Il lavoro sulla ricerca di occhio in questo laboratorio è sostenuto da American Health Foundation Assistant, Azalea (1972) fondo di dotazione, e il Fondo Progetto HKU Small (20097176185).

1. Tools Microscopio, Nikon Investigator Stereo, MBF Bioscience - MicroBrightField, Inc. 2. Preparazione <ol><li> Prima di lavorare su qualsiasi analisi morfometrica, ogni campione è sezionato retina a 4 micron di spessore e subisce la colorazione H &amp; E. </li><li> La sezione retina è suddivisa in regioni 4 - superiore regione periferica, la regione centrale superiore, la regione periferica inferiore, e la regione centrale inferiore. </li><li> Dopo aver definito le regioni, l&#39;immagine di ogni regione viene acquisito ad ingrandimenti 10x40 sotto il microscopio luminoso raggio di Nikon. </li><li> Ora, siamo pronti ad analizzare morfometria della retina. </li></ol> 3. Misurare lo spessore INL <ol><li> Quattro misurazioni dello spessore INL in ciascuna regione sono stati misurati. Quattro le linee saranno disegnate perpendicolarmente dalla linea di base contorno al confine esterno della INL. </li><li>Aprire il software chiamato Investigator Stereo e scegliere la lente 40x dalla barra degli strumenti principale. </li><li> Selezionare &quot;Apri immagine&quot; sotto il &quot;File&quot; tab. Scegli l&#39;immagine di una regione desiderata della retina che si desidera analizzare. </li><li> Scegliere il tipo di linea di contorno dalla casella di riepilogo a discesa della barra degli strumenti principale e premere il tasto &quot;Move Auto&quot; pulsante. </li><li> Tasto destro del mouse nella finestra di rintracciare e selezionare &quot;Semplice Click Tracing&quot;. </li><li> Tracciare la linea di contorno lungo il bordo interno della INL. </li><li> Quando il disegno è fatto, tasto destro del mouse nella finestra di rintracciare e scegliere &quot;Contour Open End&quot;. </li><li> Selezionare un marker la barra degli strumenti marcatore e aggiungere marcatori nei punti di giunzione. </li><li> Dopo aver aggiunto tutti i marcatori, deselezionare l&#39;icona nella barra degli strumenti marker Marker. </li><li> Posizionare un cursore al centro di uno dei marcatori. </li></ol><img src="/files/ftp_upload/3377/3377fig1.jpg" alt="Figura 1" /><ol start="11"><li> Selezionare &quot;QuickMisurare l&#39;angolo &quot;nella scheda&quot; Strumenti &quot;. </li></ol><img src="/files/ftp_upload/3377/3377fig2.jpg" alt="Figura 2" /><ol start="12"><li> Tasto destro del mouse nella finestra di tracciare e deselezionare l&#39;opzione &quot;continua&quot;. </li><li> Misurare un angolo facendo clic al centro di un marcatore, che è adiacente al marker selezionato per una estremità del segmento di linea. </li></ol><img src="/files/ftp_upload/3377/3377fig3.jpg" alt="Figura 3" /><ol start="14"><li> Quindi, spostare il mouse verso il centro del marcatore in questione e cliccate di nuovo. </li><li> Estendere la linea di elastico al bordo esterno della INL. </li><li> Regolare l&#39;angolo fino a 90 ° è stata raggiunta. </li></ol><img src="/files/ftp_upload/3377/3377fig4.jpg" alt="Figura 4" /><ol start="17"><li> Sinistra click nella posizione desiderata. Una finestra di dialogo che mostra una misurazione dell&#39;angolo si aprirà subito dopo lo scatto. </li><li> Premere il tastoTasto &quot;Enter&quot;, mentre tenendo premuto il mouse ancora. </li></ol><img src="/files/ftp_upload/3377/3377fig5.jpg" alt="Figura 5" /><ol start="19"><li> Fai clic sinistro della finestra di analisi in modo che una linea viene disegnata da un capo alla nuova fine selezionato. </li><li> Tasto destro del mouse nella finestra di rintracciare e scegliere &quot;Contour Open End&quot;. </li></ol><img src="/files/ftp_upload/3377/3377fig6.jpg" alt="Figura 6" /><ol start="21"><li> Disegnare le altre linee in posizioni differenti esattamente nello stesso modo descritto prima. </li><li> Dopo aver tracciato tutte le linee, selezionare la modalità di modifica e rinominare i 4 segmenti di linea di conseguenza. </li><li> Quattro le misure possono essere ottenuta premendo il tasto &quot;Measurement Contour&quot; pulsante. </li><li> Selezionare queste 4 misure e incollarli in un foglio di calcolo facendo clic su &quot;Copy to Clipboard&quot;. </li><li> Selezionare &quot;Salva con nome&quot; sotto &quot;File&quot; tab. I dati di tracciamento possono essere salvati come file di dati. </li></ol><ol><li> Selezionare &quot;Apri immagine&quot; sotto il &quot;File&quot; tab. Scegli l&#39;immagine di una regione desiderata della retina che si desidera analizzare. </li><li> Scegliere il tipo di linea di contorno dalla casella di riepilogo a discesa della barra degli strumenti principale. </li><li> Tracciare una linea di contorno lungo il confine dello strato delle fibre nervose (NFL). </li><li> Terminare il tracciato con la &quot;Contour End Open&quot; opzione. </li><li> A ciascuna estremità, aggiungere un marcatore sulla punta della linea e un altro in corrispondenza della posizione vicino alla punta. </li><li> Deselezionate l&#39;icona alla barra degli strumenti marker marcatore. </li><li> Posizionare un cursore al centro dei marcatori alla punta. </li><li> Selezionare &quot;Angle rapida Misura&quot; sotto &quot;Strumenti&quot; nella scheda. </li><li> Tasto destro del mouse nella finestra di tracciare e deselezionare l&#39;opzione &quot;continua&quot;. </li><li> Utilizzare le linee di gomma banda e misurare un angolo di 90 °. </li><li> Premere il tasto &quot;Invio&quot;, dopo una finestra di dialogo è spuntato. </li><li> Tasto sinistro tegli tracciando finestra in modo che una nuova linea può essere esteso perpendicolarmente dalla punta. </li><li> Tracciare un&#39;altra linea nuova esattamente nello stesso modo in un&#39;altra estremità. </li><li> Due confini sono creati all&#39;interno della quale il numero di RGC viene contato. </li><li> Scegli un confine come linea di esclusione che i tocchi di cella non viene conteggiato. </li><li> Selezionare un tipo di marcatore e allegare uno per ogni cella dal vivo. </li><li> Selezionare un altro tipo di marcatore per le cellule morte. </li><li> Il numero di ciascun tipo di marker posizionati verrà visualizzato insieme l&#39;icona marcatore conseguenza alla barra marcatore. </li><li> Selezionare la modalità Editing e prendete la linea di base del profilo. Tasto destro del mouse nella finestra di tracciare e rinominare la linea. </li><li> La lunghezza della linea di base contorno può essere ottenuta premendo &quot;Measurement Contour&quot; pulsante. </li><li> Il numero delle cellule possono quindi essere espresso per mm di lunghezza. </li><li> Selezionare &quot;Salva con nome&quot; sotto &quot;File&quot; tab. Ilrintracciamento e ai dati dei marcatori possono essere salvati come file di dati. </li></ol> 5. Misurare le dimensioni della cella di una cella RGC <ol><li> Selezionare &quot;Apri immagine&quot; sotto il &quot;File&quot; tab. Scegli l&#39;immagine di una regione desiderata della retina che si desidera analizzare. </li><li> Zoom in una cella dal vivo e scegliere il tipo di linea di contorno dalla casella di riepilogo a discesa della barra degli strumenti principale. </li><li> Disegnare una linea di contorno lungo la circonferenza della cella. </li><li> Verso la fine, tasto destro del mouse la finestra di rintracciare e selezionare &quot;contorno vicino End&quot;. </li><li> L&#39;area della cella verrà visualizzato sotto la voce di Area nella finestra di dialogo di misurazione Contour. </li></ol>

<ol>
	<li>Lam, T. T., Abler, A. S., Tso, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-Methyl-D-Aspartate+(NMDA)â&#x20AC;&#x201C;Induced+Apoptosis+in+Rat+Retina.">N-Methyl-D-Aspartate (NMDA)â&#x20AC;&#x201C;Induced Apoptosis in Rat Retina.</a> Investigative Ophthalmology &amp; Visual Science. 40, 2391-2397 (1999).</li><li>Lam, T. T., Abler, A. S., Tso, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+and+caspases+after+ischemia-reperfusion+injury+in+rat+retina.">Apoptosis and caspases after ischemia-reperfusion injury in rat retina.</a> Invest. Ophthalmol. Vis. Sci. 40, 967-975 (1999).</li><li>Luo, X. G., Chiu, K., Lau, H. S., Lee, V. W. H., Yung, K. K. L., So, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Selective+Vulnerability+of+Retinal+Ganglion+Cells+in+Rat+Chronic+Ocular+Hypertension+Model+at+Early+Phase.">The Selective Vulnerability of Retinal Ganglion Cells in Rat Chronic Ocular Hypertension Model at Early Phase.</a> Cellular and Molecular Neurobiology. 29 (8), 1143-1151 (2009).</li><li>Han, Y. S., Chung, I. Y., Park, J. M., Yu, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+effect+of+citicoline+on+retinal+cell+damage+induced+by+kainic+acid+in+rats.">Neuroprotective effect of citicoline on retinal cell damage induced by kainic acid in rats.</a> Korean J. Ophthalmol. 19, 219-226 (2005).</li><li>Hernandez, M., Urcola, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+ganglion+cell+neuroprotection+in+a+rat+model+of+glaucoma+following+brimonidine,+latanoprost+or+combined+treatments.">Retinal ganglion cell neuroprotection in a rat model of glaucoma following brimonidine, latanoprost or combined treatments.</a> Exp. Eye Res. 86, 798-806 (2008).</li><li>Chan, H. C., Chang, R. C. C., Ip, A. K. C., Chiu, K., Yuen, W. H., Zee, S. Y., So, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+effects+of+Lycium+barbarum+Lynn+on+protecting+retinal+ganglion+cells+in+an+ocular+hypertension+model+of+glaucoma.">Neuroprotective effects of Lycium barbarum Lynn on protecting retinal ganglion cells in an ocular hypertension model of glaucoma.</a> Experimental Neurology. 203, 269-273 (2007).</li></ol>

Gli autori non hanno nulla da rivelare.

1. Come ottenere una misurazione più accurata dello spessore? È possibile ingrandire l&#39;immagine facendo clic su &quot;Zoom in&quot; e il pulsante sinistro del mouse nella finestra di tracciamento. Il bordo INL può essere visto chiaramente. Se la punta della linea non è al bordo esterno della INL, siamo in grado di regolare la linea sotto la modalità Editing. Dopo aver scelto la linea interessata, tasto destro del mouse la finestra di rintracciare e selezionare &quot;Inserisci Point in ...

A correction was made to <a href="http://www.jove.com/video/3377">Morphometric Analyses of Retinal Sections</a>. There was an error with an author's name.

Erratum: Morphometric Analyses of Retinal Sections

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Tipo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Investigator Stereo </td><td> MBF software per l&#39;analisi Bioscience </td><td> Campo MicroBright </td><td></td></tr><tr><td> Microscopio </td><td> Olympus BX51 </td><td> Olympus Corporation </td><td> BX51 </td></tr></tbody></table>

morphometric analyses of retinal sections

Analisi morfometriche delle sezioni retiniche sono stati utilizzati in esame malattie retiniche. Per esempio, cellule neuronali sono stati significativamente perduto nello strato di cellule del ganglio retinico (RGCL) in modelli di ratto con N-metil-D-aspartato (NMDA)-indotta eccitotossicità 1, retinica ischemia-riperfusione 2 e 3 glaucoma. Riduzione della INL e strato interno plessiforme (IPL) spessori sono stati invertiti con il trattamento citicolina negli occhi dei ratti sottoposti a cainico acido-mediata eccitotossicità glutammato 4. Alterazione della densità e le dimensioni RGC soma è stata osservata con trattamenti farmacologici diversi gli occhi con pressione intraoculare elevata 3,5,6. Pertanto, con metodi oggettivi di analisi dei morphometries della retina possono essere di grande importanza nella valutazione delle patologie retiniche e l&#39;efficacia di strategie terapeutiche. La struttura della retina è multi-strati e diversi tipi di neuroni exist nella retina. I parametri morfometrici della retina come il numero di cellule, la dimensione della cella e spessore degli strati differenti sono più complesso rispetto al sistema di coltura cellulare. All&#39;inizio, questi parametri possono essere rilevati utilizzando altri software commerciali imaging. I valori sono normalmente di valore relativo, e cambiando il valore preciso può essere necessario ulteriore calcolo accurato. Inoltre, l&#39;analisi della dimensione e la morfologia cellulare può non essere corretta e sufficientemente sensibile per l&#39;analisi statistica, soprattutto nel modello glaucoma cronico. Le misure utilizzate in questo protocollo fornito un modo più preciso e veloce. E la lunghezza assoluta della linea e le dimensioni della cella può essere segnalato direttamente e facilmente essere copiati ad altri file. Ad esempio, abbiamo tracciato il margine dei nuclei più interna ed esterna nel INL formando una linea quindi utilizzando il software per disegnare un angolo di 90 gradi per misurare lo spessore. Pur senza l&#39;aiuto del software, la linea obliqua e forse l&#39;evoluzione dello spessore della retina puònon essere ripetibile tra gli osservatori individuali. Inoltre, il numero e la densità di RGC può anche essere quantificato. Questo protocollo diminuisce con successo la variabilità in quantificazione caratteristiche della retina, aumenta la sensibilità nel rilevare modifiche minime. Questo video dimostra tre tipi di analisi morfometriche delle sezioni retiniche. Essi comprendono misurare lo spessore INL, quantificare il numero di RGC e misurando le dimensioni delle RGC in valore assoluto. Queste tre analisi vengono effettuate con Investigator Stereo (MBF Bioscience - MicroBrightField, Inc.). La tecnica in grado di offrire una piattaforma semplice, ma scientifico per le analisi morfometriche.

Watch this Scientific Journal Video about Morphometric Analyses of Retinal Sections at JoVE.com

Morphometric Analyses of Retinal Sections

Questo video mostra tre tipi di analisi morfometrica della retina, che comprendono la misurazione dello spessore interno strato nucleare, quantificare il numero delle cellule gangliari retiniche (RGC) e misurando le dimensioni delle RGC. La tecnica in grado di offrire una piattaforma semplice, ma scientifico per le analisi morfometriche.

Le analisi morfometriche delle sezioni della retina

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

morphometric-analyses-of-retinal-sections

Research

JoVE Journal

Neuroscience

10.2K Views.  The University of Hong Kong. Questo video mostra tre tipi di analisi morfometrica della retina, che comprendono la misurazione dello spessore interno strato nucleare, quantificare il numero delle cellule gangliari retiniche (RGC) e misurando le dimensioni delle RGC. La tecnica in grado di offrire una piattaforma semplice, ma scientifico per le analisi morfometriche.

Video: Morphometric Analyses of Retinal Sections

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

Una preparazione isolati retina per registrare Risposta Luce da Geneticamente etichetta cellule gangliari della retina

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Ricerca

Neuroscienze

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

Protocollo per tridimensionale confocale morfometrica Analisi di astrociti

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

Immunocolorazione delle sezioni biocitina-riempite e trasformati per neurochimici Marcatori

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...

Microstate and Omega Complexity Analyses of the Resting-state Electroencephalography

Microstate and omega complexity are two reference-free electroencephalography (EEG) measures that can represent the temporal and spatial complexities of EEG data and have been widely used to investigate the neural mechanisms in some brain disorders. The goal of this article is to describe the protocol underlying EEG microstate and omega complexity analyses step by step. The main advantage of these two measures is that they could eliminate the reference-dependent problem inherent to traditional spectrum analysis. In addition, microstate analysis makes good use of high time resolution of resting-state EEG, and the four obtained microstate classes could match the corresponding resting-state networks respectively. The omega complexity characterizes the spatial complexity of the whole brain or specific brain regions, which has obvious advantage compared with traditional complexity measures focusing on the signal complexity in a single channel. These two EEG measures could complement each other to investigate the brain complexity from the temporal and spatial domain respectively.

This article describes the protocol underlying electroencephalography (EEG) microstate analysis and omega complexity analysis, which are two reference-free EEG measures and highly valuable to explore the neural mechanisms of brain disorders.

Microstato e analisi di complessità Omega dell'elettroencefalografia dello stato di riposo

Microstate and omega complexity are two reference-free electroencephalography (EEG) measures that can represent the temporal and spatial complexities of ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

Preparazione delle sezioni ricrello della retina

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial organization of synaptic vesicles. In order to obtain high-quality electron micrographs that can yield the degree of morphological detail necessary for quantitative analysis of pre-synaptic vesicle distribution, optimal specimen preparation is critical. Chemical fixation is the first step in the process of specimen preparation, and of utmost importance to preserve fine ultrastructure. Vascular fixation with a glutaraldehyde-formaldehyde solution, followed by treatment of vibratome-sectioned specimens with osmium tetroxide, stabilizes the maximum number of molecules, especially proteins and lipids, and results in superior conservation of ultrastructure. Tissue is then processed with counterstaining, sequential dehydration and resin-embedding. En bloc staining with uranyl acetate (i.e., staining of vibratome-sectioned tissue before resin embedding) enhances endogenous contrast and stabilizes cell components against extraction during specimen processing. Contrast can be further increased by applying uranyl acetate as a post-stain on ultrathin sections. Double-staining of ultrathin sections with lead citrate after uranyl acetate treatment also improves image resolution, by intensifying electron-opacity of nucleic acid-containing structures through selective binding of lead to uranyl acetate. Transmission electron microscopy is a powerful tool for characterization of the morphological details of synaptic vesicles and quantification of their size and spatial organization in the terminal bouton. However, because it uses fixed tissue, transmission electron microscopy can only provide indirect information regarding living or evolving processes. Therefore, other techniques should be considered when the main objective is to study dynamic or functional aspects of synaptic vesicle trafficking and exocytosis.

We describe procedures for processing newborn rat brain tissue to obtain high-resolution electron micrographs for morphometric analysis of synaptic vesicle distribution at nerve terminals. The micrographs obtained with these methods can also be used to study the morphology of a number of other cellular components and their dimensional structural relationships.

Preparazione del tessuto cerebrale del ratto neonato per l'analisi Morfolometrica ultrastrutturale della distribuzione delle vescicole sinaptiche nei terminali nervosi

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial ...

Collection of Frozen Rodent Brain Regions for Downstream Analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

Raccolta di regioni cerebrali del roditore congelato per analisi a valle

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

Preparation of the Rat Vocal Fold for Neuromuscular Analyses

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures for rat laryngeal dissection, flash-freezing, and cryosectioning of the vocal folds. This study describes how to cryosection vocal folds in both longitudinal and cross-sectional planes. A novelty of this protocol is the laryngeal tracking during cryosectioning that ensures accurate identification of the intrinsic laryngeal muscles and reduces the chance of tissue loss. Figures demonstrate the progressive cryosectioning in both planes. Twenty-nine rat hemi-larynges were cryosectioned and tracked from the emergence of the thyroid cartilage to the appearance of the first section that included the full vocal fold. The full vocal fold was visualized for all animals in both planes. There was high variability in the distance from the appearance of the thyroid cartilage to the appearance of the full vocal fold in both planes. Weight was not correlated to depth of laryngeal landmarks, suggesting individual variability and other factors related to tissue preparation may be responsible for the high variability in the appearance of landmarks during sectioning. This study details a methodology and presents morphological data for preparing the rat vocal fold for histochemical neuromuscular investigation. Due to high individual variability, laryngeal landmarks should be closely tracked during cryosectioning to prevent oversectioning tissue and tissue loss. The use of a consistent methodology, including adequate tissue preparation and awareness of landmarks within the rat larynx, will assist with consistent results across studies and aid new researchers interested in using the rat vocal fold as a model to investigate laryngeal neuromuscular mechanisms.

This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study.

Preparazione della piega vocale del ratto per le analisi neuromuscolari

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures ...

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

Dissezione di fibre muscolari scheletriche singole per analisi immunofluorescenti e morfometriche di giunzioni neuromuscolari a montaggio intero

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

Quantification of Immunostained Caspase-9 in Retinal Tissue

The family of caspases is known to mediate many cellular pathways beyond cell death, including cell differentiation, axonal pathfinding, and proliferation. Since the identification of the family of cell death proteases, there has been a search for tools to identify and expand the function of specific family members in development, health, and disease states. However, many of the currently commercially available caspase tools that are widely used are not specific for the targeted caspase. In this report, we delineate the approach we have used to identify, validate, and target caspase-9 in the nervous system using a novel inhibitor and genetic approaches with immunohistochemical read-outs. Specifically, we used the retinal neuronal tissue as a model to identify and validate the presence and function of caspases. This approach enables the interrogation of cell-type specific apoptotic and non-apoptotic caspase-9 functions and can be applied to other complex tissues and caspases of interest. Understanding the functions of caspases can help to expand current knowledge in cell biology, and can also be advantageous to identify potential therapeutic targets due to their involvement in disease.

Presented here is a detailed immunohistochemistry protocol to identify, validate, and target functionally relevant caspases in complex tissues.