Este trabajo fue apoyado por MH084494 (CJL), y MH079276 y NS060658 (CAP).

1. Preparación Cortar <ol><li> Cortar rodajas de cerebro usando un micrótomo de vibración. Hemos preparado 240 micras cortes horizontales del cerebro medio de una isoflurano anethetized ratas Sprague-Dawley (Charles River Laboratories) usando un microtomo vibrante (Microm HM 650V), de acuerdo con la Universidad de Texas en San Antonio de Cuidado de Animales institucional y el empleo. </li><li> Mantenga los segmentos en una cámara de incubación hasta que esté listo para grabar. Utilizamos un recipiente de incubación se calienta a 32 ° C y lleno de líquido cefalorraquídeo artificial (ACSF, en mM): NaCl 126, KCl 2,5, 1,25 NaH 2 PO 4, 4 MgCl 2, 2 CaCl2, 10 de dextrosa, 25 de NaHCO3, 1,3 de ácido ascórbico, 2,4 piruvato de sodio y glutatión 0,05. </li></ol> 2. Registro electrofisiológico <ol><li> La transferencia de la división para el equipo de grabación intracelular en el que un líquido cefalorraquídeo artificial (ACSF) a 35 ° C se perfundidos. Nosotros utilizamos la misma que en LCRa 1,2 excepto que 2mM de MgCl 2 fue utilizado y el glutatión se omitió. Para cortes en horizontal preparada, que normalmente atraviesan el corte a lo largo de la línea media. </li><li> Visualizar la neurona diana. Visualizamos cada sustancia negra neuronas dopaminérgicas con un sistema de gradiente de contraste de imagen. </li><li> Tirar de un electrodo con un extractor de electrodos electrónicos. Tiramos los electrodos con una resistencia de punta de 4.10 mW con un extractor P97 Micropipeta (Compañía Sutter Instrument). </li><li> Llenar un electrodo con la solución interna deseada. Utilizamos una solución que contiene (en mM): 138 K-gluconato, 10 HEPES, 2 MgCl 2, 0,2 EGTA, 0,0001 CaCl 2, 4 Na-ATP, 0,4 Na-GTP. La solución interna se ajustó a un pH de 7,3 con KOH 1M y una osmolaridad de 270-275 MOSMS. </li><li> Hacer un sello gigaohm en la neurona que desee. La ruptura con el sello de succión. Esto constituye un registro de células enteras. Un amplificador de MultiClamp 700B se utiliza en nuestra configuración. El amplificador debe ser colocado en el modo de pinza de corriente de la &#39;I = 0&#39;. </li></ol> 3. Conductancia de aplicación con pinza dinámica <ol><li> RTXI (www.rtxi.org) fue ejecutado en la computadora pinza dinámica. Un modelo personalizado por escrito que contiene un receptor de NMDA se ha cargado en la memoria. La corriente que se inyecta en las células en tiempo real se calcula por la siguiente ecuación: I = NMDA NMDA-g * [1 / (1 ​​+ ([Mg] / 3,57) * e (-V m * 0.062))] * (V m - E NMDA) donde g es la conductancia NMDA deseada (en ns; por defecto es 0 ns), [Mg] es la concentración de magnesio (se establece en 1,5 mM en el ejemplo a continuación), E NMDA es el potencial de inversión para el receptor de NMDA (a 0 mV), y V m es el potencial de membrana de la célula de medida desde el amplificador (en milivoltios). </li><li> La salida de la computadora pinza dinámica se conecta a la entrada de comandos del amplificador a través de un convertidor de analógico a digital. </li><li> El amplificador se coloca en modo de pinza de corriente de la &quot;IC&quot;. </li><li> Ingrese su conductividad deseada del receptor NMDA en RTXI (por ejemplo, 40 ns). Usted debe ver una explosión fásica de potenciales de acción. Por otra parte, la conductancia se puede dar a través de RTXI una salida analógica (Figura 1, &#39;g (t)&#39;). Un factor de escala apropiado debe ser utilizado dentro de RTXI para convertir la señal de voltios a Siemens. </li></ol> 4. Resultados representante Una configuración de éxito para la aplicación de una conductividad usando la pinza dinámica se muestra en la Figura 1. Con esta instalación, hemos hecho una célula somática toda la grabación de una neurona dopaminérgica en la sustancia negra pars compacta. Las células dopaminérgicas general inflama espontáneamente en las tarifas bajas con un patrón similar al marcapasos. Una ráfaga de potenciales de acción puede ser provocado por la aplicación fásica de una conductancia del receptor NMDA con pinza dinámica (fig. 1B). <img src="/files/ftp_upload/2275/2275fig1.jpg" alt="Figura 1" /> Figura 1: Aplicación de una conductancia de los receptores NMDA mediante la técnica de la pinza dinámica. Configuración de hardware A. ilustra las conexiones entre el equipo de registro intracelular y el ordenador pinza dinámica. B. Una ráfaga de potenciales de acción es provocada por la aplicación de una conductancia de los receptores NMDA 40ns en una grabación de células enteras de una sustancia negra pars compacta dopaminérgicos neurona.

<ol>
	<li>Robinson, H. P., Kawai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+of+digitally+synthesized+synaptic+conductance+transients+to+measure+the+integrative+properties+of+neurons.">Injection of digitally synthesized synaptic conductance transients to measure the integrative properties of neurons.</a> J Neurosci Methods. 49, 157-165 (1993).</li><li>Sharp, A. A., O'Neil, M. B., Abbott, L. F., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+clamp:+artificial+conductances+in+biological+neurons.">The dynamic clamp: artificial conductances in biological neurons.</a> Trends Neurosci. 16, 389-394 (1993).</li><li>Sharp, A. A., O'Neil, M. B., Abbott, L. F., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+clamp:+computer-generated+conductances+in+real+neurons.">Dynamic clamp: computer-generated conductances in real neurons.</a> J Neurophysiol. 69, 992-995 (1993).</li><li>Prinz, A. A., Abbott, L. F., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+clamp+comes+of+age.">The dynamic clamp comes of age.</a> Trends Neurosci. 27, 218-224 (2004).</li><li>Deister, C. A., Teagarden, M. A., Wilson, C. J., Paladini, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+intrinsic+neuronal+oscillator+underlies+dopaminergic+neuron+bursting.">An intrinsic neuronal oscillator underlies dopaminergic neuron bursting.</a> J Neurosci. 29, 15888-15897 (2009).</li><li>Lobb, C. J., Wilson, C. J., Paladini, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dynamic+role+for+GABA+receptors+on+the+firing+pattern+of+midbrain+dopaminergic+neurons.">A dynamic role for GABA receptors on the firing pattern of midbrain dopaminergic neurons.</a> J Neurophysiol. 104, 403-413 (2010).</li></ol>

La técnica de la pinza dinámica demostrado aquí mejora de la técnica tradicional de la inyección de corriente continua al permitir que el experimentador para imitar los efectos eléctricos de la activación de un receptor. En este video, nos han demostrado que se puede añadir los efectos de la activación de un receptor de NMDA en la actividad espontánea de las neuronas dopaminérgicas, es decir, una ráfaga de potenciales de acción son evocados. Debido a la flexibilidad de la aplicación de hardware / software, una variedad de extensiones se pueden utilizar. El signo de la corriente inyectada se puede cambiar de negativo a positivo, lo que representa un escenario donde los efectos del receptor activado se elimina de una neurona. Neuronas modelo, representado en la forma de una serie de ecuaciones diferenciales, también puede ser resuelto numéricamente y permitir que el experimentador para investigar las redes pequeñas.

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<td>K-gluconate anhydrous</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
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<td>HEPES</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
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<td>CaCl2 X 2H2O</td>
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<td>Multiclamp 700B with CV-7B headstage</td>
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<td>Molecular Devices</td>
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<td>P-97 Flaming/Brown Micropipette Puller</td>
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<td>Sutter Instrument Company</td>
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<td>Microfil syringe needles</td>
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<td>World Precision Instruments</td>
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<td>Micromanipulator</td>
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<td>Siskiyou, Inc.</td>
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<td>Monitor</td>
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<td>Triview</td>
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application of a nmda receptor conductance in rat midbrain dopaminergic neurons using the dynamic clamp technique

Los neurocientíficos estudiar la función del cerebro mediante la investigación de cómo las neuronas del cerebro se comunican. Muchos investigadores observar los cambios en la actividad eléctrica de una o más neuronas en respuesta a una entrada experimental controlado. La actividad eléctrica de las neuronas se pueden grabar en rodajas de cerebro aislado utilizando técnicas de patch clamp con micropipetas de vidrio. Tradicionalmente, los investigadores pueden simular la entrada neuronal mediante la inyección de corriente a través de la pipeta, la estimulación eléctrica de las células de otros o resto de conexiones axonal en el corte, o la manipulación farmacológica de los receptores situados en la membrana de la célula neuronal registrada. Inyección de corriente directa tiene la ventaja de transmitir una forma de onda predeterminada actual con una precisión temporal de alta en el lugar de la grabación (por lo general el soma). Sin embargo, eso no cambia la resistencia de la membrana neuronal, como no hay canales de iones son físicamente abierto. Inyección de corriente por lo general emplea pulsos rectangulares y por lo tanto no modelar la cinética de los canales iónicos. Por último, la inyección de corriente no pueden imitar los cambios químicos en la célula que se produce con la apertura de los canales iónicos. Receptores pueden ser físicamente activa por estimulación eléctrica o farmacológica. El experimentador tiene una buena precisión temporal de la activación del receptor con la estimulación eléctrica de la división. Precisión sin embargo, no se limita espacial de la activación del receptor y la naturaleza exacta de lo que se activa por estimulación es desconocida. Este último problema puede reducirse en parte mediante agentes farmacológicos específicos. Por desgracia, el curso temporal de la activación de los agentes farmacológicos suele ser lenta y la precisión espacial de las entradas registradas en la celda se desconoce. La técnica de la pinza dinámica permite un experimentador para cambiar la corriente pasa directamente a la celda sobre la base de información en tiempo real del potencial de membrana de la célula (Robinson y Kawai 1993, Sharp et al, 1993a, b;. Para revisión, ver Prinz et al. 2004). Esto permite que un experimentador para imitar los cambios eléctricos que ocurren en el lugar de la grabación en respuesta a la activación de un receptor. En tiempo real los cambios en la corriente aplicada se determina por una ecuación matemática implementado en el hardware. Recientemente, hemos utilizado la técnica de la pinza dinámica para investigar la generación de ráfagas de potenciales de acción por la activación fásica de los receptores NMDA en las neuronas dopaminérgicas de la sustancia nigra pars compacta (Deister et al, 2009;. Lobb et al, 2010).. En este vídeo, que muestran los procedimientos necesarios para aplicar una conductancia de los receptores de NMDA en la neurona dopaminérgica.

Watch this Scientific Journal Video about Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique at JoVE.com

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

En este video, que muestra cómo aplicar una conductancia en una neurona dopaminérgica registrado en la configuración de célula entera, en rodajas de cerebro de rata. Esta técnica se llama la pinza dinámica.

Aplicación de una conductancia del receptor NMDA en las neuronas dopaminérgicas del mesencéfalo de rata con la técnica de sujeción dinámica

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

application-nmda-receptor-conductance-rat-midbrain-dopaminergic

Research

JoVE Journal

Neuroscience

12.0K vistas.  University of Texas San Antonio - UTSA. En este video, que muestra cómo aplicar una conductancia en una neurona dopaminérgica registrado en la configuración de célula entera, en rodajas de cerebro de rata. Esta técnica se llama la pinza dinámica.

Video: Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Culturas organotípicos rebanada de mesencéfalo ventral embrionario: Un sistema para estudiar el desarrollo neuronal dopaminérgica In vitro</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

Investigación

Neurociencias

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Inducidas Odorante-respuestas registradas de neuronas olfativas del receptor utilizando la técnica de aspiración con una pipeta

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

En electroporación in ovo en el mesencéfalo polluelo para estudiar la función génica en el desarrollo neuronal dopaminérgica

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

Implementar Clamp dinámico con conductancias sinápticas y artificiales en células de ratón ganglionares retinianas

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Cultivo primario de ratón neuronas dopaminérgicas

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

Modulaciones Ambientales del Número de dopamina mesencéfalo neuronas en ratones adultos

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Perforado grabación de patch-clamp de ratón neuronas sensoriales olfativas en neuroepitelio Intacto: Análisis funcional de las neuronas que expresan un odorante del receptor Identificado

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

La identificación fiable de las neuronas dopaminérgicas de estar en el mesencéfalo culturas utilizando ARN-Secuenciación y promotor impulsado TH expresión de EGFP

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system.

La evaluación simultánea de Hemodinámica cerebral y propiedades de dispersión de luz de la<em&gt; En Vivo</em&gt; Cerebro de rata utilizando multiespectral de imágenes de reflectancia difusa

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

Aplicación de la abrazadera de parche guiada por imágenes automatizada para el estudio de neuronas en cortes cerebrales

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...