Pedro Barrie de la Maza Fondazione ha sostenuto questo lavoro.

1) Amministrazione intragastrico di Rotenone <ol><li> Pesare l&#39;animale. </li><li> Calcolare il volume totale di rotenone / veicolo soluzione necessaria. In questo caso 0,01 ml per grammo di peso animale. </li><li> Caricare una siringa da 1 ml con il volume già calcolato di soluzione rotenone (0,625 mg / mL rotenone, 4% carbossimetilcellulosa cloroformio e 1,25%) o soluzione di veicoli (4% carbossimetilcellulosa cloroformio e 1,25%). In questo caso corrisponde ad un 5 mg / kg dose di rotenone. </li><li> Raccogliete il mouse e, tenendo la coda di topo con una mano, allungare la pelle del collo tirandolo con le prime due dita dell&#39;altra mano. </li><li> Una volta che la testa è fissato tra le due dita, appoggiate il palmo della mano e girarlo a testa in giù. Fissare la coda con il quarto e quinto dito. </li><li> Mantenere la testa verso la parte posteriore, premere il palato per aprire la bocca del mouse. Introdurre delicatamente la sonda gastrica in bocca. E &#39;importante utilizzare un ago sonda gastrica e non un ago normale. Lentamente, spostare la sonda gastrica nella direzione dello stomaco abbastanza lontano. Ciò eviterà materiale trovato nei polmoni. </li><li> Somministrare la soluzione rotenone o di un veicolo premendo delicatamente l&#39;embolo. Una volta completato, lentamente estrarre la sonda gastrica. </li><li> Posizionare il nuovo animale in un&#39;altra gabbia fino a quando tutti gli animali da una gabbia è fatto. </li></ol> 2) L&#39;immunocolorazione Procedure <ol><li> Dopo il trattamento, i topi vengono anestetizzati utilizzando Ketamin 100 mg / kg und Xylazin 10 mg / kg ip e intracardially perfusi con paraformaldeide 4% in PBS. </li><li> Il fegato viene estratto da dissezione, e collocato nel 15% saccarosio durante la notte. Il giorno successivo, il tessuto viene trasferito al 30% di saccarosio e incubate di nuovo una notte a 4 ° C. </li><li> Il tessuto viene poi messo in tessuto-tek e conservati in un congelatore ° -80 C fino all&#39;utilizzo. </li><li> Utilizzando un criostato, 20 micron intestino sezioni vengono trasferite alle diapositive Superfrost. </li><li> Blocco e immunostaining procedure utilizzando anti-βIII-tubulina e anti-alfa-sinucleina anticorpi vengono poi eseguite come descritto in precedenza 9. </li></ol> 3) Analisi dell&#39;immagine alfa-sinucleina Inclusione Si raccomanda che una persona esterna a conoscenza di origine immagini &#39;esegue l&#39;analisi. Pertanto, i dati devono essere codificati. Analisi delle immagini è stata effettuata utilizzando il software FIGI. <ol><li> Aprire il file immagine confocale. Poi, dividere i canali in modo che sia possibile lavorare su ogni canale in modo indipendente. </li><li> Chiudere quei canali che non stanno per essere utilizzare e selezionare Analisi&gt; Imposta Scala e impostare la dimensione del pixel a 1. Impostare la distanza nota a 0,13 (questo dipenderà l&#39;obiettivo e la risoluzione utilizzata) e cliccare su globale. La dimensione dell&#39;immagine sarà ora mostrato in micron. </li><li> Seleziona alfa-sinucleina e selezionate ganglio, assicurarsi che la selezione racchiude il ganglio in tutte le sezioni. </li><li> Image Press&gt; Duplica per duplicare l&#39;immagine. Con il ganglio selezionato, solo un&#39;immagine delle dimensioni del ganglio verranno duplicati. </li><li> Premere Modifica&gt; Fuori Cancella. La parte dell&#39;immagine di fuori della regione selezionati diventeranno neri. </li><li> Duplicare l&#39;immagine di nuovo per determinare in fasi successive come pure la procedura è andato. Le immagini che hanno un segnale alto in rapporto al rumore di fondo non deve essere utilizzato per l&#39;analisi delle immagini. </li><li> Seleziona Immagine&gt; Regolazioni&gt; Soglia e quindi selezionare MaxEntropy. Quindi spostare sulle fette nella immagine ritagliata e l&#39;originale per garantire che la procedura è andata bene. La soglia entropica seleziona un livello di segnale di intensità in base alla istogramma dell&#39;immagine e solo i pixel con intensità superiore a questa soglia sarà visualizzato in un bianco su un&#39;immagine nera. </li><li> Selezionare Elabora, quindi fare clic su Smooth. Questo passo pixeled trasforma i bordi delle immagini in bordi lisci. </li><li> Selezionare Elabora, poi clicca sul binario, e scegliete Crea binari. L&#39;immagine verrà visualizzata come immagine nero su bianco. </li><li> Duplica immagine per eseguire il passaggio successivo sull&#39;immagine duplicato. </li><li> Fare clic su Analizza&gt; Analizzare particelle. Quindi selezionare Mostra: Contorni e cliccare su Riassumere. Il resto delle impostazioni dovrebbe essere lasciato in default. Questa funzione riconosce il numero totale di pixel con un segnale e quelli che sono raggruppati insieme. La superficie totale di pixel con il segnale più il numero di gruppi di pixel e la dimensione media sono segnalati. </li><li> Confrontare e selezionare la fetta più grande che si adatta bene. La fetta sarà segnato. </li><li> Copiare la tabella di dati con il totale alfa-sinucleina superficie, il numero di inclusioni e la dimensione media delle particelle. Incollare i dati in un foglio excel o annotato altrove. </li><li> Torna a FIJI e trovare la fetta precedentemente selezionato nella β-tubulina di canale, e selezionarlo. </li><li> Seleziona area ganglio e cliccare su Analizza quindi selezionare Misura. Un&#39;altra tabella apparirà mostrando lasuperficie totale del ganglio. </li><li> Estrarre i dati dalla tabella al foglio di eccellere nei pressi della alfa-sinucleina misura o copiare il basso altrove. </li><li> Utilizzando Excel, dividere i vari parametri ottenuti nelle alfa-sinucleina misura dalla superficie ganglio di ottenere totale alfa-sinucleina numero superficie e inclusione normalizzato in base alle dimensioni ganglio. </li></ol> 4) Rappresentante Risultati Quando il protocollo amministrazione sonda gastrica viene eseguita correttamente i disagi per l&#39;animale sono minimi. Trattamento con questa concentrazione di rotenone permette un effetto locale di rotenone sulle ENS senza rotenone livelli nel sangue e nessuna inibizione del complesso muscolo o cervello mitocondriale ho anche dopo 1,5 mesi di trattamento (vedi Figura 1). Se l&#39;analisi delle immagini viene eseguita correttamente una rigorosa analisi di alfa-sinucleina modello dovrebbe essere possibile. Fornisce dati affidabili sulla quantità totale di alfa-sinucleina (superficie totale), alfa-sinucleina modello nella cella (numero di inclusioni) e la presenza di alfa-sinucleina inclusioni (dimensione inclusione) (Figura 2). <img src="/files/ftp_upload/2123/2123fig1.jpg" alt="Figura 1" /> Figura 1. Disfunzione motoria in topi trattati con rotenone senza riconoscimento del rotenone nel sangue o nel sistema nervoso centrale. A, di serie (50 ng / mL) e cromatogramma da campioni di cervello di 20, 10 e 5 mg / kg topi trattati. B e C, la quantificazione dei livelli di rotenone nel sangue (B) e sistema nervoso centrale (C). B, i livelli di sangue 1 , 2 e 3 ore dopo il trattamento. I topi sono stati divisi in tre gruppi (n = 3) e trattati con 2,5, 5, 10 e 20 mg / kg rotenone (n = 3), 300 ml di sangue è stato estratto ore 1, 2 e 3 dopo la somministrazione rotenone e raccolto insieme per analisi HPLC. C, i topi sono stati trattati per una settimana una volta al giorno con 5 (n = 3), 10 (n = 3) e 20 (n = 1) mg / kg rotenone, cervello e tronco encefalico sono stati estratti 1 e 2 ore dopo ultima somministrazione e preparati per l&#39;analisi HPLC. D, Complesso I mitocondriale attività in campioni di muscolo e nel cervello dei topi trattati con 1,5 mesi. <img src="/files/ftp_upload/2123/2123fig2.jpg" alt="Figura 2" /> Figura 2. Rotenone amministrato localmente induce alfa-sinucleina fosforilazione, l&#39;accumulo e l&#39;aggregazione con gliosi nei gangli ENS. (Bar scala 20 micron). A, B, C, anti βIII-tubulina, l&#39;alfa-sinucleina e colorazione DAPI nel duodeno (B) e ileo ( A, C) sezioni. Freccia in B, 1,5 mesi di trattamento ha indotto un aumento di alfa-sinucleina modello puntata all&#39;interno dei gangli del sistema nervoso enterico rispetto ai 3 controlli mesi (A). Freccia in C, 3 mesi di trattamento ha indotto la formazione di grandi alfa-sinucleina inclusioni (ø&gt; 6 micron). D, colorazione di immunofluorescenza utilizzando anticorpi anti-alfa-sinucleina, Thioflavine S e DAPI. Freccia in D, solo 3 mesi i topi trattati hanno mostrato l&#39;aggregazione di più questi alfa-sinucleina accumuli. E, E &#39;, E&#39;&#39;, immagine passi quantificazione analisi. E, esempio di immagine utilizzati nel quantificazione mostrando un ganglio ENS immunostained contro l&#39;alfa sinucleina mostrando inclusioni diverse. E &#39;, esempio di immagine ottenuta dopo il protocollo di analisi delle immagini. E&#39;&#39;, sovrapposizione tra alcune delle inclusioni originale (verde) e la rappresentazione ottenuto dopo aver eseguito il protocollo (linee gialle). FG, quantificazione di questo esperimento mostrato in AC è stata fatta utilizzando la segmentazione automatica e l&#39;entropia metodi basati soglia. Singolo asterisco, P &lt;0,05, e doppio asterisco, P &lt;0,01. F, ogni colonna rappresenta il totale di alfa-sinucleina superficie / ganglio superficie. G, ogni colonna rappresenta il numero totale di alfa-sinucleina inclusioni / superficie ganglio. Tutti i grafici mostrano media ± 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 ha una domanda di brevetto in sospeso per questo modello animale (numero Application PCT / EP 2009/005688).

Amministrazione intragastrica è stato precedentemente effettuato 12. Tuttavia, secondo il manoscritto Inden, il rotenone è stato somministrato sciolto in carbossimetilcellulosa sale dello 0,5% di sodio (CMSS) da solo. Rotenone è una sostanza ad alta liphophylic. Pertanto, rotenone non può essere sciolto in 0,5% CMSS da solo e si precipitano se fatto così. L&#39;uso del cloroformio crea una sospensione rotenone distribuito uniformemente evitando precipitazioni. Inoltre, a causa della maggiore concentrazio...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Rotenone</td>
<td>&nbsp;</td>
<td>SIGMA-Aldrich</td>
<td>R8875</td>
<td>Germany</td>
</tr>
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<td>Chloroform</td>
<td>&nbsp;</td>
<td>Carl Roth</td>
<td>3313.1</td>
<td>Germany</td>
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<td>Carboxymethylcellulose</td>
<td>&nbsp;</td>
<td>SIGMA-Aldrich</td>
<td>C5678</td>
<td>Germany</td>
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<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>
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<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

Nella malattia di Parkinson (PD) pazienti, la patologia associata segue un modello caratteristico che coinvolge tra l&#39;altro il sistema nervoso enterico (ENS) 1,2, il bulbo olfattivo (OB), il nucleo motore dorsale del vago (DMV) 3, l&#39;intermedio- nucleo dei 4 midollo spinale e la substantia nigra, fornendo la base per la messa in scena neuropatologici della malattia 4,5. L&#39;ENS e l&#39;OB sono le strutture più esposte nervoso e le prime ad essere colpite. È interessante notare che PD è stata correlata ai pesticidi 6-8. Qui vi mostriamo nel dettaglio due metodi utilizzati nel nostro studio precedente 9. Per analizzare gli effetti del rotenone agire localmente sulle ENS, abbiamo somministrato rotenone utilizzando una sonda gastrica per un anno vecchio C57/BL6 topi. Rotenone è un pesticida largamente usato che inibisce fortemente complesso mitocondriale I 10. E &#39;altamente lipofile e scarsamente assorbito nel tratto gastrointestinale 11. I nostri risultati hanno mostrato che la somministrazione di 5 mg / kg di rotenone non ha inibito l&#39;attività Complesso I mitocondriale nel muscolo o del cervello. Così, suggerendo che usando il nostro metodo di rotenone amministrazione non ha attraversato il sistema epatoportale e agiva esclusivamente sulla ENS. Qui vi mostriamo un metodo di amministrare con una sonda gastrica pesticidi e il protocollo di analisi delle immagini utilizzate per analizzare gli effetti del pesticida in alfa-sinucleina accumulo nel ENS. La prima parte illustra un metodo che consente la somministrazione intragastrica di pesticidi (rotenone) ad una concentrazione desiderata precisa. Il secondo metodo mostra una semi-automatica protocollo di analisi delle immagini per analizzare alfa-sinucleina accumulo nel ENS utilizzando un software di analisi dell&#39;immagine.

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

Morbo di Parkinson è stata correlata all&#39;esposizione ai pesticidi. Qui vi mostriamo un metodo per fornire pesticidi utilizzando un tubo gastrico alla concentrazione desiderata e un metodo per analizzare il loro effetto in alfa-sinucleina accumulo nel sistema nervoso enterico.

La somministrazione orale di Rotenone utilizzando un Gavage e Image Analysis di alfa-sinucleina inclusioni nel sistema nervoso enterico

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

16.9K Views.  Technische Universität Dresden. Morbo di Parkinson è stata correlata all'esposizione ai pesticidi. Qui vi mostriamo un metodo per fornire pesticidi utilizzando un tubo gastrico alla concentrazione desiderata e un metodo per analizzare il loro effetto in alfa-sinucleina accumulo nel sistema nervoso enterico.

Video: 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.

Visualizzazione e manipolazione genetica di dendriti e spine nella corteccia cerebrale e nell&#39;ippocampo mouse utilizzando<em&gt; In utero</em&gt; Elettroporazione

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 ...

Ricerca

Neuroscienze

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.

Consegna diretta intraventricolare di farmaci per il sistema nervoso centrale, Roditore

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 optogenetic stimolazione del sistema nervoso centrale Roditore

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 del sistema nervoso enterico

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.

Estrazione sequenziale di solubile e insolubile sinucleina alfa da Brains parkinsoniani

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.

Bioluminescenza Imaging di Neuroinflammation in topi transgenici Dopo periferica inoculazione di alfa-sinucleina fibrille

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.

Metodi e consigli per la somministrazione endovenosa di Virus Adeno-associato ai ratti e valutazione di trasduzione del sistema nervoso centrale

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.

Somministrazione esogena di microsomi-collegato dell'alfa-synuclein aggregati di neuroni primari come un modello potente delle cellule di formazione di fibrille

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.

Trapianto di progenitori interneuroni corticali chemiogeneticamente ingegnerizzati nei primi cervelli dei topi postnatali

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.

Generazione di fibrille preformate alfa-sinucleina da monomeri e uso 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 ...