Name: Analysis of Dendritic Spine Morphology in Cultured CNS Neurons (Video) | JoVE Journal
Uploaded: 2011-07-13T12:00:00.000+00:00
Duration: 11 min 48 s
Description: Learn to analyze dendritic spine morphology and motility in primary cultured cortical neurons using GFP transfection and confocal microscopy. This method enables quantitative assessment of synaptic structure dynamics.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

playerTrialDesktop

We thank Kelly Jones for careful editing. This work was supported by NIH grant R01MH 071316, Alzheimer's Association, the 
National Alliance for Research on Schizophrenia and Depression (NARSAD), and the National Alliance for Autism Research (NAAR) (P.P.); American Heart 
Association Postdoctoral Fellowship (D.P.S.); American Heart Association Predoctoral Fellowship (K.M.W.).

<ol><li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developments+in+neuronal+cell+culture%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Developments in neuronal cell culture.</a> Nature. 336, 185-186 (1988).</li>
<li>Xie, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kalirin-7+controls+activity-dependent+structural+and+functional+plasticity+of+dendritic+spines%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Kalirin-7 controls activity-dependent structural and functional plasticity of dendritic spines.</a> Neuron. 56, 640-656 (2007).</li>
<li>Spear, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modeling+adolescent+development+and+alcohol+use+in+animals%5BTitle%5D)+AND+%22Alcohol+Res+Health%22%5BJournal%5D)">Modeling adolescent development and alcohol use in animals.</a> Alcohol Res Health. 24, 115-123 (2000).</li>
<li>Srivastava, D. P., Woolfrey, K. M., Liu, F., Brandon, N. J., Penzes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estrogen+receptor+ss+activity+modulates+synaptic+signaling+and+structure%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Estrogen receptor ss activity modulates synaptic signaling and structure.</a> J Neurosci. 30, 13454-13460 (2010).</li>
<li>Srivastava, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+enhancement+of+two-step+wiring+plasticity+by+estrogen+and+NMDA+receptor+activity%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">Rapid enhancement of two-step wiring plasticity by estrogen and NMDA receptor activity.</a> Proc Natl Acad Sci USA. 105, 14650-14655 (2008).</li>
<li>Woolfrey, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epac2+induces+synapse+remodeling+and+depression+and+its+disease-associated+forms+alter+spines%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Epac2 induces synapse remodeling and depression and its disease-associated forms alter spines.</a> Nat Neurosci. 12, 1275-1284 (2009).</li>
<li>Allison, D. W., Gelfand, V. I., Spector, I., Craig, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+actin+in+anchoring+postsynaptic+receptors+in+cultured+hippocampal+neurons%3A+differential+attachment+of+NMDA+versus+AMPA+receptors%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Role of actin in anchoring postsynaptic receptors in cultured hippocampal neurons: differential attachment of NMDA versus AMPA receptors.</a> J Neurosci. 18, 2423-2436 (1998).</li>
<li>Harms, K. J., Tovar, K. R., Craig, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synapse-specific+regulation+of+AMPA+receptor+subunit+composition+by+activity%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Synapse-specific regulation of AMPA receptor subunit composition by activity.</a> J Neurosci. 25, 6379-6388 (2005).</li>
<li>Xie, Z., Huganir, R. L., Penzes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activity-dependent+dendritic+spine+structural+plasticity+is+regulated+by+small+GTPase+Rap1+and+its+target+AF-6%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Activity-dependent dendritic spine structural plasticity is regulated by small GTPase Rap1 and its target AF-6.</a> Neuron. 48, 605-618 (2005).</li>
<li>Jones, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+modulation+of+spine+morphology+by+the+5-HT2A+serotonin+receptor+through+kalirin-7+signaling%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">Rapid modulation of spine morphology by the 5-HT2A serotonin receptor through kalirin-7 signaling.</a> Proc Natl Acad Sci USA. 106, 19575-19580 (1957).</li>
<li>Dunaevsky, A., Tashiro, A., Majewska, A., Mason, C., Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developmental+regulation+of+spine+motility+in+the+mammalian+central+nervous+system%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">Developmental regulation of spine motility in the mammalian central nervous system.</a> Proc Natl Acad Sci USA. 96, 13438-13443 (1999).</li>
<li>Lichtman, J. W., Conchello, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescence+microscopy%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Fluorescence microscopy.</a> Nat Methods. 2, 910-919 (2005).</li>
<li>Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Do+spines+and+dendrites+distribute+dye+evenly%5BTitle%5D)+AND+%22Trends+Neurosci%22%5BJournal%5D)">Do spines and dendrites distribute dye evenly.</a> Trends Neurosci. 27, 445-446 (2004).</li>
</ol>

Production of this work was supported by MetaMorph (Molecular Devices, Inc.).

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...

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

analysis of dendritic spine morphology in cultured cns neurons

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 ...

The overall goal of the following experiment is to assess the effects of various molecular and pharmacological manipulations on dendritic spine morphology and motility. This is achieved by transecting primary cultured cortical neurons with GFP, with or without exogenous expression of DNA plasmids to outline neuronal morphology and to manipulate endogenous protein expression and function. In the second step, cells can be treated pharmacologically and subsequently fixed into immuno stain or prepared for time-lapse imaging.And then pharmacological treatment, which will allow for the visualization of dendritic spines and the induced changes in dendritic spine morphology and motility. Next Z Series images of fixed cells or images of live cells taken at specific time points are acquired using a confocal microscope. Detailed measurements of dendritic spine morphology can be made from 2D projections of Z series images.The results obtained show the effect of molecular or pharmacological manipulations on dendritic spine morphology or motility based on quantitative morphological analysis of dendritic spine shape or motility. This method can help answer key questions in neuroscience such as ho

analysis-of-dendritic-spine-morphology-in-cultured-cns-neurons

Research

JoVE Journal

Neuroscience

Source: Pekarek, B. T., et. al., Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing. J. Vis. Exp. (2021)In this video, we demonstrate the preparation, imaging, and analysis of a cleared mouse brain to examine dendritic arbors, spine morphology, and neuronal distribution using confocal microscopy and 3D reconstruction techniques.

Confocal Microscopy Imaging of Intact Dendritic Arbors and Spine Morphology in a Cleared Mouse Brain

Source: Gouder, et. al. Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells. J. Vis. Exp. (2015).This video demonstrates the imaging and analysis of dendritic spines in transduced, immunolabeled pyramidal neurons using confocal microscopy. Background noise is reduced, and dendrites are traced by estimating their diameter and length. Automated spine segmentation involves setting parameters for dendrite and spine dimensions...

Quantification of Dendritic Spines Using Confocal Microscopy Imaging

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.

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

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.

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

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.

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

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

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.

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

Dendritic spines are important cellular features of the nervous system. Here live imaging methods are described for assessing the structure and function of dendritic spines in C. elegans. These approaches support the development of mutant screens for genes that define dendritic spine shape or function.

Imaging Dendritic Spines in Caenorhabditis elegans

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.

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

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.

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

Learn to analyze dendritic spine morphology and motility in primary cultured cortical neurons using GFP transfection and confocal microscopy. This method enables quantitative assessment of synaptic structure dynamics.

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Explore More Videos