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 stem cells followed by their differentiation into neurons. This protocol was retained since it allows the selective production of pyramidal glutamatergic neurons.
<ol>
	<li>Treat 6-well culture plates with glass coverslips with poly-ornithine (diluted to 1/6 in DPBS, stock concentration 0.01%) O/N, followed by three washes in DPBS. Then add laminin (stock concentration 1 mg/ml, diluted 500 times in DPBS) for at least 10 hr under the flow hood.</li>
	<li>Plate and dispatch NSC at low density (50,000 cells/cm2) in 6-well culture plates with glass coverslips in 3 ml of culture medium consisting of DMEM/F12 (500 ml), 2 vials (5 ml each) of N2 supplement, 2 vials (10 ml each) of B27 supplement, 10 ml of Pen-Streptomycin (Penicillin = 10,000 units/ml and Streptomycin = 10,000 units/ml), 1 ml of 2-mercaptoethanol (Stock solution: 50 mM) and laminin (1/500), without growth factors. CRITICAL STEP: Carry out this step carefully by adding cells with slow rotating movements in order to reduce cell clustering.</li>
	<li>Remove the culture medium. Add fresh N2B27 medium containing 2 &#181;g/ml of fresh laminin solution to keep the neuron attached on the glass coverslips and avoid clumping. Change the medium every 3 days. Keep some of the remaining medium (200 &#181;l) before adding fresh medium in order to prevent the cell from drying. Alternatively, proceed rapidly and change the total volume (3 ml).</li>
</ol>
2. Lentiviral Transduction
Note: GFP lentiviral vectors were kindly provided by Dr. Uwe Maskos Laboratory at Institut Pasteur (Paris) and prepared according to the published protocol 5. GFP expression is driven by the mouse phosphoglycerate kinase (PGK) promoter. For this study, viral titer was of 400 ng/&#181;l (stock solution in PBS 1x).
<ol>
	<li>Transduce human iPSC-derived neurons at any step of maturation by viral particles by adding 1 &#181;l of the stock solution containing 40 ng of GFP lentiviral vector per culture well (6-well plates) and incubate for 48 hr in fresh culture medium. Note: For this protocol, the duration of incubation allows a good labeling of spine structures.</li>
</ol>
3. Immunofluorescence
Note: In order to improve the labeling of the whole spine morphology, immunofluorescence labeling was performed using an anti-GFP antibody in permeabilized conditions.
<ol>
	<li>Remove culture medium and fix transduced cells on coverslips in 4% paraformaldehyde for 10 min at RT, then wash 3 times in 1x&#160;PBS (10 min each).</li>
	<li>Immerge coverslips in PBS supplemented with 0.05% Triton (100x) and 10% horse serum for 1 hr at RT, then wash 3 times in 1x&#160;PBS.</li>
	<li>Add 100 &#181;l of 1x PBS supplemented with 4% horse serum and primary antibody (1/1,000) raised against GFP and diluted by a factor of 1,000 on each coverslip. Incubate in a dark box O/N at 4&#176;C, then wash 3 times with 1x PBS.</li>
	<li>Dilute Alexa Fluor 488-conjugated antibody (1/200) in PBS supplemented with 0.5% of Tween 20 and incubate for 1 hr at RT. Then wash 3 times in 1x PBS and mount coverslips on glass slides with mounting medium for fluorescence microscopy.</li>
</ol>
4. Dendritic Spine Imaging
<ol>
	<li>Perform confocal Imaging through a confocal laser-scanning microscope.</li>
	<li>Select healthy neurons with pyramidal morphology and a full dendritic arborization, and quantify at least 10 neurons per condition from separate experiments. Quantify 60 - 100 &#181;m per dendrite.</li>
	<li>Acquire images using a 40X oil NA = 1.3 objective and a 488&#160;nm laser line for GFP excitation, with a typical peak power at sample level around 20&#160;&#181;W. Set pixel size around 80&#160;nm to properly sample dendritic spines. 
	Note: The subsequent analysis call for an image in which the noise does not compromise the proper segmentation of dendrites and spines. With the spatial sampling and power settings mentioned above, we observed that a pixel dwell-time of 3.15&#160;&#181;s is sufficient to collect enough photons to build such an image. For dim samples, image quality can be improved by averaging 2 to 4 scans. The acquisition of several XY tiles may be needed to cover the region of interest, which then must be stitched together before processing.</li>
	<li>To sample the whole neuron volume, acquire a Z-stack, with a Z spacing ranging from 150&#160;nm to 300&#160;nm, yielding 20 to 30 Z slices. 
	Note: The lateral spatial resolution achieved with these settings is 234 nm and the axial resolution is 591 nm. The sampling chosen here is adequate, but as the axial resolution is larger than the smallest size of the spines to be imaged, the analysis favors the spines that extend laterally from the dendrites. 
	&#160;</li>
</ol>
5. 3D Quantification of Dendritic Spines
Note: The following sections specifically describe the use of the Imaris software for analysis. Alternative implementations exist, including NeuronStudio15 or Metamorph8, which can provide similar results.
Use the following key settings:
<ol>
	<li>As a pre-processing stage, use Gaussian filtering through the image processing facilities offered by the software. Perform Gaussian filtering via the Image Processing &#62; Smoothing &#62; Gaussian Filter. Set the filter width to be equal to the pixel size in XY.</li>
	<li>Perform a Semi-automatic tracing of dendrites, using the Filament Tracer module of the software.
	<ol>
		<li>First, estimate the dendrite diameter, by using the distance tool in the Slice&#160;tab of the software.&#160;</li>
		<li>In the Surpass&#160;tab, click on the Filaments tool. For a better robustness, this protocol relies on semi-automated tracking; click on Skip automatic creation. The module interface is now showing the Draw&#160;tab.&#160;Here, select AutoPath&#160;as a method, Dendrite as a type,&#160;and input the estimated dendrite diameter.</li>
		<li>Use the Select&#160;mode of the pointer, turning the cursor into a box. Shift-right-click on the dendrite starting point. Note: the software performs initial calculations.</li>
		<li>Move the pointer along the dendrite. From the starting point (represented as a blue sphere), a yellow line representing the most likely dendrite path is shown. Shift-left-click on the dendrite endpoint.</li>
	</ol>
	</li>
	<li>Perform the automated spines segmentation. Note: The spines on the traced dendrite are to be found automatically by the module interface.&#160;
	<ol>
		<li>In the module interface, click on the Creation&#160;tab. In the Rebuild&#160;drop-list, pick Rebuild Dendrite Diameter&#160;and check the Keep data&#160;box. Then click Rebuild.</li>
		<li>Set the Threshold&#160;so that the segmented volume corresponds to the actual dendrite volume. As an Algorithm, select Shortest Distance from Distance Map.&#160;Click the Next button.</li>
		<li>Determine the smallest spine head diameter and the maximal length, again by using the distance tool in the&#160;Slice&#160;tab of the software, then come back to the Surpass tab and enter the parameters. For this protocol,&#160;values around 200 - 300&#160;nm for minimal diameter and 4&#160;&#181;m for maximal length are good starting points.&#160;Do not check the Allow Branch Spines&#160;box.&#160;Click the&#160;Next&#160;button.</li>
		<li>Adjust the Seed Points Threshold&#160;so that the blue points representing spines localize to actual spine heads.&#160;Click the&#160;Next&#160;button. Note: The core calculation is done now and can be lengthy.</li>
	</ol>
	</li>
	<li>Classify spines by going to the Tools tab of the module interface. Click on Classify Spines. In the prompted user interface, make sure that there are four classes, defined by their morphology as follows: Stubby: length&#160;&#60;1 &#181;m; Mushroom: Length (spine) &#62;3 and Max width (head) &#62;mean width (neck) x&#160;2; Long thin: Mean width (head) &#8805;&#160;Mean width (neck); Filopodia-like: Length &#8804;&#160;4 &#181;m (no head). &#160; 
	Note: The module interface generates four new Filament objects containing the results of the classification.&#160;</li>
	<li>Export Statistical data: on any of these four object interfaces, go to the Statistics tab.&#160; 
	Click on the Export All Statistics to File&#160;button. 
	Note: Other statistical values can be exported for dendrites (e.g., length, area, mean diameter, branch depth, branching angle, volume, etc.), and for spines (e.g.,&#160;straightness, area of attachment, length and volume of distinct spine parts, spine diameter, densities, etc.)</li>
</ol>

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I., Espinosa, A., Fairen, A., Yuste, R., Defelipe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-synaptic+dendritic+spines+in+neocortex.">Non-synaptic dendritic spines in neocortex.</a> Neuroscience. 145, 464-469 (2007).</li><li>Benavides-Piccione, R., Fernaud-Espinosa, I., Robles, V., Yuste, R., DeFelipe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-based+comparison+of+human+dendritic+spine+structure+using+complete+three-dimensional+reconstructions.">Age-based comparison of human dendritic spine structure using complete three-dimensional reconstructions.</a> Cerebral Cortex. 23 (8), 1798-1810 (2013).</li><li>Boissart, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+from+human+pluripotent+stem+cells+of+cortical+neurons+of+the+superficial+layers+amenable+to+psychiatric+disease+modeling+and+high-throughput+drug+screening.">Differentiation from human pluripotent stem cells of cortical neurons of the superficial layers amenable to psychiatric disease modeling and high-throughput drug screening.</a> Translational Psychiatry. 3, 1-11 (2013).</li><li>Avale, M. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Afadin+is+required+for+maintenance+of+dendritic+structure+and+excitatory+tone.">Afadin is required for maintenance of dendritic structure and excitatory tone.</a> Journal of Biological Chemistry. 287 (43), 35964-35974 (2012).</li><li>Srivastava, D. P., Woolfrey, K. M., Penzes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+dendritic+spine+morphology+in+cultured+CNS+neurons.">Analysis of dendritic spine morphology in cultured CNS neurons.</a> Journal of Visualized Experiments. (53), e2794 (2011).</li><li>Brennand, K. J., Gage, F. 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The authors have nothing to disclose.

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.

<table><tbody><tr><td>D-PBS (1x), sans Calcium, Magnesium et Phenol Red</td><td>Gibco/ Life Technologies</td><td>14190169</td><td></td></tr><tr><td>Poly-L-Ornithine Solution Bioreagent</td><td>Sigma Aldrich</td><td>P4957</td><td></td></tr><tr><td>Mouse laminin</td><td>Dutscher Dominique</td><td>354232</td><td></td></tr><tr><td>N&lt;sub&gt;2&lt;/sub&gt; Supplement</td><td>Gibco/ Life Technologies</td><td>17502048</td><td></td></tr><tr><td>B-27 Supplement w/o Vitamin A (50x)</td><td>Gibco/ Life Technologies</td><td>12587010</td><td></td></tr><tr><td>DMEM/NUT.MIX F-12 W/GLUT-I</td><td>Gibco/ Life Technologies</td><td>31331028</td><td></td></tr><tr><td>Neurobasal Med SFM</td><td>Gibco/ Life Technologies</td><td>21103049</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Gibco/ Life Technologies</td><td>31350-010</td><td></td></tr><tr><td>Penicillin-Steptomycin</td><td>Gibco/ Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>GFP Rabbit Serum Polyclonal Antibody</td><td>Gibco/ Life Technologies</td><td>A-6455</td><td></td></tr><tr><td>Horse serum</td><td>Gibco/ Life Technologies</td><td>16050130</td><td></td></tr><tr><td>Alexa Fluor 488 Goat Anti-Rabbit&amp;nbsp;</td><td>Gibco/ Life Technologies</td><td>A11034</td><td></td></tr><tr><td>Polyclonal Anti-betaIII tubulin Antibody</td><td>Millipore</td><td>AB9354</td><td></td></tr><tr><td>Coverglass 13 mm</td><td>VWR</td><td>631-0150</td><td></td></tr><tr><td>Prolong Gold Antifade Reagent with DAPI</td><td>Gibco/ Life Technologies</td><td>P36931</td><td></td></tr><tr><td>Tween(R) 20 Bioextra, Viscous Liquid</td><td>Sigma Aldrich Chimie</td><td>P7949</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich Chimie</td><td>X100-100ML</td><td></td></tr><tr><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></tr><tr><td>Confocal laser scanning microscope</td><td>Zeiss (Germany)</td><td>LSM 700</td><td></td></tr><tr><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></tr><tr><td>Huygens Software</td><td>Huygens software, SVI, Netherlands</td><td>Pro version</td><td>Optional (for deconvolution testing)</td></tr></tbody></table>

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 ...

three-dimensional-quantification-dendritic-spines-from-pyramidal

Research

JoVE Journal

Developmental Biology

11.5K Views.  Institut Pasteur.  Dendritische stekels van piramidale neuronen zijn de sites van de meest prikkelende synapsen in de hersenen van zoogdieren cortex. Deze methode beschrijft een 3D-kwantitatieve analyse van de wervelkolom morfologie in menselijke corticale piramidale glutamaat neuronen afgeleid van geïnduceerde pluripotente stamcellen. 

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

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

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.

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.

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Neuroscience

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

3D Modeling of Dendritic Spines with Synaptic Plasticity

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the mechanisms of synaptic plasticity in dendritic spines. In this protocol, the detailed 3D structure of the dendrites and dendritic spines is modeled with meshes on the software Blender with CellBlender. The synaptic and extrasynaptic regions are defined on the mesh. Next, the synaptic receptor and synaptic anchor molecules are defined with their diffusion constants. Finally, the chemical reactions between synaptic receptors and synaptic anchors are included and the computational model is solved numerically with the software MCell. This method describes the spatiotemporal path of every single molecule in a 3D geometrical structure. Thus, it is very useful to study the trafficking of synaptic receptors in and out of the dendritic spines during the occurrence of synaptic plasticity. A limitation of this method is that the high number of molecules slows the speed of the simulations. Modeling of dendritic spines with this method allows the study of homosynaptic potentiation and depression within single spines and heterosynaptic plasticity between neighbor dendritic spines.

The protocol develops a three-dimensional (3D) model of a dendritic segment with dendritic spines for modeling synaptic plasticity. The constructed mesh can be used for computational modeling of AMPA receptor trafficking in the long-term synaptic plasticity using the software program Blender with CellBlender and MCell.

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Rapid Golgi Stain for Dendritic Spine Visualization in Hippocampus and Prefrontal Cortex

Golgi impregnation, using the Golgi staining kit with minor adaptations, is used to impregnate dendritic spines in the rat hippocampus and medial prefrontal cortex. This technique is a marked improvement over previous methods of Golgi impregnation because the premixed chemicals are safer to use, neurons are consistently well impregnated, there is far less background debris, and for a given region, there are extremely small deviations in spine density between experiments. Moreover, brains can be accumulated after a certain point and kept frozen until further processing. Using this method any brain region of interest can be studied. Once stained and cover slipped, dendritic spine density is determined by counting the number of spines for a length of dendrite and expressed as spine density per 10 &#181;m dendrite.

The protocol describes a modification of the rapid Golgi method, which can be adapted to any part of the nervous system, for staining neurons in the hippocampus and medial prefrontal cortex of the rat.

Golgi impregnation, using the Golgi staining kit with minor adaptations, is used to impregnate dendritic spines in the rat hippocampus and medial ...

Protocol for Dendritic Spine Analysis Using Neurolucida 360

Dendritic Spine Quantification Using an Automatic Three-Dimensional Neuron Reconstruction Software

Synaptic connections allow for the exchange and processing of information between neurons. The post-synaptic site of excitatory synapses is often formed on dendritic spines. Dendritic spines are structures of great interest in research centered around synaptic plasticity, neurodevelopment, and neurological and psychiatric disorders. Dendritic spines undergo structural modifications during their lifespan, with properties such as total spine number, dendritic spine size, and morphologically defined subtype altering in response to different processes. Delineating the molecular mechanisms regulating these structural alterations of dendritic spines relies on morphological measurement. This mandates accurate and reproducible dendritic spine analysis to provide experimental evidence. The present study outlines a detailed protocol for dendritic spine quantification and classification using Neurolucida 360 (automatic three-dimensional neuron reconstruction software). This protocol allows for the determination of key dendritic spine properties such as total spine density, spine head volume, and classification into spine subtypes thus enabling effective analysis of dendritic spine structural phenotypes.

Author Spotlight: Optimizing Dendritic Spine Analysis for Balanced Manual and Automated Assessment in the Hippocampus CA1 Apical Dendrites

Dendritic spines are post-synaptic compartments of most excitatory synapses. Alterations to dendritic spine morphology occur during neurodevelopment, aging, learning, and many neurological and psychiatric disorders, underscoring the importance of reliable dendritic spine analysis. This protocol describes quantifying dendritic spine morphology accurately and reproducibly using automatic three-dimensional neuron reconstruction software.

Synaptic connections allow for the exchange and processing of information between neurons. The post-synaptic site of excitatory synapses is often formed ...

Generating Cortical Pyramidal Neurons from Human Neural Stem Cells

Source: Gouder, L., et al,. <a href="https://app.jove.com/v/53197">Three-dimensional quantification of dendritic spines from pyramidal neurons derived from human induced pluripotent stem cells.</a> J. Vis. Exp. (2015)
This video describes a method for preparing glass coverslips coated with polyornithine and laminin to enhance the adhesion and growth of neural stem cells. The technique involves sequential incubations and washes, facilitating the formation of a supportive matrix that promotes neuronal cell growth and differentiation, mimicking structures found in the neurons.


	Neuronal Culture
	Note: Fibroblast reprogramming in pluripotent stem cells, commitment to the dorsal telencephalon lineage, derivation, amplification, ...

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

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