Die Pedro Barrie de la Maza-Stiftung unterstützt diese Arbeit.

1) Intragastric Verwaltung von Rotenon <ol><li> Wiegen des Tieres. </li><li> Berechnen Sie das Gesamtvolumen der Rotenon / Fahrzeug-Lösung benötigt. In diesem Fall 0,01 ml pro Gramm des Tieres Gewicht. </li><li> Laden Sie eine 1 ml-Spritze mit den zuvor berechneten Volumen von Rotenon Lösung (0,625 mg / mL Rotenon, 4% Carboxymethylcellulose und 1,25% Chloroform) oder Vehikel-Lösung (4% Carboxymethylcellulose und 1,25% Chloroform). In diesem Fall entspricht es einem 5 mg / kg Rotenon Dosis. </li><li> Pick-up mit der Maus und die Maus am Schwanz mit einer Hand, erstrecken sich die Haut des Halses, indem Sie es mit den ersten zwei Fingern der anderen Hand. </li><li> Sobald der Kopf zwischen den beiden Fingern fixiert ist, lehnen sie gegen die Handfläche und drehen Sie ihn auf den Kopf. Fix der Schwanz mit dem vierten und fünften Finger. </li><li> Keeping den Kopf nach hinten, drücken Sie den Gaumen mit der Maus in den Mund zu öffnen. Vorsichtig einführen Magensonde in den Mund. Es ist wichtig, eine Magensonde Nadel und nicht einer regulären Nadel verwenden. Langsam bewegen sich die Sonde in Richtung des Magens weit genug. Dadurch wird vermieden, Material immer in der Lunge. </li><li> Verwalten Sie die Rotenon Lösung oder Fahrzeug durch sanftes Drücken der Embolus. Wenn Sie fertig sind, langsam extrahieren Sie die Magensonde. </li><li> Legen Sie das Tier wieder in einem anderen Käfig, bis alle Tiere aus einem Käfig fertig sind. </li></ol> 2) Immunfärbung Procedures <ol><li> Nach der Behandlung werden die Mäuse betäubt mit Ketamin 100 mg / kg und Xylazin 10 mg / kg ip und intrakardial mit 4% Paraformaldehyd in PBS perfundiert. </li><li> Die Eingeweide werden dann durch Dissektion extrahiert, und in 15% Saccharose über Nacht. Am nächsten Tag wird das Gewebe auf 30% Saccharose überführt und erneut über Nacht bei 4 ° C. </li><li> Das Gewebe wird dann in Tissue-tek gelegt und in einem -80 ° C Gefrierschrank bis zur Verwendung. </li><li> Mit einem Kryostaten, 20 micron Darm Querschnitte Superfrost Objektträger übertragen. </li><li> Blocking und Immunfärbung Verfahren mit anti-βIII-Tubulin-und anti-alpha-Synuclein-Antikörper sind dann wie zuvor beschrieben 9 durchgeführt. </li></ol> 3) Alpha-Synuclein Inclusion Image Analysis Es wird empfohlen, eine externe Person nicht bewusst images &#39;Herkunft der Analyse durchführt. Daher sollten die Daten zu kodifizieren. Die Bildanalyse erfolgte mit FIJI Software. <ol><li> Öffnen Sie die konfokale Bilddatei. Dann teilen Sie die Kanäle, so dass es möglich ist, auf jedem Kanal unabhängig voneinander arbeiten. </li><li> Schließen Sie die Kanäle, die nicht gehen, um zu verwenden, und wählen Sie Analyse&gt; Set Scale und setzen Sie die Pixelgröße auf 1. Setzen Sie die bekannte Strecke bis 0,13 (dieser wird auf der objektiven und der Auflösung abhängen) und klicken Sie auf global. Die Größe des Bildes wird nun in Mikron gezeigt werden. </li><li> Wählen Sie Alpha-Synuclein-Kanal und wählen Ganglion, stellen Sie sicher, dass die Auswahl das Ganglion umschließt in allen Schichten. </li><li> Drücken Sie Bild&gt; Duplizieren, um das Bild zu duplizieren. Mit dem Ganglion gewählt, nur ein Bild der Größe des Ganglion dupliziert werden. </li><li> Drücken Sie Bearbeiten&gt; Löschen Outside. Der Teil des Bildes außerhalb der gewählten Region wird schwarz. </li><li> Duplizieren Sie das Bild erneut in späteren Schritten, wie gut die Verfahren ging zu bestimmen. Bilder, die ein hohes Signal-Rausch-Verhältnis in den Hintergrund haben, sollten nicht für die Bildanalyse verwendet werden. </li><li> Wählen Sie Bild&gt; Anpassen&gt; Schwellenwert und wählen Sie dann MaxEntropy. Dann über die Scheiben in das freigestellte Bild und das Original zu bewegen, um sicherzustellen, dass das Verfahren gut gelaufen ist. Die entropischen Schwelle wählt eine Intensität Signalpegel auf das Bild Histogramm und nur die Pixel mit Intensitäten größer als dieser Schwellenwert wird in einem weißen über schwarzes Bild angezeigt basiert. </li><li> Wählen Sie Prozess, dann auf glatte klicken. Dieser Schritt wandelt die pixeligen Kanten der Bilder in glatte Kanten. </li><li> Wählen Sie Prozess, dann auf Binary klicken, und wählen Sie Binary. Das Bild wird als ein schwarzer über Weiß-Bild angezeigt werden. </li><li> Duplizieren Bild zum nächsten Schritt auf dem duplizierten Bild durchzuführen. </li><li> Klicken Sie auf Analysieren&gt; Analyze Partikel. Dann wählen Sie Zeigen: Outlines und klicken Sie auf Auswerten. Der Rest der Einstellungen sollten leaved in Verzug ist. Diese Funktion erkennt die Gesamtzahl der Pixel mit einem Signal und diejenigen, die gruppiert werden. Die Gesamtfläche der Pixel mit Signal plus der Anzahl der Gruppen von Pixeln und die mittlere Größe gemeldet werden. </li><li> Vergleichen Sie und wählen Sie das größte Segment, das gut passt. Die Scheibe markiert. </li><li> Kopieren Sie die Daten-Tabelle mit der gesamten Alpha-Synuclein Oberfläche, die Anzahl der Einschlüsse und die mittlere Partikelgröße. Fügen Sie die Daten in eine Excel-Tabelle oder Anmerkungen an anderer Stelle. </li><li> Return to FIJI und finden Sie die zuvor ausgewählten Slice in der β-Tubulin-Kanal, und wählen Sie es. </li><li> Wählen Ganglion Bereich und klicken Sie auf Analysieren und wählen Sie dann messen. Eine andere Tabelle erscheint und zeigt dieGesamtfläche des Ganglion. </li><li> Entpacken Sie die Daten aus der Tabelle der Excel-Tabelle in der Nähe des Alpha-Synuclein-Messung oder kopieren Sie es an anderer Stelle. </li><li> Mit Excel, teilen sich die verschiedenen Parameter in der alpha-Synuclein Messung durch das Ganglion Oberfläche erhalten, um insgesamt alpha-Synuclein Oberfläche und die Einbeziehung Anzahl von Ganglienzellen Größe normiert zu erhalten. </li></ol> 4) Repräsentative Ergebnisse Wenn eine Magensonde Verwaltung Protokoll korrekt durchgeführt wird, die Nachteile für das Tier sind minimal. Die Behandlung mit dieser Konzentration von Rotenon ermöglicht eine lokale Wirkung von Rotenon auf die ENS ohne Rotenon-Spiegel im Blut und keine Hemmung des Muskel oder Gehirn mitochondrialen Komplex I auch nach 1,5 Monaten der Behandlung (siehe Abbildung 1). Wenn Bildanalyse korrekt durchgeführt wird, eine strenge Analyse von Alpha-Synuclein-Muster sollte möglich sein. Es gibt verlässliche Daten über den Gesamtbetrag von Alpha-Synuclein (Gesamtfläche), alpha-Synuclein-Muster in der Zelle (Anzahl der Einschlüsse) und das Vorhandensein von Alpha-Synuclein Einschlüsse (Einbeziehung Größe) (Abbildung 2). <img src="/files/ftp_upload/2123/2123fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Motor Dysfunktion bei Rotenon behandelten Mäuse ohne Nachweis von Rotenon in Blut-oder ZNS. A, Standard (50 ng / mL) und Chromatogramm vom Gehirn Proben von 20, 10 und 5 mg / kg behandelten Mäuse. B und C, die Quantifizierung von Rotenon-Spiegel im Blut (B) und CNS (C). B-, Blut-Level 1 , 2 und 3 Stunden nach der Behandlung. Mäuse in drei Gruppen (n = 3) wurden geteilt und mit 2,5, 5, 10 und 20 mg / kg Rotenon (n = 3) wurden 300 ul Blut 1, 2 und 3 Stunden nach Rotenon Verwaltung extrahiert und zusammen gebündelt HPLC-Analyse. C, wurden die Mäuse für eine Woche behandelt werden einmal täglich mit 5 (n = 3), 10 (n = 3) und 20 (n = 1) mg / kg Rotenon, Gehirn und Hirnstamm 1 extrahiert wurden und 2 Stunden nach letzten Verabreichung und vorbereitet für die HPLC-Analyse. D, mitochondriale Komplex I-Aktivität in den Muskeln und Gehirn Proben von 1,5 Monate behandelten Mäuse. <img src="/files/ftp_upload/2123/2123fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Lokal verwaltete Rotenon induziert alpha-Synuclein Phosphorylierung, Akkumulation und Aggregation mit Gliose in ENS Ganglien. (Maßstab 20 mu m). A, B, C, anti βIII-Tubulin, alpha-Synuclein und DAPI-Färbung in Zwölffingerdarm (B) und Ileum ( A, C) Abschnitte. Pfeil in B, induziert 1,5 Monate der Behandlung eine erhöhte Alpha-Synuclein punktförmige Muster innerhalb enterale Nervensystem Ganglien, wenn bis 3 Monate kontrolliert (A) verglichen. Pfeil in C, 3 Monate Behandlung induzierte Bildung von größeren alpha-Synuclein Einschlüsse (ø&gt; 6 um). D, Immunfluoreszenzfärbung mit anti-alpha-Synuclein, Thioflavin S und DAPI. Pfeil in D, nur 3 Monate behandelten Mäuse zeigten Aggregation dieser größeren alpha-Synuclein Ansammlungen. E, E &#39;, E&#39;&#39;, Bildanalyse Quantifizierung Schritte. E, immungefärbt Beispiel Bild in Quantifizierung zeigt eine ENS Ganglion eingesetzt gegen alpha Synuclein zeigen verschiedene Einschlüsse. E &#39;, beispielsweise der Bild für Bild-Analyse-Protokoll erhalten. E&#39;&#39;, Überschneidungen zwischen einigen der ursprünglichen Einschlüsse (grün) und die Darstellung nach der Durchführung des Protokolls (gelbe Linien) erhalten. FG, Quantifizierung des Experiments gezeigt, in AC wurde mit Hilfe der automatischen Segmentierung und Entropie-basierte Schwellwertverfahren. Single-Sternchen, P &lt;0,05, und doppelklicken Sie Sternchen, P &lt;0,01. F, jede Spalte entspricht insgesamt alpha-Synuclein Oberfläche / ganglion Oberfläche. G repräsentiert jede Spalte Gesamtzahl von Alpha-Synuclein Einschlüsse / ganglion Oberfläche. Alle Diagramme zeigen Mittelwerte ± SEM

<ol>
	<li>Braak, H., de Vos, R. A., Bohl, J., Del Tredici, K., K, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastric+alpha-synuclein+immunoreactive+inclusions+in+Meissner's+and+Auerbach's+plexuses+in+cases+staged+for+Parkinson's+disease-related+brain+pathology.">Gastric alpha-synuclein immunoreactive inclusions in Meissner's and Auerbach's plexuses in cases staged for Parkinson's disease-related brain pathology.</a> Neurosci Lett. 396, 67-72 (2006).</li><li>Wakabayashi, K., Takahashi, H., Ohama, E., Ikuta, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease:+an+immunohistochemical+study+of+Lewy+body-containing+neurons+in+the+enteric+nervous+system.">Parkinson's disease: an immunohistochemical study of Lewy body-containing neurons in the enteric nervous system.</a> Acta Neuropathol. 79, 581-583 (1990).</li><li>Wakabayashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restricted+occurrence+of+Lewy+bodies+in+the+dorsal+vagal+nucleus+in+a+patient+with+late-onset+parkinsonism.">Restricted occurrence of Lewy bodies in the dorsal vagal nucleus in a patient with late-onset parkinsonism.</a> J Neurol Sci. 165, 188-191 (1999).</li><li>Braak, H., Sastre, M., Bohl, J. R., de Vos, R. A., Del Tredici, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease:+lesions+in+dorsal+horn+layer+I,+involvement+of+parasympathetic+and+sympathetic+pre-+and+postganglionic+neurons.">Parkinson's disease: lesions in dorsal horn layer I, involvement of parasympathetic and sympathetic pre- and postganglionic neurons.</a> Acta Neuropathol. 113, 421-429 (2007).</li><li>Braak, H., Ghebremedhin, E., Rub, U., Bratzke, H., Del Tredici, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+in+the+development+of+Parkinson's+disease-related+pathology.">Stages in the development of Parkinson's disease-related pathology.</a> Cell Tissue Res. 318, 121-134 (2004).</li><li>Gorell, J. M., Johnson, C. C., Rybicki, B. A., Peterson, E. L., Richardson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+risk+of+Parkinson's+disease+with+exposure+to+pesticides,+farming,+well+water,+and+rural+living.">The risk of Parkinson's disease with exposure to pesticides, farming, well water, and rural living.</a> Neurology. 50, 1346-1350 (1998).</li><li>Semchuk, K. M., Love, E. J., Lee, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease+and+exposure+to+agricultural+work+and+pesticide+chemicals.">Parkinson's disease and exposure to agricultural work and pesticide chemicals.</a> Neurology. 42, 1328-1335 (1992).</li><li>Butterfield, P. G., Valanis, B. G., Spencer, P. S., Lindeman, C. A., Nutt, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+antecedents+of+young-onset+Parkinson's+disease.">Environmental antecedents of young-onset Parkinson's disease.</a> Neurology. 43, 1150-1158 (1993).</li><li>Pan-Montojo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+Parkinson's+disease+pathology+is+reproduced+by+intragastric+administration+of+rotenone+in+mice.">Progression of Parkinson's disease pathology is reproduced by intragastric administration of rotenone in mice.</a> PLoS One. 5, e8762-e8762 (2010).</li><li>Sherer, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+toxicity+in+rotenone+models+of+Parkinson's+disease.">Mechanism of toxicity in rotenone models of Parkinson's disease.</a> J Neurosci. 23, 10756-10764 (2003).</li><li>Bove, J., Prou, D., Perier, C., Przedborski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxin-induced+models+of+Parkinson's+disease.">Toxin-induced models of Parkinson's disease.</a> NeuroRx. 2, 484-494 (2005).</li><li>Inden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodegeneration+of+mouse+nigrostriatal+dopaminergic+system+induced+by+repeated+oral+administration+of+rotenone+is+prevented+by+4-phenylbutyrate,+a+chemical+chaperone.">Neurodegeneration of mouse nigrostriatal dopaminergic system induced by repeated oral administration of rotenone is prevented by 4-phenylbutyrate, a chemical chaperone.</a> J Neurochem. 101, 1491-1504 (2007).</li></ol>

Francisco Pan-Montojo hat ein Patent angemeldet für dieses Tiermodell (Anmeldenummer PCT / EP 2009/005688).

Intragastric Verwaltung wurde bereits 12 durchgeführt. Doch nach Inden Manuskript, Rotenon verabreicht wurde in 0,5% Carboxymethylcellulose-Natriumsalz (CMSS) allein aufgelöst. Rotenon ist ein High liphophylic Substanz. Daher kann Rotenon nicht in 0,5% CMSS aufgelöst allein sein und Niederschlag, wenn getan. Der Einsatz von Chloroform entsteht eine gleichmäßig verteilte Rotenon Federung vermeiden Niederschlag. Außerdem wird durch den höheren Konzentrationen des Pestizids in der CMSS ist das endgültige...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Rotenone</td>
<td>&nbsp;</td>
<td>SIGMA-Aldrich</td>
<td>R8875</td>
<td>Germany</td>
</tr>
<tr>
<td>Chloroform</td>
<td>&nbsp;</td>
<td>Carl Roth</td>
<td>3313.1</td>
<td>Germany</td>
</tr>
<tr>
<td>Carboxymethylcellulose</td>
<td>&nbsp;</td>
<td>SIGMA-Aldrich</td>
<td>C5678</td>
<td>Germany</td>
</tr>
<tr>
<td>Gavage 1,2 mm x 60 mm</td>
<td>&nbsp;</td>
<td>Unimed</td>
<td>&nbsp;</td>
<td>Switzerland</td>
</tr>
<tr>
<td>LSM 510</td>
<td>&nbsp;</td>
<td>Zeiss Microscopes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>FIJI</td>
<td>&nbsp;</td>
<td>http://pacific.mpi-cbg.de</td>
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oral administration of rotenone using a gavage and image analysis of alpha-synuclein inclusions in the enteric nervous system

In der Parkinson-Krankheit (PD)-Patienten, folgt der damit verbundenen Pathologie ein charakteristisches Muster mit unter anderem die enterale Nervensystem (ENS) 1,2, der Riechkolben (OB), der Nucleus dorsalis des Vagus (DMV) 3, die intermediolateralen Kern des Rückenmarks 4 und der Substantia nigra, die die Grundlage für die neuropathologischen Inszenierung der Krankheit 4,5. Die ENS und der OB sind die am stärksten exponierten Nerven Strukturen und die ersten, die betroffen sein. Interessanterweise hat PD um Pestizidbelastung 6-8 in Verbindung gebracht. Hier zeigen wir im Detail zwei Methoden in unserer vorherigen Studie 9 verwendet. Um die Effekte von Rotenon lokal handeln auf der ENS zu analysieren, wir Rotenon mit einer Magensonde zur einjährigen C57/BL6-Mäusen verwaltet. Rotenon ist eine weit verbreitete Pestizid, das stark hemmt mitochondrialen Komplex I 10. Es ist sehr lipophilen und schlecht im Magen-Darm-Trakt 11 absorbiert. Unsere Ergebnisse zeigten, dass die Gabe von 5 mg / kg Rotenon nicht hemmen mitochondrialen Komplex I-Aktivität in den Muskeln oder dem Gehirn. So, was darauf hindeutet, dass die Verwendung unserer Verwaltung Methode Rotenon nicht über die hepatoportal und war ausschließlich auf die ENS tätig. Hier zeigen wir eine Methode, um Pestizide mit Hilfe eines Magensonde und die Bildanalyse-Protokoll verwendet, um die Auswirkungen des Pestizids in alpha-Synuclein Anreicherung in der ENS analysieren zu verwalten. Der erste Teil zeigt eine Methode, intragastrische Verabreichung von Pestiziden (Rotenon) ermöglicht bei einer gewünschten genaue Konzentration. Die zweite Methode ist eine semi-automatische Bildanalyse-Protokoll zu alpha-Synuclein Anreicherung in der ENS analysieren mit Hilfe eines Bildanalyse-Software.

Watch this Scientific Journal Video about Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System at JoVE.com

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

Parkinson-Krankheit ist die Exposition gegenüber Pestiziden in Zusammenhang gebracht. Hier zeigen wir eine Methode, um Pestizide mit einer Magensonde in der gewünschten Konzentration und eine Methode, um ihre Wirkung in alpha-Synuclein Anreicherung zu analysieren, die enterale Nervensystem liefern.

Die orale Verabreichung von Rotenon mit einem Gavage und Bildanalyse von Alpha-Synuclein Einschlüsse in den enterischen Nervensystem

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

oral-administration-rotenone-using-gavage-image-analysis-alpha

Research

JoVE Journal

Neuroscience

16.8K Aufrufe.  Technische Universität Dresden. Parkinson-Krankheit ist die Exposition gegenüber Pestiziden in Zusammenhang gebracht. Hier zeigen wir eine Methode, um Pestizide mit einer Magensonde in der gewünschten Konzentration und eine Methode, um ihre Wirkung in alpha-Synuclein Anreicherung zu analysieren, die enterale Nervensystem liefern.

Serie: Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Visualisierung und genetische Manipulation von Dendriten und Dornen in der Maus Hirnrinde und im Hippocampus mit<em&gt; In utero</em&gt; Die Elektroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Forschung

Neurowissenschaften

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Direkt Intraventrikuläre Abgabe von Arzneimitteln an die Nager Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

In vivo optogenetische Stimulation des Nager Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

In Situ Ca 2+ Imaging des enterischen Nervensystems

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

Sequentielle Extraktion von löslichen und unlöslichen Alpha-Synuclein von Parkinson-Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein prionoids, which cause neuropathology within the central nervous system (CNS). Here we describe that intraglossal or intraperitoneal injection of alpha-synuclein fibrils into bigenic Tg(M83+/-:Gfap-luc+/-) mice, which overexpress human alpha-synuclein with the A53T mutation from the prion protein promoter and firefly luciferase from the promoter for glial fibrillary acidic protein (Gfap), is sufficient to induce neuropathologic disease. In comparison to homozygous Tg(M83+/+) mice that develop severe neurologic symptoms beginning at an age of 8 months, heterozygous Tg(M83+/-:Gfap-luc+/-) animals remain free of spontaneous disease until they reach an age of 22 months. Interestingly, injection of alpha-synuclein fibrils via the intraperitoneal route induced neurologic disease with paralysis in four of five Tg(M83+/-:Gfap-luc+/-) mice with a median incubation time of 229 &#177;17 days. Diseased animals showed severe deposits of phosphorylated alpha-synuclein in their brains and spinal cords. Accumulations of alpha-synuclein were sarkosyl-insoluble and colocalized with ubiquitin and p62, and were accompanied by an inflammatory response resulting in astrocytic gliosis and microgliosis. Surprisingly, inoculation of alpha-synuclein fibrils into the tongue was less effective in causing disease with only one of five injected animals showing alpha-synuclein pathology after 285 days. Our findings show that inoculation via the intraglossal route and more so via the intraperitoneal route is suitable to induce neurologic illness with relevant hallmarks of synucleinopathies in Tg(M83+/-:Gfap-luc+/-) mice. This provides a new model for studying prion-like pathogenesis induced by alpha-synuclein prionoids in greater detail.

Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83+/-:Gfap-luc+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system.

Biolumineszenz-Bildgebung von Neuroinflammation in transgenen Mäusen nach peripherer Inokulation von Alpha-Synuclein

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

Methoden und Tipps für die intravenöse Gabe von Adeno-assoziierten Virus an Ratten und Bewertung des zentralen Nervensystems Transduktion

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

Exogene Verabreichung von Alpha-Synuclein Microsomen verbundenen Aggregate zur primären Neuronen als eine mächtige Zellmodell der Fibrillen Bildung

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Transplantation von chemogenetisch hergestellten kortikalen Interneureuren in frühpostnatale Maushirne

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson&#39;s disease synucleinopathy and nigrostriatal degeneration. The standardization of &#945;-syn PFF generation and in vivo application is critical in order to ensure consistent, robust &#945;-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric &#945;-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of &#945;-syn PFFs into rats or mice. Starting with monomeric &#945;-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed &#945;-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated &#945;-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.&#160;

The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.

Generation von Alpha-Synuclein vorgeformten Fibrillen aus Monomers und Einsatz In Vivo

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model ...