The authors would like to thank the MRC-LMB workshop for the production of the enhanced gene gun barrel. We would also like to thank Dr. David R. Hampson at the University of Toronto for the use of equipment and resources.

El uso de animales y tejidos animales deben adherirse estrictamente a la aprobación del comité ético bajo las reglas y regulaciones locales. Todos los tejidos obtenidos durante este estudio adhirieron a la experimentación animal, el MRC-LMB. 1 Preparación de Materiales y Medios de Cultivo <ol><li> Preparar todas las soluciones con agua Millipore; utilizar únicamente reactivos de grado analítico. Preparar y almacenar todos los reactivos a temperatura ambiente (a menos que se indique lo contrario). </li><li> Hacer 500 ml de solución salina tamponada con HEPES (HEPES 10 mM, NaCl 120 mM pH 7,2). Filtrar a través de una unidad de filtro estéril de 0,2 micras. Almacenar a 4 ° C. </li><li> Hacer 500 ml de solución salina tamponada con fosfato (PBS; 10 mM Na 2 HPO 4, 2 mM KH 2 PO 4, NaCl 137 mM, KCl 2,7 mM, pH 7,4). Esterilizar en autoclave. </li><li> Preparar medio neuronal completándolo DMEM (medio de Eagle modificado de Dulbecco) con 25 mM HEPES, 10% de suero de ternera fetal, glucosa 30 mM, 1: 100 N2 dopplement, penicilina-estreptomicina 1.100 U / ml; pH 7,2. Filtro con una unidad de filtro estéril de 0,2 micras. Almacenar a 4 ° C. </li><li> Hacer disolución de polivinilpirrolidona (PVP) stock con 20 mg de PVP en 1 ml de etanol al 100%. Alícuota de esto en 1 ml cantidades, congelación y almacenar a -20 ° C. Solución de trabajo es de 0,05 mg / ml, por lo tanto, añadir 10 l de solución de PVP de stock para cada 4 ml de etanol al 100%. </li><li> Preparar una solución madre 0,05 M espermidina en agua Millipore; ajustar el pH a 7,2. </li><li> Hacer una solución de agarosa al 2% en DMEM (2 g de agarosa de grado electroforesis en 100 ml de DMEM en una botella de tapón de rosca esterilizada). Microondas esto hasta que la agarosa se haya disuelto. Guarde en la nevera 4 ° C durante varias semanas, si es necesario. </li></ol> 2 Preparación de Organotípicos rebanadas <ol><li> La eutanasia a C57 Negro 6 ratones de la edad deseada por asfixia de CO 2 seguido por decapitación. Retire el cerebro usando recortes laterales del cráneo a partir de la foramen magnum y terminando enlos lóbulos olfativos. Levante suavemente el cráneo de la parte posterior para exponer el cerebro. </li><li> Cortar entre los lóbulos olfativos y la corteza frontal, y a rostral del cerebelo. Levante ligeramente la parte rostral del cerebro y cortar los nervios ópticos. Por último, retire el cerebro y lo coloca en una placa de Petri llena con solución salina tamponada con HEPES frío de hielo en hielo. </li><li> El uso de un microscopio de disección, retire la pia utilizando unas pinzas finas; retirar con cuidado los vasos sanguíneos y meninges alrededor del cerebro. </li><li> Coloque el cerebro fresco en un molde receptor pequeño y cubra con la solución de agarosa enfriado ligeramente por encima de RT. Luego enfriar rápidamente el molde a 4 ° C colocándola en hielo. NOTA: Procesar las rebanadas lo más rápidamente posible para evitar la pérdida de viabilidad. </li><li> Cuando la agarosa se ha fijado (5 - 10 min), retire el cerebro agarosa embebidos del molde (recortar si es necesario) y, a continuación, péguelo en la plataforma vibroslicer. Coloque esto en una cámara vibroslicer contiene hielo estéril PBS frío. Disinfect la cámara de vibroslicer antes de ser utilizado con etanol al 70% para reducir al mínimo las contaminaciones posteriores. </li><li> Cortar el cerebro usando un vibroslicer hecha a la medida o su equivalente comercial. El concepto de modelo era diseñar una máquina de cortar el tejido que podría generar movimientos de gran amplitud y alta frecuencia horizontal hasta el borde de la cuchilla y reducir al mínimo las vibraciones verticales. </li><li> Usa la frecuencia de oscilación de 90 Hz, a una amplitud de 1,5 - 2,0 mm, y la cuchilla de corte situado en un ángulo de 15 ° con el plano horizontal; establecer el bloque de montaje sosteniendo el cerebro de agarosa embebidos para mover a 1,7 mm / min hacia la cuchilla. Recoger secciones (150 micras) en una cámara que contiene un medio de cultivo frío en hielo (DMEM Pen / Strep + 10% FCS). Ver corte y manipulación de las rodajas de tejido como un video complementario en Arsenault y O&#39;Brien 13. </li><li> Con suavidad, coloque las rebanadas en insertos de cultivo celular (0,4 m, diámetro 30 mm) en una bandeja de 6 pocillos múltiples con las med culturaia en el exterior de la pieza de inserción, y se incuban en un incubador humidificado a 37 ° C con 5% de CO 2. NOTA: Para la viabilidad del tejido adecuado, ser muy suave, mientras que la transferencia y la manipulación de las rebanadas. También, demasiado líquido evitará que las rebanadas organotípicos se adhiera a la membrana del inserto de filtro. Si esto ocurre, retire el exceso de líquido o placa de nuevo. </li><li> Aunque rebanadas organotípicos pueden ser cultivadas durante un par de semanas, procederá a la transfección no más de 4 días después de la siembra para obtener los mejores rendimientos de transfección. NOTA: Para evitar el crecimiento excesivo de la glía en condiciones de cultivo a largo plazo, el uso AraC o medio libre de suero 2. </li></ol> 3 Preparación de ADN recubierto Micro &amp; Nano-proyectiles <ol><li> Preparar nano-proyectiles que utilizan partículas de oro de 40 nm de diámetro. Brevemente, se añaden 50 l de 0,05 M espermidina y 10 l de ADN en 1 mg / ml (pEYFP-N1) a 10 mg de partículas de oro. <ol><li> Añadir calcio y espermidina en la mezcla de solucióntura durante ADN micro-partículas de oro preparación con el fin de ayudar en la unión de moléculas de ADN a las nanopartículas de oro. NOTA: La cantidad de ADN utilizado por mg de portadores de oro (DLR), debe oscilar entre 1 y 5 g de ADN por 1 mg de oro. Además, la cantidad de partículas de soporte de oro que se rodará por cartucho (MLQ), debe sí variar de 0,1 a 0,5 mg de oro. El DLR y MLQ deben ser juzgados y optimizados para cada usuario dependiendo del sistema particular bajo investigación, así como el tipo de cañón de partículas y el gen utilizado. </li></ol></li><li> Mezclar utilizando un vórtice mientras que lentamente la adición de 50 l de 1 M de CaCl2 en fracciones de 10 - 15 l. Después de 5 min de agitación con vórtex ocasional, centrifugar a 1000 xg durante 30 segundos y luego eliminar el sobrenadante. Resuspender el precipitado de partículas de oro en 3,5 ml de 0,075 M PVP. </li><li> Dibuje esta suspensión en un tubo (diámetro 2,36 mm interna) usando una jeringa. Colocar el tubo en la estación de preparación de tubos. Permitir elpartículas de oro se asienten y eliminar el sobrenadante por aspiración con una jeringa. Gire el tubo para repartir uniformemente las partículas de oro, que posteriormente fueron secados bajo un flujo constante de nitrógeno a 5 litros por min. </li><li> Para crear ADN-balas, cortar el tubo con un cortador de tubo en longitudes de 1 cm. Cualquiera de insertar inmediatamente en el cartucho de pistola de genes o mantener desecado (4 ° C) hasta que se requiera. </li></ol> 4. biolística transfección en Organotípicos rebanadas <ol><li> Lleve a cabo los siguientes pasos dentro de una campana de flujo laminar estéril. </li><li> Inserte una batería de 9 V y un soporte de cartucho vacío en la pistola de genes Helios. </li><li> Coloque la pistola de genes para el tanque de helio con la manguera de helio. Fuego 2 - 3 espacios en blanco (ranuras vacías) a 50 psi para presurizar la manguera de helio y los depósitos en la pistola. </li><li> Cargar los cartuchos que contienen el oro recubiertas de ADN en los soportes de los cartuchos y cargar el soporte en la pistola de genes. </li><li> Desatornille el gen arma espaciador y adjuntarel cañón del arma gen modificado a la pistola de genes. </li><li> Usando una pinza estéril, coloque un inserto de filtro que contiene la rebanada organotípico en un plato de plástico estéril. </li><li> Retire el medio de rebanadas organotípicos. </li><li> Ajuste la presión del gas a 50 psi. NOTA: La protección ocular es esencial y protección para los oídos aconsejable. </li><li> Coloque la pistola de genes a las distancias apropiadas utilizando el montaje medido a 10 mm. Apuntar con cuidado el centro de la barril en la región deseada para la entrega genética. </li><li> Después de la cocción, vuelva a colocar los insertos de nuevo en su plato con medio fresco y devolverlos a las condiciones de crecimiento normales, es decir, 37 ° C con 5% de CO 2 incubadora. </li><li> Una vez terminado, cierre el tanque de helio, liberar la presión de la pistola de genes y separar la pistola de genes desde el tanque de helio. </li><li> Retire el soporte del cartucho y deseche los cartuchos y limpiar la pistola de genes. </li><li> Compruebe el tejido de la morfología y de la penetración exitosa de visualizing las rodajas usando un microscopio invertido. Si el uso de micropartículas, de manera uniforme dispersar el oro; no debe haber áreas densas de partículas de oro se ven en la división. Las nanopartículas debido a su pequeño tamaño no pueden ser vistos como unidades individuales por técnicas de microscopía convencionales. </li><li> Después de que el intervalo de tiempo apropiado (normalmente 1 - 2 días), fijar las rebanadas en el 4% de paraformaldehído (PFA) para la observación de la microscopía. NOTA: La fijación de PFA no siempre se desea. Para imágenes de células vivas evitar este paso. </li></ol> 5. Fijación y Visualización de cerebro rebanadas <ol><li> Después de la transfección biolística, lavar rodajas dos veces en PBS durante 2 min. </li><li> Fijar rebanadas incubando en recién hecho, hielo frío PFA al 4% en PBS durante 20 min. </li><li> Lavar rodajas dos veces en PBS durante 2 min. </li><li> Cortar suavemente alrededor de la membrana admite el uso de un bisturí, sin perturbar las rodajas montados en ellos. El uso de fórceps para colocar cada membrana de soporte / rebanada en un portaobjetos de microscopio. Descanse el organotyprebanadas ic en la parte superior de la membrana en el portaobjetos de vidrio. </li><li> Añadir una gota de medio de montaje en la parte superior de la rodaja y añadir un cubreobjetos suavemente sin ninguna fuerza directamente en contacto con la rebanada organotípicos. </li><li> Asegure el cubreobjetos con una fina capa de barniz de uñas. </li><li> Ver las rebanadas en un microscopio adecuado. </li></ol> 6. Aplicaciones Confocal <ol><li> Visualizar las rodajas mediante el uso de un microscopio confocal láser de barrido vertical con una 60X / 1,4 apertura numérica (NA) de inmersión en aceite de lente de objetivo. NOTA: La característica más problemática del microscopio confocal es el tamaño del agujero de alfiler (puesta a 1 UA), que es capaz de aislar y recoger un plano de enfoque, eliminando así el fuera de foco &#39;bruma&#39; se ve normalmente con una muestra fluorescente. Los detalles finos a menudo son oscurecidas por esta bruma y no pueden ser detectados en un microscopio nonconfocal. </li><li> Ajuste el láser de argón a una longitud de onda de excitación de 488 nm y optimizar para la recogida de emitted luz en la banda de entre 500 y 520 nm. Típicamente, recoger imágenes a 1024 x 1024 píxeles y con las pilas de z secciones a intervalos de 0,5 m para abarcar la anchura completa de cada célula nerviosa que se analizó. <ol><li> Para observar la morfología del núcleo, contador mancha estas células con DAPI (longitud de onda de excitación fijado en 405 nm y de emisión de luz recogida entre 420-4 40 nm). Por último, para el examen de fluorescencia roja, ajuste el láser a 561 nm para la excitación y 565-620 nm para la emisión. </li></ol></li><li> Por lo general, la obtención de imágenes mediante el uso de un zoom 4X exploración que cubrió un área de 57,6 m 2; tamaños de las imágenes estaban en la región de 1.024 x 1.024 píxeles. Record pilas de z secciones en intervalos de 0,5 micras y proyecciones de 5-8 cuadros combinados. </li></ol>

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P., Simonato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+cell+damage+in+organotypic+hippocampal+slice+culture+from+adult+mouse:+a+potential+model+system+to+study+neuroprotection.">Evaluation of cell damage in organotypic hippocampal slice culture from adult mouse: a potential model system to study neuroprotection.</a> Brain Research. 1385, 68-76 (2011).</li><li>Mewes, A., Franke, H., Singer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures+of+adult+transgenic+P301S+mice--a+model+for+tauopathy+studies.">Organotypic brain slice cultures of adult transgenic P301S mice--a model for tauopathy studies.</a> PloS One. 7, 45017 (2012).</li><li>Ito, T., Ibe, K., Uchino, T., Ohshima, H., Otsuka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+DNA/Gold+Nanoparticle+Encapsulated+in+Calcium+Phosphate.">Preparation of DNA/Gold Nanoparticle Encapsulated in Calcium Phosphate.</a> Journal of Drug Delivery. 2011, 647631 (2011).</li><li>Rinberg, D., Simonnet, C., Groisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pneumatic+capillary+gun+for+ballistic+delivery+of+microparticles.">Pneumatic capillary gun for ballistic delivery of microparticles.</a> Appl Phys Lett. 87, 014103 (2005).</li><li>Meijering, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron+tracing+in+perspective.">Neuron tracing in perspective.</a> Cytometry. Part A : the Journal of the International Society for Analytical Cytology. 77, 693-704 (2010).</li><li>Sasaki, T., Matsuki, N., Ikegaya, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+axon-attached+recording+with+fluorescent+patch-clamp+pipettes+in+brain+slices.">Targeted axon-attached recording with fluorescent patch-clamp pipettes in brain slices.</a> Nature Protocols. 7, 1228-1234 (2012).</li><li>Gan, W. B., Grutzendler, J., Wong, W. T., Wong, R. O., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+'DiOlistic'+labeling+of+the+nervous+system+using+lipophilic+dye+combinations.">Multicolor 'DiOlistic' labeling of the nervous system using lipophilic dye combinations.</a> Neuron. 27, 219-225 (2000).</li><li>Livet, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+strategies+for+combinatorial+expression+of+fluorescent+proteins+in+the+nervous+system.">Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system.</a> Nature. 450, 56-62 (2007).</li><li>Gholizadeh, S., Tharmalingam, S., Macaldaz, M. E., Hampson, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transduction+of+the+central+nervous+system+after+intracerebroventricular+injection+of+adeno-associated+viral+vectors+in+neonatal+and+juvenile+mice.">Transduction of the central nervous system after intracerebroventricular injection of adeno-associated viral vectors in neonatal and juvenile mice.</a> Human Gene Therapy Methods. 24, 205-213 (2013).</li><li>Arsenault, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Botulinum+protease-cleaved+SNARE+fragments+induce+cytotoxicity+in+neuroblastoma+cells.">Botulinum protease-cleaved SNARE fragments induce cytotoxicity in neuroblastoma cells.</a> Journal of Neurochemistry. 129, 781-791 (2014).</li><li>Arsenault, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stapling+of+the+botulinum+type+A+protease+to+growth+factors+and+neuropeptides+allows+selective+targeting+of+neuroendocrine+cells.">Stapling of the botulinum type A protease to growth factors and neuropeptides allows selective targeting of neuroendocrine cells.</a> Journal of Neurochemistry. 126, 223-233 (2013).</li></ol>

Dr. John A. O'Brien holds patents for the improved gene gun barrel. US patent number 10/380,452 and European patent Number 01974517.3.

The protocol describes approaches to deliver dyes and genetic materials into adult organotypic slices by using an enhanced gene gun. Essentially, the types of dyes and the variety of possible genes make this method multifaceted and amenable to answer a wide range of biological questions. The regioselective delivery method used, in this case to specific brain region, opens new avenues for experimentation as heterologous gene modulation of localized areas within a biologically relevant architecture hasn&#39;t been easily feasible before. We have previously determined that this method can be used on adult organotypic slices, where mature synapses are fully formed, as it showed improved cell survival and heterologous gene expression for many weeks13. With the advent of new and improved culture conditions for mammalian brains as well as other tissues, the use of regioselective genetic modulations will become increasingly useful for a wide variety of biochemical analyses.
As we shall highlight here and previously mentioned by others24, many different factors could readily affect the accuracy and reliability of biolistic delivery. Firstly, the preparation of the gold particle linked to dyes or DNA must be adequately balanced to prevent aggregation and to facilitate the cartridge cargo expulsion. Calcium and spermidine in the DNA-gold micro particles solution can aid the binding of DNA molecules to the gold nanoparticles. The ability for the nanoparticles to penetrate the cells under reduced pressure was previously determined17. Secondly, the gene gun must be mounted as steady as possible with the correct angle and distance. A reliable pressure gauge is also necessary for reproducibility, to maintain tissue integrity, and to properly accelerate the gold particles to their intended targets. Indeed, the distance, orientation, and stability of the equipment are also a crucial factors for the targeting and precision of regioselective delivery. As these brain structures are very small, minor variations in the firing angle could easily render the entire procedure less reliable. And finally, the preparation and maintenance of viable organotypic slices have been fraught with difficulties for many decades yet abundant and reliable protocols for different animals, brain regions, ages and cocultures are currently available2,4,25,26. These procedures should be user-optimized before submitting the tissues to biolistic procedures. This will more readily permit the user to unbiasedly ascertain the impact of the biolistic particles and the effect of the heterologous gene on their own cultures. To this end, the use of fluorescent genes such as EYFP and EGFP can both permit a novel user to test the targeting precision and also follow the cellular viability through heterologous gene expression over a sustained period of time13. Many of the critical steps for reliable and reproducible use of this protocol are highlighted in the following three paragraphs.
For efficient transfection, the DNA concentration must be appropriate. Concentrations that are too low would hinder the transfection yield while concentrations that are too high can cause agglomeration of the nanoparticles. Aggregates of gold can dramatically reduce transfection efficiency, cause uneven distributions, and can lead to increased cytotoxicity and oxidative stress. Problems with coating efficiency are likely due to the expiration of the spermidine solution (should be replaced every 2 to 3 months). For an adequate biolistic spread, the labeling must be even. A non-homogeneous labeling of the gold nanoparticle/DNA suspension may be due to the PVP solution. It should also be replaced every 2 to 3 months. Coating of the gold nanoparticles can be seen on agarose gel electrophoresis as a higher MW band27.
Each biological system under investigation as well as each different instrument used might require small changes in gas pressure to reach optimized levels of transfection and biolistic spread. The critical parameter is the pressure of the helium pulse required to strip the micro- or nano-carriers from the plastic cartridge and propel them into the tissue. A high gas pressure will affect cell survival, because it will create shock waves across the target and disrupt or detach the tissue from the matrix17. Aiming the gene gun barrel and having the apparatus exactly perpendicular to the tissue is important for accurate biolistic delivery. The selected area should be placed in the direct center of the barrel at precisely 10 mm from the outer aperture. This results in a 3 mm diameter of gold particle delivery around the epicenter. The gene gun barrel should be cleaned with 70% ethanol after each use. To protect the tissue from large particle aggregates, the barrel also contains a nylon mesh that should be replaced regularly to maintain optimum performance. In order to replace the mesh unscrew the cap then insert a new nylon mesh between the cap and the O-ring.
Fluorescently labeled cells should be visible at least 1 - 2 days after transfection around the epicenter. Absence or misalignment of labeling can result from inadequate preparation and instability of the gene gun mounting. The observations of cells using confocal microscopy depends on a number of factors: the wavelength of the excitation/emission light, pinhole size, NA of the objective lens, refractive index of components in the light path, depth of the tissue, and the alignment of the instrument. These factors should be optimized for each system under investigation.
Recent advances in biolistics were exploited for the success of this method. The gene gun barrel used in this study was modified in order to reduce the pressure necessary as well as funnel the biolistic scatter pattern into a more constrained and specific area17,19. Other barrel modifications have tried to restrict the biolistic spread and reduce the outflow pressure28 yet this custom designed gene gun barrel designed by Dr. O&#39;Brien is a single component fitted to the standard Helios Gene gun. Subsequently, sub-micrometer gold particles were used to reduce the invasiveness of micrometer particles that we had previously determined caused cellular damage and ultimately, loss of sustained heterologous gene expression. These changes to the biolistic procedure improved the overall feasibility and reproducibility of the method13. Other improvements on slice preparation such as in-depth troubleshooting of the slicing procedure, using a DMEM-Agarose matrix to preserve the brain, and numerous other useful advances in organotypic brain slice preparation previously reported13 enabled us to explore the use of adult slices that have developed mature synaptic networks.
In our study we mainly used dyes and fluorescent heterologous genes to visualize cell morphology as a readout on transfection efficiency and an ability to image the morphological characteristics of neurons in mouse brain tissues. The signal to noise ratio of the stochastic delivery within a defined radius enabled us to completely isolate a single cell&#39;s dentritic harbor within its native context. As numerous efforts are used to investigate these morphological characteristics, mapping &#39;connectomics&#39; or to label single cells for various analyses, this method could easily be combined with existing protocols such as electrophysiology and neurite measurements29,30. Evidently, combinations of fluorescent heterologous genes could enable a much more robust delineation of single cells as the stochastic distribution of fluorescent proteins, and their combination would determine the color of the individual cells31,32. The use of cell specific promoters such as synapsin and glial fibrillary acid protein upstream of the heterologous gene exon can also restrict the expression to subpopulations33. Indeed, the unending variety of genetic material could also be delivered in a regioselective manner by gold nanoparticles using this same procedure. The total transfected regions could thus be analyzed with traditional methods, such as dissection and submitting that region to western immunoblotting to analyze the localized effect of the heterologous gene.
Improvements in organotypic slice procedures will also permit wider use of brain tissues for long-term experimentation as well as permit the use of other tissues and cocultures and would facilitate transfection procedures. Lipid and polymer transfection have been previously reported to affect membrane homeostasis, internalization, protein permeability34 and frequently show very low efficiency to transfect terminally differentiated neurons. Cell specific cargo targeting is also amenable only to cells that express the cell surface receptors needed for internalization, and although this method can be highly selective, it can lack targeted regioselectivity35. Viral vectors otherwise are often difficult and costly to produce, require stringent regulations and have on occasion demonstrated safety concerns. Biolistic delivery thus has an unparalleled ability to regioselectively transfect neurons and other cell types within viable tissues. As gold is non toxic, further advances in the use of biolistics could pave the way for biomedical uses such as transdermal pharmaceutical delivery, noninvasive in vivo gene delivery, as well as localized nonviral gene therapies.

Originalmente la técnica biolistic, una frase a su vez-de-para &quot;balística biológicos&quot;, se estableció para la transferencia génica mediada por partículas en las células vegetales 1. Este método físico de la transformación celular micro o nanopartículas acelera a alta velocidad para superar las barreras físicas de las membranas impermeables a las células con el fin de entregar cargamentos tales como ADN o colorantes. Debido a que no depende de ligando de receptores específicos y / o las propiedades bioquímicas en las membranas de la superficie celular, transferencia de genes mediada por partículas se puede aplicar fácilmente a una variedad de sistemas biológicos, tales como cortes de cerebro organotípicos. Usando rebanadas organotípicos tienen ventajas sobre otros en las plataformas in vitro ya que mantienen muchas de las propiedades anatómicas y bioquímicas que son pertinentes a la biología in vivo 2-4. Las rebanadas mayoría conservan las características arquitectónicas locales desde donde se han originado y preserve la actividad neuroquímica y la conectividad de las sinapsis. El uso de cortes de cerebro para la investigación básica, y en los esfuerzos farmacéuticos, ha aumentado de forma concomitante con el número de posibles manipulaciones biotecnológicas para medir y monitorear los comportamientos neurobiológicas del cerebro en un in vivo como contexto 3,5-7. Las principales ventajas para el uso de sistemas de ensayo a base de cortes organotípicos es que proporciona control experimental fácil y permite manipulaciones precisas de ambientes extracelulares. Fructíferamente, sistemas de cultivo de cortes organotípicos se han establecido a partir de una variedad de regiones cerebrales, tales como, pero no limitado a, la corteza, la médula espinal y cerebelo 8-10. Además, un número de cocultivos se ha demostrado, que permiten la evaluación de la comunicación intercelular entre las regiones cerebrales distales así como entre las neuronas y las células patológicas 11,12. Muchos protocolos ya han sido establecidos para successfully rebanadas cultura organotípicos y puede mantener la viabilidad a largo plazo y muchos estudios recientes ahora utilizan los métodos de interfaz de membrana y varias modificaciones a la misma 13. Este principio mantiene las rebanadas organotípicos en la interfaz entre el medio y la atmósfera humidificada de la incubadora colocando las rodajas en un filtro de membrana porosa. El medio puede así proporcionar suficientes nutrientes para alimentar a las rebanadas organotípicos mediante movimiento capilar. Típicamente rebanadas se han preparado a partir de animales postnatales tempranos (3 - 9 días de edad; P3 - 9). Sin embargo, los tejidos del cerebro de estas rebanadas mostrar un alto nivel de plasticidad celular y tienen una resistencia inherente a los esfuerzos mecánicos, que es útil para obtener cultivos viables, sin embargo, las sinapsis maduras y circuitos neuroanatómico no se han desarrollado completamente in vivo hasta 2 a 3 semanas de edad 14. Por ejemplo, las observaciones anteriores habían demostrado que los cortes de hipocampo obtenidas de P0 - 1 neonatos, aunque muy viable siguiente preparación, perdiendo algunas características morfológicas. Esencialmente, ellos demostraron ser inadecuados para cultivos a largo plazo sugieren células inmaduras fueron más propensos a des-diferenciar en comparación con cultivos organotípicos de animales mayores 15,16. Por esta razón, nuestro método ha sido optimizado para los cortes de cerebro organotípicos adultos en la que la maduración y el desarrollo arquitectónico han llegado a sus etapas terminales 13,17-21. Sin embargo, este método también es adecuado para el recién nacido y rebanadas organotípicos juveniles. Una vez que se han producido las rebanadas organotípicos viables toda la placa que contiene las rebanadas puede ser llevado al monte biolistic y sometido a regioselectiva entrega y transfección. El montaje correcto de la pistola de genes (como se describe en la Figura 1), orientado 90 ° a una distancia de 10 mm directamente sobre las rebanadas (desde la abertura en el tejido), permite el rápido suministro biolístico de los 40 nm irld partículas recubierto cargas. Estas cargas tales como tintes y vectores de ADN fluorescentes, así como cualquier gen de interés, se entregan fácilmente en las rodajas viables con facilidad. Así pues, este método describe el protocolo necesario para ofrecer, de una manera regioselectiva, carga recubierto las nanopartículas de oro en cortes de cerebro organotípicos viables. El cañón de la pistola de genes y montaje óptimo se puede ver en la Figura 1. Para más información sobre las mejores balística barril y nanopartículas, por favor ver O&#39;Brien, et al., 17. Refinamientos posteriores también están indicados para el usuario para optimizar las condiciones tales como la cantidad apropiada de oro entregado por área objetivo, definida como microportadores Cantidad de carga (MLQ), y para determinar la cantidad de ADN cargado por mg de oro, definido como de relación de ADN Cargando (DLR). Antes de la precipitación de ADN en partículas de oro y cargarlos en el tubo Tefzel que comprende cartuchos de la pistola de genes real &#39;9 ;, es necesario para calcular la cantidad de ADN y el oro requerida para cada transfección que puede diferir ligeramente entre los tejidos y las condiciones (relación debe mantenerse entre 1 mg de oro y 1 g de ADN a 1: 5). Es vital para preparar la proporción adecuada de MLQ a DLR en contrario partículas de oro recubiertas de ADN podrían adherirse entre sí formando más grande que la aglomeración se esperaba, lo que podría reducir la homogeneidad global de la propagación biolistic así como daños incremento del tejido y la citotoxicidad. Este método muestra las recientes mejoras en el suministro biolístico, que han sido probados y decididos a ser susceptibles de cortes de cerebro organotípicos neonatales, juveniles y adultos. Además, mediante la explotación de propagación reducida biolístico el gen arma de barril, el usuario es ahora capaz de transfectar de forma selectiva una región puntual en el cerebro. Tras el tiempo de incubación apropiado, la expresión de las proteínas fluorescentes, que alcanza sus tasas máximas entre 24 y 48hr, se puede visualizar mediante epifluorescencia y microscopía confocal. Estos fluoróforos permiten el análisis morfológico y la localización de las células individuales dentro de las estructuras biológicamente relevantes. Sin embargo, la modulación regioselectiva de las rebanadas organotípicos por otros genes heterólogos también puede ser controlado por cualquier otra prueba susceptibles de estas preparaciones. Posibles estrategias de trabajo para la entrega de genes localizados se presentan en la Figura 2.

<table border="0" cellpadding="0" cellspacing="0" width="656">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Name of Material/ Equipment</td>
			<td style="width:141px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:173px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Helios gene gun system</td>
			<td style="width:141px;">Bio Rad</td>
			<td>165-2431</td>
			<td>Gene gun and tube prep station</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">HEPES&#160;</td>
			<td style="width:141px;">Sigma</td>
			<td>83264-100ML-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">NaCl</td>
			<td style="width:141px;">Sigma</td>
			<td>S7653-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">KCl</td>
			<td style="width:141px;">Sigma</td>
			<td>P9333-500G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:223px;">Na2HPO4</td>
			<td style="width:141px;">Sigma</td>
			<td>S7907-100G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">KH2PO4</td>
			<td style="width:141px;">Sigma</td>
			<td>P9791-100G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">0.2 &#181;m sterile filter unit</td>
			<td style="width:141px;">Millipore</td>
			<td>SCGPU05RE</td>
			<td>to filter media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">DMEM</td>
			<td style="width:141px;">Gibco</td>
			<td>21885-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Fetal calf serum</td>
			<td style="width:141px;">Gibco</td>
			<td>10270-098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">D-Glucose</td>
			<td style="width:141px;">Sigma</td>
			<td>G8270-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">N2</td>
			<td style="width:141px;">Life Technologies</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Penicillin/Streptomycin</td>
			<td style="width:141px;">Sigma</td>
			<td>p4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyvinylpyrrolidone</td>
			<td style="width:141px;">Sigma</td>
			<td>856568-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Ethanol&#160;</td>
			<td style="width:141px;">Sigma</td>
			<td>277649-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Spermidine</td>
			<td style="width:141px;">Sigma</td>
			<td>S2626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Agarose</td>
			<td style="width:141px;">Bio Rad</td>
			<td>161-3100EDU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Vibroslicer</td>
			<td style="width:141px;">Laica</td>
			<td style="width:119px;">VT1200</td>
			<td>We used a custom design</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Gene gun mount</td>
			<td style="width:141px;"></td>
			<td style="width:119px;"></td>
			<td>Built in house at U of T</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Humidified cell culture incubator</td>
			<td style="width:141px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Gold nanoparticles</td>
			<td style="width:141px;">cytodiagnostics</td>
			<td style="width:119px;">G-40-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pEYFP-N1</td>
			<td style="width:141px;">Clontech</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tubing cutter</td>
			<td style="width:141px;">Bio Rad</td>
			<td>165-2422</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Tefzel tubing</td>
			<td style="width:141px;">Bio Rad</td>
			<td>165-2441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Barrel</td>
			<td style="width:141px;"></td>
			<td style="width:119px;"></td>
			<td>Custom made by the LMB</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cell culture insert</td>
			<td style="width:141px;">Millipore</td>
			<td>PICM0RG50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Paraformaldehyde</td>
			<td style="width:141px;">Sigma</td>
			<td>76240 Fluka</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Dissecting microscope</td>
			<td style="width:141px;"></td>
			<td style="width:119px;"></td>
			<td>for convenience</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Confocal microscope</td>
			<td style="width:141px;"></td>
			<td style="width:119px;"></td>
			<td>user defined for usage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Glass slide</td>
			<td style="width:141px;">VWR</td>
			<td>2588-CA48323-185</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Microcover glass</td>
			<td style="width:141px;">VWR</td>
			<td colspan="2">2448-CA48366-067-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Vectashield mounting media</td>
			<td style="width:141px;">Vector laboratories</td>
			<td>H-1400&#160;&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

regioselective biolistic targeting in organotypic brain slices using a modified gene gun

La transfección de ADN ha sido de gran valor para las ciencias biológicas y con los últimos avances en preparaciones de cortes de cerebro organotípicos, el efecto de varios genes heterólogos por lo tanto podría ser investigado fácilmente manteniendo muchos aspectos de la biología in vivo. Ha habido un interés creciente para transfectar neuronas terminales diferenciadas para las que los métodos de transfección convencionales han estado plagado de dificultades, tales como los bajos rendimientos y pérdidas significativas en la viabilidad. Transfección biolística puede eludir muchas de estas dificultades sin embargo, sólo recientemente se ha modificado esta técnica de modo que es susceptible para su uso en tejidos de mamíferos. Nuevas modificaciones a la cámara de acelerador han mejorado la precisión de disparo de la pistola de genes y el aumento de sus profundidades de penetración mientras que también permite el uso de presión de gas inferior (50 psi) sin pérdida de eficiencia de la transfección así como permitir un margen regioselectiva enfocada del paARTÍCULOS para dentro de 3 mm. Además, esta técnica es sencillo y rápido de realizar que microinyecciones tediosas. Tanto expresión transitoria y estable son posibles con nanopartículas bombardeo donde la expresión episomal puede ser detectado dentro de 24 horas y la supervivencia de las células ha demostrado ser mejor que, o al menos igual a, métodos convencionales. Esta técnica tiene sin embargo una ventaja crucial: permite la transfección a ser localizados dentro de un solo radio restringido permitiendo así al usuario aislar anatómicamente efectos del gen heterólogo. Aquí presentamos un protocolo detallado para preparar adultos viable rebanadas organotípicos y someterlos a regioselectiva transfección utilizando una pistola de genes mejorada.

Organotypic slice viability can be monitored with lactate dehydrogenase activity, propidium iodide labeling, and dUTP staining as was previously reported13,22. Evidently, the viability and integrity of the slices are important for the long-term sustained expression of the delivered genetic cargo. Following biolistic delivery into the organotypic slices, the expression of the fluorescent heterologous gene was monitored by confocal microscopy. The tightness of the biolistic spread using the modified barrel can be seen in Figure 3A. Figure 3B shows a representative image of the transfected hippocampus region using DNA coated gold nanoparticles. Figure 4 shows representative pictures of transfected adult mouse organotypic slices. As can be seen in Figure 4A, a Purkinje neuron with a remarkable dendritic harbor found at the interface of the Purkinje layer and the molecular layer of the cerebellum shows pronounced labeling following transient biolistic transfection with pEGFP-N1. Figure 4B shows a higher magnification of these dendrites where spines can be observed. Figure 4C shows a pyramidal cell found in the CA1 region of the hippocampus following biolistic delivery of pDSredFP coated gold nanoparticles.
Dyes instead of fluorescent encoded DNA cargos can also be used in a similar manner to visualize morphological characteristics in organotypic slices. DiO, which labels the plasma membranes, can more rapidly delineate individual cell morphologies or can be used in conjunction with a DNA coated biolistic delivered to the same region to confirm the regioselection. As can be seen in Figure 5A a neuron stained with DiO in the hippocampus also shows long dendrites with numerous spines. An arrow indicates the axon which is identified by the presence of synaptic boutons in comparison to the dentritic spines on the other neurites. Figure 5B shows another example of the CA1, which highlights numerous hippocampal projections with a DAPI counter stain to label the nucleus. Figure 5C shows a higher magnification of these types of parallel dendrites with numerous spines.
Evidently, these images illustrate morphological characteristics following cellular labeling. Nothing precludes the same methodology from being applied to deliver any number of exogenous genes coated onto the gold nanoparticles. To permit a visual confirmation of successful transfection, a single plasmid can be constructed to co-express both the gene of interest and a fluorescent reporter protein on the same vector.
<img alt="Figure 1" src="/files/ftp_upload/52148/52148fig1highres.jpg" /> 
Figure 1.&#160;Gene gun barrel and mounting.&#160;(A) The improved barrel developed by Dr. O&#39;Brien at the MRC-LMB that has a restricted biolistic spread that minimizes tissue damage by lowering the required pressure for particle acceleration. (B) The side view of an optimized gene gun mounting developed by Dr. Henderson and Dr. Andras Nagy at the University of Toronto. The mounting holds the gene gun at precisely 90&#730; perpendicular to the organotypic slices while permitting the user to accurately control the distance of biolistic delivery. For optimal biolistic conditions the barrel aperture should be placed directly over the region of interest at a 10 mm distance from the organotypic slices. (C) Back view of the gene gun mounting. The region of interest to be transfected needs to be placed directly underneath the barrel aperture. Targeting can be confirmed with dye (diolistic) or fluorescent protein tranfection (biolistic). <a href="https://www.jove.com/files/ftp_upload/52148/52148fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52148/52148fig2highres.jpg" /> 
Figure 2.&#160;Organotypic slice preparation and regioselective targeting.&#160;(A) A DMEM embedded adult mouse brain ready for slicing. (B) The mould was glued onto the vibroslicer mounting with the blade ready to cut a slice. (C) Regioselective heterologous gene expression strategies for biolistic delivery into coronal organotypic brain slices. Images prepared from pictures obtained from the Allen Brain Atlas anatomical reference atlas23. Left panel shows two different brain regions targeted as an example. To restrict the gene expression (1) to the cortical area of the left hemisphere. To restrict the heterologous gene expression (2) to the hypothalamus region of the brain. Middle panel shows that the expression of two heterologous genes (3 and 4) can be isolated and compares the hippocampus of two hemispheres in the same organotypic slice, essentially eliminating any bias from using different sections and/or granting the possibility to address distal crosstalk from the effects of those genes. Right panel shows the possible overlap of transgenic delivery to express the first gene (5) in a precise cortical region while expressing another gene (6) in a disparate yet overlapping cortical area. This permits to analyze the effect of genetic modulation of each heterologous gene alone and also in combination within the same slice. <a href="https://www.jove.com/files/ftp_upload/52148/52148fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52148/52148fig3highres.jpg" /> 
Figure 3.&#160;Biolistic scatter pattern seen on filter paper and low magnification image.&#160;(A) The improved barrel was fired onto filter paper to determine the spread of gold particles. Left side shows the distribution of the improved barrel while the right panel shows the normal barrel. Black bar: 1 cm. Image taken from O&#39;Brien, et al.17. (B) Low magnification image showing the stochastic transfection patter within the 3 mm biolistic spread within the hippocampal region of 6 week old mice. White bar: 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52148/52148fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52148/52148fig4highres.jpg" /> 
Figure 4.&#160;Biolistic delivery of fluorescent protein encoded DNA into organotypic slices of 6 week old mice.&#160;(A) Inverted confocal images of a fixed organotypic slice of the cerebellum of a pEGFP-N1 coated gold particles transfected Purkinje cell. Image was taken at 40X magnification. Black bar: 30 &#181;m. (B) Shows a 60x magnification of another Purkinje cell&#39;s dentritic harbor to highlight the spines. Black bar: 5 &#181;m. (C) Shows a confocal image of fixed slices taken from the hippocampal region at 60x magnification of four DSredFP transfected pyramidal cells. Black bar: 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52148/52148fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52148/52148fig5highres.jpg" /> 
Figure 5.&#160;Diolistic delivery of membrane labeling fluorescent dyes into organotypic slices.&#160;(A) shows the live imaging of a hippocampal neuron labeled with DiO under 20X magnification. The dentritic spines can clearly be observed as well as the axon as identified by the absence of dentritic spines and the presence of synaptic boutons indicated by a white arrow. White bar: 30 &#181;m. (B) Pronounced staining of the hippocampal region labeled with DiO and counterstained with DAPI. White bar: 30 &#181;m. (C) Higher magnification (60x) of the dentritic spines found in the CA1 region of the hippocampus. White bar: 5 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52148/52148fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun at JoVE.com

Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

Recent improvements in organotypic brain slice preparations have permitted their exploitation for biotechnological applications. Organotypic slices maintain local structural characteristics of in vivo biology, including functional synaptic connections. Here we present a regioselective biolistic delivery method to label and genetically manipulate these slices.

Regioselectiva biolística Focalización en Organotípicos cerebro rebanadas Usando una pistola de genes Modificado

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various ...

regioselective-biolistic-targeting-organotypic-brain-slices-using

Research

JoVE Journal

Neuroscience

16.2K vistas.  University of Toronto. Recent improvements in organotypic brain slice preparations have permitted their exploitation for biotechnological applications. Organotypic slices maintain local structural characteristics of in vivo biology, including functional synaptic connections. Here we present a regioselective biolistic delivery method to label and genetically manipulate these slices. 

Video: Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 &mu;m (length, rostrocaudal axis) x 424 &mu;m (width) x 390 &mu;m (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

Preparación Cortar, cultivo de tejidos organotípicos y grabación de la actividad luciferasa de reloj de genes en el núcleo supraquiasmático

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

Investigación

Neurociencias

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when neural circuits are matured and refined. Misregulation at this stage may lead to neurological and psychiatric disorders such as autism 
and schizophrenia1,2. Many genes have been studied in the prenatal brain and found crucial to many developmental processes3-5. However, their 
function in the postnatal brain is largely unknown, partly because their deletion in mice often leads to lethality during neonatal development, and partly because their requirement in early development hampers the postnatal analysis. To overcome these obstacles, floxed alleles of these genes are currently being generated in mice 6. When combined with transgenic alleles that express Cre recombinase in specific cell types, conditional deletion can be achieved to study gene function in the postnatal brain. However, this method requires additional alleles and extra time (3-6 months) to generate the mice with appropriate genotypes, thereby limiting the expansion of the genetic analysis to a large scale in the mouse brain.
Here we demonstrate a complementary approach that uses virally-expressed Cre to study these floxed alleles rapidly and 
systematically in postnatal brain development. By injecting recombinant adeno-associated viruses (rAAVs)7,8 encoding Cre into the neonatal brain, 
we are able to delete the gene of interest in different regions of the brain. By controlling the viral titer and coexpressing a fluorescent 
protein marker, we can simultaneously achieve mosaic gene inactivation and sparse neuronal labeling. This method bypasses the requirement of 
many genes in early development, and allows us to study their cell autonomous function in many critical processes in postnatal brain development,
 including axonal and dendritic growth, branching, and tiling, as well as synapse formation and refinement. This method has been used successfully 
in our own lab (unpublished results) and others8,9, and can be extended to other viruses, such as lentivirus 9, as well as to the expression of 
shRNA or dominant active proteins 10. Furthermore, by combining this technique with electrophysiology as well as recently-developed optical 
imaging tools 11, this method provides a new strategy to study how genetic pathways influence neural circuit development and function in mice 
and rats.

An in vivo method to test gene function in postnatal brain is described. Recombinant AAVs expressing Cre and/or a fluorescent protein are injected into neonatal mouse brain. Mosaic gene inactivation and sparse neuronal labeling are achieved, allowing rapid analysis of gene function in processes critical to neural circuit development.

Mosaico de Análisis de la función de genes en el desarrollo postnatal del cerebro del ratón mediante el uso de virus basada en la recombinación Cre

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

Electrodos múltiples grabaciones matriz de avalanchas neuronal en cultivos organotípicos

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

Entrega génica eficaz en los territorios del SNC múltiples utilizando In Utero La electroporación

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and amphetamine), the role it plays in psychiatric illnesses (e.g. schizophrenia and Attention Deficit Hyperactivity Disorder), and its involvement in degenerative disorders like Parkinson's and Huntington's disease. Under normal physiological conditions, dopamine is known to regulate locomotor activity, cognition, learning, emotional affect, and neuroendocrine hormone secretion. One of the largest densities of dopamine neurons is within the striatum, which can be divided in two distinct neuroanatomical regions known as the nucleus accumbens and the caudate-putamen. The objective is to illustrate a general protocol for slice fast-scan cyclic voltammetry (FSCV) within the mouse striatum. FSCV is a well-defined electrochemical technique providing the opportunity to measure dopamine release and uptake in real time in discrete brain regions. Carbon fiber microelectrodes (diameter of ~ 7 &mu;m) are used in FSCV to detect dopamine oxidation. The analytical advantage of using FSCV to detect dopamine is its enhanced temporal resolution of 100 milliseconds and spatial resolution of less than ten microns, providing complementary information to in vivo microdialysis.

Using fast-scan cyclic voltammetry to measure electrically evoked presynaptic dopamine dynamics in striatal brain slices.

Dinámica presináptica de dopamina estriatal en rodajas de cerebro de voltametría cíclica rápida de exploración

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Pareadas Grabaciones de células enteras en organotípicos de hipocampo rebanadas

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Grabación de la plasticidad sináptica en rebanadas Hippocampal agudas mantenidas en un pequeño volumen reciclado - perfusión- y sistema de cámara de tipo inmersión

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced tools currently used to study neurons, have only partly been taken on by oligodendrocyte researchers. Cell type-specific staining by viral transduction is a useful approach to study live organelle dynamics. This paper describes a protocol for visualizing oligodendrocyte mitochondria in organotypic brain slices by transduction with adeno-associated virus (AAV) carrying genes for mitochondrial targeted fluorescent proteins under the transcriptional control of the myelin basic protein promoter. It includes the protocol for making organotypic coronal mouse brain slices. A procedure for time-lapse imaging of mitochondria then follows. These methods can be transferred to other organelles and may be particularly useful for studying organelles in the myelin sheath. Finally, we describe a readily available technique for visualization of unstained myelin in living slices by Confocal Reflectance microscopy (CoRe). CoRe requires no extra equipment and can be useful to identify the myelin sheath during live imaging.

Myelinating oligodendrocytes promote rapid action potential propagation and neuronal survival. Described here is a protocol for oligodendrocyte-specific expression of fluorescent proteins in organotypic brain slices with subsequent time-lapse imaging. Further, a simple procedure for visualizing unstained myelin is presented.

Visualización y en proyección de imagen del Oligodendrocyte organelos en rebanadas de cerebro Organotypic mediante Virus Adeno-asociado y Microscopía Confocal

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Un enfoque alternativo para estudiar acontecimientos primarios en neurodegeneración con rodajas de cerebro de rata Ex Vivo

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Evaluación de la inducción a largo plazo de la depresión en rodajas de cerebelosores adultos

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...