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

Summary

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.

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.

Introduction

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 minim....

Protocol

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

1. Preparation for primary cortical neuron culture
   1. Solutions:
      1. Prepare Poly-L-Lysine stock solution by dissolving 5 mg of Poly-L-Lysine in 10 ml Borate buffer.
      2. Prepare working solution by diluting 1 ml Poly-L-Lysine Stock in 49 ml Borate Buffer.
      3. 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 Bo....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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