El financiamiento para el trabajo de este, y 2,10, como se cita, fue proporcionada, en parte, por unos premios de investigación de subvención a Salvatore MF de la Federación Americana para la Investigación del Envejecimiento, el Edward P. Stiles Fondo Fiduciario y la Fundación para la Investigación Biomédica del Noroeste de Louisiana, y BS Pruett de la beca predoctoral Muslow Ike, LSU Health Sciences Center-Shreveport.

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	<li>Trevitt, J. T., Carlson, B. B., Nowend, K., Salamone, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substantia+nigra+pars+reticulate+is+a+highly+potent+site+of+action+for+the+behavioral+effects+of+the+D1+antagonist+SCH23390+in+rat.">Substantia nigra pars reticulate is a highly potent site of action for the behavioral effects of the D1 antagonist SCH23390 in rat.</a> Psychopharmacology. 156, 32-41 (2001).</li><li>Salvatore, M. F., Pruett, B. S., Spann, S. L., Dempsey, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+reveals+a+role+for+nigral+tyrosine+hydroxylase+ser31+phosphorylation+in+locomotor+activity+generation.">Aging reveals a role for nigral tyrosine hydroxylase ser31 phosphorylation in locomotor activity generation.</a> PLoS ONE. 4, e8466 (2009).</li><li>Rossato, J. 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M., Shaham, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+infusion+of+brain-derived+neurotrophic+factor+into+the+ventral+tegmental+area+induces+long-lasting+potentiation+of+cocaine+seeking+after+withdrawal.">A single infusion of brain-derived neurotrophic factor into the ventral tegmental area induces long-lasting potentiation of cocaine seeking after withdrawal.</a> J. Neurosci. 24, 1604-1611 (2004).</li><li>Hoffer, B. J., Hoffman, A., Bowenkamp, K., Huettl, P., Hudson, J., Martin, D., Lin, L. F., Gerhardt, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cell+line-derived+neurotrophic+factor+reverses+toxin-induced+injury+to+midbrain+dopaminergic+neurons+in+vivo.">Glial cell line-derived neurotrophic factor reverses toxin-induced injury to midbrain dopaminergic neurons in vivo.</a> Neurosci. Lett. 182, 107-111 (1994).</li><li>Salvatore, M. F., Zhang, J. L., Large, D. M., Wilson, P. E., Gash, C. R., Thomas, T. C., Haycock, J. W., Bing, G., Stanford, J. A., Gash, D. M., Gerhardt, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Striatal+GDNF+administration+increases+tyrosine+hydroxylase+phosphorylation+in+the+rat+striatum+and+substantia+nigra.">Striatal GDNF administration increases tyrosine hydroxylase phosphorylation in the rat striatum and substantia nigra.</a> J. Neurochem. 90, 245-254 (2004).</li><li>Lu, L., Wang, X., Wu, P., Xu, C., Zhao, M., Morales, M., Harvey, B. K., Hoffer, B. J., Shaham, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+ventral+tegmental+area+glial+cell-line+derived+neurotrophic+factor+in+incubation+of+cocaine+craving.">Role of ventral tegmental area glial cell-line derived neurotrophic factor in incubation of cocaine craving.</a> Biol. Psychiatry. 66, 137-145 (2009).</li><li>Lavicky, J., Dunn, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corticotropin-releasing+factor+stimulates+catecholamine+release+in+hypothalamus+and+prefrontal+cortex+in+freely+moving+rats+as+assessed+by+microdialysis.">Corticotropin-releasing factor stimulates catecholamine release in hypothalamus and prefrontal cortex in freely moving rats as assessed by microdialysis.</a> J. Neurochem. 60, 602-612 (1993).</li><li>Salvatore, M. F., Pruett, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dichotomy+of+tyrosine+hydroxylase+and+dopamine+regulation+between+somatodendritic+and+terminal+field+areas+of+nigrostriatal+and+mesoaccumbens+pathways.">Dichotomy of tyrosine hydroxylase and dopamine regulation between somatodendritic and terminal field areas of nigrostriatal and mesoaccumbens pathways.</a> PLoS ONE. 7, e29867 (2012).</li><li>Salvatore, M. F., Garcia-Espana, A., Goldstein, M., Deutch, A. Y., Haycock, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry+of+tyrosine+hydroxylase+phosphorylation+in+the+nigrostriatal+and+mesolimbic+systems+in+vivo:+Effects+of+acute+haloperidol+and+related+compounds.">Stoichiometry of tyrosine hydroxylase phosphorylation in the nigrostriatal and mesolimbic systems in vivo: Effects of acute haloperidol and related compounds.</a> J. Neurochem. 75, 225-232 (2000).</li><li>Salvatore, M. F., Waymire, J. C., Haycock, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depolarization-stimulated+catecholamine+biosynthesis:+involvement+of+protein+kinases+and+tyrosine+hydroxylase+phosphorylation+sites+in+situ.">Depolarization-stimulated catecholamine biosynthesis: involvement of protein kinases and tyrosine hydroxylase phosphorylation sites in situ.</a> J. Neurochem. 79, 349-360 (2001).</li><li>Haycock, J. W., Lew, J. Y., Garcia-Espana, A., Lee, K. Y., Harada, K., Meller, E., Goldstein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+serine-19+phosphorylation+in+regulating+tyrosine+hydroxylase+studied+with+site-+and+phosphospecific+antibodies+and+site-directed+mutagenesis.">Role of serine-19 phosphorylation in regulating tyrosine hydroxylase studied with site- and phosphospecific antibodies and site-directed mutagenesis.</a> J. Neurochem. 71, 1670-1675 (1998).</li><li>Lindgren, N., Xu, Z. Q., Linskog, M., Herrera-Marschitz, M., Goiny, M., Haycock, J. W., Goldstein, M., Hokfelt, T., Fisone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+tyrosine+hydroxylase+activity+and+phosphorylation+at+ser19+and+ser40+via+activation+of+glutamate+NMDA+receptors+in+rat+striatum.">Regulation of tyrosine hydroxylase activity and phosphorylation at ser19 and ser40 via activation of glutamate NMDA receptors in rat striatum.</a> J. Neurochem. 74, 2470-2477 (2000).</li></ol>

No hay conflictos de interés declarado.

Como se indica en la Figura 1, los métodos detallados anteriormente deben producir lecturas múltiples de dopamina y su TH proteínas reguladoras, DAT, y VMAT2 de una muestra de SN o VTA obtenerse a partir de la rata o el ratón. Una vez más, los beneficios de la realización de este protocolo es que el investigador puede obtener lecturas operativamente con ajuste de cómo se regula la dopamina in vivo bajo virtualmente cualquier paradigma experimental y, al hacerlo, ahorrar considerables recursos experimenta...

Sistema de HPLC: El sistema básico consiste en un Shimadzu LC10-ADVP bomba HPLC, Waters WISP un 717 inyector automático de muestras, de 250 X 4,4 mm 5 micras Spherosorb SAO-1 C18 columna de fase inversa (Waters), un Bioanalytical Systems (BAS) TL12 de doble cristalino electrodo de carbón, dos detectores de BAS LC4B electroquímicas, y las aguas de un Empower 2 de recopilación de datos e integración de sistemas. La columna se mantiene a 30-45 ° C (BAS calentador columna LC22A). La fase móvil es fosfato de sodio 0,1 M (pH 3,0), 0,1 mM EDTA, 0.2-0.4 mM 1-octano sulfónico (Eastman-Kodak), y 0,35% de acetonitrilo (v / v), se filtró a través de un filtro de 0,45 micras. El caudal es de 1,2 ml / min. Cuatro lotes litros de fase móvil están optimizados para las separaciones por ajuste del pH, octano ácido sulfónico y temperatura de la columna. La fase móvil se recicla, y se purgó continuamente con gas helio para eliminar el oxígeno disuelto. El reciclado de la fase móviles casi imprescindible para mantener una buena resolución para un período de tiempo razonable. La fase móvil la vida útil se mantiene mediante el uso de un interruptor de flujo (controlada por el integrador) para desviar a perder el primero 2-7 minutos de cada carrera. Los electrodos se mantienen a potenciales de aproximadamente 0,78 y 0.95V con respecto a un electrodo de referencia Ag/AgC1. El electrodo en el potencial más alto se utiliza exclusivamente para la determinación de triptófano (y el estándar de NMDA interna). El potencial de 0,78 V proporciona una señal superior a ruido para la detección de las monoaminas y compuestos, excepto triptófano. Los cromatogramas se almacenan en el disco duro de la estación de trabajo Empower, y posteriormente se procesa y transfiere los datos directamente en una hoja de cálculo Excel para el cálculo de las cantidades de metabolitos y la compilación de datos del grupo. Bomba: Shimadzu LC-10AD Celular: BAS flujo cruzado. Carbono vidrioso electrodo de trabajo a 0,780 y 0,9Potencial V 50. Detector: BAS LC-4B, que opere en modo de doble canal. Los datos Adq. Sistema: Aguas Empower Pro 2. Inyector: 717 WISP Waters Columna: Aguas Spherosorb SAO-1, ​​5 mM de partícula, 4,4 mm x 250 mm. <table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Dodecilsulfato sódico (SDS) </td><td> - JT Baker </td><td> 4095-02 </td></tr><tr><td> Trizma Base </td><td> Sigma </td><td> T1503-1kg </td></tr><tr><td> Trizma HCl </td><td> Sigma </td><td> T3253-1kg </td></tr><tr><td> Glicerol </td><td> Sigma </td><td> G8773-500 ml </td></tr><tr><td> PVP-40 </td><td> Sigma </td><td> PVP40-1kg </td></tr><tr><td> DPBS </td><td> Gibco </td><td> 21600-069 </td></tr><tr><td> Tween 20 </td><td> Sigma </td><td> P1379-500 ml </td></tr><tr><td> Glicina </td><td> Sigma </td><td> G8898-1kg</td></tr><tr><td> Ponceau S </td><td> Fluka </td><td> 81460 </td></tr><tr><td> Azul de bromofenol </td><td> Sigma </td><td> B8026-5G </td></tr><tr><td> Ditiotreitol </td><td> Sigma </td><td> D-9163 </td></tr><tr><td> Proteína estándar 2 mg de BSA </td><td> Sigma </td><td> P5619-25VL </td></tr><tr><td> Pierce BCA Protein ensayo Reactivo A </td><td> Thermo Fisher Scientific- </td><td> 23223 </td></tr><tr><td> Precision Plus estándar Proteína </td><td> Bio Rad </td><td> 161-0373 </td></tr><tr><td> [125I]-proteína A, la actividad específica </td><td> Perkin-Elmer </td><td></td></tr></tbody></table> Tabla 2. Reactivos específicos. <table border="1"><tbody><tr><td> Reactivos </td><td> Fórmulas </td></tr><tr><td> 10% de SDS </td><td> 10 g de SDS, 100 ml de H 2 0 </td></tr><tr><td> SDS al 1% (pH 8,2) </td><td><ol><li> 10 ml10% de SDS </li><li> 60,5 mg de base Trizma </li><li> 37,22 mg de EDTA </li><li> 90 ml de H 2 0 </li></ol></td></tr><tr><td> Cobre II solución de sulfato </td><td><ol><li> 1 g de cobre II sulfato de </li><li> 25 ml de H 2 0 </li></ol></td></tr><tr><td> Tampón de muestra 3X </td><td><ol><li> Trizma base 2,27 g </li><li> SDS 6 g </li><li> Ditiotreitol 0,463 g </li><li> Glicerol 30 g </li><li> 10 mg de azul de bromofenol </li><li> DIH 2 0 (añadir inicialmente por encima de los reactivos a 40 mL de H 2 0 en un cilindro graduado, a continuación, agregar H 2 0 hasta que el volumen llegue a 100 ml) </li><li> HCl (añadir según sea necesario para alcanzar un pH de 6,85) </li><li> Congelar solución en 50 tubos de 2,0 ml. </li></ol> (Hace 100 ml): Volumen de tampón de muestra 3X necesario = volumen de la mitad de SDS utilizado en la muestra. </td></tr><tr><td> 1X tampón de muestra </td><td> Diluir tampón de muestra 3X abajo a 1X tampón de muestra utilizando DI H 2 0 </td></tr><tr><td> 10 veces<html> Funcionamiento de amortiguación (Rinde 4 L) </td><td><ol><li> Trizma base 121,1 g </li><li> La glicina 577 g </li><li> 40 g de SDS </li></ol></td></tr><tr><td> Tampón de transferencia 10 veces: (para 4 L) </td><td><ol><li> La glicina 360 g </li><li> Trizma base 96 g </li></ol></td></tr><tr><td> Ponceau </td><td><ol><li> Ponceau S. 0,5 g </li><li> Ácido acético 5 ml </li><li> DI H 2 0 95 ml </li></ol></td></tr><tr><td> 0,2% solución de HCl </td><td> 5,2 ml de HCl en 500 ml de H 2 0 </td></tr><tr><td> PVP-T20 bloqueo Soln. (Hace 4 L) </td><td><ol><li> PVP-40 40 g </li><li> DPBS 38,2 g </li><li> Tween20 2 g </li><li> Timerisol 0,4 g </li><li> 1M Tris pH 7,6 (60,6 g de Tris HCl + 13,9 g de Tris base en 500 ml de H DI 2 0) - 50 ml </li></ol></td></tr><tr><td> Buffer 10X Blot (Rinde 4 L) </td><td><ol><li> Tween 20 20 g </li><li> Tris Base 14 g </li><li> Tris-HCl 61 g </li></ol></td></tr></tbody></table>CONTENIDO &quot;&gt; la Tabla 3. de ensayo de proteínas y Western Blotting fórmulas. Las normas de la tirosina hidroxilasa TH: Los calibrados los estándares y la fosforilación de proteínas utilizadas por este laboratorio se derivan de extractos de células PC12, que se analizaron el contenido de proteína TH y estequiometría de fosforilación de las normas en contra de un TH previamente calibrados que en última instancia se originaron en el laboratorio del Dr. John Haycock 11 .

comprehensive profiling of dopamine regulation in substantia nigra and ventral tegmental area

La dopamina es un neurotransmisor con fuerza estudió en el SNC. De hecho, su participación en la actividad locomotora y la conducta relacionada con la recompensa ha promovido cinco décadas de investigación sobre las deficiencias moleculares asociados con la regulación de la dopamina. La mayoría de estas investigaciones de la regulación de la dopamina en el centro del cerebro sobre la base molecular para su regulación en las regiones terminales de campo de las vías y nigroestriatales mesoaccumbens, cuerpo estriado y núcleo accumbens. Además, estos estudios se han concentrado en el análisis del contenido de tejido dopamina con la normalización de peso sólo tejido húmedo. La investigación de las proteínas que regulan la dopamina, como la tirosina hidroxilasa (TH) de proteínas, la fosforilación de TH, transportador de dopamina (DAT), y el transportador vesicular de monoaminas 2 (VMAT2) de proteínas a menudo no incluyen el análisis de contenido de tejido de la dopamina en la misma muestra. La capacidad de analizar tanto el contenido de tejido dopamina y sus proteínas reguladoras (incluyendo el post-TRAmodificaciones nslational) no sólo le da el poder inherente a la interpretación de la relación de la dopamina con el nivel de proteínas y la función de TH, DAT, o VMAT2, sino que también se extiende a la economía de la muestra. Esto se traduce en un menor costo, y sin embargo, produce ideas sobre la regulación molecular de la dopamina en prácticamente cualquier paradigma de la elección de los investigadores. Nos centramos el análisis en el cerebro medio. Aunque el SN y VTA son típicamente descuidado en la mayoría de los estudios de regulación de dopamina, estos núcleos son fácilmente disecados con la práctica. Una lectura completa del contenido de los tejidos la dopamina y la TH, DAT, o VMAT2 puede llevarse a cabo. Existe un creciente literatura sobre el impacto de la función de la dopamina en el VTA SN y en el comportamiento y las Tropezaciones de sustancias exógenas o procesos de enfermedades en ellos 1-5. Además, los compuestos tales como factores de crecimiento tiene un profundo efecto sobre las proteínas de dopamina y la dopamina-regulación, en un grado relativamente mayor en el SN o VTA <sup&gt; 6-8. Por lo tanto, esta metodología se presenta para referencia a los laboratorios que deseen ampliar sus investigaciones sobre cómo los tratamientos específicos modulan el comportamiento y la regulación de la dopamina. Aquí, un método de varios pasos que se presentan para el análisis de contenido de tejido de dopamina, los niveles de proteína de TH, DAT, o VMAT2 y TH fosforilación de la sustancia negra del mesencéfalo y VTA de los roedores. El análisis de la fosforilación de TH puede proporcionar importantes conocimientos sobre no sólo cómo la actividad de TH se regula, sino también las cascadas de señalización en los núcleos afectados somatodendrítica en un paradigma determinado. Vamos a ilustrar la técnica de disección para separar estos dos núcleos y el procesamiento de la muestra de tejido disecado que produce un perfil de revelar los mecanismos moleculares de regulación de dopamina in vivo, específicos para cada núcleos (Figura 1).

Watch this Scientific Journal Video about Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area at JoVE.com

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

La dopamina está claramente regulado en los núcleos del cerebro medio, que contiene la celda de los cuerpos y las dendritas de las neuronas de dopamina. Aquí se describe un método de disección y manejo de la muestra-para maximizar los resultados, y por lo tanto las conclusiones y puntos de vista, sobre la regulación de la dopamina en el núcleo del cerebro medio de la sustancia negra (SN) y el área tegmental ventral (VTA) en los roedores.

Perfiles Integral del Reglamento de la dopamina en la sustancia negra y el área tegmental ventral

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

comprehensive-profiling-dopamine-regulation-substantia-nigra-ventral

Research

JoVE Journal

Neuroscience

25.6K vistas.  Louisiana State University Health Sciences Center. La dopamina está claramente regulado en los núcleos del cerebro medio, que contiene la celda de los cuerpos y las dendritas de las neuronas de dopamina. Aquí se describe un método de disección y manejo de la muestra-para maximizar los resultados, y por lo tanto las conclusiones y puntos de vista, sobre la regulación de la dopamina en el núcleo del cerebro medio de la sustancia negra (SN) y el área tegmental ventral (VTA) en los roedores.

Video: Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

Medición única célula de la liberación de dopamina simultánea con fijación de voltaje y Amperometría

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

Investigación

Neurociencias

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

En Expression in ovo de microARN en ventral del polluelo Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

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, ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Microdisección de captura por láser - Una demostración del aislamiento individual de dopamina neuronas y el Área ventral tegmental completo

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

Evaluación de la Homeostasis dopaminérgica en ratones por medio de análisis de cromatografía líquida de alto rendimiento y absorción de la dopamina Synaptosomal

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...

Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the substantia nigra, is essential. To estimate the total dopaminergic neuron number, unbiased stereology using the optical fractionator method is currently considered the gold standard. Because the theory behind the optical fractionator method is complex and because stereology is difficult to achieve without specialized equipment, several commercially available complete stereology systems that include the necessary software do exist, purely for cell counting reasons. Since purchasing a specialized stereology setup is not always feasible, for many reasons, this report describes a method for the stereological estimation of dopaminergic neuronal cell counts using standard microscopy equipment, including a light microscope, a motorized object table (x, y, z plane) with imaging software, and a computer for analysis. A step-by-step explanation is given on how to perform stereological quantification using the optical fractionator method, and pre-programmed files for the calculation of estimated cell counts are provided. To assess the accuracy of this method, a comparison to data obtained from a commercially available stereology apparatus was performed. Comparable cell numbers were found using this protocol and the stereology device, thus demonstrating the precision of this protocol for unbiased stereology.

This work presents a step-by-step protocol for the unbiased stereological estimation of dopaminergic neuronal cell numbers in the mouse substantia nigra using standard microscopy equipment (i.e., a light microscope, a motorized object table (x, y, z plane), and public domain software for digital image analysis.

Estereológicos estimación de número de la neurona dopaminérgica en el Nigra del Substantia de ratón usando el fraccionador óptico y equipo de microscopía estándar

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

Identificación del Receptor de dopamina D1-alfa en roedores núcleo Accumbens por una innovador ARN In Situ la tecnología de detección

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

Preparación de láminas agudas de la médula espinal para celulares Patch-clamp de grabación en las neuronas de la sustancia Gelatinosa

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 days a year. Participants consent to donate their brain and spinal cord. Most patients were followed by neurologists at the Cleveland Clinic Mellen Center for MS Treatment and Research. Their clinical courses and neurological disabilities are well-characterized. Soon after death, the body is transported to the MS Imaging Center, where the brain is scanned in situ by 3 T magnetic resonance imaging (MRI). The body is then transferred to the autopsy room, where the brain and spinal cord are removed. The brain is divided into two hemispheres. One hemisphere is immediately placed in a slicing box and alternate 1 cm-thick slices are either fixed in 4% paraformaldehyde for two days or rapidly frozen in dry ice and 2-methylbutane. The short-fixed brain slices are stored in a cryopreservation solution and used for histological analyses and immunocytochemical detection of sensitive antigens. Frozen slices are stored at -80 &#176;C and used for molecular, immunocytochemical, and in situ hybridization/RNA scope studies. The other hemisphere is placed in 4% paraformaldehyde for several months, placed in the slicing box, re-scanned in the 3 T magnetic resonance (MR) scanner and sliced into centimeter-thick slices. Postmortem in situ MR images (MRIs) are co-registered with 1 cm-thick brain slices to facilitate MRI-pathology correlations. All brain slices are photographed and brain white-matter lesions are identified. The spinal cord is cut into 2 cm segments. Alternate segments are fixed in 4% paraformaldehyde or rapidly frozen. The rapid procurement of postmortem MS tissues allows pathological and molecular analyses of MS brains and spinal cords and pathological correlations of brain MRI abnormalities. The quality of these rapidly-processed postmortem tissues (usually within 6 h of death) is of great value to MS research and has resulted in many high-impact discoveries.

Multiple sclerosis is an inflammatory demyelinating disease with no cure. Analysis of brain tissue provides important clues to understanding the pathogenesis of the disease. Here we discuss the methodology and downstream analysis of MS brain tissue collected through a unique rapid autopsy program in operation at the Cleveland Clinic.

Programa integral de autopsias para individuos con esclerosis múltiple

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 ...

Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry (CIS-FSCV) to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

Phasic dopamine (DA) release from the ventral tegmental area (VTA) to the nucleus accumbens plays a pivotal role in reward processing and reinforcement learning. Understanding how the diverse neuronal inputs into the VTA control phasic DA release can provide a better picture of the circuitry that controls reward processing and reinforcement learning. Here, we describe a method that combines intra-VTA cannula infusions of pharmacological agonists and antagonists with stimulation-evoked phasic DA release (combined infusion and stimulation, or CIS) as measured by in vivo fast-scan cyclic voltammetry (FSCV). Using CIS-FSCV in anesthetized rats, a phasic DA response can be evoked by electrically stimulating the VTA with a bipolar electrode fitted with a cannula while recording in the nucleus accumbens core. Pharmacological agonists or antagonists can be infused directly at the stimulation site to investigate specific VTA receptors&#39; roles in driving phasic DA release. A major benefit of CIS-FSCV is that VTA receptor function can be studied in vivo, building on in vitro studies.

Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry CIS-FSCV to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

The goal of this protocol is to directly manipulate ventral tegmental area receptors to study their contribution to subsecond dopamine release.

Infusión y estimulación combinadas con voltamperometría cíclica de exploración rápida (CIS-FSCV) para evaluar la regulación del receptor del área tegmental ventral de la dopamina fásica

Phasic dopamine (DA) release from the ventral tegmental area (VTA) to the nucleus accumbens plays a pivotal role in reward processing and reinforcement ...