This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (23700412, 25122707 and 26670090 to S.Y.).

Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee at Hamamatsu University School of Medicine.
1. Preparation of Matrices
<ol>
	<li>Boil 4-8 silicone matrices in a microwave or on a hot plate for 5 min and allow them to dry completely for 1 h under laminar flow (striped side up). 
	Note: The following procedures should be performed under laminar flow.</li>
	<li>Blow compressed air or use transparent sticky tape to remove any dust from the striped side of the matrices. Keep the striped side clean so it can firmly adhere to the plate surface.</li>
	<li>Carefully place the matrices onto 6-cm plastic dishes by pressing with a finger (one matrix per dish; stripe side down). Avoid getting air bubbles between the matrix and the dish. Mark the location of the stripes on the bottom side of the culture dish.</li>
	<li>If the matrix fails to attach, repeat from step 1.2.</li>
</ol>
2. Stripe Generation and Laminin Coating for Neuron Culture
<ol>
	<li>Prepare fluorescently labeled recombinant protein (25 &#181;l each for injection [step 2.2] or 100 &#181;l each for the placement method [step 2.2.1]) by mixing the Fc-tagged protein (10-50 &#181;g/ml) and a fluorescent dye-conjugated anti-human Fc antibody (1 to 3 ratio: 30-150 &#181;g/ml) in phosphate buffered saline (PBS). Then, incubate the mixture for 30 min at RT.</li>
	<li>Using a 22-G syringe, inject 25 &#181;l of the recombinant protein prepared in the previous step (2.1) through the small hole on the side of the matrix (arrows in Figure 1A, 1C, and 1E).
	<ol>
		<li>Alternatively, place 100 &#181;l of the recombinant protein into the top slit of the matrix and aspirate approximately half of the solution from the small hole (arrows in Figure 1A, 1C, and 1E), so that the recombinant protein remains in the striped regions.</li>
	</ol>
	</li>
	<li>Incubate the dish for 30 min at 37 &#176;C in a cell culture incubator.</li>
	<li>Place 300 &#181;l of PBS into the top slit (Figure 1B) and aspirate from the small hole located on the side of the matrix (arrows in Figure 1A, C and E) to remove unattached recombinant proteins. Do not aspirate the PBS completely, because the proteins will dry. Repeat this step twice. (3 times total)</li>
	<li>Carefully remove the matrix and immediately place ~100 &#181;l of control protein (10-50 &#181;g/ml; Fc) to cover the entire striped area, creating an alternate coating. Then, incubate the dish for 30 min at 37 &#176;C in the cell culture incubator.</li>
	<li>After washing the dish surface three times with PBS, coat it with ~100 &#181;l of 20 &#181;g/ml laminin in PBS. 
	Note: Laminin should be thawed on ice to prevent solidification.</li>
	<li>Incubate the dish for 1 h at 37 &#176;C in the cell culture incubator.</li>
	<li>Wash the dish surface three times with PBS and add culture medium. Place the coated dish with culture medium in a 5% CO2 incubator at 37 &#176;C until use.</li>
</ol>
3. Culturing Hippocampal Neurons from E15.5 Mouse Embryos
<ol>
	<li>Euthanize mother via cervical dislocation and isolate three E15.5 embryos.</li>
	<li>Cut the skin and skull sagittally at the midline of the head with scissors, and remove the brain using a small spoon. Transfer the brain carefully to a 60-mm Petri dish containing 3 ml of Hank&#39;s Balanced Salt Solution (HBSS).</li>
	<li>Using a scalpel, cut out the hemispheres including cortex, hippocampus and striatum (Figure 2A). Then, dissect out the hippocampal region by carefully removing the meninges, and transfer the dissected hippocampal tissues to a 15-ml centrifuge tube containing 2 ml of HBSS solution, on ice (Figure 2B-C). Collect 6 hippocampi from 3 embryos in the tube, which are sufficient for culturing neurons in 8 dishes.</li>
	<li>Replace the HBSS solution with trypsin/EDTA solution (2 ml) and incubate the tube at 37 &#176;C for 15 min in a water bath. 
	Note: Aspirate HBSS without centrifugation and do not decant the HBSS solution by tilting the tube.</li>
	<li>Neutralize the trypsin activity by adding 500 &#181;l of fetal bovine serum (FBS).</li>
	<li>Centrifuge the mixture at 100 x g for 5 min. Remove the supernatant and wash the pellet with 2 ml of culture medium. Repeat this step once more.</li>
	<li>Pipet the hippocampi up and down 10 times in culture medium using a 1,000-&#181;l tip with a large hole (cut-tip) to dissociate the tissue.</li>
	<li>Change to a regular (uncut) 1,000-&#181;l tip and pipet the dissociated tissue up and down to obtain a single-cell suspension.</li>
	<li>Separate the single cells by passing them through an 80-100-&#181;m mesh cell strainer.</li>
	<li>Centrifuge the filtered single cell suspension at 150 x g for 5 min.</li>
	<li>Remove the supernatant by aspirating, and resuspend the pellet (neurons) in 2 ml of culture medium.</li>
	<li>Count the cells using a hemocytometer with trypan blue staining to determine the density and viability. Then, plate 10,000 neurons in 150 &#181;l of culture medium for each set of stripes.The suspension should be carefully placed to cover the entire stripe region.</li>
</ol>
4. Visualization of Cell Bodies and Axons by Immunofluorescence Staining using an Anti-Tau1 Antibody
<ol>
	<li>After 24 h of culture, aspirate the medium without disturbing the adherent cells, and fix the neurons with pre-warmed 4% paraformaldehyde (PFA)/4% sucrose/PBS at RT&#160;for 15 min.</li>
	<li>Wash the plate surface 3 times with PBS. If desired, use a water-repellant pen to draw a circle around the striped region to decrease the amount of antibody needed.</li>
	<li>Permeabilize the neurons with 0.3% Triton X-100/PBS for 5 min.</li>
	<li>Incubate the neurons with blocking solution containing 3% donkey serum and 0.1% Triton X-100 for 30 min followed by incubation with an anti-Tau1 antibody (1:200) in 1% donkey serum/0.1% Triton X-100/PBS O/N at 4 &#176;C.</li>
	<li>Wash the plate surface 3 times with PBS. Incubate the neurons with a fluorophore-conjugated anti-mouse IgG antibody in 1% bovine serum albumin (BSA)/0.1% Triton X-100/PBS for 30 min.</li>
	<li>Wash the plate surface 3 times with PBS. Cover the striped region with an 18 mm x 18 mm cover glass using 50 &#181;l of antifade solution. avoid air bubbles, which interfere with the imaging.</li>
	<li>Obtain fluorescent images with a fluorescence microscope using the appropriate system, e.g., x10 objective lens, filter sets for GFP and RFP, and 1,000-msec exposure time (Figure 3).</li>
</ol>

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The authors have nothing to disclose.

This protocol describes a stripe assay that uses recombinant protein and dissociated neurons from the E15.5 mouse hippocampus. This assay allows the efficient observation of repulsive, attractive, or neutral responses of neurons to a recombinant protein of interest placed in a striped pattern. A major advantage of this protocol is the simple method for generating the stripes, in which the protein is directly printed onto the surface of a plastic dish, compared to the traditional method, which requires special matrices, a...

Axon guidance is the process by which newly formed neurons send axons to their target during development of the nervous system1,2. Developing axons carry a highly motile structure at their tip called the growth cone. The growth cone senses extracellular cues to navigate the axon&#39;s path. Guidance molecules, such as slit, semaphorin, and ephrins, can attract or repel axons depending on their interaction with suitable receptors and co-receptors on the axon1,3,4. The activated receptors transfer signals to the growth cone that affect its cytoskeletal organization for axon and growth-cone movements.
Various methods have been developed to evaluate the action of attractant and repellent molecules. Chemo-attractants and repellents can be administered into the growth/culture medium with a gradient concentration (e.g., Dunn&#39;s chamber or &#181;-slides)5,6, in a highly concentrated spot by micro-pipette (e.g., turning assay)7 or at a homogenous concentration by bath application (e.g., growth cone collapse assay)8,9.
Other methods include a stripe assay or microcontact printing (&#181;CP), in which a chemo-attractant or repellent is coated on the surface of a plate as a substrate10-12. Thestripe assay was originally developed by Bonhoeffer and colleagues in 1987 to analyze topographical mapping in the chick retino-tectal system13. The original method required a vacuum system to coat proteins onto polycarbonate nucleopore membranes using striped and meshed matrices. In later versions, the recombinant proteins were directly printed on the surface of a culture plate in a striped pattern using narrow slit silicon matrices14,15. Recently, various research groups have successfully applied this stripe assay to the analysis of axon guidance molecule activities16-21.
Here, we present the detailed protocol for a stripe assay that measures the attraction or repulsion of axon guidance molecules for dissociated hippocampal neurons. Notably, this method can be applied in minimally equipped laboratory settings. For this assay, alternating stripes of a fluorescently labeled substrate and a control protein are generated on a plastic dish using a silicon matrix with 90-&#181;m slits and coated with laminin. In our demonstration, dissociated hippocampal neurons from E15.5 mice were cultured on alternating stripes of recombinant ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control Fc protein21. After 24 h of culture, both the axons and cell bodies of the neurons were strongly repelled from the FLRT2 stripes. Staining with an anti-Tau1 antibody revealed that ~90% of the neurons were distributed on the Fc-coated regions, compared to ~10% on the FLRT2-Fc, indicating that FLRT2 has a strong repulsive function for hippocampal neurons21.

<table><tbody><tr><td>15 ml centrifuge tube</td><td>Violamo&amp;nbsp;</td><td>1-3500-01</td><td></td></tr><tr><td>4% Paraformaldehyde (PFA)</td><td>Nacalai</td><td>01954-85</td><td></td></tr><tr><td>Alexa Fluor 488 Goat anti-human IgG antibody</td><td>Thermo Scientific</td><td>A11013</td><td></td></tr><tr><td>Alexa Fluor 594 Donkey anti-mouse IgG antibody</td><td>Thermo Scientific</td><td>A-21203</td><td>Dilution 1/500</td></tr><tr><td>Anti-Tau1 antibody</td><td>Chemicon</td><td>MAB3420</td><td>Dilution 1/200</td></tr><tr><td>Antifade</td><td>Thermo Scientific</td><td>P7481</td><td>Alternative mounting media may be used</td></tr><tr><td>B27 supplement</td><td>Thermo Scientific</td><td>17504-044</td><td>Dilution 1/50</td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>01-2030-2</td><td></td></tr><tr><td>Cell strainer 100 um</td><td>BD Falcon</td><td>352360</td><td></td></tr><tr><td>Centrifugation machine</td><td>Kubota</td><td>2410</td><td></td></tr><tr><td>Cover glass 18mmx18mm</td><td>Matsunami</td><td>18x18 mm No. 1</td><td></td></tr><tr><td>DAKO pen</td><td>DAKO</td><td>S2002</td><td>Alternative water-repellent pen may be used</td></tr><tr><td>Disposable scalpel</td><td>Feather</td><td>2975#11</td><td></td></tr><tr><td>FBS</td><td>Thermo Scientific</td><td>10437-028</td><td></td></tr><tr><td>Fluorecent microscope</td><td>Nikon</td><td>E600</td><td></td></tr><tr><td>Forceps No. 5</td><td>Fine Science Tools</td><td>11254-20</td><td></td></tr><tr><td>GlutaMAX</td><td>Thermo Scientific</td><td>35050-061</td><td>Dilution 1/100</td></tr><tr><td>Hamilton Syringe</td><td>Hamilton</td><td>805N</td><td>22 gauge, 50 uL</td></tr><tr><td>HBSS</td><td>Thermo Scientific</td><td>14170-112</td><td></td></tr><tr><td>Human IgG, Fc Fragment</td><td>Jackson</td><td>009-000-008</td><td></td></tr><tr><td>Laminin</td><td>Thermo Scientific</td><td>23017-015</td><td></td></tr><tr><td>Neurobasal</td><td>Thermo Scientific</td><td>21103-049</td><td></td></tr><tr><td>Normal Donkey Serum</td><td>Jackson</td><td>017-000-121</td><td></td></tr><tr><td>PBS</td><td>Nacalai</td><td>14249-24</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Scientific</td><td>15070-063</td><td>Dilution 1/100</td></tr><tr><td>Plastic culture dish, 60 mm</td><td>Thermo Scientific</td><td>150288</td><td></td></tr><tr><td>Silicone Matrices</td><td></td><td></td><td>Available and purchasable from Prof. Martin Bastmeyer (bastmeyer@kit.edu)</td></tr><tr><td>Stereo Microscope</td><td>Olympus</td><td>SZ61</td><td></td></tr><tr><td>Tip, 1000 uL</td><td>Watson</td><td>125-1000S</td><td></td></tr><tr><td>Transparent sticky tape</td><td>Tesa</td><td>57315</td><td>Alternative sticky tape may be used</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Trypan blue, 0.4%</td><td>Bio-Rad</td><td>145-0013</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Thermo Scientific</td><td>25300-054</td><td></td></tr><tr><td>Culture medium</td><td></td><td></td><td>Neurobasal supplemented with B27, GlutaMAX and Penicillin-Streptomycin.</td></tr></tbody></table>

stripe assay to study the attractive or repulsive activity of a protein substrate using dissociated hippocampal neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Dissociated hippocampal neurons from E15.5 mice were plated and cultured for 24 h on stripes of fluorescently labeled control Fc (Figure 3A-C) or FLRT2-Fc (Figure 3D-F) alternating with non-labeled control Fc. In both cases, the neurons were aggregated and extended their axons as bundles. On the control Fc/Fc stripes, the neurons were distributed evenly on the fluorescently labeled and non-labeled stripes, and they extended their axons in random direction...

Watch this Scientific Journal Video about Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons at JoVE.com

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

stripe-assay-to-study-attractive-or-repulsive-activity-protein

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

10.5K Views.  Hamamatsu University School of Medicine. Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons. 

Video: Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings.&#160;The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is characteristic of disease states like major depressive disorder (MDD) and Alzheimer's disease. Traditional techniques to investigate synapse numbers include genetic expression of fluorescent markers (e.g., green fluorescent protein (GFP)), dyes that fill a neuron (e.g., carbocyanine dye, DiI), and immunofluorescent detection of spine markers (e.g., postsynaptic density 95 (PSD95)). A major caveat to these proxy techniques is that they only identify postsynaptic changes. Yet, a synapse is a connection between a presynaptic terminal and a postsynaptic spine. The gold standard for measuring synapse formation/elimination requires time-consuming electron microscopy or array tomography techniques. These techniques require specialized training and costly equipment. Further, only a limited number of neurons can be assessed and are used to represent changes to an entire brain region. DetectSyn is a rapid fluorescent technique that identifies changes to synapse formation or elimination due to a disease state or drug activity. DetectSyn utilizes a rapid proximity ligation assay to detect juxtaposed pre- and postsynaptic proteins and standard fluorescent microscopy, a technique readily available to most laboratories. Fluorescent detection of the resulting puncta allows for quick and unbiased analysis of experiments. DetectSyn provides more representative results than electron microscopy because larger areas can be analyzed than a limited number of fluorescent neurons. Moreover, DetectSyn works for in vitro cultured neurons and fixed tissue slices. Finally, a method is provided to analyze the data collected from this technique. Overall, DetectSyn offers a procedure for detecting relative changes in synapse density across treatments or disease states and is more accessible than traditional techniques.

DetectSyn is an unbiased, rapid fluorescent assay that measures changes in relative synapse (pre- and postsynaptic engagement) number across treatments or disease states. This technique utilizes a proximity ligation technique that can be used both in cultured neurons and fixed tissue.

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is ...

Biochemistry

A High-content Assay for Monitoring AMPA Receptor Trafficking

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different stimuli. The &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are responsible for fast excitatory synaptic transmission in neurons, are trafficked to and from the postsynaptic surface to dynamically alter neuronal excitability. AMPA receptor trafficking is essential for synaptic plasticity and can be disrupted in neurological disease. However, prevalent approaches for quantifying receptor trafficking ignore entire receptor pools, are overly time- and labor-intensive, or potentially disrupt normal trafficking mechanisms and therefore complicate the interpretation of resulting data. We present a high-content assay for the quantification of both surface and internal AMPA receptor populations in cultured primary hippocampal neurons using dual fluorescent immunolabeling and a near-infrared fluorescent 96-well microplate scanner. This approach facilitates the rapid screening of bulk internalized and surface receptor densities while minimizing sample material. However, our method has limitations in obtaining single-cell resolution or conducting live cell imaging. Finally, this protocol may be amenable to other receptors and different cell types, provided proper adjustments and optimization.

Neuronal excitability can be modulated through a dynamic process of endo- and exocytosis of excitatory ionotropic glutamate receptors. Described here is an accessible, high-content assay for quantifying surface and internal receptor population pools.

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin occurs in equilibrium in its two forms: monomeric globular (G-actin) and polymerized filamentous (F- actin). At the synaptic terminals, actin cytoskeleton forms the basis for critical pre- and post-synaptic functions. Moreover, dynamic changes in the actin polymerization status (interconversion between globular and filamentous forms of actin) are closely linked to plasticity-related alterations in synaptic structure and function. We report here a modified fluorescence-based methodology to assess polymerization status of actin in ex vivo conditions. The assay employs fluorescently labelled phalloidin, a phallotoxin that specifically binds to actin filaments (F-actin), providing a direct measure of polymerized filamentous actin. As a proof of principle, we provide evidence for the suitability of the assay both in rodent and post-mortem human brain tissue homogenates. Using latrunculin A (a drug that depolymerizes actin filaments), we confirm the utility of the assay in monitoring alterations in F-actin levels. Further, we extend the assay to biochemical fractions of isolated synaptic terminals wherein we confirm increased actin polymerization upon stimulation by depolarization with high extracellular K+.

We report a simple, time-efficient and high-throughput fluorescence spectroscopy-based assay for the quantification of actin filaments in ex vivo biological samples from brain tissues of rodents and human subjects.

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin ...

Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in preparation for detailed downstream analysis such as flow cytometry or single-cell sequencing. Neural dissociation can be conducted via mechanical dissociation (such as using filters, chopping techniques, or pipette trituration), enzymatic digestion, or a combination thereof. The delicate nature of neuronal cells can complicate efforts to obtain the highly viable, true single-cell suspension with minimal cellular debris that is required for single-cell analysis. The data demonstrate that this combination of automated mechanical dissociation and enzymatic digestion consistently yields a highly viable (&gt;90%) single-cell suspension, overcoming the aforementioned difficulties. While a few of the steps require manual dexterity, these steps lessen sample handling and potential cell loss. This manuscript details each step of the process to equip other laboratories to successfully dissociate small quantities of neural tissue in preparation for downstream analysis.

This neural cell dissociation protocol is intended for samples with a low amount of starting material and yields a highly viable single-cell suspension for downstream analysis, with optional fixation and staining steps.

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in ...

Visualizing Proteins in Primary Hippocampal Neurons by Live&#45;Cell Imaging

Using Live&#45;Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal transport is disrupted in multiple neurodegenerative diseases, and projection neurons are particularly vulnerable because of the need to transport cellular materials over long distances and sustain substantial axonal mass. Pathological modifications of several disease-related proteins negatively affect transport, including tau, amyloid-&#946;, &#945;-synuclein, superoxide dismutase, and huntingtin, providing a potential common mechanism by which pathological proteins exert toxicity in disease. Methods to study these toxic mechanisms are necessary to understand neurodegenerative disorders and identify potential therapeutic interventions.
Here, cultured primary rodent hippocampal neurons are co-transfected with multiple plasmids to study the effects of pathological proteins on fast axonal transport using live-cell confocal imaging of fluorescently-tagged cargo proteins. We begin with the harvest, dissociation, and culturing of primary hippocampal neurons from rodents. Then, we co-transfect the neurons with plasmid DNA constructs to express fluorescent-tagged cargo protein and wild-type or mutant tau (used as an exemplar of pathological proteins). Axons are identified in live cells using an antibody that binds an extracellular&#160;domain of neurofascin, an axon initial segment protein, and an axonal region of interest is imaged to measure fluorescent cargo transport.
Using KymoAnalyzer, a freely available ImageJ macro, we extensively characterize the velocity, pause frequency, and directional cargo density of axonal transport, all of which may be affected by the presence of pathological proteins. Through this method, we identify a phenotype of increased cargo pause frequency associated with the expression of pathological tau protein. Additionally, gene-silencing shRNA constructs can be added to the transfection mix to test the role of other proteins in mediating transport disruption. This protocol is easily adaptable for use with other neurodegenerative disease-related proteins and is a reproducible method to study the mechanisms of how those proteins disrupt axonal transport.

Author Spotlight: Understanding the Impact of Pathological Proteins on Axonal Transport in Neurodegenerative Diseases

Here, we demonstrate how to combine transfection of primary hippocampal rodent neurons with live-cell confocal imaging to analyze pathological protein-induced effects on axonal transport and identify mechanistic pathways mediating these effects.

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal ...

Assessing Attractive or Repulsive Activity of Proteins Using Hippocampal Neurons

Source: Yamagishi, S. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/54096">Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons.</a> J. Vis. Exp. (2016)
This video demonstrates a method to assess protein activity using hippocampal neurons over patterned protein zones. Neurons exhibit repulsion in test protein zones and attachment in control protein zones, highlighting the effects of attractive or repulsive protein activity.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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