We thank Souvik Maiti for help in designing the antagomirs. We also acknowledge Rangeetha J. Naik, Rakesh Dey, and Bijay Pattnaik for their help with experimental methods. This work was funded by the Council of Scientific and Industrial Research (BSC0123). HS, MV and RR acknowledge fellowship from the Council of Scientific and Industrial Research, India. MAS acknowledge fellowship from the University Grants Commission, India.

Nota: Todo el procedimiento incluyendo sujetos animales han sido aprobados por el Comité Institucional de Ética Animales (AICE) en el Instituto de Genómica y Biología Integrativa, Nueva Delhi (IGIB / AEC / 10/2013). Este protocolo se ajusta específicamente para la ejecución selectiva de antagomir-29 en el cerebro y desmontables de miR-29. 1. antagomir Estrategia Diseño <ol><li> Recuperar la secuencia de los genes miARN maduro de miRBase 11 <a href="http://www.mirbase.org/">(http://www.mirbase.org/).</a> </li><li> Recuperar las secuencias de miembros de la familia miARN relacionados utilizando el enlace de &quot;familia de genes&quot; en el registro miRBase <a href="http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000009">(Http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000009).</a> </li><li> Invertir complementar la secuencia: <a href="http://www.bioinformatics.org/sms/rev_comp.html">http://www.bioinformatics.org/sms/rev_comp.html</a> proporciona una herramienta conveniente pararevertir secuencias del complemento. </li><li> Marque las posiciones que ser modificados por LNA utilizando las siguientes pautas: <ol><li> Elige residuos de timina colocados 3-4 bases aparte para la modificación LNA, ya pirimidinas LNA son más estables que las purinas 12. </li><li> Elija residuos de citosina separadas por 3-4 residuos si paso anterior produce menos de cinco modificaciones. </li><li> Dar prioridad a las cinco modificaciones en el extremo 5 &#39;del antagomir, ya que esto evita la secuencia de semillas. </li></ol></li></ol> Preparación Complejo 2. antagomir-neuropéptido Nota: Este es el paso más crítico en el protocolo y que necesita la estandarización. Para estandarizar el protocolo de cualquier oligonucleótido marcado con fluorescencia (FLO) se puede utilizar en lugar de la antagomir 9. Los péptidos deben ser de alta pureza (calidad HPLC, pureza 98%) y .Sequences cargo de péptido y antagomir se dan en la Tabla 1. <ol><li> Decidir el número de mesesles de antagomir a inyectar en base a la carga molar en antagomir, su toxicidad y el peso del ratón. Calcular el número de moles de péptido a inyectar basa en relación de carga molar de antagomir: péptido. NOTA: Estandarizar relación de carga molar de antagomir: péptido de la toxicidad y la entrega efectiva en el cerebro del ratón. Utilice diferentes relaciones de carga molar de oligonucleótido marcado con fluorescencia: péptido de la normalización. Encontramos que antagomir-29 y el control eran ambos tóxicos en 4microgram / gramo de peso corporal del ratón. Antagomir-29 y el control se utilizaron a 2microgram / gramo de peso corporal, pero es probable que varíe para cada microRNA esta concentración efectiva. </li><li> Diluir el péptido y antagomir en tubos de microcentrífuga separados de soluciones madre estéril con 10% de D-glucosa a la concentración final deseada como calculada en el paso 1. </li><li> Mantenga el tubo de microcentrífuga con la solución de péptido en un vórtice a velocidad moderada vórtex. </li><li> Añadir 1/3 de la unantagomir solución al tubo de solución de péptido, lentamente, gota a gota, mientras se mezcla a fondo en un mezclador de vórtice. </li><li> Continuar mezclando de soluciones en el vórtice para el próximo 1 min y deje reposar la mezcla durante 1 minuto. </li><li> Repita el paso 4 y 5 de dos veces más para mezclar el restante antagomir solución con la solución de péptido en el mismo tubo de microcentrífuga. La adición lenta es crítica para la formación de complejos de mono-dispersa que son eficaces en la transfección. Además rápido puede conducir a la agregación de los complejos y su precipitación. </li><li> Incubar el complejo durante 30 min a TA sin agitación con vórtex. Durante este tiempo de incubación, aclimatarse a los ratones el laboratorio donde se llevarán a cabo inyecciones. </li></ol> 3. cola vena Inyección de Complejo antagomir-neuropéptido <ol><li> Para frenar el animal, coloque el ratón en un retenedor o decapicone del tamaño adecuado. </li><li> Limpie la superficie de la cola utilizando pr hisopo de algodóne-empapado en agua caliente (aproximadamente 40 ° C). Esto promoverá la vasodilatación y aumentar la visibilidad de la vena. </li><li> Acércate a la cola con un volumen pequeño (0,5-1,0 cc) jeringa de insulina en un ángulo de 15 a 20 °. Tenga cuidado de no introducir el aire en la jeringa. Comience en la parte distal de la cola. Inyectar el complejo. Retire la aguja y aplique una gasa con antiséptico directamente en el sitio de inyección (aproximadamente 5 a 10 segundos) para detener cualquier sangrado. </li><li> Vuelva a colocar el ratón en una jaula de ventilación individual en el grupo de 3-5, con la mazorca de maíz y papel de seda como enriquecimiento. No guarde los ratones no inyectados e inyectados en una misma jaula. </li></ol> 4. Los ensayos de comportamiento Nota: Mantenga el intervalo de tiempo de 3-4 horas entre ensayos de inyección y de comportamiento para permitir la recuperación después de la inyección. Traiga el ratón para una habitación tranquila. No moleste a los animales durante este período de aclimatación. <ol><li> Aclimatar el ratón a lanuevo ambiente durante 30 minutos y luego comenzar los siguientes ensayos de comportamiento. Mantenga una distancia de 10 minutos entre cada ensayo conductual. </li><li> Juntando las extremidades posteriores: NOTA: Esta prueba indica disfunción motora y deterioro neurológico. <ol><li> Levante el ratón con cuidado mediante la celebración de cerca de la base de la cola en el fondo claro. </li><li> Compruebe la posición de las extremidades posteriores de 10-15 seg. Ensancha ratón tipo salvaje extremidades posteriores lejos de abdomen, mientras que un ratón atáxica tiende a retraer uno o ambos miembros posteriores hacia el abdomen más de 50% del tiempo de 13 suspendido. </li><li> Devuelva el ratón a la jaula </li></ol></li><li> Prueba Ledge: <ol><li> Levante el ratón con cuidado mediante la celebración de la cola y mantenerlo en la cornisa de una jaula. </li><li> Observe el ratón específicamente en la esquina de la jaula. Wild tipo de ratón puede pasar las esquinas fácilmente sin miedo y perder el equilibrio. Ratón atáxica pierde su equilibrio, congelación o sacude mientras camina por la cornisa de la jaula y en las esquinas. </li></ol></li><li&gt; ensayo huella del ratón: NOTA: Para este ensayo mantener la lámina absorbente listo, en el suelo de una pista estrecha (~ 70 cm de largo, ~ 5 cm de ancho, con ~ 5 cm de altos muros.) <ol><li> Mantenga el ratón suavemente con una mano y aplicar la tinta en las extremidades traseras por un cepillo. </li><li> Coloque la lámina absorbente en el suelo de una pista estrecha. </li><li> Permita ratón para caminar o correr sobre una lámina absorbente en una línea recta en una pista de aterrizaje de un extremo a otro. </li><li> Repetir el proceso dos veces más con cada ratón utilizando lámina absorbente fresco. </li><li> Medir la distancia entre dos pasos consecutivos en el movimiento hacia adelante. No incluya primeros y últimos huellas donde el animal se acaba de iniciar y terminar su recorrido, respectivamente. </li></ol></li></ol>

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The authors have nothing to disclose.

Here we demonstrate a widely accessible methodology to study the effects of miRNA modulation. Currently, most attempts at in vivo characterization of miRNA functions involve the creation of knockout mice or a transgenic that expresses a miRNA sponge. Most miRNAs, even the cell type specific ones are expressed in more than one organ. For instance, miRNAs initially thought to be specific to the hematopoietic system are also expressed in the brain, due to the presence of microglia. Thus even a cell type specifi...

Los microARNs se han convertido en nuevas dianas terapéuticas debido a su papel universal en la regulación de la expresión génica y pruebas directas de la participación en la enfermedad. MiRNAs se están explorando activamente por su potencial como fármaco dirigido a 1,2. Además, alteraciones en la expresión de los genes miARN están asociados con varias enfermedades 3 y simulación de estos cambios por la perturbación artificial de expresión de los genes miARN se puede utilizar para estudiar las vías celulares implicadas en la manifestación de la enfermedad. Entrega específica de tejido de miARN fármacos dirigidos es actualmente un gran desafío para el desarrollo de fármacos basados ​​miARN. Antagomirs e imita miARN son agentes prometedores para perturbar los niveles de miRNA 4-6. Sin embargo, las características especiales que mejoran su especificidad y eficacia tienen que ser incorporado en el diseño de antagomirs antes de que puedan ser utilizados para la perturbación in vivo de la expresión de los genes miARN. Los microARN son especialmente relevantes<html> como objetivos en neurodegenerativa actualmente incurable y enfermedades del neurodesarrollo. La barrera sangre-cerebro plantea una restricción a la entrega de antagomirs en el cerebro. Inyecciones estereotácticas son ampliamente utilizados en modelos de roedores para entregar moléculas a lugares específicos en el cerebro 7. Se requiere habilidad, amplia inversión en instrumentación y el tiempo. Inyecciones estereotáxicas son invasivas, implica cirugía, causar al menos leve lesión y se limita a la entrega local. El uso de péptidos penetrantes de células con una preferencia por las neuronas de orientación puede contrarrestar estas limitaciones, ya que pueden ser entregados a través de la ruta trans-vascular, pero romper la barrera hematoencefálica. Tal péptido derivado de la rabia Virus de la glucoproteína (RVG), se utilizó previamente para entregar siRNA contra virus de encefalitis japonesa en ratones 8. Encontramos que el uso del péptido para antagomir entrega, miRNAs puede ser efectivamente derribado en el cerebro del ratón 9. ontenido &quot;&gt; El segundo gran reto de miARN knock-down surge desde el pequeño tamaño de miRNAs y la presencia de isoformas de secuencias estrechamente relacionadas. Tomamos el ejemplo de la MMU-miR-29 familiar que consta de tres isoformas estrechamente relacionados, miR-29a , b y c. antagomirs son también en general modificada a lo largo de la columna vertebral para aumentar su estabilidad y hacerlas resistentes al ataque de nucleasas. Locked Nucleic Acids (LNA) ofrecen una ventaja adicional que mejoran la estabilidad térmica e incluso conducir a la degradación orientar sobre y más allá impedimento estérico 10. La introducción de modificaciones a lo largo de la columna vertebral puede ser eficaz, pero caro. Hemos visto anteriormente que las modificaciones más allá de un número óptimo pueden no mejorar aún más la eficacia. El diseño de la antagomir por lo tanto, implica la modificación óptima de la antagomir. Para antagomir complejo no covalente con el péptido neurotrópico, un hepta- cargada a la extensión nona-arginina se utiliza el. D-argininaresiduos se utilizan ya que confieren mayor estabilidad ya que no son susceptibles a la escisión por proteasas. Hepta a estiramientos-nona arginina actúan agentes celulares penetrante como eficientes, a pesar de que no confieren especificidad de tipo celular. Al enlazar covalentemente el péptido RVG al enlazador nona-arginina, un neurotrópico, péptido celular penetrante se generó. Los residuos cargados positivamente del péptido interactúan con la columna vertebral de ácido nucleico cargado negativamente, para formar complejos. Estos complejos se pueden utilizar para transfectar eficazmente ADN o ARN en células cultivadas e in vivo en los tejidos.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Vortex</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Restrainer or Decapicone</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow runway</td>
			<td></td>
			<td></td>
			<td>~70-cm-long, ~5-cm-wide with ~5-cm-high walls.</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescently labelled oligonucleotides (siGLO)</td>
			<td>GE Healthcare Dharmacon INC</td>
			<td>D0016300120</td>
			<td></td>
		</tr>
		<tr>
			<td>10% sterile D-glucose</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antagomir-29</td>
			<td>Exiqon</td>
			<td>custom synthesis</td>
			<td></td>
		</tr>
		<tr>
			<td>Antagomir-control</td>
			<td>Exiqon</td>
			<td>custom synthesis</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuropeptide RVG</td>
			<td>G.L.Biochem (Shanghai) Ltd.</td>
			<td>custom synthesis</td>
			<td>&#62;98% purity</td>
		</tr>
		<tr>
			<td>Neuropeptide RVM</td>
			<td>G.L.Biochem (Shanghai) Ltd.</td>
			<td>custom synthesis</td>
			<td>&#62;98% purity</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Warm water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbent sheets</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ink</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antiseptic</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple alternative to stereotactic injection for brain specific knockdown of mirna

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

Usando el procedimiento que aquí se presenta, complejos de oligonucleótido 50microgram marcado con fluorescencia (FLO) y ~ 850microgram RVG péptido 1:15 relación de carga molar: se prepararon (FLO péptido) y se inyecta una sola vez a través de la vena de la cola. Complejo de la no-neurotrópico Matrix Virus de la Rabia (RVM) péptido y FLO fue utilizado como un control de entrega. Al día siguiente, los ratones se aislaron cerebro y el hígado y se prepararon suspensiones de células individuales. Las células se ...

Watch this Scientific Journal Video about A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA at JoVE.com

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

Una alternativa sencilla para inyección estereotáctica para el Cerebro Derribo específico de miARN

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

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12.6K vistas.  CSIR-Institute of Genomics and Integrative Biology, New Delhi, India. MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

Video: A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

El grano y Pruebas de Manejo de Capellini: Simple medidas cuantitativas de la función de las patas delanteras diestro en ratas y ratones

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

Investigación

Neurociencias

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Gen de entrega de cerebro de rata postnatal por la inyección no ventricular plásmido y electroporación

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

La cirugía estereotáxica para la lesión excitotóxica de áreas específicas del cerebro en la rata adulta

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).
The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre- and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.
Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

Combinados Optogenética y congelación-fractura Réplica Immunolabeling para Analizar Arreglo-entrada específica de glutamato receptores en el ratón amígdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

Un método alternativo de inyección y validado para acceder al espacio subretiniano<i&gt; vía</i&gt; Un enfoque posterior transescleral

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

Una manera sencilla de medir alteraciones en el comportamiento de búsqueda de recompensa Uso<em&gt; Drosophila melanogaster</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

Microdisección de captura láser Atlas estereotáctico de regiones del cerebro afectadas por la lesión traumática

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Sencilla generación de una cultura de alto rendimiento de las neuronas inducidas de fibroblastos de piel humana adulta

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

Combinando Optogenetics con microRNAs artificiales para caracterizar los efectos de Gene caída en la función presináptica en circuitos neuronales intactas

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

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, ...