Name: quantification of filamentous actin (f-actin) puncta in rat cortical neurons
Uploaded: 2016-02-10T14:00:00.000+00:00
Duration: 10 min 13 s
Description: Watch this Scientific Journal Video about Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons at JoVE.com

This work was funded by NIH grants DA013137, DA031604, and HD043680. Partial support was provided by a NIH T32 training grant in Biomedical-Behavioral science.

All animal protocols were reviewed and approved by the Animal Care and Use Committee at the University of South Carolina (assurance number: A3049-01).
1. Low-density Embryonic Neuronal Culture
<ol>
	<li>Preparation for primary cortical neuron culture
	<ol>
		<li>Solutions:
		<ol>
			<li>Prepare Poly-L-Lysine stock solution by dissolving 5 mg of Poly-L-Lysine in 10 ml Borate buffer.</li>
			<li>Prepare working solution by diluting 1 ml Poly-L-Lysine Stock in 49 ml Borate Buffer.</li>
			<li>Prepare borate Buffer, pH 8.4 by starting with 395 ml dH2O and then adding all ingredients (1.24 g boric acid + 1.9 g of Borax). Adjust pH to 8.4 by 1 M NaOH. Adjust to 400 ml and filter with 0.2 &#181;m nylon membrane filter under hood.</li>
			<li>Prepare HBSS solution, pH 7.2 by starting with 445 ml dH2O and adding all other ingredients (50 ml 10X HBSS Stock + 1.2 g HEPES). Adjust pH to 7.2 by 1 N HCl, bring to 500 ml and filter with 0.2 &#181;m nylon membrane filter under hood.</li>
			<li>Prepare plating medium: DMEM/F12 with 10% fetal bovine serum. Filter with 0.2 &#181;m nylon membrane filter under hood.</li>
			<li>Prepare complete growth medium: To 50 ml Neurobasal medium, add the supplements (500 &#181;l GlutaMax (100X) + 500 &#181;l Glucose + 1 ml B-27 (50X) + 500 &#181;l of Antibiotic-Antimycotic solution (100X) + 100 &#181;l 7.5% Sodium Bicarbonate. Filter with 0.2 &#181;m nylon membrane filter under hood.</li>
		</ol>
		</li>
		<li>Two days before culture:
		<ol>
			<li>Coat twelve 35 mm glass-bottom dishes with 2 ml of Poly-L-Lysine working solution, and keep under the hood O/N.</li>
		</ol>
		</li>
		<li>One day before culture:
		<ol>
			<li>Empty coating agent from the dishes and rinse with ddH2O. Allow dishes to dry for an hour under hood.</li>
			<li>Prepare plating medium (DMEM/F12 with 10% fetal bovine serum (10% of total solution). Pipette 2 ml medium into each dish, and incubate O/N at 37 oC, 5% CO2. 
			Note: It is important to avoid plastic culture dishes when culturing. Use of glass-bottom dishes (for inverted microscopes) or glass coverslips (for upright microscopes) yields sharp and clear images. Compared to a plastic-bottom dish, the glass-bottom dish avoids unwanted hues or glare during fluorescent microscopy.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Cell culture protocol:
	<ol>
		<li>Put refrigerated HBSS Buffer (100-150 ml), 100 mm petri dishes, forceps, scissors, 50 ml tubes, and 15 ml tubes under UV light in hood.</li>
		<li>Euthanize pregnant rat with lethal inhalation of 5% sevoflurane under the hood outside of culture room.</li>
		<li>Sterilize with 70% EtOH, tent the skin of the lower abdomen using forceps while cutting from the tail up in the middle of abdominal cavity.Open the uterus using scissors to expose the placenta.</li>
		<li>Remove 8-10 E18 fetuses. Place 8-10 fetuses in petri dishes containing HBSS solution.</li>
		<li>Hold the back of the head of the fetus with forceps, then use scissors to sever the head from body. Place it in another petri dish filled with 5-7 ml of HBSS. Transfer dish to hood.</li>
		<li>Fill two additional 100 mm petri dish with cold HBSS.</li>
		<li>Peel aside skull, and scoop brain into a new petri dish filled with HBSS.</li>
		<li>Use sharp curved forceps, separate cerebellum and brain stem from brain, then transfer brain to a new petri dish with cold HBSS in it.</li>
		<li>Use tweezers to secure, separate the hemispheres with curved forceps. Remove meninges; isolate frontal cortex and transfer pieces into marked 15 ml centrifuge tubes.</li>
		<li>Fill the 15 ml tube with fresh 2 ml HBSS and add 20 &#181;l Trypsin EDTA (10x concentrated mixture with 0.5% Trypsin and 5.3 mM EDTA.) to each 15 ml tube.</li>
		<li>Incubate 10-15 min at RT. Gently swirl tube every few minutes, do not allow settling.</li>
		<li>After 10-15 min, use glass pipette to remove old HBSS and rinse twice with new HBSS.</li>
		<li>Immerse in trypsin inhibitor (1 mg/ml, 2 mg Trypsin inhibitor in 2 ml HBSS), mix well and let it sit 5 min. Wash twice with fresh HBSS.</li>
		<li>Using glass pipette with rubber bulb, triturate brain pieces 10-15 times very slowly so as to avoid bubbles.Triturate again using a calibrated pipette and a sterile tip with a reduced tip diameter very slowly until homogeneous.</li>
		<li>Transfer dissociated cells at desired density to pre-prepared coated culture dishes (50 cells/mm2). Incubate O/N at 37 oC, 5% CO2.</li>
		<li>After 24 hr, replace DMEM/F12 medium with freshly made complete growth serum-free Neurobasal medium.</li>
		<li>Maintain the cells at all times in a 5% CO2/95% room air humidified incubator for at least 12-14 days before experimentation. Supplement the cells every 5-6 days with fresh Neurobasal medium replacing approximately 50% of the old medium.</li>
	</ol>
	</li>
</ol>
2. Fluorescent Labeling and Immunocytochemistry
Note: The immunofluorescent labeling of primary cortical cell cultures was carried out in glass bottom 35 mm cell culture dishes with a working volume of 1 ml.
<ol>
	<li>Wash the glass-bottom dish twice with phosphate-buffered saline pH 7.4 (PBS).</li>
	<li>Fix cells with 4% paraformaldehyde for 15 min at RT.</li>
	<li>Wash cells twice with PBS and permeabilize with 0.1% Triton X-100 in PBS for 5 min.</li>
	<li>Treat cells for 20 min at RT with an F-actin specific stain, AlexaFluor 488 Phalloidin (1:40, 25 &#181;l of Phalloidin in 1 ml PBS).</li>
	<li>Rinse cells twice with PBS and block with 10% normal goat serum at RT for 1-2 hr.</li>
	<li>Dilute the chicken polyclonal anti-MAP2 antibody (1:2,500) in PBS with 2% normal goat serum and incubate O/N at 4 oC.</li>
	<li>Following incubation, rinse in PBS twice.</li>
	<li>Dilute the secondary antibody (Alexa Red 594-conjugated goat anti-chicken IgG (1:500) with 2% normal goat serum and incubate for 2 hr at RT.</li>
	<li>Rinse with PBS and add 10 &#181;l/dish of Hoescht dye.</li>
	<li>Incubate 3 min at RT and wash with PBS twice. 
	Note: The labeled cells now ready for step 3 (F-actin puncta counting).</li>
	<li>Preserve cell sample with 100 &#181;l of antifade reagent as a coverslipping agent and keep at 4 oC in the dark for long term storage. 
	Note: Phalloidin is relatively stable in PBS at +4 &#176;C in the dark for up to 1 week and fluorescence can be preserved with antifade coverslipping reagents. However, optimal images are obtained from 1-3 days after staining since Phalloidin may start to diffuse out with extended storage.</li>
</ol>
3. F-actin Puncta Counting
<ol>
	<li>Turn on fluorescent microscope and switch to 20&#215; objective. Open microscope software, and set up program at 1280&#215;960 pixel image size, and 0.17 &#181;m/pixel image resolution at 1&#215; zoom.</li>
	<li>Acquire images of co-labeled F-actin/MAP2 neurons under green (495 nm)/red (613 nm) fluorescent channels.</li>
	<li>Choose 5 Green (F-actin)/Red (MAP2) immunolabeled/Blue (Hoescht) fluorescent images of individual neuron with clearly defined dendritic arbors.</li>
	<li>Identify the F-actin rich structures in second order dendritic segment (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence.</li>
	<li>Rotate the selected region of images to a horizontal level. Copy and paste as a new image.</li>
	<li>Subtract the background of images, using a consistent adjustments for each dish.</li>
	<li>Count the bright green F-actin puncta and trace the length of selected dendritic segment manually by trained independent observers. Export the data to a spreadsheet file.</li>
	<li>Calculate the density by dividing total F-actin labeled puncta (N) by the length (L) of the MAP2 labeled dendrites. Express data as number of F-actin puncta per 10 &#181;m of dendrite 
	Note: Puncta (size &#8804;1.5 &#181;m) of F-actin fluorescence with a peak intensity of at least 50% above the average intensity of staining in the dendritic shaft were included in each selected dendritic segment.</li>
</ol>

<ol>
	<li>Heller, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biochemical+anatomy+of+cortical+inhibitory+synapses.">The biochemical anatomy of cortical inhibitory synapses.</a> PLoS One. 7, e39572 (2012).</li><li>Craig, A. M., Blackstone, C. D., Huganir, R. L., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+clustering+of+glutamate+and+gamma-aminobutyric+acid+receptors+opposite+terminals+releasing+the+corresponding+neurotransmitters.">Selective clustering of glutamate and gamma-aminobutyric acid receptors opposite terminals releasing the corresponding neurotransmitters.</a> Proc Natl Acad Sci USA. 91, 12373-12377 (1994).</li><li>Johnson, O. L., Ouimet, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+regulatory+role+for+actin+in+dendritic+spine+proliferation.">A regulatory role for actin in dendritic spine proliferation.</a> Brain Res. 1113, 1-9 (2006).</li><li>Rao, A., Craig, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+between+the+actin+cytoskeleton+and+the+postsynaptic+density+of+dendritic+spines.">Signaling between the actin cytoskeleton and the postsynaptic density of dendritic spines.</a> Hippocampus. 10 (5), 527-541 (2000).</li><li>Matus, A., Ackermann, M., Pehling, G., Byers, H. R., Fujiwara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+actin+concentrations+in+brain+dendritic+spines+and+postsynaptic+densities.">High actin concentrations in brain dendritic spines and postsynaptic densities.</a> Proc Natl Acad Sci USA. 79, 7590-7594 (1982).</li><li>Allison, D. W., Gelfand, V. I., Spector, I., Craig, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+actin+in+anchoring+postsynaptic+receptors+in+cultured+hippocampal+neurons:+differential+attachment+of+NMDA+versus+AMPA+receptors.">Role of actin in anchoring postsynaptic receptors in cultured hippocampal neurons: differential attachment of NMDA versus AMPA receptors.</a> J Neurosci. 18, 2423-2436 (1998).</li><li>Sekino, Y., Kojima, N., Shirao, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+actin+cytoskeleton+in+dendritic+spine+morphogenesis.">Role of actin cytoskeleton in dendritic spine morphogenesis.</a> Neurochem Int. 51, 92-104 (2007).</li><li>Zhang, W., Benson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+synapse+development+defined+by+dependence+on+F-actin.">Stages of synapse development defined by dependence on F-actin.</a> J Neurosci. 21 (14), 5169-5181 (2001).</li><li>Bertrand, S. J., Aksenova, M. V., Mactutus, C. F., Booze, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+Tat+protein+variants:+Critical+role+for+the+cysteine+region+in+synaptodendritic+injury.">HIV-1 Tat protein variants: Critical role for the cysteine region in synaptodendritic injury.</a> Exp Neurol. 248, 228-235 (2013).</li><li>Bertrand, S. J., Mactutus, C. F., Aksenova, M. V., Espensen-Sturges, T. D., Booze, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptodendritic+recovery+following+HIV+Tat+exposure:+neurorestoration+by+phytoestrogens.">Synaptodendritic recovery following HIV Tat exposure: neurorestoration by phytoestrogens.</a> J Neurochem. 128 (1), 140-151 (2014).</li><li>Lau, P. M., Zucker, R. S., Bentley, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+filopodia+by+direct+local+elevation+of+intracellular+calcium+ion+concentration.">Induction of filopodia by direct local elevation of intracellular calcium ion concentration.</a> J Cell Biol. 145, 1265-1275 (1999).</li></ol>

None of the authors have conflicts of interest to declare.

In this protocol, we describe culturing rat cortical neurons at low density in 35 mm glass-bottom dishes which allows us to identify dendrites of individual neurons. Next, we use Phalloidin and MAP2 staining to detect dendritic changes. Then, we used specialized software to quantify changes in F-actin puncta.
To determine changes in F-actin puncta the entire neuronal network of an individual neuron must be clearly visible, this allows selection of appropriate second order dendritic segments fr...

The primary goal of this study is to develop a reliable method of measurement (estimation) of synaptic integrity of the neuronal dendritic network. Here we describe quantification of F-actin puncta in primary rat cultured neurons using a combination of Phalloidin staining and immunocytochemical (ICC) detection of dendrites with subsequent analysis using specialized (NIS-Elements) software.
Labeled phallotoxins have similar affinity for both large and small filaments (F-actin) but do not bind to monomeric globular actin (G-actin), unlike some actin antibodies 1. Nonspecific binding of Phalloidin is negligible, thus providing minimal background during cellular imaging. Phalloidin is much smaller than antibodies that would typically be used to label cellular proteins for fluorescent microscopy, which allows for much more intense labeling of F-actin by Phalloidin. Thus, detailed images of F-actin localization in neurons can be obtained through the use of labeled Phalloidin.
Phalloidin (F-actin) staining of neuronal dendrites generates discrete &#34;hot spots&#34; or bright &#34;puncta&#34;, which represent a variety of dendritic structures, including mature spines, non-spiny synapses 2 and immature spines. Immature spines include thin filopodia and some forms of patch morphology, and may represent the initiation of spinogenesis 3. Immature spines and non-spiny patches lack PSD95 4. Changes in production of F-actin lead to subsequent changes in not only spines but also additional dendritic structures, thus making Phalloidin an important tool for investigating synaptodendritic integrity 5-7. In general, numbers of Phalloidin-positive (F-actin) puncta reflect a balance among active synapses (excitatory and inhibitory), actin dynamics and synapse stability 8.
Although it is important to study specific types of synapses (i.e., excitatory spines), when the target of a treatment is unknown it is necessary to first estimate the general integrity of a variety of dendritic structures. Since F-actin is a major component of dendritic spines and other structures, including inhibitory synapses, an altered number of F-actin puncta may indicate a synaptopathy. This synaptopathy may then be investigated further for more specific alterations. Our quantification method for detecting multiple synaptic types/structures yields an overall estimate of dendritic synaptic alterations (increases and decreases) following various experimental treatments.

<table><tbody><tr><td>35 mm Glass Bottom Dishes No. 1.5 coverglass</td><td>MatTek Corporation</td><td>P35G-1.5-20-C</td><td></td></tr><tr><td>DMEM/F12 medium</td><td>Life Technologies</td><td>10565-018</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Life Technologies</td><td>15400-054</td><td></td></tr><tr><td>Poly-L-Lysine</td><td>Sigma</td><td>P9155</td><td></td></tr><tr><td>Boric acid</td><td>Sigma</td><td>B0252</td><td></td></tr><tr><td>Borax</td><td>Sigma</td><td>B9876</td><td></td></tr><tr><td>GlutaMax</td><td>Life Technologies</td><td>35050-061</td><td>100X</td></tr><tr><td>Glucose</td><td>VWR</td><td>101174Y</td><td></td></tr><tr><td>HBSS</td><td>Sigma</td><td>H4641</td><td>10X</td></tr><tr><td>Neurobasal medium</td><td>Life Technologies</td><td>21103-049</td><td></td></tr><tr><td>B-27 supplement</td><td>Life Technologies</td><td>17504-044</td><td>50X</td></tr><tr><td>Antibiotic-Antimycotic solution</td><td>Cellgro</td><td>30004CI</td><td>100X</td></tr><tr><td>Sodium Bicarbonate</td><td>Life Technologies</td><td>25080</td><td></td></tr><tr><td>Vannas Scissors</td><td>World Precision Instruments</td><td>500086</td><td></td></tr><tr><td>Iris Scissors</td><td>World Precision Instruments</td><td>500216</td><td></td></tr><tr><td>Iris Forceps</td><td>World Precision Instruments</td><td>15914</td><td></td></tr><tr><td>Dumont #7 Forceps</td><td>World Precision Instruments</td><td>14097</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>World Precision Instruments</td><td>14095</td><td></td></tr><tr><td>ProLong Gold</td><td>Life Technologies</td><td>P36930</td><td></td></tr><tr><td>Paraformaldehyde&amp;nbsp;</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Cover glass</td><td>VWR</td><td>631-0137</td><td></td></tr><tr><td>AlexaFluor 488 Phalloidin</td><td>Life Technologies</td><td>A12379</td><td></td></tr><tr><td>Normal horse serum</td><td>Life Technologies</td><td>26050-070</td><td></td></tr><tr><td>Chicken polyclonal anti-MAP2</td><td>abcam</td><td>Ab92434</td><td></td></tr><tr><td>Alexa Red 594-conjugated goat anti-chicken IgG</td><td>Life Technologies</td><td>A11042</td><td></td></tr><tr><td>NucBlue Live cell stain ReadyProbes Reagent Hoescht 33342</td><td>Life Technologies</td><td>R37605</td><td></td></tr><tr><td>NIS-Elements software package</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>Nikon Eclipse TE2000-E inverted fluorescent computer-controlled microscope&amp;nbsp;</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>0.2 &amp;mu;m Nalgene nylon membrane filter</td><td>Fishersci</td><td>151-4020</td><td></td></tr></tbody></table>

quantification of filamentous actin (f-actin) puncta in rat cortical neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

In the present methods, we first culture rat cortical neurons at low density in 35 mm glass-bottom dishes, which allows us to identify the dendrites of individual neurons. In Figure 1, the differential interference contrast (DIC) images show the morphological changes in developing fetal rat cortical neurons at days 4, 6, 10, 14, 21 and 27 in vitro. Note that the length and number of dendrites increase with maturation of cultured rat primary neurons. Neurons are u...

Watch this Scientific Journal Video about Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons at JoVE.com

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

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Neuroscience

12.0K Views.  University of South Carolina. Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Video: Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally expressed molecular guidance cues that bind growth cone receptors and regulate the dynamics and organization of the growth cone cytoskeleton3-6. The main target of these navigational signals is the actin filament meshwork that fills the growth cone periphery and that drives growth cone motility through continual actin polymerization and dynamic remodeling7. Positive or attractive guidance cues induce growth cone turning by stimulating actin filament (F-actin) polymerization in the region of the growth cone periphery that is nearer the source of the attractant cue. This actin polymerization drives local growth cone protrusion, adhesion of the leading margin and axonal elongation toward the attractant.
Actin filament polymerization depends on the availability of sufficient actin monomer and on polymerization nuclei or actin filament barbed ends for the addition of monomer. Actin monomer is abundantly available in chick retinal and dorsal root ganglion (DRG) growth cones. Consequently, polymerization increases rapidly when free F-actin barbed ends become available for monomer addition. This occurs in chick DRG and retinal growth cones via the local activation of the F-actin severing protein actin depolymerizing factor (ADF/cofilin) in the growth cone region closer to an attractant8-10. This heightened ADF/cofilin activity severs actin filaments to create new F-actin barbed ends for polymerization. The following method demonstrates this mechanism. Total content of F-actin is visualized by staining with fluorescent phalloidin. F-actin barbed ends are visualized by the incorporation of rhodamine-actin within growth cones that are permeabilized with the procedure described in the following, which is adapted from previous studies of other motile cells11, 12. When rhodamine-actin is added at a concentration above the critical concentration for actin monomer addition to barbed ends, rhodamine-actin assembles onto free barbed ends. If the attractive cue is presented in a gradient, such as being released from a micropipette positioned to one side of a growth cone, the incorporation of rhodamine-actin onto F-actin barbed ends will be greater in the growth cone side toward the micropipette10.
Growth cones are small and delicate cell structures. The procedures of permeabilization, rhodamine-actin incorporation, fixation and fluorescence visualization are all carefully done and can be conducted on the stage of an inverted microscope. These methods can be applied to studying local actin polymerization in migrating neurons, other primary tissue cells or cell lines.

A method to visualize and quantify F-actin barbed ends in neuronal growth cones is described. After culturing neurons on glass coverslips, cells are permeabilized with a saponin-containing solution. Then, a short incubation with the saponin buffer containing rhodamine-actin incorporates fluorescent actin onto free actin barbed ends.

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally ...

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

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of their normal in vivo interactions. Slices obtained from a variety of transgenic mouse lines or use of viral vectors for expression of fluorescently tagged proteins or reporters in wild type brain slices allow for high-resolution imaging by fluorescence microscopy. Although several methods have been developed for imaging brain slices, combining slice culture with the ability to perform repetitive high-resolution imaging of specific cells in live slices over long time periods has posed problems. This is especially true when viral vectors are used for expression of exogenous proteins since this is best done in a closed system to protect users and prevent cross contamination. Simple modifications made to the roller tube brain slice culture method that allow for repetitive high-resolution imaging of slices over many weeks in an enclosed system are reported. Culturing slices on photoetched coverslips permits the use of fiducial marks to rapidly and precisely reposition the stage to image the identical field over time before and after different treatments. Examples are shown for the use of this method combined with specific neuronal staining and expression to observe changes in hippocampal slice architecture, viral-mediated neuronal expression of fluorescent proteins, and the development of cofilin pathology, which was previously observed in the hippocampus of Alzheimer's disease (AD) in response to slice treatment with oligomers of amyloid-&#946; (A&#946;) peptide.

Presented here is a modified roller tube method for culturing and intermittent high-resolution imaging of rodent brain slices over many weeks with precise repositioning on photoetched coverslips. Neuronal viability and slice morphology are well maintained. Applications of this fully enclosed system using viruses for cell-type specific expression are provided.

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of ...

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 ...

Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy (3D-SIM)

The axon initial segment (AIS) is the site at which action potentials initiate and constitutes a transport filter and diffusion barrier that contribute to the maintenance of neuronal polarity by sorting somato-dendritic cargo. A membrane periodic skeleton (MPS) comprising periodic actin rings provides a scaffold for anchoring various AIS proteins, including structural proteins and different ion channels. Although recent proteomic approaches have identified a considerable number of novel AIS components, details of the structure of the MPS and the roles of its individual components are lacking. The distance between individual actin rings in the MPS (~190 nm) necessitates the employment of super-resolution microscopy techniques to resolve the structural details of the MPS. This protocol describes a method for using cultured rat hippocampal neurons to examine the precise localization of an AIS protein in the MPS relative to sub-membranous actin rings using 3D-structured illumination microscopy (3D-SIM). In addition, an analytical approach to quantitively assess the periodicity of individual components and their position relative to actin rings is also described.

Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy 3D-SIM

The present protocol describes a method to visualize and measure actin rings and other components of the membrane periodic skeleton of the axon initial segment using cultured rat hippocampal neurons and 3D-structured illumination microscopy (3D-SIM).

The axon initial segment (AIS) is the site at which action potentials initiate and constitutes a transport filter and diffusion barrier that contribute to ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Biology

Quantification of Filamentous Actin Puncta in Rat Cortical Neurons

Source: Li, H., et al. <a href="https://app.jove.com/v/53697">Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons.</a> J. Vis. Exp. (2016)
This video demonstrates the procedure for fluorescently staining rat cortical neurons for microscopy. It targets F-actin and dendritic proteins to analyze structural integrity and calculate F-actin density, which is crucial for studying neuronal function and health.

1. Fluorescent Labeling and Immunocytochemistry
Note: The immunofluorescent labeling of primary cortical cell cultures was carried out in glass bottom 35 ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

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