Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Summary

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced pluripotent stem cells.

Abstract

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis ¹. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Introduction

Dendritic spines of cortical pyramidal neurons are small and thin protrusions which are distributed along the basal and apical dendrites of these neuronal subtypes in rodent, primate, and human brain. They are the sites of most excitatory synapses and display key functions in learning and cognitive processes. The detailed structures of human dendritic spines have been technically studied by electron microscopy ². However, such approach is time-consuming and represents heavy workload. More recently, a three-dimensional (3D) reconstruction of the morphology of dendritic spines has been reported in human brain cortex using specific software combined to large m....

Protocol

1. Neuronal Culture

Note: Fibroblast reprogramming in pluripotent stem cells, commitment to the dorsal telencephalon lineage, derivation, amplification, and banking of late cortical progenitors (LCP) were described in Boissart et al⁴. Neuronal differentiation of LCP-like cells was also performed according to Boissart et al⁴ with slight modifications. Other procedures have been developed for direct reprogramming of fibroblasts into induced pluripotent stem cells followed by their differentiation into neurons. This protocol was retained since it allows the selective production of pyramidal glutamater....

Results

The present study describes a standardized protocol for spine quantification of cultured dendrites of pyramidal neurons derived from iPSC. This protocol allows the analysis of spine maturation on human neurons and its possible comparison with the maturation of spines in standard rodent neuronal cultures as well as in in vivo animal models.

Figure 1A represents a scheme of the different steps of culture which allow the production of cortical pyramidal neurons. Such sch.......

Discussion

The quantification of the morphological features of pyramidal neurons relied on the software. The Filament Tracer interface was used for segmentation of neurons and spines, and the XT module was used for their analysis.

To analyze the accuracy of our technique, we first compared the measured morphologic parameters (length, area, and total spine volume when applicable), with those published using rat mature pyramidal neurons in culture ^{6, 7} and human brain tissues ³. Densi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
D-PBS (1x), sans Calcium, Magnesium et Phenol Red	Gibco/ Life Technologies	14190169
Poly-L-Ornithine Solution Bioreagent	Sigma Aldrich	P4957
Mouse laminin	Dutscher Dominique	354232
N2 Supplement	Gibco/ Life Technologies	17502048
B-27 Supplement w/o Vitamin A (50x)	Gibco/ Life Technologies	12587010
DMEM/NUT.MIX F-12 W/GLUT-I	Gibco/ Life Technologies	31331028
Neurobasal Med SFM	Gibco/ Life Technologies	21103049
2-mercaptoethanol	Gibco/ Life Technologies	31350-010
Penicillin-Steptomycin	Gibco/ Life Technologies	15140-122
GFP Rabbit Serum Polyclonal Antibody	Gibco/ Life Technologies	A-6455
Horse serum	Gibco/ Life Technologies	16050130
Alexa Fluor 488 Goat Anti-Rabbit	Gibco/ Life Technologies	A11034
Polyclonal Anti-betaIII tubulin Antibody	Millipore	AB9354
Coverglass 13 mm	VWR	631-0150
Prolong Gold Antifade Reagent with DAPI	Gibco/ Life Technologies	P36931
Tween(R) 20 Bioextra, Viscous Liquid	Sigma Aldrich Chimie	P7949
Triton X-100	Sigma Aldrich Chimie	X100-100ML
Human Fibroblasts	Coriell Cell Line Biorepository	GM 4603 and GM 1869	Coriell Institute for Medical Research, Camden, NJ, USA
Confocal laser scanning microscope	Zeiss (Germany)	LSM 700
Imaris Software	Bitplane AG, Zurich	6.4.0 version	Filament Tracer and Imaris XT modules are necessary
Huygens Software	Huygens software, SVI, Netherlands	Pro version	Optional (for deconvolution testing)