OG-P fue apoyado por CONACyT s de subvención (CB-2008 a 101.476) y FRABA (686 décimos). AQ-H el apoyo del Instituto Nacional de Salud, el Instituto Médico Howard Hughes, la Fundación Robert Wood Johnson y la Fundación Madre Maryland celular.

<ol><li> Hacen que las agujas de vidrio antes de la cirugía del ratón: <ol class="lalpha"><li> Coloque el tubo capilar de vidrio en un extractor de micropipeta. </li><li> Calentar la mitad del tubo de vidrio para suavizar el tubo de vidrio en el área localizada. </li><li> Estire el tubo de vidrio a lo largo de su eje longitudinal por una distancia inicial suficiente para causar una reducción en el diámetro del tubo de vidrio en el área localizada. </li><li> Mantenga el estiramiento del tubo de vidrio hasta que se rompe. De esta manera, dos agujas idénticas barril se obtienen. </li></ol></li><li> Coloque la aguja de vidrio en un microforge. Un ángulo de 30 ° es suficiente para bisel de la punta. </li><li> Compruebe con el microscopio que cada aguja tiene el diámetro interior correcto. Para la mayoría de los fármacos hidrófilos un diámetro de 30-50 micras interior es ideal (figura 1). </li><li> Llene la aguja con aceite mineral de la más amplia extremo de la aguja de vidrio. Deje que el aceite mineral (MSDS, cat. M7700) entra por capilaridad hasta que llega a aproximadamente el 50% de la longitud del tubo capilar. Ponga la grasa de alto vacío (Dow Corning, cat. 05054-AB) alrededor del pistón para sellar el extremo más ancho de la aguja. </li><li> Inserte el émbolo por el extremo más ancho de la aguja y presione suavemente el aceite mineral hasta que una pequeña gota de aceite se ve saliendo de la punta de la aguja de vidrio. </li><li> Asegurar la aguja de vidrio en el titular de la microinyector. </li><li> Anestesiar el ratón con el 2,5% Avertin (2,2,2-tribromoetanol + terc-amil alcohol, 1:1 w / v). Dosis de 25 30μL por gramo intraperitoneal. </li><li> Ponga una almohadilla de calefacción en el dispositivo de estereotaxia para mantener la temperatura corporal del animal a 37 ° C. </li><li> Colocar y fijar la cabeza del ratón en el dispositivo de estereotaxia con barras de oído y un soporte para los dientes. </li><li> Limpie la cabeza del ratón con una solución de clorhexidina al 0,1% de gluconato, seguido por el 70% de etanol y 0,1% de gluconato de clorhexidina por segunda vez. </li><li> Incisión en la piel con una hoja de bisturí # 15 de las orejas del ratón a la línea del cráneo Lamba. </li><li> Pulir el cráneo con un algodón para secar la piel y tirar hacia fuera del campo quirúrgico. </li><li> Use la punta de la aguja de vidrio a punto en el vértice de la sutura Bregma. No tienes que definir la posición inicial del inyector (la coordenada &quot;cero&quot;). </li><li> Establecer el punto de ser perforado por el movimiento de la &quot;X&quot; e &quot;Y&quot; eje del dispositivo de estereotaxia sobre el cráneo. </li><li> Taladro con mucho cuidado los agujeros en la coordenada seleccionada. Microdrills y otras herramientas de metal fueron esterilizadas previamente a 250 ° C durante 60 segundos con un paño seco de cuentas de vidrio esterilizador (Cole Palmer, cat. N º EW-10779-00). </li><li> Con unas pinzas finas retirar con cuidado la capa más profunda de los huesos y romper la membrana duramatter (verá un poco de líquido cefalorraquídeo se escape). </li><li> Confirmar que la aguja se puede ir a través de los agujeros perforados. </li><li> Pipeta de 1 l de la droga que desee inyectar y lo puso en un pequeño trozo de parafilm. </li><li> Coloque la parafina en el cráneo. </li><li> Aspirar la droga haciendo girar la rueda microinyector, mientras se revisa bajo el microscopio que el líquido se va para arriba en la aguja de vidrio. </li><li> Retire la parafina y volver a configurar el sistema de cero. </li><li> Mover la aguja de las coordenadas seleccionadas, por el titular hasta que la punta de la aguja de vidrio tocar suavemente la superficie del cerebro y establecer la &quot;Z&quot; coordinar a cero. </li><li> Introducir la aguja de vidrio en el parénquima cerebral a una profundidad seleccionada. </li><li> Se inyectan la droga lentamente a una velocidad de ~ 1 nl / seg. </li><li> Cuando se inyecta el volumen deseo, dejar que el medicamento difusa en el parénquima durante 2 min. A continuación, proceder a retirar la aguja suavemente y poco a poco. </li><li> Cola de la herida quirúrgica y remover al animal del dispositivo de estereotaxia y el sacrificio del animal en una jaula de pre-calentado que contiene una cama limpia para la recuperación de la anestesia. </li><li> Para proporcionar analgesia postoperatoria todos los animales recibieron 5 mg / kg por vía subcutánea cada hora Ketorolaco 12 por 24 h. </li></ol> Los resultados representativos: Cuando se sigue este protocolo, una inyección muy precisa y se obtiene un trayecto de la aguja muy estrecha minimiza la lesión cerebral. Como un resultado representativo de este método, se inyectan lisofosfatidil colina (lisolecitina) en el cuerpo calloso que produce la desmielinización de los tractos de sustancia blanca 1-4. Para minimizar el daño cerebral producido por la aguja de vidrio, se inyecta sólo el 20 nl de lisolecitina en el cuerpo calloso, pero si los volúmenes necesarios mayores de hasta 200 nl se puede inyectar con el mismo método. Desmielinización se detecta por la ausencia de expresión de proteína básica de mielina en los tractos de sustancia blanca (Figura 2). <img src="/files/ftp_upload/2082/2082fig1.jpg" alt="Figura 1" /> Figura 1. Una aguja de vidrio con un diámetro de 50 micras. Distancia entre dos ticks cortoen la escala representa una longitud de 50 micras. <img src="/files/ftp_upload/2082/2082fig2.jpg" alt="Figura 2" /> Figura 2. Lisolecitina inyección en el cuerpo calloso. Desmielinización se muestra como una expresión de la proteína básica de mielina no (área punteada). Tenga en cuenta el pequeño tamaño del trayecto de la aguja de cristal (flechas). Bar = 100 m

<ol>
	<li>Menn, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+of+oligodendrocytes+in+the+subventricular+zone+of+the+adult+brain.">Origin of oligodendrocytes in the subventricular zone of the adult brain.</a> J Neurosci. 26, 7907-7918 (2006).</li><li>Gonzalez-Perez, O., Romero-Rodriguez, R., Soriano-Navarro, M., Garcia-Verdugo, J. M., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+Growth+Factor+Induces+the+Progeny+of+Subventricular+Zone+Type+B+Cells+to+Migrate+and+Differentiate+into+Oligodendrocytes.">Epidermal Growth Factor Induces the Progeny of Subventricular Zone Type B Cells to Migrate and Differentiate into Oligodendrocytes.</a> Stem Cells. 27, 2032-2043 (2009).</li><li>Hall, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+aspects+of+remyelination+after+demyelination+produced+by+the+intraneural+injection+of+lysophosphatidyl+choline.">Some aspects of remyelination after demyelination produced by the intraneural injection of lysophosphatidyl choline.</a> J Cell Sci. 13, 461-477 (1973).</li><li>Webster, G. R., Thompson, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+presence+of+lysolecithin+in+nervous+tissues.">Observations on the presence of lysolecithin in nervous tissues.</a> Biochim Biophys Acta. 63, 38-45 (1962).</li><li>Doetsch, F., Caille, I., Lim, D. A., Garc&#237;a-Verdugo, J. M., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subventricular+Zone+Astrocytes+Are+Neural+Stem+Cells+in+the+Adult+mammalian+Brain.">Subventricular Zone Astrocytes Are Neural Stem Cells in the Adult mammalian Brain.</a> Cell. 97, 1-20 (1999).</li><li>Doetsch, F., Petreanu, L., Caille, I., Garcia-Verdugo, J. M., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGF+converts+transit-amplifying+neurogenic+precursors+in+the+adult+brain+into+multipotent+stem+cells.">EGF converts transit-amplifying neurogenic precursors in the adult brain into multipotent stem cells.</a> Neuron. 36, 1021-1034 (2002).</li><li>Baraban, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+seizures+by+transplantation+of+cortical+GABAergic+interneuron+precursors+into+Kv1.1+mutant+mice.">Reduction of seizures by transplantation of cortical GABAergic interneuron precursors into Kv1.1 mutant mice.</a> Proc Natl Acad Sci U S A. 106, 15472-15477 (2009).</li><li>Richardson, R. M., Barbaro, N. M., Alvarez-Buylla, A., Baraban, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+cell+transplantation+for+temporal+lobe+epilepsy.">Developing cell transplantation for temporal lobe epilepsy.</a> Neurosurg Focus. 24, E17-E17 (2008).</li><li>Alvarez-Dolado, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+inhibition+modified+by+embryonic+neural+precursors+grafted+into+the+postnatal+brain.">Cortical inhibition modified by embryonic neural precursors grafted into the postnatal brain.</a> J Neurosci. 26, 7380-7389 (2006).</li></ol>

El método demostró en este video es muy útil para proporcionar la mayor parte de los fármacos o vectores virales en lugares muy precisos en el cerebro. Algunas de las principales ventajas de esta técnica son la confiabilidad del punto de focalización, la exactitud de las inyecciones y el pequeño tamaño de la lesión cerebral y daños en las vías 1, 2, 5, 6. Una vez que la técnica se estandarizó el rango de mala focalización que 50 micras o menos 1, 2. Los trasplantes de células también puede hacerse mediante el uso más amplio, 100-150 micras 7-9, agujas. Por lo tanto, la entrega eficiente de células pueden ser depositados en los daños colaterales lesión muy específica minimizar inducida por el trasplante de células. Hay tres pasos cruciales en esta técnica: <ol><li> Nosotros siempre recomendamos rascarse suavemente el cráneo con una aguja de insulina para etiquetar las coordenadas seleccionadas antes de perforar. </li><li> Introducir la aguja de vidrio con un movimiento rápido para penetrar rápidamente en la superficie del cerebro. </li><li> Una vez que la profundidad óptima se alcanza se debe introducir la aguja de 0,1 mm más en el parénquima cerebral para hacer un &quot;canal&quot; que permiten a las drogas para difundir libremente en el cerebro. </li></ol>

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		<div>
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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Capillary glass tube</td>
<td>&nbsp;</td>
<td>Wiretrol I</td>
<td>5-000-1001</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette puller</td>
<td>&nbsp;</td>
<td>Kopf</td>
<td>Model 730</td>
<td>&nbsp;</td>
<tr>
<td>Microforge</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>Model 48000</td>
<td>&nbsp;</td>
<tr>
<td>Mineral oil</td>
<td>&nbsp;</td>
<td>MSDS</td>
<td>M7700</td>
<td>&nbsp;</td>
<tr>
<td>High-vacuum grease</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>05054-AB</td>
<td>&nbsp;</td>
<tr>
<td>Anesthesia: 2.5% Avertin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>2,2,2-tribromoethanol + tert-amyl alcohol, 1:1 w/v</td>
</tr>
<tr>
<td>Heater pad</td>
<td>&nbsp;</td>
<td>Mastex</td>
<td>Model 900</td>
<td>&nbsp;</td>
<tr>
<td>Stereotactic device</td>
<td>&nbsp;</td>
<td>Kopf</td>
<td>Model Kopf-900</td>
<td>&nbsp;</td>
<tr>
<td>Surgical scalpel blade # 15</td>
<td>&nbsp;</td>
<td>Medi-Cut</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micro driller</td>
<td>&nbsp;</td>
<td>Ideal Micro Drill</td>
<td>67-1000</td>
<td>&nbsp;</td>
<tr>
<td>Fine forceps</td>
<td>&nbsp;</td>
<td>Fine Scientific Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1-&mu;L Micropipette</td>
<td>&nbsp;</td>
<td>Rainin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Parafilm M.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Surgical microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>Vasiorkop with Contraves system</td>
<td>&nbsp;</td>
<tr>
<td>Microinjector</td>
<td>&nbsp;</td>
<td>Narishige</td>
<td>Model MO-10</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

targeting of deep brain structures with microinjections for delivery of drugs, viral vectors, or cell transplants

Microinyecciones en el parénquima cerebral son procedimientos importantes para llevar medicamentos, vectores virales o los trasplantes de células. La lesión cerebral que produce una aguja de la inyección durante su trayectoria es una gran preocupación especialmente en el cerebro del ratón, no sólo para el cerebro es pequeño, pero también a veces se necesitan múltiples inyecciones. Mostramos aquí un método para producir agujas capilar de vidrio con una luz de 50 m que reduce significativamente el daño cerebral y permite una segmentación precisa en el cerebro de roedores. Este método permite una entrega de volúmenes pequeños (20 a 100 nl), reduce los riesgos de sangrado y reduce al mínimo la difusión pasiva de drogas en el parénquima cerebral. Mediante el uso de diferentes tamaños de tubos capilares, o cambiar la luz de aguja, varios tipos de sustancias y las células se pueden inyectar. Microinyecciones con un tubo capilar de vidrio representan una mejora significativa en las técnicas de inyección y cerebral profunda orientación con mínimo daño colateral en los roedores pequeños.

Watch this Scientific Journal Video about Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants at JoVE.com

Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants

En este artículo, se muestra un método para fabricar agujas capilar de vidrio con una luz de 50 micras. Esta técnica reduce significativamente el daño cerebral, minimiza la difusión pasiva de las drogas y permite una segmentación precisa en el cerebro de roedores.

Focalización de estructuras profundas del cerebro con microinyecciones de entrega de medicamentos, vectores virales, o trasplantes de células

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an ...

targeting-deep-brain-structures-with-microinjections-for-delivery

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.5K vistas.  University of Colima. En este artículo, se muestra un método para fabricar agujas capilar de vidrio con una luz de 50 micras. Esta técnica reduce significativamente el daño cerebral, minimiza la difusión pasiva de las drogas y permite una segmentación precisa en el cerebro de roedores.

Video: Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

Escalable neumático matrices inyector para Viral focalización de los circuitos cerebrales intactas en 3-D

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

Investigación

Neurociencias

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

Inyección intracraneal de adeno-asociado vectores virales

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

La estimulación cerebral profunda con simultánea fMRI en roedores

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Non-restraining EEG Radiotelemetry: Epidural and Deep Intracerebral Stereotaxic EEG Electrode Placement

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and neurodegenerative diseases as well as epilepsies. This powerful technique does not only provide valuable insights into the underlying pathophysiological mechanisms, i.e., the etiopathogenesis of CNS related diseases, it also facilitates the development of new translational, i.e., therapeutic approaches. Whereas competing techniques that make use of recorder systems used in jackets or tethered systems suffer from their unphysiological restraining to semi-restraining character, radiotelemetric EEG recordings overcome these disadvantages. Technically, implantable EEG radiotelemetry allows for precise and highly sensitive measurement of epidural and deep, intracerebral EEGs under various physiological and pathophysiological conditions. First, we present a detailed protocol of a straight forward, successful, quick and efficient technique for epidural (surface) EEG recordings resulting in high-quality electrocorticograms. Second, we demonstrate how to implant deep, intracerebral EEG electrodes, e.g., in the hippocampus (electrohippocampogram). For both approaches, a computerized 3D stereotaxic electrode implantation system is used. The radiofrequency transmitter itself is implanted into a subcutaneous pouch in both mice and rats. Special attention also has to be paid to pre-, peri- and postoperative treatment of the experimental animals. Preoperative preparation of mice and rats, suitable anesthesia as well as postoperative treatment and pain management are described in detail.

Non-restraining EEG radiotelemetry is a valuable methodological approach to record in vivo long-term electroencephalograms from freely moving rodents. This detailed protocol describes stereotaxic epidural and deep intracerebral electrode placement in different brain regions in order to obtain reliable recordings of CNS rhythmicity and CNS related behavioral stages.

No restricción EEG radiotelemetría: epidural y Deep intracerebral estereotáxica colocación de electrodos EEG

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and ...

Personalized Needles for Microinjections in the Rodent Brain

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

We describe here a protocol for microinjection in the rodent brain that uses quartz needles. These needles do not produce detectable tissue damage and ensure reliable delivery even in deep regions. Moreover, they can be adapted to research needs by personalized designs and can be re-used.

Agujas personalizadas para microinyecciones en el cerebro de roedor

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this challenging. Because the ventricles are negative spaces located deep within the brain, the only way to understand their anatomy is by appreciating their boundaries formed by related structures. Looking at a 2D representation of these spaces, in any of the cardinal planes, will not enable visualisation of all of the structures that form the boundaries of the ventricles. Thus, using 2D sections alone requires students to compute their own mental image of the 3D ventricular spaces. The aim of this study was to develop a reproducible method for dissecting the human brain to create an educational resource to enhance student understanding of the intricate relationships between the ventricles and periventricular structures. To achieve this, we created a video resource that features a step-by-step guide using a fiber dissection method to reveal the lateral and third ventricles together with the closely related limbic system and basal ganglia structures. One of the advantages of this method is that it enables delineation of the white matter tracts that are difficult to distinguish using other dissection techniques. This video is accompanied by a written protocol that provides a systematic description of the process to aid in the reproduction of the brain dissection. This package offers a valuable anatomy teaching resource for educators and students alike. By following these instructions educators can create teaching resources and students can be guided to produce their own brain dissection as a hands-on practical activity. We recommend that this video guide be incorporated into neuroanatomy teaching to enhance student understanding of the morphology and clinical relevance of the ventricles.

This paper demonstrates the effective use of a fiber dissection method to reveal the superficial white matter tracts and periventricular structures of the human brain, in three-dimensional space, to aid student comprehension of ventricular morphology.

Explorar el espacio profundo - descubrimiento de la anatomía de las estructuras periventriculares para revelar los ventrículos laterales del cerebro humano

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

Biología celular cuantitativa de neurodegeneración en Drosophila mediante análisis imparcial de fluorescencia etiquetas proteínas con ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

Producción, purificación y Control de calidad de vectores de base de Virus Adeno-asociado

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

Three-Dimensional Shape Modeling and Analysis of Brain Structures

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. We have developed a software package for accurate and robust shape modeling and group-wise analysis. Here, we introduce an pipeline for the shape analysis, from individual 3D shape modeling to quantitative group shape analysis. We also describe the pre-processing and segmentation steps using open software packages. This practical guide would help researchers save time and effort in 3D shape analysis on brain structures.

We introduce a semi-automatic protocol for shape analysis on brain structures, including image segmentation using open software, and further group-wise shape analysis using an automated modeling package. Here, we demonstrate each step of the 3D shape analysis protocol with hippocampal segmentation from brain MR images.

Modelado de formas tridimensionales y análisis de estructuras cerebrales

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. ...

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative cell replacement therapies while also creating tools to study cell fate in situ. To date, it has been possible to obtain neurons via in vivo reprogramming, but the precise phenotype of these neurons or how they mature has not been analyzed in detail. In this protocol, we describe a more efficient conversion and cell-specific identification of the in vivo reprogrammed neurons, using an AAV-based viral vector system. We also provide a protocol for functional assessment of the reprogrammed cells&#8217; neuronal maturation. By injecting flip-excision (FLEX) vectors, containing the reprogramming and synapsin-driven reporter genes&#160;to specific cell types in the brain that serve as the target for cell reprogramming. This technique allows for the easy identification of newly reprogrammed neurons. Results show that the obtained reprogrammed neurons functionally mature over time, receive synaptic contacts and show electrophysiological properties of different types of interneurons. Using the transcription factors Ascl1, Lmx1a and Nurr1, the majority of the reprogrammed cells have properties of fast-spiking, parvalbumin-containing interneurons.

This protocol aims at generating directly reprogrammed interneurons in vivo, using an AAV-based viral system in the brain and a FLEX synapsin-driven GFP reporter, which allows for cell identification and further analysis in vivo.

En Vivo Reprogramación Directa de Células Gliales Residentes en Interneuronas por Inyección Intracerebral de Vectores Virales

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...