</td>
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			<td style="width:187px;">12CIR-1.5;must be nitric acid treated for 24 hours, rinsed in DI H2O 2X, and dried prior to use. Must be coated with 1mg/mL Poly-d-lysine and rinsed prior to plating cells</td>
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			<td height="41" style="height:41px;width:179px;">Amaxa nucleofection solution</td>
			<td style="width:99px;">Lonza</td>
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			<td style="width:187px;">100ml/transfection</td>
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			<td height="61" style="height:61px;width:179px;">Amaxa Nucleofector/electroporator</td>
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			<td height="81" style="height:81px;width:179px;">35mm Glass bottom live cell imaging dishes</td>
			<td style="width:99px;">Matek Corporation</td>
			<td style="width:76px;">p356-1.5-14-C</td>
			<td style="width:187px;">must be coated with 1mg/mL Poly-d-lysine and rinsed prior to plating cells</td>
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			<td height="41" style="height:41px;width:179px;">Olympus IX81-ZDC2 inverted microscope</td>
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			<td height="41" style="height:41px;width:179px;">100x 1.49 NA TIRF objective</td>
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			<td style="width:76px;"></td>
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			<td style="width:187px;">Used for scanning blots</td>
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			<td style="width:187px;">Used for scanning on the Odyssey Infrared system; Image studio lite used for offline analysis of blots</td>
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			<td height="61" style="height:61px;width:179px;">Metamorph for Olympus</td>
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			<td height="41" style="height:41px;width:179px;">40x 1.4 NA Plan Apochromat objective</td>
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			<td style="width:76px;"></td>
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			<td height="61" style="height:61px;width:179px;">Neurobasal media</td>
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			<td style="width:187px;">Base solution for both serum free and trypsin quenching media</td>
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			<td height="62" style="height:62px;width:179px;">Supplement B27</td>
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			<td style="width:187px;">500ml/50mLs Serum free media and Trypsin Quenching media</td>
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			<td height="21" style="height:21px;width:179px;">10X&#160; trypsin</td>
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			<td height="21" style="height:21px;width:179px;">HEPES</td>
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			<td height="61" style="height:61px;width:179px;">Dulbecco&#39;s Phosphate Buffered Saline (DPBS)+ Ca + Mg</td>
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			<td height="42" style="height:42px;width:179px;">Poly-D-Lysine</td>
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			<td style="width:187px;">Dissolved in sterile water at 1mg/mL</td>
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		<tr height="81">
			<td height="81" style="height:81px;width:179px;">Botulinum A toxin BoNTA</td>
			<td style="width:99px;">List Biological Laboratories</td>
			<td style="width:76px;">128-A</td>
			<td style="width:187px;"></td>
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		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Rabbit polyclonal anti human VAMP2</td>
			<td style="width:99px;">Cell signaling</td>
			<td style="width:76px;">11829</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:179px;">Mouse monoclonal anti rat Syntaxin1A</td>
			<td style="width:99px;">Santa Cruz Biotechnology</td>
			<td>sc-12736</td>
			<td style="width:187px;"></td>
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		<tr height="61">
			<td height="61" style="height:61px;width:179px;">Goat polyclonal anti human SNAP-25</td>
			<td style="width:99px;">Santa Cruz Biotechnology</td>
			<td>sc-7538</td>
			<td style="width:187px;"></td>
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			<td height="41" style="height:41px;width:179px;">Mouse monoclonal anti human &#946;III-tubulin&#160;</td>
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			<td height="81" style="height:81px;width:179px;">Alexa Fluor 568 and Alexa Fluor 488 phalloidin, or Alexa Fluor 647</td>
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			<td style="width:76px;"></td>
			<td style="width:187px;"></td>
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		<tr height="121">
			<td height="121" style="height:121px;width:179px;">LI-COR IR-dye secondary antibodies</td>
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			<td style="width:76px;">P/N 925-32212,P/N 925-68023, P/N 926-68022</td>
			<td style="width:187px;">800 donkey anti-mouse, 680 donkey anti rabbit, 680 donkey anti goat</td>
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		<tr height="61">
			<td height="61" style="height:61px;width:179px;">0.2um pore size nitrocellulose membrane</td>
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			<td style="width:76px;">9004-70-0</td>
			<td style="width:187px;"></td>
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			<td height="41" style="height:41px;width:179px;">Sucrose</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Paraformaldehyde</td>
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			<td style="width:76px;">O-4042-500</td>
			<td style="width:187px;"></td>
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			<td height="41" style="height:41px;width:179px;">Triton-X100</td>
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			<td height="41" style="height:41px;width:179px;">40% Bis-Acrylimide</td>
			<td style="width:99px;">Fisher scientific</td>
			<td style="width:76px;">BP1408-1</td>
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			<td height="42" style="height:42px;width:179px;">Name</td>
			<td style="width:99px;">Company</td>
			<td style="width:76px;">Catalog Number</td>
			<td style="width:187px;">Comments</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Alternative Validated Antibodies</td>
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			<td style="width:76px;"></td>
			<td style="width:187px;"></td>
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		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Mouse Monoclonal Anti-Syntaxin HPC-1 clone</td>
			<td style="width:99px;">Sigma Aldrich</td>
			<td style="width:76px;">S0664</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:179px;">&#160;Mouse Monoclonal Synaptobrevin 2 (VAMP2)</td>
			<td style="width:99px;">Synaptic Systems</td>
			<td style="width:76px;">104-211</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:179px;">Mouse Monoclonal SNAP25</td>
			<td style="width:99px;">Synaptic Systems</td>
			<td style="width:76px;">111-011</td>
			<td style="width:187px;"></td>
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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.

Utilizing in vitro biochemical techniques to assay the amount of SDS-resistant SNARE complexes in a population of neurons. Figure 1 shows the resulting western blot following completion of the SDS-resistant SNARE complex assay probed for SNAP-25, syntaxin1A and VAMP2.
TIRF microscopy at the basal cell membrane provides high resolution images of individual exocytic fusion events in single cells. ...

Watch this Scientific Journal Video about Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis at JoVE.com

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

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 ...

The overall goal of this procedure is to make quantifiable observations of the temporal and spatial occurrence of neuronal exocytic vesicle fushion and axon branching. This method can help answer key questions in the neuroscience field, such as when and where vesicle fusion inserts membrane material into the expanding plasma membrane during neuronal development. The main advantage of this technique is that it combines live cell imaging to acquire both high spatial and temporal resolution in single cells and biochemical approaches to acquire population based information.After culturing and simulating cortical neurons with Netrin-1 or a control according to the protocol text. Aspirate PBS from the first well of a condition and replace with 250 microliters of homogenization buffer. Incubate on ice for five minutes, and then using a cell lifter, homogenize the cells on ice.Aspirate the PBS from the next well of the same condition and add the recently homogenized mixture to the well which will increase protein concentrations. Next, transfer the homogenized solution to a pre-cooled 1.6 milliliter microfuge tube on ice, and add 20%Triton X-100 to reach a final concentration of 1%Then with a 1, 000 microliter pipette, triturate the mixture ten times while minimizing bubbles. Incubate the tubes on ice for two minutes to solubilize the proteins.Then centrifuge at 6, 010 RCF at 4 degrees Celsius for 10 minutes to pellet the non-solubilized material. Transfer the lie state supernatant into a new chilled 1.6 milliliter tube on ice. Next, use the Bradford assay to determine protein concentration.Then, use homogenization buffer with 1%Triton X-100 and 5X sample buffer supplemented with BME to dilute the samples to a final concentration of 3-5 milligrams per milliliter. Divide each experimental sample evenly into two tubes. Then incubate one sample at 37 degrees Celsius for 30 minutes, and the other at 100 degrees Celsius for 30 minutes.Invert the tubes intermittently to keep samples in solution. Then carry out SDS page and Western blotting according to the text protocol. After setting up the TIRF microscope and the sample and finding a neuronal focal plane according to the text protocol.Start the laser software and connect to the laser control software. Set the illumination to wide field and select the objective. Then set the refractive index of the sample and adjust the laser intensity by unchecking TTL for the 491 laser.Imaging parameter optimization is important during imaging of floor intact exocytic vesicles to avoid focus drift and phototoxicity while producing images of high signal to noise ratio sufficient for analysis. Adjust the slider to 100 and then bring it back down to a value between 20 and 40. Then recheck TTL.Next, focus on the sample again in transmitted light illumination. Then in the imaging software, select the 491 laser illumination and open the shutter. Place the condenser upside down on the optical bench so as not to scratch the lens.Fine adjust the focal point of the laser on the ceiling and with the condenser removed, center the point to the center of the closed field diaphragm. Then replace the condenser and in the TIRF software sent the penetration depth to 110 nanometers. Then switch from wide field illumination to TIRF illumination mode for imaging.Now, using wide field epifluorescence find VAMP2 fluorent expressing cells through the oculars. Then to reduce photobleaching and phototoxicity, adjust the imaging parameters to maximize the signal to noise ratio and dynamic range using minimal exposure time and laser intensity. Set continuous auto focus per cell.Then acquire a time lapse image set with acquisition occurring every 0.5 seconds for five minutes. For the Netrin-1 stimulated condition in a laminar flow hood, add to a final concentration of 500 nanograms per milliliter of Netrin-1 to a dish of cells and return the dish to the incubator for one hour prior to imaging. To quantify the frequency of exocytic events normalized per cell area and time.In ImageJ, open image stacks by dragging the file into the window or by choosing File, Open, file name. To remove stable fluorescent signals that do not represent vesicle fusion events, use Image, Stack, Z Project, Projection type Average Intensity. To create an average Z Projection of the entire stack.Subtract the resulting mean image from each image in the timelapse using Process, Image Calculator, Image 1 your stack, Operation Subtract image two. Resulting in a newly created Z Projection. This will emphasize exocytic events.Now by eye, count exocytic fushion events, which can be identified as spots of diffraction limited fluorescent signals that rapidly diffuse as VAMP2 fluoren diffuses within the plasma membrane. Highlight the first image with Image, Adjust, Threshold, and adjust the slider until the entire cell is threshold highlighted. Then under Analyze, choose Set Measurements and check the area and display label.With the wand tracing tool, set the thresholded cellular area and press M to measure. Transfer the collected data to a spreadsheet according to the text protocol. At two days in-vitro, place the glass bottom imaging dish containing untransfected neurons in a pre-warmed humid environmental chamber.Utilize a microscope equipped with a six DX plan apochromat 1.4 NA DIC objective lens and a high numerical aperture condenser for the best image quality and resolution. Next, with transmitted light illumination adjust the focal plane to find neurons through oculars. For DIC imaging of axon branching, proper setup of cooler illumination will be integral for image optimization and is critical to producing analyzable results.Then, with the neurons of interest within the field of view, use the multi-area acquisition function in the imaging software to find and save the XYZ locations of at least six cells. Now, add to a final concentration of 250 nanograms per milliliter of Netrin-1 or other factor of interest to simulate the cells and press start multi-area acquisition. Sequentially acquire images at each position every 20 seconds for 24 hours.Pausing acquisition and refocusing as necessary. Within the imaging software, review images by choosing Apps, Review Multi Dimensional Data and open the desired file. Identify stable axon branches that formed during the imaging session.Use the trace region tool to measure a stable axon branch from the base of the axon to the tip. After treating neurons with Netrin and botulinum toxin and fixing and immunostaining, and immunostaining for beta-3 tubolin and actin according to the text protocol. Use an inverted microscope to collect wide field epifluorescence images.Open an image stack in ImageJ and to manually analyze the branching use line, segmented line and trace the axon which is defined as the longest neurite extending from the soma. With Analyze, Tools, ROI Manager, save the axon tracing as a region of interest. Then trace and save each axon branch, which is defined as a neurite greater than or equal to 20 micrometers in length.Include only primary branches in the analysis. Shown here is a Western blot following the completion of the SDS resistant SNARE complex assay. Probed for SNAP25, Syntaxin 1A, and VAMP2.SNARE proteins in complex, an identifiable monomers are shown here. An example of a VAMP2 fluroen mediated exocytic event occurring over time in a cortical neuron is shown here. The zoomed insets denote the soma, an axon branch, and an axonal growth cone showing the spatial utility of this assay.Circles detonate single exocytic fusion events, which can be seen via TIRF microscopy. This time lapse DIC imaging shows the Netrin stimulated formation of an axon branch in real time. These locations denote the initial protrusion from a branch site.These represent fully formed stable branches of at least 20 micrometers measured from the main axon to the branch tip. In this experiment, cortical neurons at 3 DIV were either treated with Netrin-1, which stimulates branching or botulinum A toxin, which cleaves SNAP25, a component of the SNARE complex and blocks exocytosis. The results demonstrate that SNARE mediated exocytosis is requisite for Netrin dependent branching.Once mastered, the DIC imaging can be completed in a single overnight imaging session. TIRF imaging of individual exocytic events can be managed in 4-5 hours. The SNARE complex formation biochemistry protocol can be performed in two hours.While attempting these procedures, it's important to remember to pace yourself and not rush any single step. Each procedure requires attention and patience for the best results. Following this procedure, other methods like cotransfection of tagged vesicle components with tagged candidate cargos can be performed to answer additional questions.Like what is the cargo of the exocytic vesicles? After it's development, this technique paved the way for researchers in the field of neuroscience to explore neuritogenesis, axon branching, and other stages in morphogensis in any dissociated neuronal culture. After watching this video, you should have a good understanding of how to observe and quantify the spatial and temporal nature of neuronal exocytic vesicle fusion events corresponding to changes in neuronal morphogensis.Don't forget that working with botulinum toxin or high powered lasers can be extremely hazardous and precautions such as wearing proper safety garments and using the microscope safety lock mechanism should always be taken while performing this procedure.

utilizing-combined-methodologies-to-define-role-plasma-membrane

Research

JoVE Journal

Neuroscience

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960&#8217;s, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

This article details the enrichment of proteins associated with the synaptic plasma membrane by ultracentrifugation on a discontinuous sucrose gradient. The subsequent preparation of post-synaptic density proteins is also described. Protein preparations are suitable for western blotting or 2D DIGE analysis.

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and genetic mechanisms that contribute to hearing loss. Introduction of vectors into the inner ear must be done in a way that widely distributes the agent throughout the cochlea while minimizing injury to the existing structures. This manuscript describes a post-auricular surgical approach that can be used for mouse cochlear therapy using molecular, pharmacologic, and viral delivery to mice postnatal day 10 and older via the round window membrane (RWM). This surgical approach enables rapid and direct delivery into the scala tympani while minimizing blood loss and avoiding animal mortality. This technique involves negligible or no damage to essential structures of the inner and middle ear as well as neck muscles while wholly preserving hearing. To demonstrate the efficacy of this surgical technique, the vesicular glutamate transporter 3 knockout (VGLUT3 KO) mice will be used as an example of a mouse model of congenital deafness that recovers hearing after delivery of VGLUT3 to the inner ear using an adeno-associated virus (AAV-1).

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

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) ...

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our understanding of the molecular mechanisms underlying axon degeneration remains limited. Injury-induced axon degeneration serves as a simple model to study how severed axons execute their own disassembly (axon death). Over recent years, an evolutionarily conserved axon death signaling cascade has been identified from flies to mammals, which is required for the separated axon to degenerate after injury. Conversely, attenuated axon death signaling results in morphological and functional preservation of severed axons and their synapses. Here, we present three simple and recently developed protocols that allow for the observation of axonal morphology, or axonal and synaptic function of severed axons that have been cut-off from the neuronal cell body, in the fruit fly Drosophila. Morphology can be observed in the wing, where a partial injury results in axon death side-by-side of uninjured control axons within the same nerve bundle. Alternatively, axonal morphology can also be observed in the brain, where the whole nerve bundle undergoes axon death triggered by antennal ablation. Functional preservation of severed axons and their synapses can be assessed by a simple optogenetic approach coupled with a post-synaptic grooming behavior. We present examples using a highwire loss-of-function mutation and by over-expressing dnmnat, both capable of delaying axon death for weeks to months. Importantly, these protocols can be used beyond injury; they facilitate the characterization of neuronal maintenance factors, axonal transport, and axonal mitochondria.

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Here, we provide protocols to perform three simple injury-induced axon degeneration (axon death) assays in Drosophila melanogaster to evaluate the morphological and functional preservation of severed axons and their synapses.

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our ...