El Pedro Barrie de la Maza Fundación apoya este trabajo.

1) la administración intragástrica de la rotenona <ol><li> Pesa el animal. </li><li> Calcular el volumen total de la solución de rotenona / vehículo necesario. En este caso, 0,01 ml por gramo de peso del animal. </li><li> Cobro de una jeringa de 1 ml con el volumen calculado previamente de la solución de rotenona (0,625 mg / mL rotenona, el 4% y el 1,25 carboximetilcelulosa cloroformo%) o solución vehículo (4% carboximetilcelulosa y 1,25% cloroformo). En este caso corresponde a una dosis de rotenona 5 mg / kg. </li><li> Levante el ratón y, sosteniendo la cola del ratón con una mano, estirar la piel del cuello tirando de él con los dos primeros dedos de la otra mano. </li><li> Una vez que la cabeza se fija entre los dos dedos, es apoyarse en la palma de la mano y la coloque boca abajo. Fijar la cola con los dedos cuarto y quinto. </li><li> Mantener la cabeza hacia atrás, pulse el paladar para abrir la boca del ratón. Introducir la sonda suavemente en la boca. Es importante utilizar una aguja de alimentación forzada y no una aguja normal. Poco a poco, mover la sonda en la dirección del estómago lo suficientemente lejos. Esto evitará que el material consiguiendo en los pulmones. </li><li> Administrar la solución para la rotenona o el vehículo presionando suavemente el émbolo. Cuando esté terminado, poco a poco extraer la sonda. </li><li> Vuelva a colocar el animal en otra jaula hasta que todos los animales de una jaula se llevan a cabo. </li></ol> 2) Procedimientos de inmunotinción <ol><li> Después del tratamiento, los ratones son anestesiados utilizando ketamina 100 mg / kg ip und Xylazin 10 mg / kg y perfusión intracardíaca con paraformaldehído al 4% en PBS. </li><li> Las tripas se extraen por la disección, y se coloca en el 15% de sacarosa durante la noche. Al día siguiente, el tejido se transfiere al 30% de sacarosa y se incuba de nuevo la noche a 4 ° C. </li><li> El tejido es colocado entonces en Tissue-Tek y se almacenan en un congelador de -80 º C hasta su uso. </li><li> El uso de un criostato, de 20 micras intestino secciones se transfieren a las diapositivas Superfrost. </li><li> El bloqueo y la inmunotinción procedimientos utilizando anticuerpos anti-βIII-tubulina y anti-alfa-sinucleína se realiza como se ha descrito previamente 9. </li></ol> 3) Imagen de la alfa-sinucleína Inclusión Análisis Se recomienda que una persona externa desconoce el origen de imágenes &quot;realiza el análisis. Por lo tanto, los datos deben ser codificados. Análisis de imágenes se realizó utilizando el software FIJI. <ol><li> Abra el archivo de imagen confocal. Luego, dividir los canales de modo que es posible trabajar en cada canal de forma independiente. </li><li> Cerca de los canales que no se van a utilizar y seleccionar Analizar&gt; Definición de la escala y establecer el tamaño del pixel a 1. Establecer la distancia conocida a 0,13 (esto dependerá de los objetivos y la resolución utilizada) y haga clic en el global. El tamaño de la imagen se mostrará ahora en micrones. </li><li> Seleccione la alfa-sinucleína del canal y seleccione ganglionares, asegúrese de que la selección encierra el ganglio en todos los cortes. </li><li> Imagen de prensa&gt; Duplicar para duplicar la imagen. Con el ganglio seleccionado, sólo una imagen del tamaño de los ganglios se duplicará. </li><li> Pulse Editar&gt; Borrar fuera. La parte de la imagen, fuera de la región seleccionada se convertirá en negro. </li><li> Duplicar la imagen de nuevo para determinar en qué tan bien los pasos posteriores del procedimiento fue. Las imágenes que tienen una señal al ruido de fondo no relación se debe utilizar para el análisis de imágenes. </li><li> Seleccione Imagen&gt; Ajustes&gt; Umbral y luego seleccione MaxEntropy. A continuación, se mueven sobre las rebanadas de la imagen recortada y la original para garantizar que el procedimiento fue bien. El umbral de entropía selecciona un nivel de intensidad de señal basado en el histograma de la imagen y sólo los píxeles con intensidades superiores a este umbral se muestran en una imagen blanco sobre negro. </li><li> Seleccione Proceso, a continuación, haga clic en liso. Este paso se transforma los bordes de las imágenes pixeladas en los bordes lisos. </li><li> Seleccione Proceso, a continuación, haga clic en binario, y elija la opción de binarios. La imagen se muestra como un negro sobre blanco la imagen. </li><li> Duplicar la imagen para realizar el siguiente paso en la imagen duplicada. </li><li> Haga clic en Análisis&gt; Análisis de partículas. A continuación, seleccione Mostrar: Contornos y haga clic en Resumir. El resto de ajustes se deben a falta de hojas. Esta función reconoce el número total de píxeles con una señal y los que se agrupan. La superficie total de píxeles de la señal más el número de grupos de píxeles y el tamaño medio se informó. </li><li> Comparar y seleccionar la mayor porción que se ajusta bien. El corte será marcado. </li><li> Copia de la tabla de datos con el total de la alfa-sinucleína superficie, el número de inclusiones y el tamaño medio de partícula. Pegue los datos en una hoja de cálculo Excel o anotado en otra parte. </li><li> Volver a Fiji y encontrar el trozo seleccionado previamente en el canal de β-tubulina, y seleccionarlo. </li><li> Seleccionar el área del ganglio y haga clic en Analizar a continuación, seleccione Medir. Otra tabla aparecerá mostrando elsuperficie total de los ganglios. </li><li> Extraer los datos de la tabla de la hoja de Excel, cerca de la medición de la alfa-sinucleína o copiarlo en otra parte. </li><li> El uso de Excel, se dividen los diferentes parámetros obtenidos en la medición de la alfa-sinucleína en la superficie del ganglio para obtener el total de alfa-sinucleína número de superficie y la inclusión normalizada por el tamaño del ganglio. </li></ol> 4) Los resultados representativos Cuando protocolo de administración de alimentación forzada se lleva a cabo correctamente las molestias para el animal son mínimas. El tratamiento con esta concentración de rotenona permite un efecto local de la rotenona en la ENS, sin los niveles de la rotenona en la sangre y no la inhibición de los músculos o el cerebro complejo I mitocondrial, incluso después de 1,5 meses de tratamiento (ver figura 1). Si el análisis de imágenes se realiza correctamente un análisis riguroso de la alfa-sinucleína patrón debe ser posible. Se da datos fiables sobre la cantidad total de la alfa-sinucleína (superficie total), la alfa-sinucleína en el patrón de células (número de inclusiones) y la presencia de alfa-sinucleína inclusiones (tamaño inclusión) (Figura 2). <img src="/files/ftp_upload/2123/2123fig1.jpg" alt="Figura 1" /> Figura 1. Disfunción motora en ratones tratados con rotenona sin detección de la rotenona en la sangre o del SNC. Un estándar, (50 ng / ml) y cromatograma de las muestras de cerebro de 20, 10 y 5 mg / kg los ratones tratados. B y C, la cuantificación de los niveles de la rotenona en la sangre (B) y sistema nervioso central (C). B, los niveles de una , 2 y 3 horas después del tratamiento. Los ratones se dividieron en tres grupos (n = 3) y tratados con 2,5, 5, 10 y 20 mg / kg rotenona (n = 3), 300 l de sangre se extrajo de 1, 2 y 3 después de la administración y la rotenona agruparon para El análisis por HPLC. C, los ratones fueron tratados durante una semana una vez al día con 5 (n = 3), 10 (n = 3) y 20 (n = 1) mg / kg rotenona, el cerebro y el tallo cerebral se obtuvieron 1 y 2 horas después la última administración y preparados para el análisis por HPLC. D, complejo I mitocondrial actividad en muestras de músculo y el cerebro de los ratones tratados 1,5 meses. <img src="/files/ftp_upload/2123/2123fig2.jpg" alt="Figura 2" /> Figura 2. Rotenona administrada localmente induce la fosforilación de la alfa-sinucleína, la acumulación y agregación con gliosis en los ganglios de la ENS. (Barras de escala de 20 micras). A, B, C, anti-tubulina βIII, alfa-sinucleína y la DAPI en el duodeno (B) y el íleon ( A, C) secciones. Flecha en B, 1,5 meses de tratamiento indujo un aumento de la alfa-sinucleína patrón punteado dentro de los ganglios del sistema nervioso entérico, en comparación con 3 controles meses (A). Flecha en C, la formación de 3 meses de tratamiento inducida de más alfa-sinucleína inclusiones (ø&gt; 6 mm). D, la tinción de inmunofluorescencia utilizando anticuerpos anti-alfa-sinucleína, S tioflavina y DAPI. Flecha en D, sólo tres meses los ratones tratados mostraron una agregación de estos grandes acumulaciones de alfa-sinucleína. E, E &#39;, E&#39;&#39;, la imagen de los pasos análisis y cuantificación. E, ejemplo de la imagen que se utiliza en la cuantificación mostrando un ganglio ENS immunostained contra la alfa-sinucleína muestra inclusiones diferentes. E &#39;, ejemplo de la imagen obtenida tras el protocolo de análisis de imagen. E&#39;&#39;, la superposición entre algunas de las inclusiones original (verde) y la representación obtenida tras la realización del protocolo (líneas amarillas). FG, la cuantificación del experimento se muestra en AC se realizó mediante la segmentación automática basada en la entropía y los métodos de umbralización. Solo asterisco, p &lt;0,05, y el doble asterisco, p &lt;0,01. F, cada columna representa el total de la alfa-sinucleína superficie / superficie del ganglio. G, cada columna representa el número total de alfa-sinucleína inclusiones / superficie ganglionares. Todos los gráficos muestran la 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 tiene una solicitud de patente pendiente para este modelo animal (número de la solicitud PCT / EP 2009/005688).

La administración intragástrica se ha realizado previamente 12. Sin embargo, según el manuscrito de Inden, rotenona se administró la sal disuelta en carboximetilcelulosa 0,5% de sodio (CMSS) por sí sola. La rotenona es una sustancia liphophylic alta. Por lo tanto, la rotenona no puede ser disuelto en el CMSS 0,5% solo y se precipitará si se hace así. El uso de cloroformo crea una suspensión rotenona uniformemente distribuida para evitar la precipitación. Además, debido a las mayores concentraciones ...

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		<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

En la enfermedad de Parkinson (EP) de los pacientes, la patología asociada sigue un patrón característico implican entre otras cosas, el sistema nervioso entérico (SNE) 1,2, el bulbo olfatorio (OB), el núcleo motor dorsal del vago (DMV) 3, la intermediolateral núcleo de las 4 de la médula espinal y la sustancia negra, proporcionando la base para la puesta en escena neuropatológicos de la enfermedad 4,5. La ENS y OB son las estructuras más expuestas nervioso y los primeros en verse afectados. Curiosamente, PD se ha relacionado con la exposición a plaguicidas 6-8. Aquí se muestra en detalle dos métodos utilizados en nuestro estudio anterior 9. Con el fin de analizar los efectos de la rotenona actúa localmente en el ENS, se administró utilizando una sonda rotenona a un año de edad C57/BL6 ratones. La rotenona es un pesticida muy utilizado que inhibe fuertemente complejo I mitocondrial 10. Es muy lipofílicos y absorbe mal en el tracto gastrointestinal 11. Nuestros resultados mostraron que la administración de 5 mg / kg de la rotenona no inhibió la actividad de complejo I mitocondrial en el músculo o en el cerebro. Por lo tanto, lo que sugiere que el uso de rotenona nuestro método de administración no cruzó el sistema hepatoportal y se actúa solamente en la ENS. Aquí se muestra un método para administrar los pesticidas con una alimentación por sonda y el protocolo de análisis de imágenes para analizar los efectos de los plaguicidas en la acumulación de alfa-sinucleína en la ENS. La primera parte muestra un método que permite la administración intragástrica de plaguicidas (rotenona) a una concentración deseada precisión. El segundo método muestra la imagen de un protocolo semi-automático de análisis para analizar la acumulación de alfa-sinucleína en la ENS utilizando un software de análisis de imagen.

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

La enfermedad de Parkinson se ha relacionado con la exposición a plaguicidas. Aquí se muestra un método para entregar los pesticidas mediante un tubo gástrico a la concentración deseada y un método para analizar su efecto en la acumulación de alfa-sinucleína en el sistema nervioso entérico.

La administración oral de la rotenona con una sonda y Análisis de Imágenes de la alfa-sinucleína inclusiones en el sistema nervioso entérico

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 vistas.  Technische Universität Dresden. La enfermedad de Parkinson se ha relacionado con la exposición a plaguicidas. Aquí se muestra un método para entregar los pesticidas mediante un tubo gástrico a la concentración deseada y un método para analizar su efecto en la acumulación de alfa-sinucleína en el sistema nervioso entérico.

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.

La visualización y la manipulación genética de las dendritas y espinas en la corteza cerebral y el hipocampo del ratón usando<em&gt; En el útero</em&gt; La electroporación

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 ...

Investigación

Neurociencias

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.

Entrega intraventricular directa de medicamentos al roedor Sistema Nervioso Central

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 optogenético la estimulación del sistema nervioso central de roedores

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 nervioso entérico

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.

Extracción secuencial de soluble e insoluble alfa-sinucleína de cerebros parkinsonianos

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.

Imágenes de bioluminiscencia de la neuroinflamación en ratones transgénicos Después periférica La inoculación de la alfa-sinucleína fibrillas

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.

Métodos y consejos para la administración intravenosa del Virus Adeno-asociado a las ratas y la evaluación de la transducción de señales del sistema nervioso Central

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.

Administración exógena de alfa-sinucleína asociada a microsomas agregados a las neuronas primarias como un modelo de celular de gran alcance de la formación de las fibrillas de

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.

Trasplante de progenitores interneuronas corticales de ingeniería química en cerebros de ratón postnatal temprano

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.

Generación de fibrillas alfa-Synucleinas preformadas de monómeros y 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 ...