Name: three-dimensional quantification of dendritic spines from pyramidal neurons derived from human induced pluripotent stem cells
Uploaded: 2015-10-10T14:00:00
Description: Watch this Scientific Journal Video about Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells at JoVE.com

This work was funded by the Institut Pasteur, the Bettencourt-Schueller foundation, Centre National de la Recherche Scientifique, University Paris Diderot, Agence Nationale de la Recherche (ANR-13-SAMA-0006; SynDivAutism), the Conny-Maeva Charitable Foundation, the Cognacq Jay Foundation, the Orange Foundation, and the Fondamental Foundation. L.G. is supported by an undergraduate fellowship from the Health Ministry. We acknowledge the help of BitPlane in particular Georgia Golfis, in the early stage of this work.

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&#160;pluripotent ...

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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...

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 manual spine analysis3.
Green fluorescence protein (GFP) technology coupled to immunofluorescence represents an accurate tool for spine identification and shape measurement by fluorescence microscopy. This approach can be easily applied to cultured neurons. However, no data have been reported on the analysis of spine maturation and morphology on human neurons derived from induced&#160;pluripotent stem cells (iPSC).
The objective of this study was to describe a protocol, which allows dendritic spine imaging from cultured human neurons in vitro. GFP labeling, confocal microscopy and 3D analysis with the Filament Tracer module of Imaris software were used in the present protocol. Culture steps that are necessary to obtain cortical glutamatergic neurons of layers II to IV from neural stem cells (NSC) are also briefly described here.&#160;The entire protocol for human NSC production has already been published elsewhere 4.

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			<td>Gibco/ Life Technologies</td>
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			<td>Sigma Aldrich</td>
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			<td>Mouse laminin</td>
			<td>Dutscher Dominique</td>
			<td>354232</td>
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			<td>N2 Supplement</td>
			<td>Gibco/ Life Technologies</td>
			<td>17502048</td>
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			<td>B-27 Supplement w/o vit A (50X)</td>
			<td>Gibco/ Life Technologies</td>
			<td>12587010</td>
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			<td>DMEM/NUT.MIX F-12 W/GLUT-I</td>
			<td>Gibco/ Life Technologies</td>
			<td>31331028</td>
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			<td>Neurobasal Med SFM</td>
			<td>Gibco/ Life Technologies</td>
			<td>21103049</td>
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			<td>2-mercaptoethanol</td>
			<td>Gibco/ Life Technologies</td>
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			<td>Gibco/ Life Technologies</td>
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			<td>GFP Rabbit Serum Polyclonal Antibody</td>
			<td>Gibco/ Life Technologies</td>
			<td>A-6455</td>
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			<td>Horse serum</td>
			<td>Gibco/ Life Technologies</td>
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			<td>Alexa Fluor 488 Goat Anti-Rabbit&#160;</td>
			<td>Gibco/ Life Technologies</td>
			<td>A11034</td>
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			<td>Polyclonal Anti-betaIII tubulin antibody</td>
			<td>Millipore</td>
			<td>AB9354</td>
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			<td>Coverglass 13 mm</td>
			<td>VWR</td>
			<td>631-0150</td>
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			<td>Prolong Gold Antifade Reagent avec DAPI</td>
			<td>Gibco/ Life Technologies</td>
			<td>P36931</td>
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			<td>Tween(R) 20 Bioextra, Viscous Liquid</td>
			<td>Sigma Aldrich Chimie</td>
			<td>P7949</td>
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			<td>Triton X-100</td>
			<td>Sigma Aldrich Chimie</td>
			<td>X100-100ML</td>
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			<td>Human Fibroblasts</td>
			<td>Coriell Cell Line Biorepository</td>
			<td>GM 4603 and GM 1869</td>
			<td>Coriell Institute for Medical Research, Camden, NJ, USA</td>
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			<td>Confocal laser scanning microscope</td>
			<td>Zeiss (Germany)</td>
			<td>LSM 700</td>
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			<td>Imaris Software</td>
			<td>Bitplane AG, Zurich</td>
			<td>6.4.0 version</td>
			<td>Filament Tracer and Imaris XT modules are necessary</td>
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			<td>Huygens Software</td>
			<td>Huygens software, SVI, Netherlands</td>
			<td>Pro version</td>
			<td>Optional (for deconvolution testing)</td>
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three-dimensional quantification of dendritic spines from pyramidal neurons derived from human induced pluripotent stem cells

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&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

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...

Watch this Scientific Journal Video about Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells at JoVE.com

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

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&#160;pluripotent stem cells.

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

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