Este trabajo fue apoyado por la Asociación de Distrofia Muscular, el NIH (NS062320).

1. La administración subcutánea de receptor de acetilcolina muscarínicos (mAChR) antagonistas <ol><li> Preparar en condiciones asépticas, la dosis apropiada de antagonista mAChR, (cf. cuadro) mediante la disolución del fármaco en solución salina fisiológica estéril en el tubo de reacción de 1,5 ml. Los antagonistas se utilizaron los siguientes: atropina, methoctramina, 4-DAMP, AFDX-116, AFDX-384, MT 7. </li><li> Dibuja 50μl de solución en jeringa de insulina a1cc y usar una jeringa para cada ratón. También preparan las jeringas que contienen solución salina fisiológica única para cada ratón en el grupo control. Mantenga las jeringas en el hielo hasta que esté listo para inyectar. </li><li> Anestesiar a los ratones con ketamina-xilazina cóctel (120 mg / kg de ketamina, 8 mg / kg de xilazina) Verifique que la profundidad anestésica adecuada mediante la observación de la falta de respuesta a los pies pellizco. </li><li> Después de la sedación adecuada, afeitarse el vello que cubre la región rostral de la cabeza de la región caudal del cuello, limpie el cabello lejos queda con el 70% de etanol. </li><li> Coloque el cursor bajo microscopio estereoscópico, e inserte la aguja paralela al músculo LAL mientras tira suavemente sobre la aguja de la jeringa (Fig. 1A, B). Esto asegura que la aguja se inserta en el tejido conjuntivo que recubre y no dañan directamente el músculo LAL sí mismo. Insertar la aguja de modo que la punta cubre el aspecto caudal del músculo LAL. </li><li> Muy lentamente comienzan a inyectar la solución sobre el músculo LAL derecho mientras se retira la jeringa, el tiempo total de inyección debe ser de aproximadamente 1 minuto. Si se inyecta correctamente, la solución debe formar una &quot;cúpula&quot; que es más o menos 1 cm de diámetro, en la piel que recubre que permanecerá por lo menos durante una hora. Una solución inyectada uniformemente cubrirá las regiones de todo el rostral y caudal del músculo LAL derecha. </li><li> Repita el procedimiento cada 12 horas durante 1 a 7 días. Para estudios1 otros, se utilizó el mismo protocolo para inyectar factores de crecimiento cada 12 horas durante un máximo de 21 días. No hay respuestas anormales se observaron en ratones administrados con 2 anestesia al día durante un máximo de 21 días. </li></ol> 2. Disección de los músculos LAL <ol><li> La eutanasia a los ratones con una sobredosis de pentobarbital sódico (390 mg / kg). La muerte está asegurada mediante la evaluación de la falta de ritmo cardíaco después de toracotomía. </li><li> Precisar el ratón en un plato de la disección, la parte dorsal con un alfiler en cada pata y un pin a través de la nariz. </li><li> Hacer la primera incisión en la piel solamente, con unas tijeras pequeñas para cortar la primavera a lo largo del LAL izquierda de la región proximal a la oreja a la región alrededor del omóplato izquierdo. </li><li> Retire con cuidado la piel en esta región, pero no cortar demasiado cerca de la oreja derecha, ya que es uno de los puntos de fijación para el músculo LAL derecha. </li><li> Localice el tubo de tejido adiposo en posición central de los lípidos en la línea media, en la capa superficial de la cabeza, donde los músculos LAL derecha e izquierda se encuentran. Con la tijera pequeña primavera, corte de 1 cm a la derecha del tejido adiposo (hacia la oreja izquierda) desde el borde proximal del músculo LAL izquierda hasta llegar a los hombros (Fig. 1C). </li><li> Una vez hecha la incisión, pelar el músculo derecho LAL con unas pinzas pequeñas para exponer el lado ventral del músculo. Recorte el tejido conjuntivo y la fascia, mientras que con cuidado tirando hacia arriba del músculo LAL derecha con una pinza (Fig. 1C). </li><li> Corte alrededor de la parte caudal del LAL justo en la base de la oreja. Continuar el corte, moviéndose hacia el extremo rostral de la oreja derecha, manteniendo el derecho LAL entregado (Fig. 1C).. Es mejor incluir mayor cantidad de tejido en este paso que el riesgo de dañar la propia LAL. Si el músculo comienza a secarse, pipeta 1X PBS sobre el músculo. </li><li> Lugar disecados derecho LAL en la cultura de 30 mm plato Sylgard contiene 1X PBS, la parte dorsal hacia arriba. Precisar los rincones del músculo con alfileres pequeño insecto. </li><li> El uso de pinzas y tijeras pequeñas de primavera, el tejido conectivo de limpiar superficies dorsal y ventral del músculo LAL derecha. A su vez más de los músculos y volver a pin con el fin de limpiar la superficie ventral. 03.02 músculos se pueden colocar en el plato de 30mm misma. </li></ol> 3. Triple tinción de NMJs LAL: Día 1 <ol><li> Antes de inmunotinción principio, preparar todos los reactivos necesarios. 1X PBS, 2% de albúmina sérica bovina BSA) / PBS, 0,1 M de glicina en el 2% BSA / PBS, 0,2% Triton X100 en el 2% BSA / PBS, y el 4% de paraformaldehído (PFA) en 1X PBS serán necesarios. Pre-enfriado de metanol en congelador a -20 ° C. </li><li> Deja músculos LAL clavado en la parte dorsal plato durante este proceso. Deseche 1X solución de PBS y añadir una cantidad suficiente del 4% (PFA) para cubrir el músculo. Plato a cabo el agitador a velocidad 2 o 3 por 20 minutos. A menos que se indique lo contrario, el plato debe ser colocado en un agitador durante los siguientes pasos. </li><li> Desechar 4% PFA y lavar el músculo con 1X PBS 3 veces, 10 &#39;cada uno. </li><li> Deseche última PBS 1X lavar y añadir 0,1 M de glicina en el 2% BSA / PBS solución durante 30 &#39;. </li><li> Descartar una solución de glicina 0,1 M und añadir 1X PBS con rodamina conjugado α-bungarotoxina a una concentración de 1:200 durante 15 &#39;. </li><li> Deseche α-bungarotoxina solución y lave muscular en 1X PBS, 3 veces 10 &#39;cada uno. </li><li> Permeabilizar el tejido mediante la adición de pre-enfriado metanol y colocar la cápsula en el congelador a -20 ° C, precisamente por 5 minutos, no es necesario agitarlo. </li><li> Eliminar el metanol y lavar 3 veces 10 &#39;cada uno con PBS 1X. Deseche el último lavado y el tejido bloque con 0,2% Triton X-100 en el 2% BSA / PBS durante 1 hora. Las etapas de lavado y el bloqueo se realiza a temperatura ambiente. </li><li> Incubar los músculos durante la noche, temblando, a 4 ° C en un cóctel de anticuerpos primarios diluidos en la solución de bloqueo (véase el cuadro). </li></ol> 4. Triple tinción de NMJs LAL: Día 2 <ol><li> Lavar los músculos en 1X PBS 3 veces, 10 &#39;cada uno a temperatura ambiente. </li><li> Añadir un cóctel de anticuerpos secundarios en solución de bloqueo durante 1 hora a temperatura ambiente (cf. cuadro). Los músculos deben estar protegidos de la luz a partir de este paso para evitar que la foto-blanqueo de Alexa-Fluor 647 fluorescencia. </li><li> Lavar los músculos en 1X PBS 3 veces, 10 &#39;cada uno. </li><li> Limpie cualquier resto de tejido conectivo en 1X PBS, corte los bordes laterales de los músculos de la LAL y montaje de diapositivas cara dorsal, utilizando vidrio cubreobjetos y medios de montaje. El uso de esmalte de uñas transparente para asegurar cubreobjetos en su lugar. </li><li> Proteger a las diapositivas de la luz y almacenar a -20 ° C hasta que esté listo para su análisis. </li></ol> 5. Imagen confocal de NMJs LAL <ol><li> Imágenes de alta resolución confocal se obtienen con un plan de objetivos Leica Apo aceite de 63X (1.4NA) en un microscopio confocal Leica TCS 4D. </li><li> Fluoresceína y las señales de Alexa 647 fueron escaneados de forma simultánea con las líneas de láser de 488 nm (argón, 488 nm, 20 mW) y 633 nm (He-Ne, 633 nm, 10 mW), respectivamente. Secuencial, la señal de rodamina se escanea con la línea de láser 561 nm (DPSS, 561, 20 mW). Secciones ópticas se recogieron en 0,3 intervalos de micras, y el número de imágenes en el plano z varió de 60-80. El agujero se ajusta a una Airy (107,97 um). El formato de imagen 1024 x 1024 (X, Y), con un factor de zoom de 1, en los resultados de velocidad de 400 Hz en un tamaño de imagen de 234,32 m x 234,32 m y tamaño de píxel de 229,05 x 229,05 nm nm. </li><li> Leica TCS-NT adquisición de software se utiliza para reconstruir la serie Z de las imágenes en proyecciones de máxima intensidad. </li><li> Multipanel imágenes se ajustan el brillo y contraste usando Adobe Photoshop. </li><li> Para que un músculo LAL típico, uno debe ser capaz de analizar aproximadamente 75-100 NMJs face. Estabilidad sináptica está determinada por características como la alineación espacial de pre, peri y post-sinápticas elementos (es decir, los nervios, las células de Schwann y el músculo), y las mediciones del área sináptica (es decir, el tamaño de la sinapsis). </li></ol> Los resultados representativos: En la UNM mamífero adulto, un solo axón motor desarrolla ramas finas que forman glorietas muy diferenciados de una terminación nerviosa (Fig. 2, verde) en una sola fibra muscular, precisamente, adosados ​​a grupos postsináptica de AChR nicotínico (Fig. 2; Rojo) . Perisynaptically, terminal de las células de Schwann tape bien todas las ramas de los terminales nerviosos presinápticos (Fig. 2, azul). La integridad estructural y funcional de esta organización tripartita está muy perturbado por la aplicación diaria de inhibidores específicos de los subtipos mAChR. En el ejemplo que aquí se presenta (Fig. 3), 4-DAMP, un antagonista mAChR con alta afinidad por la M1, M3, M4 y M5 subtipos mAChR, evoca la eliminación selectiva de los terminales nerviosos de NMJs numerosos a lo largo de la superficie del músculo (Fig. 3B, C). Además, la terminal de las células de Schwann son anormalmente reposo 8 como lo demuestra brillante etiquetado S100 sin extensión del proceso (Fig. 3B &quot;, 3C&quot;). Postsináptico, las fibras musculares son normales y no hay pérdida de nAChR (Fig. 3B, 3C). <img src="/files/ftp_upload/3124/3124fig1.jpg" alt="Figura 1"/> Figura 1. Localización anatómica y la organización del músculo LAL. Ubicación de rostral (rLAL), caudal (CLAL), y el nervio LAL (LALn) y las bandas correspondientes placa terminal se muestran (A, recuadro). La ubicación y orientación de la aguja con respecto al músculo LAL se muestra para el procedimiento de inyección (B). Puntos de incisión se muestran para el procedimiento de disección (C). <img src="/files/ftp_upload/3124/3124fig2.jpg" alt="Figura 2"/> Figura 2. Organización tripartita de la UNM. Gran aumento vistas confocal de un ratón MNJ. Un solo axón elabora finas ramas terminales (A), bien cubierto por el terminal de células de Schwann y sus procesos (A, punto terminal de asteriscos a los órganos de la célula de Schwann). Postsináptico en la fibra muscular, un grupo de AChR nicotínico es, precisamente, adosados ​​a las ramas terminales del nervionales y las células de Schwann del terminal. <img src="/files/ftp_upload/3124/3124fig3.jpg" alt="Figura 3"/> Figura 3. Músculos LAL tratados con 4-DAMP, un antagonista mAChR. Puntos de vista confocal de alta y baja magnificación de los músculos LAL tratados con vehículo o con 4-DAMP. En contraste con el músculo tratado con vehículo (A-A NMJs&#39;&#39;&#39;), numerosos en los terminales de los músculos 4-DAMP tratados con nervio falta (B-B&#39;&#39;&#39;, C-C&#39;&#39;&#39;). Un área del recuadro en la Figura 2B se amplía en la figura 2C.

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No hay conflictos de interés declarado.

El método aquí presentado permite la investigación de los roles previamente no reconocido de los subtipos específicos mAChR de señalización en la estabilidad y el mantenimiento de NMJs mamíferos. Este método también será útil para probar los efectos de los factores neurotróficos y agentes farmacológicos. Por ejemplo, nuestro laboratorio encontró que el factor neurotrófico ciliar (CNTF) provocó brotes de casi todas las terminales nerviosas en ratones adultos LAL <a>1</a> . Este resultado contrasta con est...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> la ketamina </td><td> Hospira </td><td> NDC0409-2051-05 </td><td> Dosis: 120mg/kg </td></tr><tr><td> xilazina </td><td> Lloyd Laboratorios </td><td> LA33806 </td><td> Dosis: 8mg/kg </td></tr><tr><td> atropina </td><td> Sigma-Aldrich </td><td> A0132 </td><td> (Pureza&gt; 98%); Dosis: 0.2mg/kg - 20mg/kg </td></tr><tr><td> atropina </td><td> Voigt de distribución global </td><td> AT105 </td><td> De calidad farmacéutica </td></tr><tr><td> Methoctramina </td><td> Sigma-Aldrich </td><td> M105 </td><td> Dosis: 100 - 400M </td></tr><tr><td> 4-DAMP </td><td> Sigma-Aldrich </td><td> D142 </td><td> Dosis: 2.5mg/kg </td></tr><tr><td> AFDX-116 </td><td> Bioscience Tocris </td><td> 1105 </td><td> 250M </td></tr><tr><td> AFDX-384 </td><td> Bioscience Tocris </td><td> 1345 </td><td> 50M - 500M </td></tr><tr><td> MT 7 </td><td> Péptidos Internacional </td><td> PMT-4340-s </td><td> 0,1 M - 1M </td></tr><tr><td> 1X buffer fosfato pH de solución salina, 7.4 </td><td> Invitrogen </td><td> 10010049 </td><td></td></tr><tr><td> Paraformaldehído </td><td> Pescador </td><td> T353-500 </td><td> Haz solución al 10% por primera vez por la disolución 10g/100mL desionizada agua destilada; hacer 4% con 1X PBS, ajustar el pH a 7.4 </td></tr><tr><td> Sodio pentobarbitol </td><td> Virbac Salud Animal </td><td> NDC-051311-050-01 </td><td> Dosis: 390mg/kg </td></tr><tr><td> Sylgard </td><td> Dow Corning </td><td> Parte # 184 </td><td> Siga las instrucciones que vienen con el kit, puede utilizar múltiples cultura plato de tamaño mediano (30mm, 60mm, 100mm) en función de las necesidades </td></tr><tr><td> 0,1 M de glicina </td><td> Sigma-Aldrich </td><td> G-7126 </td><td> Añadir 0.185g a 25 ml de 2% de BSA / PBS </td></tr><tr><td> 2% de albúmina de suero bovino (BSA al 2%) </td><td> Sigma-Aldrich </td><td> A3059-100g </td><td> Disolver 2 g de BSA en 100 ml de PBS 1X </td></tr><tr><td> 0,2% Triton X100 en el 2% BSA / PBS (tampón de bloqueo) </td><td> Sigma-Aldrich </td><td> T9284-100 ml </td><td> Disolver 0.2ml/100mL 2% de BSA / PBS </td></tr><tr><td> α-bungarotoxina </td><td> Invitrogen </td><td> T1175 </td><td> Uso a una concentración de 1:200 </td></tr><tr><td> SMI-312 </td><td> Sternberger anticuerpos monoclonales </td><td> SMI312 </td><td> Uso a una concentración de 1:1000 </td></tr><tr><td> SV2 </td><td> Estudios del Desarrollo del Banco hibridoma </td><td> SV2-Sobrenadante </td><td> Uso a una concentración de 1:10 </td></tr><tr><td> S100 </td><td> Dako </td><td> Z0311 </td><td> Uso a una concentración de 1:400 </td></tr><tr><td> FITC cabra anti-ratón IgG1 </td><td> Roche </td><td> 03117731001 </td><td> Uso a una concentración de 1:200, pero si el fondo es alto, trate de 1:400 </td></tr><tr><td> Alexa-Fluor 647 conjugado de cabra anti-conejo </td><td> Invitrogen </td><td> A21244 </td><td> Uso a una concentración de 1:200 </td></tr><tr><td> Vectashield fluorescentes montaje de media </td><td> Vector de laboratorios </td><td> H-1000 </td><td> Esto no es un medio de comunicación en un apuro, tendrá que asegurar la hoja de la cubierta con esmalte de uñas transparente. </td></tr><tr><td> Tijeras pequeñas de Primavera </td><td> Herramientas de Bellas Ciencia </td><td> 15002-08 </td><td></td></tr><tr><td> Pinzas de disección </td><td> Herramientas de Bellas Ciencia </td><td> 11295-51 </td><td></td></tr></tbody></table>

subcutaneous administration of muscarinic antagonists and triple-immunostaining of the levator auris longus muscle in mice

Los músculos de las extremidades traseras de los roedores, tales como gemelos y el músculo tibial anterior, se utilizan con frecuencia en los estudios farmacológicos in vivo de las señales esenciales para la formación y el mantenimiento de NMJs mamíferos. Sin embargo, la penetración del fármaco en estos músculos después de la administración subcutánea o intramuscular es a menudo incompleta o irregular y muchas NMJs puede quedar afectada. Aunque la administración sistémica con dispositivos tales como mini-bombas se pueden mejorar los efectos espacio-temporales, la naturaleza invasiva de este método puede causar confusión respuestas inflamatorias y / o daño muscular directo. Además, el análisis completo de la NMJs en un músculo extremidades posteriores es un reto porque requiere mucho tiempo de seccionamiento de serie y la inmunotinción extensa. La LAL del ratón es una hoja delgada y plana de músculo que se encuentra superficialmente en el dorso del cuello. Es un músculo de contracción rápida que funciona para mover el pabellón de la oreja. Que contiene partes rostral y caudal que se originan en la línea media del cráneo y se extienden lateralmente a la porción cartilaginosa de cada pabellón. El músculo está inervado por una rama del nervio facial que los proyectos de caudal a medida que sale del agujero estilomastoideo. Nosotros y otros han encontrado LAL a ser una preparación útil que ofrece ventajas para la investigación de corto y largo plazo los efectos in vivo de las drogas en NMJs y los músculos. En primer lugar, su localización superficial facilita múltiples aplicaciones locales de medicamentos bajo anestesia ligera. En segundo lugar, su delgadez (2-3 capas de las fibras musculares) permite la visualización y el análisis de casi todos los NMJs dentro del músculo. En tercer lugar, la facilidad de disección con su nervio intacto, junto con el patrón de su inervación permite el análisis complementario electrofisiológicos in vitro 9,5. Por último, y quizás lo más importante, una pequeña cantidad aplicada (~ 50μl) fácilmente cubre la superficie del músculo entero, ofrece una exposición uniforme y prolongada de todas sus NMJs a la droga y elimina la necesidad de un enfoque sistémico 1,8.

Watch this Scientific Journal Video about Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice at JoVE.com

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Se describen los procedimientos para la administración repetida de los inhibidores de la señalización de receptores muscarínicos a la largo del músculo elevador del auris (LAL) del músculo de los ratones adultos jóvenes y de inmunotinción posterior de sus uniones neuromusculares (NMJs) en wholemounts. El músculo LAL tiene ventajas únicas para revelar<em&gt; In vivo</em&gt; Efectos farmacológicos en NMJs.

La administración subcutánea de antagonistas muscarínicos y Triple-inmunotinción del músculo largo del elevador del Auris en ratones

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo  pharmacological studies of the signals ...

subcutaneous-administration-muscarinic-antagonists-triple

Research

JoVE Journal

Neuroscience

13.6K vistas.  Arcadia University. Se describen los procedimientos para la administración repetida de los inhibidores de la señalización de receptores muscarínicos a la largo del músculo elevador del auris (LAL) del músculo de los ratones adultos jóvenes y de inmunotinción posterior de sus uniones neuromusculares (NMJs) en wholemounts. El músculo LAL tiene ventajas únicas para revelar In vivo Efectos farmacológicos en NMJs.

Video: Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

Evaluación de las vocalizaciones de ultrasonidos durante autoadministración de drogas en ratas

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

Investigación

Neurociencias

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

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, the olfactory bulb (OB), the dorsal motor nucleus of the vagus (DMV)3, the intermediolateral nucleus of the spinal cord 4 and the substantia nigra, providing the basis for the neuropathological staging of the disease4,5. The ENS and the OB are the most exposed nervous structures and the first ones to be affected. Interestingly, PD has been related to pesticide exposure6-8. Here we show in detail two methods used in our previous study 9. In order to analyze the effects of rotenone acting locally on the ENS, we administered rotenone using a gavage to one-year old C57/BL6 mice. Rotenone is a widely used pesticide that strongly inhibits mitochondrial Complex I 10. It is highly lipophylic and poorly absorbed in the gastrointestinal tract 11. Our results showed that the administration of 5 mg/kg of rotenone did not inhibit mitochondrial Complex I activity in the muscle or the brain. Thus, suggesting that using our administration method rotenone did not cross the hepatoportal system and was acting solely on the ENS. Here we show a method to administer pesticides using a gavage and the image analysis protocol used to analyze the effects of the pesticide in alpha-synuclein accumulation in the ENS. The first part shows a method that allows intragastric administration of pesticides (rotenone) at a desired precise concentration. The second method shows a semi-automatic image analysis protocol to analyze alpha-synuclein accumulation in the ENS using an image analysis software.

Parkinson's disease has been related to the exposure to pesticides. Here we show a method to deliver pesticides using a gastric tube at the desired concentration and a method to analyze their effect in alpha-synuclein accumulation in the enteric nervous system.

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, ...

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

Imagen confocal espectral de marcado con fluorescencia receptores nicotínicos en Knock-en ratones con administración crónica de nicotina

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Microdiálisis de etanol durante etanol operante determinación y la auto-administración de etanol por cromatografía de gases

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

Promoción de la supervivencia y la diferenciación de células madre neurales con fibrina y factor de crecimiento cócteles después de la médula espinal severa lesión

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

Propiocepción y la tensión en los receptores de cangrejo Limbs: Ejercicios de Laboratorio Estudiante

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

Un protocolo para la administración de Real-Time fMRI entrenamiento de Neurofeedback

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Recording Brain Electromagnetic Activity During the Administration of the Gaseous Anesthetic Agents Xenon and Nitrous Oxide in Healthy Volunteers

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most neuroimaging or neurophysiological investigations in humans have been confined to the study of &#947;-Amino-Butyric-Acid-(GABA)-receptor-agonist-based anesthetics, while the effects of dissociative N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthetics ketamine, nitrous oxide (N2O) and xenon (Xe) are largely unknown. This paper describes the methods underlying the simultaneous recording of magnetoencephalography (MEG) and electroencephalography (EEG) from healthy males during inhalation of the gaseous anesthetic agents N2O and Xe. Combining MEG and EEG data enables the assessment of electromagnetic brain activity during anesthesia at high temporal, and moderate spatial, resolution. Here we describe a detailed protocol, refined over multiple recording sessions, that includes subject recruitment, anesthesia equipment setup in the MEG scanner room, data collection and basic data analysis. In this protocol each participant is exposed to varying levels of Xe and N2O in a repeated measures cross-over design. Following relevant baseline recordings participants are exposed to step-wise increasing inspired concentrations of Xe and N2O of 8, 16, 24 and 42%, and 16, 32 and 47% respectively, during which their level of responsiveness is tracked with an auditory continuous performance task (aCPT). Results are presented for a number of recordings to highlight the sensor-level properties of the raw data, the spectral topography, the minimization of head movements, and the unequivocal level dependent effects on the auditory evoked responses. This paradigm describes a general approach to the recording of electromagnetic signals associated with the action of different kinds of gaseous anesthetics, which can be readily adapted to be used with volatile and intravenous anesthetic agents. It is expected that the method outlined can contribute to the understanding of the macro-scale mechanisms of anesthesia by enabling methodological extensions involving source space imaging and functional network analysis.

Simultaneous magnetoencephalography and electroencephalography provides a useful tool to search for common and distinct macro-scale mechanisms of reductions in consciousness induced by different anesthetics. This paper illustrates the empirical methods underlying the recording of such data from healthy humans during N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthesia during inhalation of nitrous oxide and xenon.

Registro actividad electromagnética cerebral durante la administración del xenón gaseoso agentes anestésicos y óxido nitroso en voluntarios sanos

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most ...

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (&#964;m) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

The protocol described in this paper uses the mouse levator auris longus (LAL) muscle to record spontaneous and nerve-evoked postsynaptic potentials (current-clamp) and currents (voltage-clamp) at the neuromuscular junction. Use of this technique can provide key insights into mechanisms of synaptic transmission under normal and disease conditions.

Del levator Auris Longus Preparación para examen de la transmisión Neuromuscular mamífero bajo condiciones de fijación de voltaje

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

La detección de 5-Hidroximetilcitosina en células madre neuronales y cerebros de ratones

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...