title: Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons.
abstract: 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 µm) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.
authors.affiliation: Laboratory Program in Behavioral, Neuroscience, Department of Psychology, University of South Carolina.
authors.forename: Hailong
authors.initials: H
authors.lastname: Li
authors.affiliation: Laboratory Program in Behavioral, Neuroscience, Department of Psychology, University of South Carolina.
authors.forename: Marina
authors.initials: M
authors.lastname: Aksenova
authors.affiliation: Laboratory Program in Behavioral, Neuroscience, Department of Psychology, University of South Carolina.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Bertrand
authors.affiliation: Laboratory Program in Behavioral, Neuroscience, Department of Psychology, University of South Carolina.
authors.forename: Charles F
authors.initials: CF
authors.lastname: Mactutus
authors.affiliation: Laboratory Program in Behavioral, Neuroscience, Department of Psychology, University of South Carolina; booze@mailbox.sc.edu.
authors.forename: Rosemarie
authors.initials: R
authors.lastname: Booze
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D003712:Dendrites / Q000378:metabolism; D006624:Hippocampus / Q000196:embryology* / Q000378:metabolism; D007150:Immunohistochemistry; D008840:Microfilament Proteins / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009473:Neuronal Plasticity / Q000502:physiology*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D051381:Rats / Q000196:embryology; D013569:Synapses / Q000378:metabolism
chemical_list: D000199:Actins; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins