Questo lavoro è stato sostenuto dal programma di ricerca intramurale del National Institutes of Health, l&#39;Istituto Nazionale di Eunice Kennedy Shriver delle saluti infantili e dello sviluppo umano (concessione HD008913 a C.-HL), e il Centro per l&#39;Information Technology (PGM, NP, ESM , e MM).

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Y., 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=Tiling+of+R7+axons+in+the+Drosophila+visual+system+is+mediated+both+by+transduction+of+an+activin+signal+to+the+nucleus+and+by+mutual+repulsion.">Tiling of R7 axons in the Drosophila visual system is mediated both by transduction of an activin signal to the nucleus and by mutual repulsion.</a> Neuron. 56 (5), 793-806 (2007).</li><li>Peng, H., Ruan, Z., Long, F., Simpson, J. H., Myers, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=V3D+enables+real-time+3D+visualization+and+quantitative+analysis+of+large-scale+biological+image+data+sets.">V3D enables real-time 3D visualization and quantitative analysis of large-scale biological image data sets.</a> Nat Biotechnol. 28 (4), 348-353 (2010).</li><li>Takemura, S. Y., Lu, Z., Meinertzhagen, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+circuits+of+the+Drosophila+optic+lobe:+the+input+terminals+to+the+medulla.">Synaptic circuits of the Drosophila optic lobe: the input terminals to the medulla.</a> J Comp Neurol. 509 (5), 493-513 (2008).</li><li>Takemura, S. Y., 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=Cholinergic+circuits+integrate+neighboring+visual+signals+in+a+Drosophila+motion+detection+pathway.">Cholinergic circuits integrate neighboring visual signals in a Drosophila motion detection pathway.</a> Curr Biol. 21 (24), 2077-2084 (2011).</li><li>Keller, P. J., Schmidt, A. D., Wittbrodt, J., Stelzer, E. 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Gli autori non hanno nulla da rivelare.

Qui, mostriamo come immagine e analizzare pergole dendritiche di neuroni midollo Drosophila. La prima sezione, dual-view di imaging, descrive la deconvoluzione e la combinazione di due pile di immagine in una pila alta risoluzione. La seconda sezione, dendriti tracciamento e la registrazione, descrive il tracciamento e la registrazione dei dendriti dei neuroni midollo nella matrice colonna di riferimento. La terza sezione, analisi dendritica, descrive l&#39;uso di script personalizzati per analizzare i modelli ...

Durante lo sviluppo, i neuroni dendriti elaborati in complesse ma stereotipati modelli ramificati per formare sinapsi con i loro partner presinaptici. modelli di ramificazioni dendritiche correlano con l&#39;identità e le funzioni dei neuroni. Le posizioni di pergole dendritiche determinano il tipo di ingressi presinaptiche che ricevono, mentre il dendritica ramificazione complessità e dimensioni del campo governano il numero di input. Così, dendritiche proprietà morfologiche sono fattori determinanti per la connettività sinaptica e computazione neuronale. In molte regioni del cervello complessi, come le mosche lobi ottici e retina dei vertebrati, circuiti sinaptici sono organizzati in colonne e strati per facilitare l&#39;elaborazione delle informazioni 1, 2. In una tale organizzazione colonna e strato, neuroni presinaptici di un distinto assoni progetto modalità per terminare a un livello specifico (cosiddetto targeting specifico-layer) e per formare un ordinato matrice bidimensionale (i called mappa topografica), mentre i neuroni postsinaptici estendono dendriti di dimensioni adeguate a livelli specifici per ricevere gli ingressi presinaptici dei tipi e dei numeri corretti. Mentre assonale mira a strati e le colonne è stato ben studiato 3, 4, e tanto meno si sa su come dendriti vengono indirizzati a livelli specifici e si espandono opportunamente dimensionate campi recettivi per formare connessioni sinaptiche con la corretta partner presinaptica 5. La difficoltà di imaging e quantificare dendritiche mira a strati e le colonne ha ostacolato lo studio dello sviluppo dendritiche nelle strutture cerebrali colonnari e laminati. Drosophila neuroni midollo sono un modello ideale per lo studio di routing dendritiche e montaggio del circuito in colonne e strati. Il neuropil mosca midollare è organizzato come un reticolo 3D di 10 strati e circa 750 colonne. Ogni colonna è innervato da un insieme di afferenze, tra cui photoreceptors R7 / R8 e neuroni lamina L1 - L5, i cui terminali assonale formare le mappe topografiche in maniera specifica strati 6. Circa 38 tipi di neuroni midollo sono presenti in ogni colonna di midollo e dendriti elaborati in strati specifici e con adeguati dimensioni del campo per ricevere input da queste afferenze 7. I circuiti sinaptici nel midollo sono state ricostruite a livello microscopico elettronico; in tal modo, le partnership sinaptiche sono ben stabiliti 7, 8. Inoltre, strumenti genetici per l&#39;etichettatura di vari tipi di neuroni midollo sono disponibili 9, 10, 11. Esaminando tre tipi di transmedulla (TM) neuroni (Tm2, TM9 e TM20), abbiamo già individuato due-specifica delle cellule di tipo attributi dendritiche: (i) i neuroni proiettano Tm dendriti in entrambe le direzione anteriore o posteriore (Proj planareezione direzione), a seconda dei tipi di cellule e (ii) dendriti dei neuroni midollo terminare in strati midollo specifici in un modo specifico tipo cellulare (terminazione specifico layer) 12. Planar direzione di proiezione e terminazione specifici strati sono sufficienti per differenziare questi tre tipi di neuroni Tm, mentre le mutazioni che sconvolgono le risposte TM per spunti di livello e colonna riguardano aspetti distinti di questi attributi. Qui, vi presentiamo un flusso di lavoro completo per l&#39;esame della patterning dendritica dei neuroni midollo Drosophila in colonne e strati (Figura 1). Innanzitutto, mostriamo un metodo di imaging dual-view, che utilizza software personalizzato per unire due immagine confocale pile per generare immagini isotrope alta qualità. Questo metodo richiede solo la microscopia confocale convenzionale per generare immagini di alta qualità che consentono la tracciabilità affidabile di rami dendritici, senza ricorrere alla microscopia super-risoluzione, ad unas STED (emissione stimolata Depletion) o l&#39;illuminazione strutturale. In secondo luogo, vi presentiamo un metodo per tracciare pergole dendritiche e per registrare le tracce dei neuriti conseguenti ad una matrice di colonna di riferimento. Terzo, mostriamo i metodi di calcolo per estrarre informazioni sulla direzione di proiezione planare e terminazione specifico strato di dendriti, nonché per derivare stime per frequenze ramificazione e terminazione dendritiche. Insieme, questi metodi consentono la caratterizzazione di modelli dendritiche in 3D, la classificazione di tipi cellulari basate su morfologie dendritiche, e l&#39;identificazione di potenziali difetti nei mutanti.

<table border="0" cellpadding="0" cellspacing="0" width="1632">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Software</td>
			<td style="width:301px;"></td>
			<td style="width:227px;"></td>
			<td style="width:640px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Huygens Professional&#160;</td>
			<td>Scientific Volume Imaging</td>
			<td>version 16.05</td>
			<td>for image deconvolution (https://svi.nl).&#160; commercial software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MIPAV</td>
			<td></td>
			<td>version 7.3.0</td>
			<td>for image recombination and registration (http://mipav.cit.nih.gov/.).&#160; freeware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MIPAV plugin: PlugInDrosophilaRetinalRegistration.class</td>
			<td></td>
			<td></td>
			<td>freeware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MIPAV plugin: PlugInDrosophilaStandardColumnRegistration.class</td>
			<td></td>
			<td></td>
			<td>freeware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaris</td>
			<td>Bitplane</td>
			<td></td>
			<td>for tracing neurites and assigning reference points for image registration (http://www.bitplane.com). commercial software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vaa3D</td>
			<td></td>
			<td></td>
			<td>for visualizing swc files (https://github.com/Vaa3D/release/releases/).&#160; freeware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matlab</td>
			<td>Mathworks</td>
			<td>R2014b</td>
			<td>for morphometric analysis of dendrites (http://www.mathworks.com).&#160; commercial software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matlab toolbox: TREES1.14</td>
			<td></td>
			<td>v1.14</td>
			<td>for analyzing dendritic morphometric parameters (http://www.treestoolbox.org/download.html).&#160; freeware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matlab toolbox: Dendritic_Tree_Toolbox</td>
			<td></td>
			<td>v1.0</td>
			<td>for calculating morphometric parameters (https://science.nichd.nih.gov/confluence/display/snc/Data+collections+for+imagines+combination+and+standardize+column+registration). Freeware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sample files</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SWC file definition</td>
			<td></td>
			<td></td>
			<td>http://www.neuronland.org/NLMorphologyConverter/MorphologyFormats/SWC/Spec.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">The codes and sample files for image combination and registration</td>
			<td></td>
			<td></td>
			<td>https://science.nichd.nih.gov/confluence/display/snc/Data+collections+for+imagines+combination+and+standardize+column+registration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reference point example&#160;</td>
			<td></td>
			<td></td>
			<td>https://science.nichd.nih.gov/confluence/download/attachments/117216914/points.csv?version=1&#38;modificationDate=1471880596000&#38;api=v2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Computer system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MS Windows Windows 7 x64 or Macintosh OS X 10.7 or later</td>
			<td></td>
			<td></td>
			<td>3GHz 64-bit quad-core processor, 16G RAM (minimal)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optional: Quadro4000&#160; (or above) graphic card</td>
			<td>Nvidia</td>
			<td></td>
			<td>for stereographic visualization of dendrites.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optional: NVIDIA 3D vision2</td>
			<td>Nvidia</td>
			<td></td>
			<td>http://www.nvidia.com/object/3d-vision-main.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optional: 120 Hz LCD display for NVIDIA 3D vision2</td>
			<td></td>
			<td></td>
			<td>http://www.nvidia.com/object/3d-vision-system-requirements.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reagents for imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24B10 antibody</td>
			<td>The Developmental Studies Hybridoma Bank</td>
			<td>24B10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GFP Tag Antibody</td>
			<td>Thermofisher Scientific</td>
			<td>G10362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-Rabbit (H+L), Alexa Fluor 488</td>
			<td>Thermofisher Scientific</td>
			<td>A11034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-Mouse (H+L), Alexa Fluor 568</td>
			<td>Thermofisher Scientific</td>
			<td>A21124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VECTASHIELD Antifade Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounting&#160;Clay&#160;</td>
			<td>Fisher</td>
			<td>S04179</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% glycerol in 1X PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover glasses, high performance, D=0.17mm</td>
			<td>Zeiss</td>
			<td>474030-9000-000</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing dendritic morphology in columns and layers

In molte regioni del sistema nervoso centrale, come le mosche lobi ottici e corteccia vertebrato, circuiti sinaptici sono organizzati in strati e colonne per facilitare il cablaggio cervello durante lo sviluppo e l&#39;elaborazione delle informazioni in animali sviluppati. neuroni post-sinaptici dendriti elaborati nei modelli specifici del tipo a livelli specifici di sinapsi con opportuni terminali presinaptici. Il neuropil mosca midollare è composto da 10 livelli e circa 750 colonne; ogni colonna è innervato dal dendriti di oltre 38 tipi di neuroni midollo, che corrispondono con i terminali degli assoni di alcuni 7 tipi di afferenze in maniera specifica per tipo. La presente relazione illustra le procedure per l&#39;immagine e analizzare dendriti dei neuroni midollo. Il flusso di lavoro prevede tre sezioni: (i) la sezione di imaging dual-view combina due pile immagine confocale raccolti a orientamenti ortogonali ad alta risoluzione di immagini 3D di dendriti in; (Ii) il dendrite tracciamento e la sezione di registrazione tracce dendritichepergole in 3D e registri tracce dendritiche nella matrice colonna di riferimento; (Iii) la sezione di analisi dendritica analizza i modelli dendritiche rispetto alle colonne e strati, tra cui la direzione di terminazione e la proiezione planare specifico strato di gazebo dendritiche, e deriva stime di frequenze ramificazione e terminazione dendritiche. I protocolli utilizzano plug-in personalizzati costruiti sulla MIPAV open-source (Medical Imaging elaborazione, l&#39;analisi e la visualizzazione) di piattaforma e personalizzati toolbox nella lingua di laboratorio della matrice. Insieme, questi protocolli forniscono un flusso di lavoro completo per analizzare l&#39;instradamento dendritica di Drosophila neuroni midollo in strati e colonne, per identificare i tipi di cellule, e determinare difetti di mutanti.

Utilizzando la procedura di imaging dual-view qui presentato, un cervello mosca contenente neuroni TM20 scarsamente etichettati è stato ripreso in due direzioni ortogonali. Prima di imaging, il cervello era macchiato di appropriati anticorpi primari e secondari per la visualizzazione GFP e fotorecettori assoni membrana-tethered. Per l&#39;imaging, il cervello è stato montato prima secondo un orientamento orizzontale (Figura 2A, B). Un neurone TM20 GFP-etichettati e le ...

Watch this Scientific Journal Video about Analyzing Dendritic Morphology in Columns and Layers at JoVE.com

Analyzing Dendritic Morphology in Columns and Layers

Qui mostriamo come analizzare dendritica instradamento dei neuroni midollo Drosophila in colonne e strati. Il flusso di lavoro comprende una tecnica di imaging dual-view per migliorare la qualità delle immagini e strumenti computazionali per tracciare, registrare mandrini dendritiche alla matrice di colonna di riferimento e per analizzare le strutture dendritiche nello spazio 3D.

Analizzando dendritiche Morfologia in colonne e strati

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

analyzing-dendritic-morphology-in-columns-and-layers

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

9.2K Views.  National Institutes of Health (NIH).  Qui mostriamo come analizzare dendritica instradamento dei neuroni midollo Drosophila in colonne e strati. Il flusso di lavoro comprende una tecnica di imaging dual-view per migliorare la qualità delle immagini e strumenti computazionali per tracciare, registrare mandrini dendritiche alla matrice di colonna di riferimento e per analizzare le strutture dendritiche nello spazio 3D. 

Video: Analyzing Dendritic Morphology in Columns and Layers

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

Analisi automatizzata Sholl di Digitized morfologia neuronale a scale multiple

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

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Neuroscienze

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.

Analisi di Morfologia spine dendritiche in colture di neuroni del SNC

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

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

Indurre crescita dendritica in colture di neuroni simpatici

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

L&#39;analisi di morfologia dendritica delle cellule di Purkinje in Culture Slice organotipiche

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile stimulation is a positive early experience that mimics maternal licking and grooming in the rat. Exposing rat pups to this positive experience can be completed easily and cost-effectively by using highly accessible materials such as a household duster. Using a cross-litter design, pups are either stroked or left undisturbed, for 15 min, three times per day throughout the perinatal period. To measure the neuroplastic changes related to this positive early experience, Golgi-Cox staining of brain tissue is utilized. Owing to the fact that Golgi-Cox impregnation stains a discrete number of neurons rather than all of the cells, staining of the rodent brain with Golgi-Cox solution permits the visualization of entire neuronal elements, including the cell body, dendrites, axons, and dendritic spines. The staining procedure is carried out over several days and requires that the researcher pay close attention to detail. However, once staining is completed, the entire brain has been impregnated and can be preserved indefinitely for ongoing analysis. Therefore, Golgi-Cox staining is a valuable resource for studying experience-dependent plasticity.

This paper describes the procedures for tactile stimulation of rat pups and subsequent Golgi-Cox staining of neuronal morphology. Tactile stimulation is a positive experience that is administered in the perinatal period by stroking pups with a household duster. Golgi-cox staining is a reliable procedure permitting the visualization of entire neurons.

Visualizzare gli effetti di una prima esperienza positiva, tattile stimolazione, su dendritiche Morfologia e Synaptic connettività con Golgi-Cox colorazione

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Veloce micro-ionoforesi di glutammato e GABA: un utile strumento per indagare l&#39;integrazione Synaptic

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Manipolazione genetica delle cerebellare granuli neuroni<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Per studiare neuronale Morfologia e migrazione

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

La indiretto Neuron-astrociti co-coltura saggio: An<em&gt; In Vitro</em&gt; Set-up per l&#39;indagine dettagliata delle Interazioni neuroni-glia

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

Analisi quantitativa di un neurone Arborization dentritico complessità in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the &#8220;tri-partite&#8221; synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons,&#160;where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo- and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes&#8217; normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

Visualizzazione e analisi del trasporto intracellulare di organelli e di altri carichi in Astrociti

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

Analizzando dendritiche Morfologia in colonne e strati

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Analisi automatizzata Sholl di Digitized morfologia neuronale a scale multiple

Analisi di Morfologia spine dendritiche in colture di neuroni del SNC

Indurre crescita dendritica in colture di neuroni simpatici

L'analisi di morfologia dendritica delle cellule di Purkinje in Culture Slice organotipiche

Visualizzare gli effetti di una prima esperienza positiva, tattile stimolazione, su dendritiche Morfologia e Synaptic connettività con Golgi-Cox colorazione

Veloce micro-ionoforesi di glutammato e GABA: un utile strumento per indagare l'integrazione Synaptic

Manipolazione genetica delle cerebellare granuli neuroni

La indiretto Neuron-astrociti co-coltura saggio: An

Analisi quantitativa di un neurone Arborization dentritico complessità in Drosophila

Visualizzazione e analisi del trasporto intracellulare di organelli e di altri carichi in Astrociti