This work was supported by core facilities of the Center for Translational Neuroscience supported by NIH award P20 GM103425 and P30 GM110702 to Dr. Garcia-Rill. This work was also supported by grants from FONCYT-Agencia Nacional de Promoci&#242;n Cient&#236;fica y Tecnol&#242;gica; BID 1728 OC.AR. PICT-2012-1769 and UBACYT 2014-2017 #20120130101305BA (to Dr. Urbano).

Todos los protocolos experimentales fueron aprobados por el Comité de Cuidado y Uso de Animales Institucional de la Universidad de Arkansas para las Ciencias Médicas (Protocolo número # 3593) y estaban de acuerdo con los Institutos Nacionales de Salud directrices para el cuidado y uso de animales de laboratorio. 1. Preparación de líquido cefalorraquídeo Standard-artificial (ACSF) <ol><li> Preparación de la disolución de la A <ol><li> Añadir 700 ml de agua destilada a un vaso de precipitados limpio 1 L antes de la adición de productos químicos. </li><li> Mientras se agitaba continuamente un volumen de 500 ml, añadir 136,75 g de NaCl, 6,99 g de KCl, 2,89 g de MgSO 4, y 2,83 g de NaH 2 PO 4. </li><li> Añadir más agua destilada hasta alcanzar un volumen final de 1 L. Después de la dilución, la concentración final de cada compuesto es 117 mM NaCl, KCl 4,69 mM, 1,2 mM de MgSO4, y 1,18 mM NaH 2 PO 4. </li><li> Mantener refrigerado a 4 ° C durante un máximo de 2 semanas. </li></ol></li><li> Preparación de la disolución de la B <ol><li> Añadir 700 ml de agua destilada a un vaso de precipitados de 1 l limpia antes de la adición de productos químicos. </li><li> Mientras se agitaba continuamente un volumen de 500 ml, añadir 41,45 g de D-glucosa y 41,84 g de NaHCO 3. </li><li> Añadir más agua destilada hasta alcanzar un volumen final de 1 L. Después de la dilución, la concentración final de cada compuesto es de 11,5 mM de D-glucosa y 24,9 mM de NaHCO3. </li><li> Mantener refrigerado a 4 ° C durante un máximo de 2 semanas. </li><li> Antes de cada mezcla de ensayo de 50 ml de la acción A con 50 ml de solución madre B y llevar a 1 l con agua destilada para obtener una solución definitiva LCRa concentración y dejar a temperatura ambiente mientras se continua la oxigenación con Carbógeno (95% O 2 - 5% de CO 2 de la mezcla) durante al menos 30 min. Preparar LCRa concentración final sólo al comienzo de cada experimento y desechar al final del día. </li></ol></li></ol> 2. Preparación de líquido cefalorraquídeo sacarosa-artificial(Sacarosa-LCRa) <ol><li> Preparación de la solución de la C <ol><li> Añadir 700 ml de agua destilada a un vaso de precipitados limpio 1 L antes de la adición de productos químicos. </li><li> Mientras se agita continuamente a 500 ml de agua destilada, añadir 240 g de sacarosa, 6,55 g de NaHCO 3, 0,671 g de KCl, 4,88 g de MgCl 2, 0,22 g de CaCl 2 y 0,21 g de ácido ascórbico. </li><li> Añadir más agua destilada para llegar a un volumen final de 1 L. Después de la dilución, la concentración final de cada compuesto es sacarosa 701,1 mM, 78 mM NaHCO 3, 9 mM de KCl, 24 mM MgCl 2, CaCl 1,5 mM de ácido ascórbico 2 y 1,2 mM . </li><li> Mantenga disolución madre C a 4 ° C durante un máximo de una semana. </li><li> Antes de cada mezcla experimento 300 ml de 50 ml de Stock C con 600 ml de agua destilada para obtener la solución de sacarosa-LCRa a la concentración final y dejar a temperatura ambiente mientras que continuamente la oxigenación con carbógeno (95% O 2 - 5% de CO 2 de la mezcla) durante al menos 30 min. </li></ol&gt; </li></ol> 3. Preparación de la rebanada <ol><li> Coloque un vaso de precipitados limpio con 100 ml de solución de sacarosa-LCRa en hielo, mientras que la oxigenación con carbógeno y corregir el pH a 7,4 usando un medidor de pH, mientras que la adición de unas gotas de solución 0,1 M de NaOH (cuando pH &lt;7,4) o solución de HCl 0,1 M (cuando pH&gt; 7,4). </li><li> Llenar una cámara de corte de un vibratome con sacarosa-ACSF y oxigenar la misma. Encienda el sistema de refrigeración basado en glicerol acoplado a la cámara de corte y esperar 15 minutos para permitir que se enfríe a 0 - 4 ° C. </li><li> Anestesiar a las crías de rata (9 a 12 años de adulto oportuna embarazadas ratas Sprague-Dawley) con ketamina (70 mg / kg, ip, usando &lt;50 l de volumen final). Cuando las crías está en calma, vuelve a comprobar que el reflejo de pinzamiento del rabo está ausente. </li><li> Decapitar a los cachorros. <ol><li> Cortar la piel de la cabeza longitudinalmente desde el frente hacia atrás utilizando una hoja de bisturí de acero al carbono, y tire de la piel a los lados con fórceps. Cortar el hueso que cubre el cerebro se mueva el indicador scissors lateralmente y totalmente quitarlo para exponer el cerebro. </li><li> A continuación, retire rápidamente el cerebro usando una espátula (inicialmente colocado bajo el bulbo olfatorio con el fin de empujar suavemente el cerebro desde la más rostral hacia las zonas más caudal). Empuje suavemente el cerebro en el líquido enfriado con hielo oxigenada sacarosa-cefalorraquídeo artificial (ACSF-sacarosa). </li></ol></li><li> Haga un corte parasagital en el hemisferio derecho (la eliminación de aproximadamente un tercio del hemisferio), y la cola de la parte recortada del cerebro en un disco metálico que se fija magnéticamente a la cámara de corte de un vibroslicer para cortar sagitales 400 micras secciones que contienen la núcleo pedunculopontino (PPN). Mantenga rebanadas PPN a temperatura ambiente durante 45 minutos antes de patch-clamp grabaciones de células enteras. </li></ol> 4. Las oscilaciones Grabaciones gamma-banda en PPN Rebanadas <ol><li> Preparación de la solución intracelular de potasio-gluconato (alta-K Solution +) <ol><li> Colocar enhielo en un vaso de precipitados limpio con 10 ml de agua destilada. Mientras se agitaba continuamente añadir: K + -gluconate, 90.68 mg fosfocreatina di Tris sal, HEPES 47.66 mg, 1,5 mg de EGTA, 40,58 mg de Mg 2 + -ATP, y 4 mg de Na + 2 -GTP. </li><li> Ajustar el pH a 7,3 con KOH (100 mM en agua destilada). Añadir agua destilada hasta alcanzar un volumen final de 20 ml. Si es necesario, ajustar la osmolaridad con sacarosa (100 mM en agua destilada) a ser 280 a 290 mOsm. Después de la dilución, la concentración final de cada compuesto es 124 mM K + -gluconate, 10 mM fosfocreatina di sal Tris, HEPES 10 mM, EGTA 0,2 mM, Mg2 + -ATP 4, y Na 0,3 mM + 2 -GTP. </li><li> soluciones alícuota de 1 ml intracelulares en tubos y congelación a -20 ° C. Utilice una alícuota por día y mantener a 4 ° C durante los experimentos. </li></ol></li><li> De células enteras Patch Clamp Grabaciones <ol><li> Coloque las rebanadas en una cámara de inmersión y perfundir ellos (1,5 ml / min) con oxigenada(95% O 2 - 5% de CO 2) LCRa que contiene los siguientes antagonistas de los receptores: antagonista del receptor de ácido selectivo de NMDA 2-amino-5-fosfonovalérico (APV, 40 M), competitivo AMPA / kainato antagonista del receptor de glutamato 6-ciano-7- nitroquinoxalina-2,3-diona (CNQX, 10μM), antagonista del receptor de glicina estricnina (STR, 10 M), el GABA específica A gabazine antagonista del receptor (GBZ, 10 M), y sodio tetrodotoxina bloqueador de los canales (TTX, 3μM) NOTA: En los resultados y las cifras, estos antagonistas se denominan colectivamente como bloqueadores sinápticas o SBS. </li><li> Llenar las pipetas de parche de grabación (2-7 Resistencia mO; hecha de gruesos tubos capilares regulares, disponibles en el mercado de vidrio de borosilicato de pared de 1,0 mm de diámetro exterior y 0,6 mm de diámetro interior) con la solución intracelular de alto K + utilizando rellenos patch-pipetas disponibles en el mercado con una filtro de la solución. Inserte la pipeta en el soporte de su amplificador. Aplicar una pequeña pospresión itive usando una jeringa de 1 ml conectada al soporte de pipeta usando un tubo de silicona conectado a una válvula de tres vías. Conecte la parte posterior del soporte de pipeta a un amplificador de patch clamp. </li><li> Mover la pipeta de grabación utilizando un micromanipulador mecánico cerca del núcleo PPN usando un objetivo 4X combinado de interferencia diferencial óptica de contraste en el infrarrojo cercano. NOTA: núcleo PPN se puede observar en rodajas dorsal al pedúnculo cerebeloso superior (SCP; observable como un grueso manojo de los axones). Pipetas de registro se encuentran en la PPN pars compacta, que se encuentra inmediatamente dorsal al extremo posterior del pedúnculo. </li><li> Llevar la pipeta de grabación en contacto con una neurona PPN mientras visualizaron utilizando un objetivo de inmersión 40X agua, y rápidamente aplicar succión negativa para formar un sello con la célula. </li><li> Utilice el software de sellado de fijación de voltaje para controlar la resistencia de la pipeta durante la succión negativa usando el protocolo del fabricante. <ol><li> Cuando s negativosucción se aumenta lentamente y los valores de resistencia de lectura por el monitor de patch-clamp en la punta de la pipeta alcanzar 80 a 100 MOhm, cambiar rápidamente el potencial de mantenimiento de -50 mV y liberar la presión negativa. Iniciar la aplicación continua de succión negativa hasta la ruptura de membrana y eléctrica acceso de la neurona se consigue en la configuración de célula completa. </li><li> Si los valores de resistencia de acceso medidos por el software de sellado de fijación de voltaje son de 10 MOhm o superior, y luego continuar aplicando succión negativa en cantidades más pequeñas. </li></ol></li><li> Compensar capacitancia (es decir, transitorios lentos y rápidos observados después de la ruptura de la membrana de la célula) y la resistencia en serie en el modo de fijación de voltaje. Cambiar el modo de grabación de fijación de corriente, y rápidamente compensar los valores de puente (por ejemplo, mover botón del amplificador o haga clic en el menú de compensación automática a través del ratón computer&#39;s). <ol><li> Un seguimiento continuo de descanso potencial de membrana de la neurona PPN está grabando ucantar el protocolo del fabricante. Si el potencial de membrana en reposo cambio hacia valores de despolarización o hiperpolarización, a continuación, utilizar pequeñas cantidades de corriente continua (hasta 100 Pa) para mantener el valor final óptimo -50 mV. NOTA: Mantener sólo las neuronas PPN con un potencial de membrana en reposo estable (PGR) de -48 mV o más hiperpolarizado (es decir, RMP &lt;-48 mV). </li></ol></li></ol></li></ol>

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</ol>

The authors declare that they have no competing financial interests.

PPN neuronas tienen propiedades intrínsecas que les permiten disparar potenciales de acción en las frecuencias de la banda beta / gamma durante las grabaciones en vivo de los animales que están despiertos o durante el sueño REM, pero no durante el sueño de ondas lentas 2,3,5,13-17. Otros autores han mostrado que los cortes transversales del tallo cerebral en más niveles anteriores de PPN reduce frecuencias gamma durante los registros de EEG. Sin embargo, cuando las lesiones del tronco cerebral ...

núcleo PPN se incluye anatómicamente en el tegmento mesencefálico caudal. El PPN es un componente clave de RAS 1. El PPN participa en el mantenimiento de los estados activados de comportamiento (es decir, el despertar, el sueño REM) 2. La estimulación eléctrica de la PPN in vivo induce la oscilación rápida (20 - 40 Hz) en el EEG cortical 3, mientras que las lesiones bilaterales PPN en la rata reducen o eliminan el sueño REM 4. Si bien la mayoría de las neuronas PPN disparar los potenciales de acción en frecuencia / gamma-banda beta (20 - 80 Hz), algunas neuronas presentan bajas tasas de activación espontánea (&lt;10 Hz) 5. Por otra parte, la PPN parece estar implicado en otros aspectos del comportamiento tales como la motivación y la atención 6. Alta frecuencia directa (40 - 60 Hz) 7 estimulación eléctrica del núcleo PPN en animales descerebrados puede promover la locomoción. En los últimos años, la estimulación profunda del cerebro (DBS) de PPN se ha utilizado para tratar a pacientes que sufren frotrastornos que implican déficits m de la marcha, como la enfermedad (PD) 8 de Parkinson. Los informes previos demostraron que casi todas las neuronas PPN pueden disparar potenciales de acción en la frecuencia de la banda gamma cuando se despolariza el uso de pulsos de corriente 9 cuadrados. Debido a la activación drástica de los canales de potasio dependientes de voltaje durante las despolarizaciones pulsos cuadrados hasta o bajo -25 mV. Como consecuencia, no se observaron fuertes oscilaciones gamma después de bloquear la generación de potenciales de acción utilizando la tetrodotoxina 10. En un esfuerzo por evitar tal problema, 1 - Se han usado 2 seg largo de despolarización rampas de corriente. Rampas despolariza gradualmente el potencial de membrana a partir de valores de hasta 0 mV en reposo, mientras se inactiva parcialmente los canales de potasio dependientes de voltaje. Claras oscilaciones de la membrana de la banda gamma fueron evidentes dentro de la ventana de dependencia de voltaje de los canales de calcio de alto umbral (es decir, entre -25 mV y -0 mV) 10. En conclusión, la banda gamma activiTy se observó en las neuronas PPN 9, y Q y los canales de calcio dependientes de voltaje tipo N necesita / P tanto para ser activado con el fin de generar las oscilaciones de la banda gamma en el PPN 10. Una serie de estudios determinó la ubicación de los canales de calcio en las neuronas de alto umbral PPN. La inyección de la combinación de colorantes, imágenes de fluorescencia radiométrica mostró transitorios de calcio a través de los canales de calcio dependientes de voltaje que se activan en diferentes dendritas cuando despolarizado utilizando rampas de corriente 11. propiedades intrínsecas de las neuronas PPN se han sugerido para permitir la activación simultánea de estas células durante la vigilia y el sueño REM, induciendo así la actividad neuronal oscilatoria de alta frecuencia entre el SRA y bucles thalamocortical. Dicha interacción de largo alcance se considera para apoyar un estado cerebral capaz de evaluar de manera fiable el mundo que nos rodea de forma continua 12. A continuación, describimos el experimentoAL condiciones necesarias para generar y mantener la oscilación gamma banda en células in vitro PPN. Este protocolo no se ha descrito anteriormente, y ayudaría a una serie de grupos para estudiar las propiedades intrínsecas de membrana que median la actividad de la banda gamma a otras áreas del cerebro. Por otra parte, las medidas actuales podrían conducir a la conclusión errónea de que la actividad de la banda gamma no se puede generar en estas células.

<table><tbody><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S8501</td><td>C&lt;sub&gt;12&lt;/sub&gt;H&lt;sub&gt;22&lt;/sub&gt;O&lt;sub&gt;11&lt;/sub&gt;, molecular weight = 342.30</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma-Aldrich</td><td>S6014</td><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;, molecular weight = 84.01</td></tr><tr><td>Potassium Chloride</td><td>Sigma-Aldrich</td><td>P3911</td><td>KCl, molecular weight = 74.55</td></tr><tr><td>Magnesium Chloride Hexahydrate</td><td>Sigma-Aldrich</td><td>M9272</td><td>MgCl&lt;sub&gt;2&lt;/sub&gt; &amp;middot; 6H&lt;sub&gt;2&lt;/sub&gt;O, molecular weight =&amp;nbsp; 203.30</td></tr><tr><td>Calcium Chloride Dihydrate</td><td>Sigma-Aldrich</td><td>C3881</td><td>CaCl&lt;sub&gt;2&lt;/sub&gt; &amp;middot; 2H&lt;sub&gt;2&lt;/sub&gt;O, molecular weight =147.02</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G5767</td><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;12&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;, molecular weight = 180.16</td></tr><tr><td>L-Ascorbic Acid</td><td>Sigma-Aldrich</td><td>A5960</td><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;8&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;, molecular weight =176.12</td></tr><tr><td>Sodium Chloride</td><td>Acros Organics</td><td>327300025</td><td>NaCl, molecular weight =&amp;nbsp; 58.44</td></tr><tr><td>Potassium Gluconate</td><td>Sigma-Aldrich</td><td>G4500</td><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;11&lt;/sub&gt;KO&lt;sub&gt;7&lt;/sub&gt;, molecular weight =&amp;nbsp; 234.25</td></tr><tr><td>Phosphocreatine di(tris) salt</td><td>Sigma-Aldrich</td><td>P1937</td><td>C&lt;sub&gt;4&lt;/sub&gt;H&lt;sub&gt;10&lt;/sub&gt;N&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;5&lt;/sub&gt;P &amp;middot; 2C&lt;sub&gt;4&lt;/sub&gt;H&lt;sub&gt;11&lt;/sub&gt;NO&lt;sub&gt;3&lt;/sub&gt;, molecular weight =&amp;nbsp; 453.38</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td>C&lt;sub&gt;8&lt;/sub&gt;H&lt;sub&gt;18&lt;/sub&gt;N&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;4&lt;/sub&gt;S, molecular weight = 238.30</td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>E0396</td><td>[-CH&lt;sub&gt;2&lt;/sub&gt;OCH&lt;sub&gt;2&lt;/sub&gt;CH&lt;sub&gt;2&lt;/sub&gt;N(CH&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;2&lt;/sub&gt;H)&lt;sub&gt;2&lt;/sub&gt;]&lt;sub&gt;2&lt;/sub&gt;, molecular weight = 380.40</td></tr><tr><td>Adenosine 5&#039;-triphosphate magnesium salt</td><td>Sigma-Aldrich</td><td>A9187</td><td>&amp;nbsp;C&lt;sub&gt;10&lt;/sub&gt;H&lt;sub&gt;16&lt;/sub&gt;N&lt;sub&gt;5&lt;/sub&gt;O&lt;sub&gt;13&lt;/sub&gt;P&lt;sub&gt;3&lt;/sub&gt; &amp;middot; xMg2+, molecular weight = 507.18</td></tr><tr><td>Guanosine 5&#039;-triphosphate sodium salt hydrate</td><td>Sigma-Aldrich</td><td>G8877</td><td>C&lt;sub&gt;10&lt;/sub&gt;H&lt;sub&gt;16&lt;/sub&gt;N&lt;sub&gt;5&lt;/sub&gt;O&lt;sub&gt;14&lt;/sub&gt;P&lt;sub&gt;3&lt;/sub&gt; &amp;middot; xNa+, molecular weight = 523.18</td></tr><tr><td>Tetrodotoxin citrate</td><td>Alomone Labs</td><td>T-550</td><td>C&lt;sub&gt;11&lt;/sub&gt;H&lt;sub&gt;17&lt;/sub&gt;N&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;8&lt;/sub&gt;, molecular weight = 319.27</td></tr><tr><td>&amp;nbsp;DL-2-Amino-5-Phosphonovaleric Acid</td><td>Sigma-Aldrich</td><td>A5282</td><td>&amp;nbsp;C&lt;sub&gt;5&lt;/sub&gt;H&lt;sub&gt;12&lt;/sub&gt;NO&lt;sub&gt;5&lt;/sub&gt;P, molecular weight = 197.13</td></tr><tr><td>CNQX disodium salt hydrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C239</td><td>C&lt;sub&gt;9&lt;/sub&gt;H&lt;sub&gt;2&lt;/sub&gt;N&lt;sub&gt;4&lt;/sub&gt;Na&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;4&lt;/sub&gt; &amp;middot; xH2O, molecular weight = 276.12</td></tr><tr><td>Strychnine</td><td>Sigma-Aldrich</td><td>S0532</td><td>C&lt;sub&gt;21&lt;/sub&gt;H&lt;sub&gt;22&lt;/sub&gt;N&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;, molecular weight = 334.41</td></tr><tr><td>Mecamylamine hydrochloride</td><td>Sigma-Aldrich</td><td>M9020</td><td>&amp;nbsp;C&lt;sub&gt;11&lt;/sub&gt;H&lt;sub&gt;21&lt;/sub&gt;N &amp;middot; HCl, molecular weight = 203.75</td></tr><tr><td>Gabazine (SR-95531)</td><td>Sigma-Aldrich</td><td>S106</td><td>C&lt;sub&gt;15&lt;/sub&gt;H&lt;sub&gt;18&lt;/sub&gt;BrN&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt;, molecular weight = 368.23</td></tr><tr><td>Ketamine hydrochloride</td><td>Mylan</td><td>67457-001-00</td><td></td></tr><tr><td>Microscope</td><td>Nikon</td><td>Eclipse E600FN</td><td></td></tr><tr><td>Micromanipulator</td><td>Sutter Instruments</td><td>ROE-200</td><td></td></tr><tr><td>Micromanipulator</td><td>Sutter Instruments</td><td>MPC-200</td><td></td></tr><tr><td>Amplifier</td><td>Molecular Devices</td><td>Multiclamp 700B</td><td></td></tr><tr><td>A/D converter</td><td>Molecular Devices</td><td>Digidata 1440A</td><td></td></tr><tr><td>Heater</td><td>Warner Instruments</td><td>TC-324B</td><td></td></tr><tr><td>Pump</td><td>Cole-Parmer</td><td>Masterflex L/S 7519-20</td><td></td></tr><tr><td>Pump cartridge</td><td>Cole-Parmer</td><td>Masterflex 7519-85</td><td></td></tr><tr><td>Pipette puller</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr><tr><td>Camera</td><td>Q-Imaging</td><td>RET-200R-F-M-12-C</td><td></td></tr><tr><td>Vibratome</td><td>Leica Biosystems</td><td>&amp;nbsp;Leica VT1200 S</td><td></td></tr><tr><td>Refrigeration system</td><td>Vibratome Instruments</td><td>900R</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>microscope</td><td>Nikon</td><td>Eclipse E600FN</td><td></td></tr><tr><td>micromanipulator</td><td>Sutter Instruments</td><td>ROE-200</td><td></td></tr><tr><td>micromanipulator</td><td>Sutter Instruments</td><td>MPC-200</td><td></td></tr><tr><td>amplifier</td><td>Molecular Devices</td><td>Multiclamp 700B</td><td></td></tr><tr><td>A/D converter</td><td>Molecular Devices</td><td>Digidata 1440A</td><td></td></tr><tr><td>heater</td><td>Warner Instruments</td><td>TC-324B</td><td></td></tr><tr><td>pump</td><td>Cole-Parmer</td><td>Masterflex L/S 7519-20</td><td></td></tr><tr><td>pump cartridge</td><td>Cole-Parmer</td><td>Masterflex 7519-85</td><td></td></tr><tr><td>pipette puller</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr><tr><td>camera</td><td>Q-Imaging</td><td>RET-200R-F-M-12-C</td><td></td></tr></tbody></table>

recording gamma band oscillations in pedunculopontine nucleus neurons

Eferentes sinápticas de la PPN son conocidos para modular la actividad neuronal de varias regiones talámicas intralaminares (por ejemplo, la centrolateral / parafascicular; núcleo Cl / Pf). La activación de cualquiera de los PPN o núcleos Cl / PF en vivo se ha descrito para inducir el despertar del animal y un incremento en la actividad de la banda gamma en el electroencefalograma cortical (EEG). Los mecanismos celulares para la generación de oscilaciones de la banda gamma en el Sistema de Activación Reticular (RAS), las neuronas son las mismas que las que se encuentran para generar oscilaciones de la banda gamma en otros núcleos cerebros. Durante las grabaciones de corriente-clamp de neuronas (PPN de rebanadas de parasagittal 9 - ratas 25 días de edad), el uso de despolarización pasos cuadrados activa rápidamente los canales de potasio dependientes de voltaje que impedían neuronas PPN de ser despolarizado allá de -25 mV. La inyección de 1 - 2 seg tiempo de despolarización rampas de corriente despolariza gradualmente PPN potencial de membrana resting valores hacia 0 mV. Sin embargo, la inyección de despolarizantes pulsos cuadrados generan oscilaciones de banda gamma de potencial de membrana que mostraron ser más pequeña en amplitud en comparación con las oscilaciones generadas por rampas. Todos los experimentos se realizaron en presencia de los canales de sodio dependientes de voltaje y rápidas bloqueadores de receptores sinápticos. Se ha demostrado que la activación de los canales de calcio dependientes de voltaje de umbral alto subyacen la actividad oscilatoria de banda gamma en las neuronas PPN. Intervenciones farmacológicas y metodológicos específicos se describen aquí, proporcionando las herramientas necesarias para inducir y mantener la PPN por debajo del umbral de oscilación banda gamma in vitro.

Inicialmente, oscilaciones gamma fueron evocados utilizando impulsos de corriente cuadrados. Grabación abrazadera actual de las neuronas PPN en presencia de bloqueadores sinápticas y TTX se monitorizó continuamente para asegurar que el potencial de reposo de la membrana se mantuvo estable a ~ -50 mV (Figura 1A). Dos segundos impulsos de corriente largos cuadrados fueron inyectadas intracelularmente por el amplificador de pinzamiento zonal a través de la pipeta de la ...

Watch this Scientific Journal Video about Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons at JoVE.com

Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

El núcleo pedunculopontino (PPN) se encuentra en el tronco del encéfalo y sus neuronas están máximamente activa durante la vigilia y el movimiento ocular rápido (REM) del sueño cerebrales estados. Este trabajo describe el enfoque experimental para registrar en la banda gamma oscilación de la membrana por debajo del umbral vitro en neuronas PPN.

Grabación de la banda gamma oscilaciones en pedunculopontino Núcleo neuronas

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the ...

recording-gamma-band-oscillations-in-pedunculopontine-nucleus-neurons

Research

JoVE Journal

Neuroscience

8.6K vistas.  University of Buenos Aires.  El núcleo pedunculopontino (PPN) se encuentra en el tronco del encéfalo y sus neuronas están máximamente activa durante la vigilia y el movimiento ocular rápido (REM) del sueño cerebrales estados. Este trabajo describe el enfoque experimental para registrar en la banda gamma oscilación de la membrana por debajo del umbral vitro en neuronas PPN. 

Video: Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

Caracterización fisiológicas, morfológicas y neuroquímicas de las neuronas modulada por el Movimiento

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Investigación

Neurociencias

Automatic Detection of Highly Organized Theta Oscillations in the Murine EEG

Theta activity is generated in the septohippocampal system and can be recorded using deep intrahippocampal electrodes and implantable electroencephalography (EEG) radiotelemetry or tether system approaches. Pharmacologically, hippocampal theta is heterogeneous (see dualistic theory) and can be differentiated into type I and type II theta. These individual EEG subtypes are related to specific cognitive and behavioral states, such as arousal, exploration, learning and memory, higher integrative functions, etc. In neurodegenerative diseases such as Alzheimer&#39;s, structural and functional alterations of the septohippocampal system can result in impaired theta activity/oscillations. A standard quantitative analysis of the hippocampal EEG includes a Fast-Fourier-Transformation (FFT)-based frequency analysis. However, this procedure does not provide details about theta activity in general and highly-organized theta oscillations in particular. In order to obtain detailed information on highly-organized theta oscillations in the hippocampus, we have developed a new analytical approach. This approach allows for time- and cost-effective quantification of the duration of highly-organized theta oscillations and their frequency characteristics.

Theta activity in the hippocampus is related to specific cognitive and behavioral stages. Here, we describe an analytical method to detect highly-organized theta oscillations within the hippocampus using a time-frequency (i.e., wavelet analysis)-based approach.

Detección automática de muy organizada oscilaciones theta en el EEG murino

Theta activity is generated in the septohippocampal system and can be recorded using deep intrahippocampal electrodes and implantable ...

Comportamiento

Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses

In vivo recordings from single neurons allow an investigator to examine the firing properties of neurons, for example in response to sensory stimuli. Neurons typically receive multiple excitatory and inhibitory afferent and/or efferent inputs that integrate with each other, and the ultimate measured response properties of the neuron are driven by the neural integrations of these inputs. To study information processing in neural systems, it is necessary to understand the various inputs to a neuron or neural system, and the specific properties of these inputs. A powerful and technically relatively simple method to assess the functional role of certain inputs that a given neuron is receiving is to dynamically and reversibly suppress or eliminate these inputs, and measure the changes in the neuron's output caused by this manipulation. This can be accomplished by pharmacologically altering the neuron's immediate environment with piggy-back multibarrel electrodes. These electrodes consist of a single barrel recording electrode and a multibarrel drug electrode that can carry up to 4 different synaptic agonists or antagonists. The pharmacological agents can be applied iontophoretically at desired times during the experiment, allowing for time-controlled delivery and reversible reconfiguration of synaptic inputs. As such, pharmacological manipulation of the microenvironment represents a powerful and unparalleled method to test specific hypotheses about neural circuit function.
Here we describe how piggy-back electrodes are manufactured, and how they are used during in vivo experiments. The piggy-back system allows an investigator to combine a single barrel recording electrode of any arbitrary property (resistance, tip size, shape etc) with a multibarrel drug electrode. This is a major advantage over standard multi-electrodes, where all barrels have more or less similar shapes and properties. Multibarrel electrodes were first introduced over 40 years ago 1-3, and have undergone a number of design improvements 2,3 until the piggy-back type was introduced in the 1980s 4,5. Here we present a set of important improvements in the laboratory production of piggy-back electrodes that allow for deep brain penetration in intact in vivo animal preparations due to a relatively thin electrode shaft that causes minimal damage. Furthermore these electrodes are characterized by low noise recordings, and have low resistance drug barrels for very effective iontophoresis of the desired pharmacological agents.

Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses

Iontophoresis of neural agonists and antagonists during extracellular in vivo recordings is a powerful way to manipulate a neuron&#x2019;s microenvironment. These manipulations can most easily be done via piggy-back multibarrel electrodes. Here we describe how to manufacture them and use them during auditory recordings.

Fabricación y uso de Piggy-back Multibarrel Electrodos para<em&gt; En vivo</em&gt; Manipulaciones farmacológicas de las respuestas neuronales

In vivo recordings from single neurons allow an investigator to examine the firing properties of neurons, for example in response to sensory stimuli. ...

Generation of Local CA1 &#947; Oscillations by Tetanic Stimulation

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A protocol is provided to generate a model for studying CA1 &#947; oscillations (20 - 80 Hz). These &#947; oscillations are stable for at least 30 min&#160;and depend upon excitatory and inhibitory synaptic activity in addition to activation of pacemaker currents. Tetanically stimulated oscillations have a number of reproducible and easily quantifiable characteristics including spike count, oscillation duration, latency and frequency that report upon the network state. The advantages of the electrically stimulated oscillations include stability, reproducibility and episodic acquisition enabling robust characterization of network function. This model of CA1 &#947; oscillations can be used to study cellular mechanisms and to systematically investigate how neuronal network activity is altered in disease and by drugs. Disease state pharmacology can be readily incorporated by the use of brain slices from genetically modified or interventional animal models to enable selection of drugs that specifically target disease mechanisms.

Oscillations are fundamental network properties and are modulated by disease and drugs. Studying brain-slice oscillations allows characterization of isolated networks under controlled conditions. Protocols are provided for the preparation of acute brain slices for evoking CA1 &#947; oscillations.

Generación de oscilaciones γ CA1 locales por tetánica Estimulación

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A ...

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent improvements in methodologies and applications for the study of theta oscillations. A simple characterization of the isolated hippocampal preparation is presented whereby the relationship between internal hippocampal theta oscillators is examined together with the activity of pyramidal cells, and GABAergic interneurons, of the cornu ammonis-1 (CA1) and subiculum (SUB) areas. Overall, we show that the isolated hippocampus is capable of generating intrinsic theta oscillations in vitro and that rhythmicity generated within the hippocampus can be precisely manipulated by optogenetic stimulation of parvalbumin-positive (PV) interneurons. The in vitro isolated hippocampal preparation offers a unique opportunity to use simultaneous field and intracellular patch-clamp recordings from visually-identified neurons to better understand the mechanisms underlying theta rhythm generation.

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

Here, we present a protocol for recording rhythmic neuronal network theta and gamma oscillations from an isolated whole hippocampal preparation. We describe the experimental steps from extraction of the hippocampus to details of field, unitary and whole-cell patch clamp recordings as well as optogenetic pacing of the theta rhythm.

Tuning en la banda Theta Hippocampal<em&gt; In Vitro</em&gt;: Metodologías para la grabación del circuito septohippocampal de roedores aislados

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

Agudo<em&gt; In Vivo</em&gt; Grabaciones electrofisiológicas de potenciales de campo locales y actividad de múltiples unidades de la vía hiperdirecta en ratas anestesiadas

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Optogenetic de arrastre de las oscilaciones Theta Hippocampal en comportarse ratones

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

Grabación espacial restringido a oscilaciones en el hipocampo de ratones de comportarse

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

Electrophysiological Investigations of Retinogeniculate and Corticogeniculate Synapse Function

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ganglion cells and project it to the visual cortex. In addition, relay neurons receive top-down excitation from the cortex. The two main excitatory inputs to the relay neurons differ in several aspects. Each relay neuron receives input from only a few retinogeniculate synapses, which are large terminals with many release sites. This is reflected by the comparably strong excitation, the relay neurons receive, from retinal ganglion cells. Corticogeniculate synapses, in contrast, are simpler with few release sites and weaker synaptic strength. The two synapses also differ in their synaptic short-term plasticity. Retinogeniculate synapses have a high release probability and consequently display a short-term depression. In contrast, corticogeniculate synapses have a low release probability. Corticogeniculate fibers traverse the reticular thalamic nuclei before entering the lateral geniculate nucleus. The different locations of the reticular thalamic nucleus (rostrally from the lateral geniculate nucleus) and optic tract (ventro-laterally from the lateral geniculate nucleus) allow stimulating corticogeniculate or retinogeniculate synapses separately with extracellular stimulation electrodes. This makes the lateral geniculate nucleus an ideal brain area where two excitatory synapses with very different properties impinging onto the same cell type, can be studied simultaneously. Here, we describe a method to investigate the recording from relay neurons and to perform detailed analysis of the retinogeniculate and corticogeniculate synapse function in acute brain slices. The article contains a step-by-step protocol for the generation of acute brain slices of the lateral geniculate nucleus and steps for recording activity from relay neurons by stimulating the optic tract and corticogeniculate fibers separately.

Here, we present protocols for the preparation of acute brain slices containing the lateral geniculate nucleus and the electrophysiological investigation of retinogeniculate and corticogeniculate synapses function. This protocol provides an efficient way to study synapses with the high-release and low-release probability in the same acute brain slices.

Investigaciones electrofisiológicas de la función de sinapsis de retinogeniculato y corticogeniculato

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ...

Recording Gamma Band Oscillations From Pedunculopontine Nucleus Neurons in a Brain Slice

Source: Urbano, F. J., et al. <a href="https://app.jove.com/v/54685">Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons.</a> J. Vis. Exp. (2016).
The video demonstrates the method to record gamma-band oscillations in PPN neurons. Using a sagittal brain section with synaptic blockers in aCSF, it employs positive current pulses via a patch pipette to open calcium channels, inducing crucial subthreshold activity for wakefulness.

Registro de las oscilaciones de la banda gamma de las neuronas del núcleo pedunculopontino en un corte de cerebro

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Grabación de la banda gamma oscilaciones en pedunculopontino Núcleo neuronas

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