We thank Dr. Larry D. Frye for editorial help with this manuscript. This work was supported by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research 24300129 (to M.L.), 25890005 (to Y.O.) and 26640025 (to Y.T.), the National Agriculture and Food Research Organization (to Y.U.), the World Premier International Research Center Initiative (WPI) from the Ministry of Education, Culture, Sports, Science, and Technology (to Y.O., Y.T., Y.U. and M.L.) and the Nestl&#233; Nutrition Council, Japan (to M.L.).

Declaración de Ética: Procedimientos que involucran sujetos animales han sido aprobados por el Comité Experimental Animal Institucional de la Universidad de Tsukuba. 1. Preparación de electrodos y cables de EEG / EMG Grabaciones <ol><li> Preparar EEG / EMG electrodo de registro de acuerdo con el siguiente procedimiento. Nota: El electrodo es desechable y puede ser utilizado sólo para 1 animal. Planee cuidadosamente la configuración del cableado para todos los conectores. Coloque marcas en los conectores para la orientación correcta. <ol><li> Soldar cada pin de una cabecera de 4 pines a un cable de acero inoxidable de 2 cm. En resumen, sostenga un extremo del cable a la clavija, coloque un cautín caliente sobre la articulación de alambre pines y derretir un poco de soldadura para asegurar que sólo lo suficiente de soldadura se ejecuta sin problemas en la articulación. Tenga cuidado de no aplicar demasiado calor para el pasador; de lo contrario, el plástico alrededor de las clavijas se derretirá. </li><li> Suelde el extremo libre de cada uno de 2 cables conectados a la cabecera de pines a la cabezade un tornillo de acero inoxidable 1,0 mm de diámetro. En resumen, mantenga el extremo libre del alambre con el hilo por debajo de la cabeza del tornillo, coloque un cautín caliente sobre la articulación de alambre-tornillo, y derretir un poco de soldadura para asegurar que sólo lo suficiente de soldadura se ejecuta sin problemas en la articulación. Los 2 cables con tornillos sirven como electrodos de registro EEG, mientras que los 2 cables sin tornillos sirven como electrodos de registro EMG. </li><li> Use las tijeras para despojarse de 1 mm del aislamiento en el extremo de los electrodos EMG para aumentar la calidad de la señal EMG. </li><li> Cubra completamente todas las patillas soldadas con adhesivo epoxi mediante el uso de un palo o un diente de selección fina de madera para reducir el ruido eléctrico durante las grabaciones de EEG / EMG. </li></ol></li><li> Preparar un cable para conectar el electrodo con el anillo de deslizamiento tal como se describe a continuación. Este cable puede ser reutilizado. <ol><li> Soldar cada pin de un conector FFC / FPC 4 pines con un alambre de un cable plano de 30 cm. En resumen, mantenga el extremo pelado del cable a la clavija, coloque un cautín calienteen la articulación de alambre pines y derretir un poco de soldadura para asegurar que sólo lo suficiente de soldadura se ejecuta sin problemas en la articulación. Nota: Elija la longitud del cable plano que es apropiado para la altura de la jaula animal de experimentación utilizado para las grabaciones de EEG / EMG. </li><li> Soldadura pines hembra a un extremo de los cables en el otro extremo del cable plano. En resumen, mantenga un enchufe engarzado al extremo libre pelado del cable, coloque un cautín caliente sobre la articulación de alambre-socket, y derretir un poco de soldadura para asegurar que sólo lo suficiente de soldadura se ejecuta sin problemas en la articulación. </li><li> Inserte cada engarzado toma en una posición 4 engarzado vivienda. </li><li> Completamente cubrir el engarzado zócalos con adhesivo epoxi mediante el uso de un palo o un diente de selección fina de madera. </li></ol></li></ol> 2. La implantación de electrodos en la cabeza del ratón (Duración: Aprox. 20 min) <ol><li> Esterilizar todos los instrumentos quirúrgicos en un esterilizador de cuentas en caliente antes de la cirugía. Anestesiar un ratón macho (10 - 20 semanas de edad, 20 - 30 g)con una inyección intraperitoneal de pentobarbital (50 mg / kg). Después de comprobar que el ratón está profundamente anestesiado apretando un dedo del pie, afeitar el pelo en la cabeza y el cuello con una maquinilla. </li><li> Mueve el ratón para el marco estereotáxico, y fijar la cabeza entre los 2 bares del oído. Aplique vaselina en los ojos para evitar la sequedad. Limpiar la piel afeitada con alcohol y cortar a lo largo de la línea media con un escalpelo para exponer el cráneo. Clip de la piel para mantener el área quirúrgica abierta. </li><li> Mediante el uso de un cortador de carburo (tamaño de perforación: 0,8 mm de diámetro), taladro 2 agujeros en el cráneo, uno sobre el área cortical frontal (1 mm anterior al bregma, 1,5 mm lateral a la línea media) y la otra sobre la zona parietal ( 1 mm anterior a lambda, 1,5 mm lateral a la línea media) del hemisferio derecho, de acuerdo a las coordenadas estereotáxicas de Paxinos y Franklin. 23 </li><li> Mediante el uso de un destornillador de joyero, tornillos de grabación de EEG de acero inoxidable lugar en los agujeros y hacen 2 - 2.5 vueltas para cada screw para el posicionamiento epidural sobre la corteza. Nota: No inserte los tornillos demasiado profundas para evitar daños en el tejido cerebral. Compruebe que los tornillos estén bien fijos en el cráneo. Esto es importante contar con señales de EEG estables durante un largo período de varias grabaciones (por lo general, más de 1 mes). Tornillos contoneantes producen artefactos del EEG y pueden salir antes de que finalice el programa experimental. </li><li> Fije el conjunto de electrodos (véase la Sección 1.1, los pines se volvieron hacia arriba) con pegamento instantáneo en el cráneo y cubren con cemento dental. Hacer bilateralmente pequeños agujeros con unas pinzas en los trapecios (cuello) músculos e inserte los cables de acero inoxidable que sirven como EMG electrodos en los orificios. Suturar la piel con un hilo de seda (0,1 mm de diámetro) para evitar la exposición del músculo. </li><li> Retire el ratón del marco estereotáxico. Administrar ampicilina (100 mg / kg) y meloxicam (1 mg / kg) por vía intraperitoneal al ratón para evitar la infección bacteriana y para reducir el dolor post-quirúrgico, respectivamente. Keep el ratón sobre una almohadilla de calor y su seguimiento hasta que se haya recuperado el conocimiento suficiente para mantener decúbito esternal. Casa el ratón de forma individual durante la recuperación para evitar la extracción de electrodos por otros animales, y administrar meloxicam (1 mg / kg) por vía intraperitoneal en el primer día después de la cirugía. </li></ol> 3. Registro y adquisición de EEG / EMG datos <ol><li> Después de un período de recuperación de 1-semana, casa de cada ratón individualmente en una jaula experimental colocado en una cámara de registro a prueba de sonido. Mantener una temperatura ambiente de 23 ± 1 ° C y automáticamente el control de ciclos de 12 horas de luz / 12 horas de oscuridad (luces encendidas a las 08:00, la intensidad de iluminación ~ 100 lux). </li><li> Conecte el conjunto de electrodos EEG / EMG en la cabeza del ratón a un cable de grabación. Asegúrese de que el cable de grabación está conectado a un anillo deslizante (que está diseñado de tal manera que los movimientos del ratón no están restringidos por la torsión del cable) y un amplificador de señal EEG / EMG. Filtrar EEG / señales EMG (EEG, 0,5-64 Hz; EMG, 16-64 Hz), digitalizar a una velocidad de muestreo de 128 Hz por un convertidor de analógico a digital (A / D) y, finalmente, grabar en un equipo que ejecuta el software de grabación de EEG / EMG (4ª entrada &#39;Materiales&#39; Mesa, desde la parte inferior). </li><li> Habituar el ratón durante 2 - 3 días en la cámara de grabación. Si el registro de EEG / EMG incluye administraciones intraperitoneales de drogas, manejar suavemente el ratón en cada día de habituación en el momento de la administración del fármaco. </li><li> Posteriormente, iniciar el software EEG / EMG de grabación (&#39;Materiales&#39; Tabla, 4ª entrada de abajo). <ol><li> Seleccione la pestaña &quot;Archivo de datos de la información&quot; y haga clic en la casilla junto al nombre del archivo. Introduzca un nombre de archivo y haga clic en &quot;Guardar&quot;. </li><li> Seleccione la pestaña &quot;condición de grabación&quot; y seleccionar todos los canales EEG / EMG que se pueden grabar. </li><li> Seleccione la frecuencia de muestreo (128 Hz) en la pestaña de &quot;condición de grabación &#39;. </li><li> Compruebe si se muestran correctamente los canales seleccionados in thpestaña e &#39;Canal de información &quot;. </li><li> Seleccione la pestaña &quot;Ajuste del temporizador&quot; y haga clic en &quot;Monitor&quot; para mostrar EEG y EMG. </li><li> Verifique si las señales de EEG / EMG se muestran correctamente. </li><li> Seleccione la pestaña &quot;Ajuste del temporizador&quot; y establecer el tiempo de reloj para el comienzo y el final de la grabación en el área &#39;Timer principal&#39;. </li><li> Haga clic en &quot;Monitor&quot; en la pestaña de &quot;Ajuste del temporizador&quot; para iniciar la grabación. </li></ol></li><li> Registros EEG / EMG señales bajo la línea de base (es decir., La conducta de sueño / vigilia de un ratón libremente comportarse) y diferentes condiciones de tratamiento (por ejemplo., La administración de cafeína o tratamiento simulado) durante varios días. La eutanasia del ratón con una inyección intraperitoneal de pentobarbital (200 mg / kg) después de que el último día experimental. </li></ol> 4. La puntuación de la Conducta Estado Basado en EEG / EMG datos <ol><li> Inicie el software para el análisis de EEG / EMG (&#39;Materiales&#39; Mesa, 4ª entrada de abajo). Abra los datos en bruto del EEG / EMG (archivo .kcd) producidos bajo el paso 3. Haga clic en la pestaña &#39;Sleep&#39; y seleccionar el tiempo de Época. Seleccione 10 seg. </li><li> Haga clic en la pestaña &#39;Sleep&#39; y seleccione &#39;Multi-screening&#39; para anotar automáticamente todas las épocas de 10 seg en 3 etapas (es decir., NREM y sueño REM, y la vigilia) sobre la base de las amplitudes de EEG y EMG y el análisis espectral de potencia del EEG. 3 </li><li> Haga clic en &quot;condición FFT para EEG &#39;. </li><li> Establecer los parámetros para el análisis espectral de potencia [256 puntos de referencia (correspondiente a 2 segundos del EEG), función de ventana Hanning, y el promedio de 5 espectros por época]. 3 Haga clic en &#39;Aceptar&#39;. </li><li> Haga clic en &quot;Iniciar Screening &#39;para comenzar la detección automática. Abra los datos anotados (archivo .raf). </li><li> Revise los resultados de la detección automática y, si es necesario, corregir manualmente en base a los criterios estándar (ver los resultados representativos, Figura 1B y Tabla 1 </strong&gt;). 2, 3 En pocas palabras, haga clic y mantenga pulsado el botón izquierdo del ratón en una época incorrectamente anotada y arrastrar el cursor a través de la cadena de épocas incorrectamente anotados. Suelte el botón izquierdo del ratón y seleccione el estado de comportamiento correcto en la ventana emergente. Nota: En ocasiones, las épocas en la transición entre dos estados vigilantes son difíciles de conseguir de forma inequívoca a un estado. En tales casos, la época debe anotó al estado ostensible y los mismos criterios debe aplicarse a épocas similares durante todo el experimento para asegurar la reproducibilidad de los datos. </li></ol>

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The authors Yujiro Tauguchi and Sayaka Kohtoh are employees of Kissei Comtech Co., Ltd that develops SleepSign software for acquisition and automatic scoring of EEG/EMG data used in this article.

Este protocolo describe una puesta a punto para las grabaciones de EEG / EMG que permite la evaluación de sueño y la vigilia en virtud de bajo ruido, condiciones rentables y de alto rendimiento. Debido al pequeño tamaño del conjunto de cabezal de electrodos EEG / EMG, este sistema se puede combinar con otros implantes para experimentos intra-cerebrales, incluyendo la optogenética (implantación de fibra óptica) o, en conjunto con la implantación de la cánula simultánea, microinfusión de fármacos en el ratón ...

Los avances técnicos a menudo han precipitado saltos cuánticos en la comprensión de los procesos neurobiológicos. Por ejemplo, el descubrimiento de Hans Berger en 1929 que los potenciales eléctricos grabadas desde el cuero cabelludo humano tomaron la forma de ondas sinusoidales, la frecuencia de la que se relaciona directamente con el nivel de vigilia del sujeto, llevó a los rápidos avances en la comprensión de sueño-vigilia la regulación, tanto en animales y seres humanos por igual. 1 Hasta el día de la electroencephlogram (EEG), en conjunto con el electromiograma (EMG), es decir., la actividad eléctrica producida por los músculos esqueléticos, representa los datos &quot;columna vertebral&quot; de casi todas las clínicas y experimentales evaluación que busca correlacionar el comportamiento y fisiología con la actividad de las neuronas corticales en comportarse animales, incluyendo seres humanos. En la mayoría de los laboratorios de investigación del sueño de base, estos registros de EEG se llevan a cabo mediante el uso de un sistema basado en cable (Figura 1) en la que d adquiridaATA se somete fuera de línea para el análisis del patrón y el espectro de [por ejemplo., la aplicación de una transformada rápida de Fourier (FFT) algoritmo] para determinar el estado de vigilancia del sujeto que se está grabando. 2, 3 Sleep consta de movimiento ocular rápido (REM) y no REM (NREM). Sueño REM se caracteriza por una rápida baja tensión EEG, movimiento de los ojos al azar, y la atonía muscular, un estado en el que los músculos están paralizados con eficacia. Sueño REM también se conoce como sueño paradójico, porque la actividad cerebral se asemeja al de la vigilia, mientras que el cuerpo es en gran medida desconectado del cerebro y parece estar en un sueño profundo. Por el contrario, las neuronas motoras son estimulados durante el sueño NREM, pero no hay movimiento del ojo. Sueño NREM humano se puede dividir en 4 etapas, con lo que la etapa 4 se llama sueño profundo o sueño de ondas lentas y se identifica por las grandes, las ondas cerebrales lentas con la actividad delta entre 0,5 a 4 Hz en el EEG. Por otro lado, una subdivisión entre las fases de sueño NREM en animales más pequeños, como las ratas unand ratones, no se ha establecido, sobre todo porque no tienen largos períodos consolidados de sueño como se ve en los seres humanos. A través de los años, y sobre la base de la interpretación EEG, tanto de circuitos y basadas en humoral, se han propuesto varios modelos de regulación del sueño-vigilia. La neural y bases celulares de la necesidad de dormir o, alternativamente, &quot;unidad de sueño,&quot; sigue sin resolverse, pero se ha conceptualizado como una presión homeostática que se acumula durante el período de vigilia y se disipa por el sueño. Una teoría es que los factores endógenos somnogenic acumulan durante la vigilia y que su acumulación gradual es la base del sueño presión homeostática. Mientras que la primera hipótesis formal de que el sueño es regulada por factores humorales se ha acreditado al trabajo de Rosenbaum publicado en 1892 4, era Ishimori 5, 6 y Pieron 7 que de forma independiente, y hace más de 100 años, demostró la existencia de productos químicos con el sueño la promoción. Ambos investigadores propusieron, y de hecho han demostrado, que las sustancias Hypnogenic o &#39;hypnotoxins&#39; estaban presentes en el líquido cefalorraquídeo (LCR) de los perros con falta de sueño. 8 Durante el siglo pasado varias sustancias Hypnogenic putativos adicionales implicados en el proceso homeostático del sueño han sido identificados (para una revisión, ver ref. 9), incluyendo la prostaglandina (PG) D 2, 10 citocinas, 11 adenosina, la anandamida 12, 13 y el péptido de urotensina II. 14 El trabajo experimental por Ecónomo 15, 16, Moruzzi y Magoun 17, y otros en los hallazgos producidos siglo principios y mediados del 20 th que inspiró teorías basadas en circuitos de sueño y vigilia y, hasta cierto punto, eclipsó la teoría humoral entonces predominante de dormir. Hasta la fecha, se han propuesto varios &quot;modelos de circuitos&quot;, cada uno informado por los datos de diversa calidad y cantidad (para una revisión, ver Ref. 18). Uno de los modelos, Por ejemplo, propone que el sueño de onda lenta se genera a través de la inhibición de la adenosina mediada por la liberación de acetilcolina de las neuronas colinérgicas en el prosencéfalo basal, un área consisiting principalmente del núcleo de la extremidad horizontal de la banda diagonal de Broca y el inominata sustancia. 19 Otro modelo popular de la regulación del sueño / vigilia describe un mecanismo interruptor flip-flop sobre la base de las interacciones mutuamente inhibitorias entre las neuronas que inducen el sueño en el área preóptica ventrolateral y neuronas estela que inducen en el tallo del hipotálamo y el cerebro. 18, 20, 21 Por otra parte, para la conmutación en y fuera del sueño REM, un similares interacción recíprocamente inhibitoria ha sido propuesto para áreas en el tronco cerebral, que es la ventral gris periacueductal, tegmento pontino lateral, y el núcleo sublaterodorsal. 22 En conjunto, estos modelos han demostrado ser valiosa heurística y los marcos interpretativos que ofrece importantes para los estudios de investigación del sueño; sin embargo, un ost más completa comprensión de los mecanismos moleculares y circuitos que regulan el ciclo de sueño-vigilia requerirá un conocimiento más completo de sus componentes. El sistema de registro poligráfico se detalla a continuación debe ayudar en este objetivo.

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			<td height="19" style="height:19px;width:216px;">Ampicillin</td>
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			<td height="21" style="height:21px;width:216px;">Analog-to-digital converter</td>
			<td style="width:143px;">Contec</td>
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			<td height="21" style="height:21px;width:216px;">Caffeine</td>
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			<td height="21" style="height:21px;width:216px;">Carbide cutter</td>
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			<td height="19" style="height:19px;width:216px;">Crimp housing</td>
			<td style="width:143px;">Hirose</td>
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			<td height="21" style="height:21px;width:216px;">Crimp socket</td>
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			<td height="42" style="height:42px;width:216px;">Dental cement (Toughron Rebase)</td>
			<td style="width:143px;">Miki Chemical Product</td>
			<td style="width:167px;">N/A</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Epoxy adhesive</td>
			<td style="width:143px;">Konishi</td>
			<td style="width:167px;">#16351</td>
			<td></td>
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		<tr height="24">
			<td height="24" style="height:24px;width:216px;">FFC/FPC connector</td>
			<td style="width:143px;">Honda Tsushin Kogyo</td>
			<td style="width:167px;">FFC-10BMEP1(B)</td>
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			<td height="21" style="height:21px;width:216px;">Flat cable</td>
			<td style="width:143px;">Hitachi Cable</td>
			<td style="width:167px;">20528-ST LF</td>
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			<td height="20" style="height:20px;width:216px;">Instant glue (Aron Alpha A)</td>
			<td style="width:143px;">Toagosei</td>
			<td style="width:167px;">N/A</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">Meloxicam</td>
			<td style="width:143px;">Boehringer Ingelheim</td>
			<td style="width:167px;">N/A</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Pentobarbital</td>
			<td style="width:143px;">Kyoritsu Seiyaku</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Signal amplifier</td>
			<td style="width:143px;">Biotex</td>
			<td style="width:167px;">N/A</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sleep recording chamber</td>
			<td style="width:143px;">APL</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SleepSign software</td>
			<td style="width:143px;">Kissei Comtec</td>
			<td style="width:167px;">N/A</td>
			<td>for EEG/EMG recording/analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Slip ring</td>
			<td style="width:143px;">Biotex</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stainless steel screw</td>
			<td style="width:143px;">Yamazaki</td>
			<td style="width:167px;">N/A</td>
			<td>&#966;1.0&#215;2.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stainless steel wire</td>
			<td style="width:143px;">Cooner Wire</td>
			<td style="width:167px;">AS633</td>
			<td></td>
		</tr>
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polygraphic recording procedure for measuring sleep in mice

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the homeodynamics and circuitry of sleep-wake regulation. In many laboratories, a cable-based sleep recording system is used to monitor the EEG and EMG in freely behaving mice in combination with computer software for automatic scoring of the vigilance states on the basis of power spectrum analysis of EEG data. A description of this system is detailed herein. Steel screws are implanted over the frontal cortical area and the parietal area of 1 hemisphere for monitoring EEG signals. In addition, EMG activity is monitored by the bilateral placement of wires in both neck muscles. Non-rapid eye movement (Non-REM; NREM) sleep is characterized by large, slow brain waves with delta activity below 4 Hz in the EEG, whereas a shift from low-frequency delta activity to a rapid low-voltage EEG in the theta range between 6 and 10 Hz can be observed at the transition from NREM to REM sleep. By contrast, wakefulness is identified by low- to moderate-voltage brain waves in the EEG trace and significant EMG activity.

Figura 1B ilustra ejemplos del EEG del ratón en los diferentes estados de vigilancia. Como se muestra en la Tabla 1, épocas se clasifican como sueño NREM si el EEG muestra grandes, ondas cerebrales lentas con un ritmo delta por debajo de 4 Hz y la EMG tiene solamente una señal débil o no. Épocas se clasifican como sueño REM si el EEG muestra rápidas ondas cerebrales de baja tensión en el rango theta entre 6 y 10 Hz y el EMG mues...

Watch this Scientific Journal Video about Polygraphic Recording Procedure for Measuring Sleep in Mice at JoVE.com

Polygraphic Recording Procedure for Measuring Sleep in Mice

The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.

Poligráfico Procedimiento de Grabación en medición del sueño en ratones

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the ...

polygraphic-recording-procedure-for-measuring-sleep-in-mice

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Anatomy and Physiology

Neuroscience

Unit 1

Functions of the Central and Peripheral Nervous System

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23.3K vistas.  University of Tsukuba. The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.

Video: Polygraphic Recording Procedure for Measuring Sleep in Mice

Measuring Motor Coordination in Mice

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such as emotionality or cognition can be assessed, however, it is vital to determine whether the motor capabilities of e.g. a mutant or lesioned mouse allow such an assessment. Performance on a maze task requiring strength and coordination, such as the Morris water maze, might well be impaired in a mouse by motor, rather than cognitive, impairments, so it is essential to selectively dissect the latter from the former. For example, sensorimotor impairments caused by NMDA antagonists have been shown to impair water maze performance2. 
Motor coordination has traditionally been assessed in mice and rats by the rotarod test, in which the animal is placed on a horizontal rod that rotates about its long axis; the animal must walk forwards to remain upright and not fall off. Both set speed and accelerating versions of the rotarod are available.
The other three tests described in this article (horizontal bar, static rods and parallel bars) all measure coordination on static apparatus. The horizontal bar also requires strength for adequate performance, particularly of the forelimbs as the mouse initially grips the bar just with the front paws. 
Adult rats do not perform well on tests such as the static rods and parallel bars (personal observations); they appear less well coordinated than mice. I have only tested male rats, however, and male mice seem generally less well coordinated than females. Mice appear to have a higher strength:weight ratio than rats; the Latin name, Mus musculus, seems entirely appropriate. The rotarod, the variations of the foot fault test12 or the Catwalk (Noldus)15 apparatus are generally used to assess motor coordination in rats.

Protocols are presented for two established motor coordination tasks, the accelerating rotarod and horizontal bar, also two tests developed in Oxford recently, the static rods and parallel bars. These tests can detect motor impairments potentially of interest in their own right, as well as being possible variables in tests of other areas of behavior.

Medición de Coordinación motora en ratones

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such ...

Investigación

Neurociencias

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Marcaje fluorescente retrógrada Permite dirigida extracelular sola unidad de grabación de neuronas identificadas<em&gt; En vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

Registro y análisis de los ritmos circadianos en funcionamiento las ruedas Actividad en roedores

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

Medición de la columna vertebral presináptica inhibición en ratones mediante la raíz dorsal de grabación Potencial<em&gt; En Vivo</em

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Un método simple y barato para la Determinación de la sensibilidad al frío y Adaptación en ratones

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Extracelular de grabación de la actividad neuronal junto con la aplicación de sustancias Microiontophoretic neuroactivos en ratones Awake

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

Preparaciones y protocolos para todo celular parche abrazadera de neuronas Tectal de Xenopus laevis

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

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 ...

Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 (ChR2), is expressed by a virus vector in a particular type of neuronal cells in various Cre-driver mice. Activation of these opsins is triggered by application of light pulses which are delivered by laser or LED through optic cables, and the effect of activation is observed with very high time resolution. Experimenters are able to acutely stimulate neurons while monitoring behavior or another physiological outcome in mice. Optogenetics can enable useful strategies to evaluate function of neuronal circuits in the regulation of sleep/wakefulness states in mice. Here we describe a technique for examining the effect of optogenetic manipulation of neurons with a specific chemical identity during electroencephalogram (EEG) and electromyogram (EMG) monitoring to evaluate the sleep stage of mice. As an example, we describe manipulation of GABAergic neurons in the bed nucleus of the stria terminalis (BNST). Acute optogenetic excitation of these neurons triggers a rapid transition to wakefulness when applied during NREM sleep. Optogenetic manipulation along with EEG/EMG recording can be applied to decipher the neuronal circuits that regulate sleep/wakefulness states.

Here, we describe methods of optogenetic manipulation of particular types of neurons during monitoring of sleep/wakefulness states in mice, presenting our recent work on the bed nucleus of the stria terminalis as an example.

Manipulación optogenética de circuitos neuronales durante la supervisión de los estados de sueño/vigilia en ratones

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 ...

Evaluation of Hemisphere Lateralization with Bilateral Local Field Potential Recording in Secondary Motor Cortex of Mice

This article demonstrates complete, detailed procedures for both in vivo bilateral recording and analysis of local field potential (LFP) in the cortical areas of mice, which are useful for evaluating possible laterality deficits, as well as for assessing brain connectivity and coupling of neural network activities in rodents. The pathological mechanisms underlying Alzheimer&#39;s disease (AD), a common neurodegenerative disease, remain largely unknown. Altered brain laterality has been demonstrated in aging people, but whether or not abnormal lateralization is one of the early signs of AD has not been determined. To investigate this, we recorded bilateral LFPs in 3-5-month-old AD model mice, APP/PS1, together with littermate wild type (WT) controls. The LFPs of the left and right secondary motor cortex (M2), specifically in the gamma band, were more synchronized in APP/PS1 mice than in WT controls, suggesting a declined hemispheric asymmetry of bilateral M2 in this AD mouse model. Notably, the recording and data analysis processes are flexible and easy to carry out, and can also be applied to other brain pathways when conducting experiments that focus on neuronal circuits.

We present in vivo electrophysiological recording of the local field potential (LFP) in bilateral secondary motor cortex (M2) of mice, which can be applied to evaluate hemisphere lateralization. The study revealed altered levels of synchronization between the left and right M2 in APP/PS1 mice compared to WT controls.

Evaluación de la lateralización del hemisferio con registro de potencial de campo local bilateral en corteza motora secundaria de ratones

This article demonstrates complete, detailed procedures for both in vivo bilateral recording and analysis of local field potential (LFP) in the cortical ...

Poligráfico Procedimiento de Grabación en medición del sueño en ratones

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