Name: quantification of filamentous actin (f-actin) puncta in rat cortical neurons
Uploaded: 2016-02-10T14:00:00
Description: Watch this Scientific Journal Video about Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons Video 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 Buff...

<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>

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.

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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 Video at JoVE.com

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

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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