Wir danken Dr. S. Stella und Helen Vuong für Beiträge zur Kalzium-Imaging-Protokoll. Wir danken Herrn Dr. K. Blätter für Dreharbeiten für den Interview-Szenen. Wir danken Dr. Arlene Hirano für ihre Kommentare zum Manuskript. Diese Forschung und Entwicklungsprojekt wurde von den Autoren an der David Geffen School of Medicine an der UCLA durchgeführt und wird von einer vertraglichen Vereinbarung, die von der US Army Medical Research &amp; Materiel Command (USAMRMC) vergeben und verwaltet wurde möglich gemacht und die Telemedizin &amp; Advanced Technology Forschungszentrum (TATRC) in Fort Detrick, unter Vertragsnummer MD: W81XWH-10-2-0077. Unterstützung für diese Studien kam auch von NIH EY04067 und VA Merit Review (NB). NZB ist ein VA Karriere Research Scientist.

Alle Experimente wurden in Übereinstimmung mit den Richtlinien für das Wohlergehen der von der US Public Health Service Politik Menschen Pflege und Verwendung von Labortieren und der University of California-Los Angeles (UCLA) Tierforschung Committee Versuchstieren durchgeführt. Männliche und weibliche erwachsene Sprague-Dawley-Ratten (Charles River Lab, Wilmington, MA) im Alter von 3-5 Wochen wurden verwendet. 1. Tier-und Gewebepräparation für Wholemount Retina und Immunhistochemie Protokoll <ol><li> Bereiten Sie die folgenden Materialien und Werkzeuge: einem Binokular, 2 Pinzetten mit sehr feinen Spitzen, Schere, Zellulosefilterpapier, Kunststoffpipette und einem Objektträger. </li><li> Euthanize die Ratte in einer geschlossenen Kammer von tief Betäubung des Tieres mit 1-3% Isofluran gefolgt durch Enthauptung mit einer Guillotine. Die Augen mit einem Paar von Iris Schere und in eine Petrischale mit der extrazellulären Lösung zu entfernen. Hibernate A, Ames Medium oder physiologischen Lösungen empfohlen. </li><li> [Optional Schritt, wenn Verfüllung der RGZ mit Fluo-4 ist erwünscht]. Führen Sie die Fluo-4 Kennzeichnung wie in den Schritten 2.3-2.4 beschrieben. </li><li> Verwendung des Binokular (Lichtstärke: 1,6 x 10 8 Photonen / mm 2 ⋅sec), entfernen Sie die Hornhaut durch das Abschneiden der vor dem Auge Ball. Entfernen Sie die Linse und Glas von der inneren Netzhautoberfläche mit einer Pinzette. So entfernen Sie den Glaskörper, stabilisieren den Augapfel, indem die Lederhaut fest mit einer Zange. Ziehen Sie leicht dem Glaskörperbasis in Richtung der Mitte der Netzhaut, mit der anderen Zange. Die Zubereitung wird in diesem Stadium wird als Augenmuschel, die für Gefrierschnitte verwendet. Mehr Details über Gefrierschnitte können in den Referenzen 14,15 gefunden werden. </li><li> Entfernen Sie die Netzhaut von der Augenmuschel. Dann stellen vier Schnitte, damit der Netzhaut flach zu legen. Halterung sanft auf die Netzhaut auf einem Objektträger mit der Photorezeptorschicht auf. Geben Sie einen Tropfen der Lösung auf die Netzhaut und legte cellulose Filterpapier auf der Netzhaut. Sobald die Netzhaut wird an der Filterpapier, legen die Netzhaut wieder in Lösung. Wenn nötig, glätten die Netzhaut mit einem Pinsel oder einer Pinzette. </li><li> Legen Sie die Netzhäute wholemounted in 4% PFA für 10-15 min zur Fixierung. </li><li> Dreimal Waschen der wholemounted Netzhaut für 30 min in 0,1 M Phosphatpuffer (PB), pH 7,4. Sperrung der Netzhaut mit 5% normalem Ziegenserum oder Esel Serum in 0,1 PB, enthaltend 0,3% Triton X-100 und 0,1% NaN 3 bei 4 ° C über Nacht. Wir empfehlen ein Gesamtvolumen von 500 ul für alle Schritte zur Blockierung Lösungen und Antikörper zu speichern. </li><li> Am nächsten Tag, bebrüten die Netzhaut wholemounts mit dem primären Antikörper 5-7 Tage bei 4 ° C. Optimale Verdünnungen muss vom Anwender bestimmt werden. </li><li> Waschen der Netzhaut 3 x 30 min in 0,1 M PB und Inkubation über Nacht bei 4 ° C in das entsprechende Fluorophor-konjugierten sekundären Antikörper, der gegen die Spezies des primären Antikörpers (Konzentration 1: 1000) gerichtet ist. </li><li> Nach drei abschließenden Waschanlagen von 30 min mit je PB, montieren die Netzhaut in Montagemedium. Verschließen Sie die Deckgläser mit Nagellack für eine längere Lagerung. Proben bei 4 ° C und vor Licht schützen. </li></ol> 2. Kennzeichnung der Ganglienzellen und Axone in Rat Wholemount Netzhäute mit einem Calcium-Indikator-Farbstoff <ol><li> Bereiten 1000 ml Ringer-Säuger (in mM) 125 NaCl, 3 KCl, 2 CaCl 2, 1,25 NaH 2 PO 4, 1 MgCl 2, 25 NaHCO 3 enthält, und 10 Glucose mit 95% O 2/5% CO geperlt &#173; 2. </li><li> Führen Sie die Präparation wie in den Schritten 1.2-1.4 beschrieben. </li><li> Um retinalen Ganglienzellen (RGC) und ihre Axone mit einem Calcium-Indikator-Farbstoff in den Sehnerv Stumpf laden, injizieren 0,5 ul Fluo-4 Pentapotassium Salz (40 mM Stamm in H 2 O) ca. 1 mm hinter dem Augapfel mit einer Spritze . Zur Messung dynamischer Anstieg der Ganglienzelle [Ca2 + </sup&gt;] i eine Anzeige mit hoher Affinität, wie Fluo-4 mit K d von 345 nM, ist optimal. Fluo-4 bietet den zusätzlichen Vorteil, dass eine sehr hohe Quantenausbeute, was zu einer Emissionserhöhung von&gt; 100-fach, wenn Calcium gebunden. </li><li> Legen Sie die Augenmuschel in Säuger Ringer-mit 95% O sprudelte 5.2% CO &#173; 2 für 1 h bei Raumtemperatur im Dunkeln. </li></ol> 3. Calcium Imaging Protokoll zur retinalen Ganglienzellen und Axonen. <ol><li> Entfernen Sie das Auge, Linse und Glaskörper wie in den Schritten 1.1-1.4 beschrieben. Isolieren Sie die Netzhaut von der Augenmuschel und teilen sich in Quadranten. Hängen Sie eine Quadranten Ganglienzellseite nach oben auf einem Glasobjektträger und verwenden Sie eine Harfe Scheibe Raster, um es zu stabilisieren. Halten Sie die anderen Stücke dunkeladaptierten in Säuger Ringer-mit 95% O sprudelte 5.2% CO &#173; 2 auf Eis für die spätere Verwendung. </li><li> Superfuse die Aufzeichnungskammer, die mit Säuger-Ringer-Lösung undhalten Sie die Lösung kontinuierlich sprudelnde mit 95% O 2/5% CO 2. </li><li> Bild das Gewebe unter dem Mikroskop. Verhaltensexperimente bei Raumtemperatur (~ 23 ° C) zu erreichen oder physiologischen Temperaturen durch Erwärmen der Stufe Erhitzen des Wasserbades unter der Probe oder der Anwendung warmer Luft auf die Oberfläche der Probe. Hinweis: Es ist wichtig zu beachten, dass viele Zellfunktionen werden durch Temperatur, die eine Schlüsselrolle bei der Aufrechterhaltung der Homöostase intrazellulären 16 spielt geregelt. Jedoch können Einführung Erwärmung misst die Rate des Photobleichen, Verdampfung des Mediums und Fokusdrift durch thermische Ausdehnung verursachte 17 zu erhöhen. Verwendung von Raumtemperatur sinkt intrazellulären Kompartimentierung Fluo-4 18. </li><li> Durchführen einer Steuerung von zwei gepaarten hohen K +-Impulse in der Abwesenheit von Arzneimittel. Depolarisieren RGZ und RGC Axone durch die Erhöhung der [K +] o von 3 mm bis 60 mm für 33 sec bei jedem Impuls zu aktivierenVGCCs mit einem Acht-Kanal-Schwerkraft Superfusionssystem. Reduzieren Na + von 57 mM bis isosmolarity in der erhöhten K +-Lösung zu erhalten. </li><li> Den Beitrag der verschiedenen VGCCs auf die Kalzium-Signal mit der Verwendung von allgemeinen oder spezifischen Ca-Kanal-Blocker. Zum Beispiel kann die Verringerung der Calcium-Signal nach der Verabreichung einer nicht-spezifische Calciumkanal-Blocker, Kobalt, ein semi-quantitatives Maß für den Beitrag der VGCCs der hohen K + -evoked Calciumsignals. </li><li> Erwerben Fluoreszenzintensität Werte, indem Sie eine Region of Interest (ROI) um die RGZ von Interesse (Abbildung 2). </li><li> Normalisieren der Änderung der durch den zweiten Hoch K + Peak, der durch den ersten Hoch K + Peak erzeugt Änderung erzeugten Fluoreszenzintensität. Dann zum Testen spezifischer Calcium-Kanal-Blocker, gelten Medikamente nur während der zweiten Hoch K + Impuls eines Paares und normalisieren diese Spitze gegender erste Spitzenwert. So messen Sie die Auswirkungen der Droge auf der hohen K + Peak, teilen Sie die Amplitude der zweiten K + Peak durch die des ersten. Analyse sollte dieser Werte in Bezug auf ihre Übereinstimmungen Steuerungen von gepaarten hohen K + Peaks in Abwesenheit des Arzneimittels gemessen abgeleitet geführt werden. </li><li> Erwerben Sie Bilder mit 5 Sekunden-Intervallen. Auf Foto-Bleich zu vermeiden, halten Sie die Laseranregung so gering wie möglich. Bereitzustellen Anregung durch den 488 nm-Linie des Argon-Lasers, während der Photovervielfacherröhre sammelt die Fluoreszenzemission durch einen 505 nm-LP-Filter. </li></ol>

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1) Immunhistochemie auf Fluo-4-Lokalisierung in den Ganglienzellen in der Netzhautwholemounts zeigen, und 2) Calcium Imaging, Kalzium Dynamik in retinalen Ganglienzellen und ihre Axone zu analysieren: In diesem Artikel haben wir zwei verschiedene Techniken beschrieben. Immunhistochemie unter Verwendung wholemounts wurde verwendet, um die Lokalisierung von Proteinen in den Nager Retina 20-23 offenbaren. Es gibt jedoch einige Einschränkungen. Die Qualität der Färbung hängt stark...

Retinalen Ganglienzellen (RGCs) exprimieren L, N, P / Q-und T-Typ VGCC wie durch pharmakologische Blockade dieser Komponenten der gesamten Zelle bestimmt Ca-Kanalstrom 1,2. VGCC sind Trans multimeren Proteine, die in Transmitterfreisetzung, Gen-Transkription, Zellregulation und der synaptischen Plastizität 3,4,5 beteiligt sind. Groß, Trans α 1 porenbildenden Untereinheiten, die biophysikalischen und pharmakologischen Eigenschaften des Kanals, in erster Linie Hilfsextrazellulären α-Untereinheiten und 2 δ intrazellulären β-Untereinheiten zu etablieren: Funktions VGCCs bestehen aus mindestens drei verschiedene Klassen von Untereinheiten. Die beiden letztgenannten Form heteromeren Komplexe mit verschiedenen α-Untereinheiten 1 und ändern Sie die Gating-Kinetik und den Handel mit den Kanälen in der Plasmamembran 6. In den letzten Jahrzehnten wurden viele Techniken eingesetzt, um Protein expre studierenSpaltung, wie der Immunhistochemie, Enzyme-linked Immunosorbent Assay, Western-Analyse und Durchflusszytometrie. Diese Techniken erfordern die Verwendung spezifischer Antikörper zum Nachweis eines bestimmten Proteins von Interesse und liefern leistungsstarke Werkzeuge für die Lokalisierung und Verteilung spezifischer Proteine ​​in unterschiedlichen Geweben. Techniken zum Nachweis und zur Quantifizierung der mRNA Expression eines bestimmten Proteins, wie Northern-Blot-Analyse, RT-PCR, quantitative Echtzeit-RT-PCR, in situ-Hybridisierung, cDNA-Microarrays und Ribonuclease Protection Assay einen alternativen Ansatz bei der Antikörper nicht ohne weiteres verfügbar ist oder wenn die Expressionsniveaus eines bestimmten Proteins niedrig sind 7. , Eine Beschränkung auf die Verwendung von solchen molekularen Techniken ist jedoch die erforderliche Identifizierung der Gensequenz. Um Proteine ​​in der Netzhaut zu lokalisieren, kann die Immunhistochemie auf wholemount Netzhaut durchgeführt werden. Aufgrund der Zugänglichkeit der RGCs, der WholemountVorbereitung bietet eine hervorragende Plattform, um die Lokalisierung von spezifischen Proteinen auf die RGC Somata und ihre Axone zu studieren. Zusätzlich zu ihrer Lokalisierung, können einige funktionelle Eigenschaften der VGCCs in RGCs durch die Verwendung von Calcium-Imaging-Techniken nachgewiesen werden. Wir beschreiben eine Kalzium-Imaging-Protokoll, um selektive Markierung RGZ mit einem Calcium-Indikatorfarbstoff, um intrazelluläre Kalziumdynamik messen. Der Beitrag der unterschiedlichen VGCCs um das Calciumsignal in verschiedenen Zellkompartimenten können mit der Verwendung von subtypspezifischen Ca-Kanalblocker, isoliert werden. Vielleicht einer der vorteilhaften Aspekte des hier beschriebenen Calciumabbildungstechnik ist die Möglichkeit, gleichzeitig und unabhängig voneinander von mehreren RGCs und ihre Axone aufzuzeichnen. Obwohl viele physiologische Techniken, wie beispielsweise Voll Patch-Clamp-Aufnahme, eine hohe Zeitauflösung Aufnahmen Membranströme, die somatische oder axonale Quelle thesaufgezeichnet e Ströme nicht unterschieden werden und Aufnahmen können nur von einem einzelnen Neuron zu einem Zeitpunkt vorgenommen werden. Multielektroden-Arrays (MEA) sind in der Lage, gleichzeitig die Aufzeichnung Spitzen von vielen Zellen, aber weder erkennen noch zu benachteiligen die Aktivierung, beispielsweise unterschiedliche Subtypen von Calciumkanälen. MEAs bevorzugt von Zellen, die in der Nähe einer bestimmten Elektrode 8 und Satzes aus Zellen, die große Spitzen 9 erzeugen befinden aufzuzeichnen. Optischen bildgebenden Verfahren bieten eine alternative Strategie, um die gleichzeitige und unabhängige Aufnahmen von ganzen Populationen von Zellen, die mit dem von der Einzelzellmikroelektroden-und Patch-Clamp-Aufzeichnung und MEA Aufnahme gewonnenen Informationen integriert werden können ermöglichen. Obwohl die hier beschriebenen Kalzium-Imaging-Techniken wurden eingesetzt, um die Dynamik von Kalzium RGZ studieren können Patch-Clamp-und MEAs auch parallel zur weiteren Aufklärung der Ionenströme und Spick Eigenschaften der RGZ werden. <pclass = &quot;jove_content&quot;&gt; Seit verschoben Amakrinzellen machen etwa 60% der neuronalen Population in der Ganglienzellschicht in der Netzhaut der Maus 10, unser Ziel war es, eine Ladetechnik, die selektiv Etiketten RGZ mit einem synthetischen Kalzium-Indikator-Farbstoff in einer Gebrauchs wholemount Vorbereitung. Obwohl synthetische Calcium-Indikator-Farbstoffe eine ausgezeichnete Plattform für die Untersuchung der intrazellulären Calciumdynamik hat weitverbreitete Verwendung durch die Unfähigkeit behindert, um spezifische Populationen von Neuronen in einem bestimmten Netzwerk effektiv zu laden. Viele Techniken wie Bulkbeladung 11 und Elektroporation 8,12 durchgeführt, um zu laden ganze Populationen von Zellen, aber solche Techniken nicht zwischen bestimmten Zelltypen zu unterscheiden. Genetisch kodierte Calciumindikatoren liefern die Fähigkeit, selektiv zu markieren spezifischen Populationen von Zellen, jedoch erfordern solche Verfahren die Herstellung von transgenen Tieren 13. Unsere Technik beschreibt ein method selektiv beschriften RGZ in der wholemount Herstellung über Sehnerv Stumpf Injektion eines Kalzium-Indikator-Farbstoff. Zusammengenommen, strukturelle und physiologische Techniken in diesem Artikel beschrieben eine Plattform bieten, um die Lokalisierung und den Beitrag der VGCCs auf die Kalzium-Signal in RGZ und ihre Axone zu studieren.

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			<td>A rabbit polyclonal antibody&#160;was generated against the N-terminus of the RBPMS&#160;polypeptide (RBPMS4-24), GGKAEKENTPSEANLQEEEVR, by a commercial vendor (ProSci, Poway, CA).</td>
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immunohistochemical and calcium imaging methods in wholemount rat retina

In diesem Beitrag beschreiben wir die Werkzeuge, Reagenzien und die praktischen Schritte, die für notwendig: 1) erfolgreiche Vorbereitung von wholemount Netzhaut für die Immunhistochemie und 2) Kalzium-Imaging für die Untersuchung von spannungsabhängigen Calcium-Kanal (VGCC) vermittelt Calcium-Signalgebung in der Netzhaut Ganglienzellen. Die Kalzium-Imaging-Methode beschreiben wir circumvents Fragen über nicht-spezifische Belastung von Vertriebenen Amakrinzellen in der Ganglienzellschicht.

Immunmarkierung mit spezifischen Antikörpern bietet eine Plattform, um die Lokalisierung von bestimmten Proteinen von Interesse in der Netzhaut und des Calciumbildgebungstechnik zu untersuchen erlaubt die Untersuchung der Beiträge der VGCCs den Calciumdynamik in den retinalen Ganglienzellen und ihre Axone. Durch die Verwendung eines Antikörpers gegen den Ganglienzellprotein RBPMS (RNA-Bindeprotein mit mehreren Spleißen), die selektiv markiert RGCs 19, konnten wir zeigen, dass ...

Watch this Scientific Journal Video about Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina at JoVE.com

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

Immunhistochemie Protokolle werden verwendet, um die Lokalisierung eines bestimmten Proteins in der Netzhaut zu untersuchen. Calcium-Imaging-Techniken verwendet werden, um Calciumdynamik in retinalen Ganglienzellen und ihre Axone zu studieren.

Immunhistochemischen und Calcium Imaging Methoden in der Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

immunohistochemical-calcium-imaging-methods-wholemount-rat

Research

JoVE Journal

Neuroscience

14.4K Aufrufe.  University of California, Los Angeles.  Immunhistochemie Protokolle werden verwendet, um die Lokalisierung eines bestimmten Proteins in der Netzhaut zu untersuchen. Calcium-Imaging-Techniken verwendet werden, um Calciumdynamik in retinalen Ganglienzellen und ihre Axone zu studieren. 

Serie: Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

Functional Calcium Imaging in Entwicklungsländern Kortikale Netzwerke

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

Forschung

Neurowissenschaften

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

In vivo Neuronale Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

Immunhistochemische Analyse im Rat des Zentralnervensystems und peripheren Lymphknoten Gewebeschnitte

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

Nicht-invasive Parenchymale, Gefäß- und Stoffwechselhochfrequenz-Ultraschall und Photoakustische Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

In Vivo Functional Brain Imaging Ansatz, der auf Bioluminescent Calciumindikator GFP-Aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Zwei-Photonen-Kalzium Imaging in neuronalen Dendriten in Hirnschnitten

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies.
Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging.
Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm.
The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

Kultur der Hirnkapillarpericyten für zytosolische Kalziummessungen und Calcium-Imaging-Studien

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Transpupille Zwei-Photonen-In-vivo-Bildgebung der Netzhaut der Maus

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Calcium Imaging in Maus Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

Gehirn Perizyten-Kalzium und hämodynamische Bildgebung bei transgenen Mäusen in Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...