Questo lavoro è stato sostenuto da sovvenzioni dal centro nazionale per le risorse di ricerca (2G12RR03060-26A1); L'Istituto nazionale sulla salute delle minoranze e le disparità di salute (8G12MD007603-27) dal National Institutes of Health; Professional Staff Congress-City University of New York (PSC-CUNY); e facoltà Research Grant (la RFG II) American University of Sharjah. Ringraziamo Vidyasagar Sriramoju per introdurre noi a questi metodi.

<ol>
	<li>Nakashima, A., Dyck, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+and+cortical+plasticity.">Zinc and cortical plasticity.</a> Brain Res. Rev. 59, 347-373 (2009).</li><li>Frederickson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobiology+of+zinc+and+zinc-containing+neurons.">Neurobiology of zinc and zinc-containing neurons.</a> Int Rev Neurobiol. 31, 145-238 (1989).</li><li>Beaulieu, C., Dyck, R., Cynader, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+of+glutamate+in+zinc-containing+terminals+of+the+cat+visual+cortex.">Enrichment of glutamate in zinc-containing terminals of the cat visual cortex.</a> NeuroReport. 3 (10), 861-864 (1992).</li><li>Martinez-Guijarro, F. J., Soriano, E., Del Rio, J. A., Lopez-Garcia, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc-positive+boutons+in+the+cerebral+cortex+of+lizards+show+glutamate+immunoreactivity.">Zinc-positive boutons in the cerebral cortex of lizards show glutamate immunoreactivity.</a> J Neurocytol. 20 (10), 834-843 (1991).</li><li>Dyck, R., Beaulieu, C., Cynader, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemical+localization+of+synaptic+zinc+in+the+developing+cat+visual+cortex.">Histochemical localization of synaptic zinc in the developing cat visual cortex.</a> J Comp Neurol. 329 (1), 53-67 (1993).</li><li>Garrett, B., Geneser, F. A., Slomianka, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+acetylcholinesterase+and+zinc+in+the+visual+cortex+of+the+mouse.">Distribution of acetylcholinesterase and zinc in the visual cortex of the mouse.</a> Anat Embryol. (Berl). 184 (5), 461-468 (1991).</li><li>Garrett, B., Osterballe, R., Slomianka, L., Geneser, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoarchitecture+and+staining+for+acetylcholinesterase+and+zinc+in+the+visual+cortex+of+the+Parma+wallaby+(Macropus+parma).">Cytoarchitecture and staining for acetylcholinesterase and zinc in the visual cortex of the Parma wallaby (Macropus parma).</a> Brain Behav Evol. 43 (3), 162-172 (1994).</li><li>Dyck, R., Cynader, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+interdigitated+columnar+mosaic+of+cytochrome+oxidase,+zinc,+and+neurotransmitter-related+molecules+in+cat+and+monkey+visual+cortex.">An interdigitated columnar mosaic of cytochrome oxidase, zinc, and neurotransmitter-related molecules in cat and monkey visual cortex.</a> Proc. Natl. Acad. Sci. (90), 9066-9069 (1993).</li><li>Land, P. W., Akhtar, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+alteration+of+synaptic+zinc+in+rat+somatosensory+barrel+cortex.">Experience-dependent alteration of synaptic zinc in rat somatosensory barrel cortex.</a> Somatosens Mot Res. 16 (2), 139-150 (1999).</li><li>Danscher, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exogenous+selenium+in+the+brain:+a+histochemical+technique+for+light+and+electron+microscopic+localization+of+catalytic+selenium+bonds.">Exogenous selenium in the brain: a histochemical technique for light and electron microscopic localization of catalytic selenium bonds.</a> Histochemistry. 76, 281-293 (1982).</li><li>Danscher, G., Howell, G., Perez-Clausell, J., Hertel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dithizone,+Timm's+sulphide+silver+and+the+selenium+methods+demonstrate+a+chelatable+pool+of+zinc+in+CNS:+a+proton+activation+(PIXE)+analysis+of+carbon+tetrachloride+extracts+from+rat+brains+and+spinal+cords+intravitall+treated+with+dithizone.">The dithizone, Timm's sulphide silver and the selenium methods demonstrate a chelatable pool of zinc in CNS: a proton activation (PIXE) analysis of carbon tetrachloride extracts from rat brains and spinal cords intravitall treated with dithizone.</a> Histochemistry. 83, 419-422 (1985).</li><li>Gallyas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver+staining+of+myelin+by+means+of+physical+development.">Silver staining of myelin by means of physical development.</a> Neurol Res. 1 (2), 203-209 (1979).</li><li>Wong-Riley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+visual+system+of+monocularly+sutured+or+enucleated+cats+demonstrable+with+cytochrome+oxidase+histochemistry.">Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry.</a> Brain Res. 171 (1), 11-28 (1979).</li><li>Mir&#243;-Berni&#233;, N., Ichinohe, N., Perez-Clausell, J., Rockland, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc-rich+transient+vertical+modules+in+the+rat+retrosplenial+cortex+during+postnatal+development.">Zinc-rich transient vertical modules in the rat retrosplenial cortex during postnatal development.</a> J Neurosci. 138 (2), 523-535 (2006).</li><li>Ichinohe, N., Rockland, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+synaptic+zinc+in+the+macaque+monkey+amygdala.">Distribution of synaptic zinc in the macaque monkey amygdala.</a> J Comp Neurol. 489 (2), 135-147 (2005).</li><li>Innocenti, G. M., Manger, P. R., Masiello, I., Colin, I., Tettoni, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+and+callosal+connections+of+visual+areas+17,+18,+19+and+21+in+the+ferret+(Mustela+putorius).">Architecture and callosal connections of visual areas 17, 18, 19 and 21 in the ferret (Mustela putorius).</a> Cereb Cortex. 12 (4), 411-422 (2002).</li><li>Khalil, R., Levitt, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+histochemistry+reveals+circuit+refinement+and+distinguishes+visual+areas+in+the+developing+ferret+cerebral+cortex.">Zinc histochemistry reveals circuit refinement and distinguishes visual areas in the developing ferret cerebral cortex.</a> Brain Struct Funct. 218, 1293-1306 (2013).</li><li>Manger, P. R., Masiello, I., Innocenti, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Areal+organization+of+the+posterior+parietal+cortex+of+the+ferret+(Mustela+putorius).">Areal organization of the posterior parietal cortex of the ferret (Mustela putorius).</a> Cereb Cortex. 12, 1280-1297 (2002).</li><li>Wong, P., Kaas, 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=Architectonic+subdivisions+of+neocortex+in+the+gray+squirrel+(Sciurus+carolinensis.).">Architectonic subdivisions of neocortex in the gray squirrel (Sciurus carolinensis.).</a> The anatomical record. 291, 1301-1333 (2008).</li><li>Land, P. W., Shamalla-Hannah, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+plasticity+of+zinc-containing+cortical+circuits+during+a+critical+period+of+postnatal+development.">Experience-dependent plasticity of zinc-containing cortical circuits during a critical period of postnatal development.</a> J Comp Neurol. 447 (1), 43-56 (2002).</li><li>Czupryn, A., Skangiel-Kramska, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+synaptic+zinc+in+the+developing+mouse+somatosensory+barrel+cortex.">Distribution of synaptic zinc in the developing mouse somatosensory barrel cortex.</a> J Comp Neurol. 386, 652-660 (1997).</li><li>Timm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zur+Histochemie+der+Schwermetalle.+Das+Sulfid-Silber-Verfahren.">Zur Histochemie der Schwermetalle. Das Sulfid-Silber-Verfahren.</a> Dtsch Z ges gerichtl Med. 46, 706-711 (1958).</li></ol>

Gli autori non hanno nulla a rivelare.

Lo studio corrente impiega una tecnica istochimica basata su una versione modificata del Danscher (1982) metodo10, per cui zinco sinaptica localizzazione può essere rilevato e visualizzato nel cervello. Questo metodo funziona essenzialmente iniettando l'animale con il selenito di sodio chelante (Na2SeO3) di zinco (15 mg/kg). A seguito di iniezione, la selenite viaggia al cervello e si lega di zinco liberi che è localizzato alle vescicole presinaptiche di zinco contenente ne...

Le macchie istologiche sono state usate tradizionalmente per aiutare nell'identificazione delle aree corticali in varie specie, rivelando le differenze nelle caratteristiche architettoniche. L'uso combinato di tecniche istochimiche come sostanza di Nissl, reattività del citocromo ossidasi (CO) o mielina può rivelarsi fruttuosa, come rivelano i limiti areali simili nel cervello adulto. Tuttavia, queste macchie istochimiche non rivelano sempre adeguatamente chiari confini tra aree corticali e livelli nel cervello immaturo.
Nel sistema nervoso centrale, lo zinco ha diverse funzioni critiche che includono la struttura del DNA, che agisce come cofattore degli enzimi, che partecipano a numerose funzioni di regolamentazione e di agire come un neuromodulatore attraverso la sua presenza in vescicole sinaptiche di stabilizzazione 1. zinco sinaptico è unico in quanto possono essere visualizzato utilizzando i metodi istologici, mentre lo zinco legato alle proteine non può essere visualizzato in quel momento2. Questa caratteristica è stata sfruttata per rivelare il disegno di zinco sinaptica in diverse regioni corticali e l'istochimica zinco sinaptica è stato utilizzato in una serie di studi. Un sottoinsieme di neuroni glutamatergici nella corteccia cerebrale contengono zinco nelle vescicole presinaptiche entro loro assoni terminali3,4. Gli studi istochimici hanno rivelato una distribuzione eterogenea di zinco sinaptica in corteccia cerebrale5,6,7. Ci sembra essere una diversa distribuzione area e laminare di zinco histochemically reattiva in diverse regioni corticali (ad es., visual contro corteccia somatosensory), o strati (ad esempio, i livelli dello zinco nella supragranular e infragranular strati della corteccia visiva primaria sono sostanzialmente superiori a livello di input thalamocortical IV con i livelli relativamente bassi dello zinco sinaptica)5,8,9. L'eterogeneità in zinco sinaptico colorazione osservata nella corteccia è particolarmente vantaggioso, in quanto facilita l'identificazione areal e laminare.
Qui presentiamo una descrizione dettagliata di una procedura di istochimica zinco sinaptica, che è una versione modificata 1982 metodo10 di Danscher. Questo metodo utilizza la selenite iniettata intraperitonealmente (IP) in animali come un agente chelante. La selenite viaggia al cervello di reagire con piscine di zinco liberi trovati in vescicole di un sottoinsieme di glutamatergic sinapsi nel cervello. Questa reazione produce un precipitato che può essere migliorato successivamente di sviluppo d'argento2,10,11.
Questa procedura rivela pattern laminare e areali di zinco sinaptico colorazione; l'analisi densitometrica può essere utilizzato per valutare questi modelli sia qualitativamente che quantitativamente nel cervello adulto e immaturo per studiare gli effetti di altri interventi, come manipolazioni sensoriale, ambientale, farmacologiche o genetiche. Inoltre, uno potrebbe anche voler valutare potenziali cambiamenti inerenti allo sviluppo nella distribuzione di zinco sinaptica in altre strutture corticale o sottocorticale in altri sistemi-modello. Le informazioni quantitative che fornisce analisi densitometrica in questo metodo possono essere vantaggiose per il seguente sviluppo del cervello nel corso del tempo. Questo protocollo fornisce un compagno ad altri marcatori istochimici e immuno - per rivelare i confini laminari e areali.

<table><tbody><tr><td>Euthasol (Euthanasia solution)</td><td>Henry Schein</td><td>710101</td><td></td></tr><tr><td>Sodium selenite</td><td>Sigma-Aldrich</td><td>214485</td><td></td></tr><tr><td>Ketamine (Ketaved)</td><td>Henry Schein</td><td>48858</td><td>100 mg/ml injectables</td></tr><tr><td>Xylazine (Anased)</td><td>Henry Schein</td><td>33198</td><td>100 mg/ml injectables</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>F8775</td><td>Dilute to 4%</td></tr><tr><td>Gum arabic</td><td>Sigma-Aldrich</td><td>G9752-500G</td><td></td></tr><tr><td>Citric acid</td><td>Sigma-Aldrich</td><td>C1909</td><td></td></tr><tr><td>Sodium citrate</td><td>Sigma-Aldrich</td><td>W302600</td><td></td></tr><tr><td>Hydroquinone</td><td>Sigma-Aldrich</td><td>H9003</td><td></td></tr><tr><td>Silver lactate</td><td>Sigma-Aldrich</td><td>85210</td><td></td></tr><tr><td>Fish gelatine</td><td>Sigma-Aldrich</td><td>G7765</td><td></td></tr><tr><td>Cytochrome c</td><td>Sigma-Aldrich</td><td>C2506</td><td>(Type III, from equine heart)</td></tr><tr><td>Catalse</td><td>Sigma-Aldrich</td><td>C10</td><td></td></tr><tr><td>Sucrose</td><td>Domino</td><td></td><td></td></tr><tr><td>Xylene</td><td>Fisher Scientific</td><td>X5P-1GAL</td><td></td></tr><tr><td>Permount</td><td>Fisher Scientific</td><td>SP15-500</td><td></td></tr><tr><td>100% Ethanol</td><td>Fisher Scientific</td><td>A406-20</td><td>Used for dehydration prior to slide mounting</td></tr><tr><td>Coverslips</td><td>Brain Research Laboratories</td><td>#3660-1</td><td></td></tr><tr><td>Frosted unsubbed slides</td><td>Brain Research Laboratories</td><td>#3875-FR</td><td></td></tr><tr><td>Microtome</td><td>American Optical Company</td><td>860</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>BX-60</td><td></td></tr><tr><td>Adope Photoshop</td><td>Adobe Systems, San Jose, CA</td><td></td><td>To assemble images</td></tr><tr><td>ImageJ</td><td>Free software can be downloaded at http://rsb.info.nih.gov/ij/</td><td></td><td>For densometric measurements</td></tr><tr><td>Plastic tray</td><td>Any standard plastic tray may be used</td><td></td><td>to immerse slides in developer solution</td></tr><tr><td>Hot plate</td><td>Any standard hotplate may be used</td><td></td><td></td></tr></tbody></table>

use of synaptic zinc histochemistry to reveal different regions and laminae in the developing and adult brain

Caratterizzazione di organizzazione anatomica e funzionale del cervello e sviluppo richiede un'identificazione accurata di circuiti neurali distinti e regioni nel cervello adulto e immaturo. Qui descriviamo una procedura di colorazione istochimica di zinco che rivela differenze nel pattern di colorazione tra diversi strati e regioni del cervello. Altri hanno utilizzato questa procedura non solo per rivelare la distribuzione di zinco-contenendo i neuroni e circuiti nel cervello, ma anche per delineare correttamente areali e laminari confini nel cervello in via di sviluppo e adulto in diverse specie. Qui illustriamo questa procedura con immagini dallo sviluppo di colorazione e furetto adulto cervelli. Abbiamo rivelato un modello di macchiatura di zinco che serve come indicatore anatomico delle aree e livelli e può essere utilizzati in modo affidabile per distinguere le aree corticali visive nella corteccia visiva in via di sviluppo e adulta. L'obiettivo principale del presente protocollo è di presente un metodo istochimico che permette l'identificazione precisa dei livelli e delle regioni nel cervello adulto e in via di sviluppo dove altri metodi non riescono a farlo. Secondariamente, in concomitanza con analisi densitometrica delle immagini, questo metodo permette di valutare la distribuzione di zinco sinaptica per rivelare potenziali modifiche nel corso dello sviluppo. Questo protocollo descrive dettagliatamente i reagenti, strumenti e passaggi necessari per successivamente macchia sezioni di cervello congelato. Anche se questo protocollo è descritto usando il tessuto di cervello di furetto, può essere facilmente adattato per l'uso in roditori, gatti o scimmie così come in altre regioni del cervello.

I passaggi principali relativi a questo protocollo per macchiare le sezioni del cervello per zinco sinaptico sono presentati in un diagramma di flusso nella Figura 1. Il protocollo può essere diviso in tre fasi: 1) aspersione e tessuto raccolta, preparazione 2) tessuto e la macchiatura e l'istochimica 3) zinco. Brevemente, durante la prima fase del protocollo, l'animale è anestetizzato e IP iniettato con la dose appropriata di selenito di sodio. Dopo un per...

Watch this Scientific Journal Video about Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain at JoVE.com

Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

Uso di zinco sinaptica istochimica per rivelare diverse regioni e lamine nel cervello in via di sviluppo ed adulto

Descriviamo una procedura istochimica che rivela la caratteristica dello zinco laminare e areal modelli in diverse regioni del cervello di macchiatura. Il modello di macchiatura di zinco può essere utilizzato in combinazione con altri marcatori anatomici per distinguere attendibilmente strati e regioni nel cervello in via di sviluppo e adulto.

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions ...

use-synaptic-zinc-histochemistry-to-reveal-different-regions-laminae

Research

JoVE Journal

Neuroscience

7.1K Views.  American University of Sharjah. Descriviamo una procedura istochimica che rivela la caratteristica dello zinco laminare e areal modelli in diverse regioni del cervello di macchiatura. Il modello di macchiatura di zinco può essere utilizzato in combinazione con altri marcatori anatomici per distinguere attendibilmente strati e regioni nel cervello in via di sviluppo e adulto.

Video: Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae1-7. Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture1-5. Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain&#39;s macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

Large-scale Scanning Transmission Electron Microscopy Nanotomy of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution EM. Here we describe a universal method for nanotomy applied to investigate the zebrafish larval brain in health and upon non-invasive brain injury.

Su larga scala scansione microscopia elettronica a trasmissione (Nanotomy) di sana e feriti Zebrafish Cervello

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously ...

Ricerca

Biologia dello sviluppo

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

Preparazione di acuta fette zona subventricolare for Imaging di calcio

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Neuroscienze

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

Valutazione dei parametri di neuroplasticità ultrastrutturali dopo In Utero trasduzione del Mouse cervello e midollo spinale

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

Stab Ferita Lesione del Zebrafish adulti Telencefalo: Un metodo per Indagare vertebrati cervello neurogenesi e rigenerazione

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

Rilevazione di assoni localizzato mRNA nelle sezioni del cervello Utilizzando ad alta risoluzione<em&gt; In Situ</em&gt; Ibridazione

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

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 ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

Valutazione di densità di sinapsi nelle fette Hippocampal del cervello del roditore

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Biocytin Recovery and 3D Reconstructions of Filled Hippocampal CA2 Interneurons

How cortical network activity processes information is of importance to a large number of basic and clinical scientific questions. The protocol described here identifies the basic building blocks of this circuitry. The in-depth studies of cortical regions will ultimately provide other scientists with the circuit components needed for an understanding of how the brain acquires, processes and stores information and what goes wrong in disease, while the electrophysiological and morphological data are widely used by computational neuroscientists in the construction of model networks that explore information processing. The protocol outlined here describes how biocytin-filled cells recorded in the CA2 region of the hippocampus are recovered and then reconstructed in&#160;3D. Additionally, the protocol describes the demonstration of calcium binding protein or peptide content in recorded interneurons.

The protocol outlined here describes the immunofluorescence analysis, biocytin recovery and high-quality reconstructions of hippocampal CA2 interneurons following the intracellular electrophysiological recordings in vitro, allowing neuronal characterization and ultimately fine neuronal anatomy to be studied.

Recupero di biocitina e ricostruzioni 3D di interneuroni riempito Hippocampal CA2

How cortical network activity processes information is of importance to a large number of basic and clinical scientific questions. The protocol described ...

Localization of the Locus Coeruleus in the Mouse Brain

The locus coeruleus (LC) is a major hub of norepinephrine producing neurons that modulate a number of physiological functions. Structural or functional abnormalities of LC impact several brain regions including cortex, hippocampus, and cerebellum and may contribute to depression, bipolar disorder, anxiety, as well as Parkinson disease and Alzheimer disease. These disorders are often associated with metal misbalance, but the role of metals in LC is only partially understood. Morphologic and functional studies of LC are needed to better understand the human pathologies and contribution of metals. Mice are a widely used experimental model, but the mouse LC is small (~0.3 mm diameter) and hard to identify for a non-expert. Here, we describe a step-by-step immunohistochemistry-based protocol to localize the LC in the mouse brain. Dopamine-&#946;-hydroxylase (DBH), and alternatively, tyrosine hydroxylase (TH), both enzymes highly expressed in the LC, are used as immunohistochemical markers in brain slices. Sections adjacent to LC-containing sections can be used for further analysis, including histology for morphological studies, metabolic testing, as well as metal imaging by X-ray fluorescence microscopy (XFM).

The locus coeruleus is a small cluster of neurons involved in a variety of physiological processes. Here, we describe a protocol to prepare mouse brain sections for studies of proteins and metals in this nucleus.

Localizzazione del Locus Coeruleus nel cervello del topo

The locus coeruleus (LC) is a major hub of norepinephrine producing neurons that modulate a number of physiological functions. Structural or functional ...

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