Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Summary

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

Abstract

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these proteins may function in vivo.

Protocol

The protocol described here can be used to examine dendritic spine morphology and dynamics in any primary cultured system.

1. Preparation of primary cortical neuron cultures

1. Prepare high-density cortical neuron cultures from Sprague-Dawley rat E18 embryos and culture in glia-conditioned serum-free medium^1-2.
2. Euthanize one pregnant rat (E18) according to ACUC procedures; quickly remove uterus (with fetuses in it) and place in a 100 mm Petri dish on ice.
3. Cut open uterus and amniotic membrane, hold fetus by the neck (umbilical cord intact) with one forceps, use another forceps to peel scalp from back....

Discussion

The techniques described above for the detailed quantitative analysis of dendritic spine morphology, linear density and motility in either fixed or live primary cortical neurons are focused on understanding the effects of post-synaptic mechanisms that may contribute to neuropathologies. A similar approach can be used to quantify spine morphology or motility in any spiny neuron, including hippocampal pyramidal, Purkinje, or medium spiny neurons.

The protocol described here can be adapted to lo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
18 mm round Cover glass No. 1.5	Warner Instruments	64-0714 (CS-18R15)
22 mm square Cover glass No. 1.5	Warner Instruments	64-0721 (CS-22S15)
Poly-D-Lysine	Sigma-Aldrich	P-0899	MW 70~150 Kda
Neurobasal Media	Invitrogen	21103049
B27	Invitrogen	17504044
Glutamine	Invitrogen	21051024
Penicillin-Streptomycin	Invitrogen	15140148
D,L-APV (AP-5)	Ascent Scientific	Asc-004
Lipofectamine 2000	Invitrogen	11668019
DMEM	Invitrogen	11965092
HEPES	Mediatech, Inc.	25-060-C 1	1M, pH 7
Formaldehyde Solution	EMD Millipore	FX0415-5	36%, Histology grade
Normal Goats Serum	VWR international	100188-514	Jackson Immunoresearch Labs
Triton X-100	Fisher Scientific	AC21568-2500	Acros Organics
Alexa Fluor 488 goat anti-mouse IgG (H+L) highly cross-adsorbed	Invitrogen	A-11029
Alexa Fluor 488 goat anti-rabbit IgG (H+L) *highly cross-adsorbed*	Invitrogen	A-11034
ProLong Gold antifade reagent	Invitrogen	P36934	Special Packaging
Enclosed imaging stage chamber	Warner Instruments	RC-30HV
Temperature controller unit	Warner Instruments	TC-344B
MetaMorph	Universal Imaging