Los autores desean agradecer a las siguientes: Dr. Joseph Bronzino, Dr. Khamis Abu-Hassaballah, Sr. RJ Austin-LaFrance, y la Sra. Jessica Koranda.

Este protocolo es adecuado para ratones de 3 y 18 meses de edad y el peso corporal aproximado de 30-50 g). Los ratones se pueden obtener de El Laboratorio Jackson (Bar Harbor, ME). Todos los protocolos quirúrgicos y experimentales fueron aprobados por el Comité de Cuidado de Animales y el empleo Trinity College y estaban de acuerdo con la Guía del NIH para el Cuidado y Uso de Animales de Laboratorio. 1. Preparación de los animales y Procedimientos Quirúrgicos <ol><li> Preparar cóctel que contiene la anestesia de ketamina (25 mg / ml), xilazina (2,5 mg / ml) y acepromacina (0,5 mg / ml). Inyectar 1 ml / kg de peso corporal en el cuadrante inferior derecho del abdomen, complementado por inyecciones de 0,2 ml / kg cada 45 minutos a partir de entonces para mantener una profundidad estable de anestesia usando el reflejo de retirada pedal. </li><li> Preparar ratón anestesiado para la cirugía por el afeitado el pelo de su cráneo usando una máquina de afeitar eléctrica. El área afeitada debe incluir el centro de los ojos a la mitad de los oídos. Asegúrese de no afeitarse la whiskers, ya que forman parte del sistema sensorial del barril del ratón. </li><li> Use hisopos de algodón para aplicar alcohol isopropílico seguido de Betadine a la zona de afeitado en la cabeza. Entonces, también el uso de bastoncillos de algodón, se aplica una pequeña cantidad de aceite mineral en los ojos para evitar que los globos oculares de secado. </li><li> Utilice manguitos del oído de ratas lactantes en lugar de barras de oreja regulares para montar la cabeza del animal en el aparato marco estereotáxico. Para ello, primero montar la oreja izquierda en el puño del oído izquierdo. Entonces aliviar un poco la oreja derecha en el puño derecho, apoyando suavemente la cabeza del animal con una mano y el avance del manguito del oído con la otra mano. Luego, coloque un cojín eléctrico bajo el cuerpo del animal y ponerlo a 86 ° C para mantener la temperatura corporal. </li><li> A continuación, compruebe si hay rigidez bilateral al intentar mover la cabeza de lado a lado. El animal está montado correctamente en el marco estereotáxico cuando la cabeza es rígida y no se puede mover de lado a lado, pero puede girar fácilmente hacia arriba y abajo. </li><li> Descansar suavemente lahocico del ratón sobre la barra de la nariz / diente de montaje asegurándose de que los incisivos están descansando suavemente pero con seguridad dentro de la abertura de los dientes de la barra de la nariz / diente. </li><li> Con un bisturí estéril practicar una incisión en la línea media a partir de la mitad de los ojos hasta la mitad de las orejas. Asegúrese de que el bisturí se llevará a cabo en un ángulo de 45 ° y aplicar suficiente presión para cortar a través de la fascia subyacente, pero no a través del cráneo ya que esto causará sangrado excesivo. </li><li> Use hisopos de algodón para separar la fascia y exponer el cráneo. Cráneo hitos bregma y lambda debe ser claramente visible (fig. 1A). Asegúrese de que el animal está en la posición cráneo plano mediante el ajuste de la barra de la nariz / diente de tal manera que bregma y lambda puntos de referencia están en la misma posición dorsoventral (DV) (+ 0,1 mm). </li><li> Aplique una pequeña cantidad de solución salina estéril al área expuesta y usar hisopos de algodón para limpiar suavemente el cráneo. Luego, esperar 2-3 minutos para que el cráneo se sequen completamente al aireantes de proceder. </li><li> Montar una aguja en el brazo estereotáxica izquierda para medir la posición de DV bregma y lambda. El posicionamiento de DV de estos dos puntos de referencia debe estar dentro de 0,1 mm el uno del otro. Si no, la barra de la nariz del marco estereotáxico se puede ajustar hacia arriba y hacia abajo para lograr esto. </li><li> Coloque la aguja en bregma para grabar su anterior-posterior (AP), lateral (LAT) y (DV) mediciones dorsoventral. Asegúrese de que la aguja toque el cráneo y no penetra en ella. </li><li> Utilice un ratón atlas del cerebro, tales como &quot;el cerebro del ratón en estereotáxica Coordenadas&quot; 1, para determinar las coordenadas de las estructuras de destino: vía medial perforante (MPP) en el haz angular y giro dentado (GD) del hipocampo, en relación con lambda y bregma, respectivamente (véase también el cuadro 1). A continuación, utilice un bolígrafo de punta fina o un lápiz para marcar estos puntos en el cráneo. </li><li> También, marque dos puntos más en el lado contralateral del cráneo. Estos puntos which servirá como la masa (GND) y de referencia (REF) debe ser de aproximadamente 3 mm de la línea media y colocado longitudinalmente con relación a cada otra (Figura 1A, también la Tabla 1). </li><li> Quite el marcador de la aguja del brazo estereotáxica izquierda y reemplazarlo con un taladro dental eléctrico equipado con un montaje estereotáxica. Coloque el taladro sobre cada punto marcado y hacer pequeños agujeros de trépano de aproximadamente 0,5 mm de diámetro. Al taladrar, use un movimiento hacia arriba y hacia abajo repetitivo y comprobar con frecuencia si el simulacro ha penetrado en el cráneo para exponer la superficie del cerebro. </li><li> Con suavidad pero con firmeza conducir un electrodo de tornillo (# 0-80 1/8 de los tornillos de máquina de acero inoxidable, ranurado fillister cabeza) en cada uno de los agujeros contralateral (GND y REF) de tal manera que sólo tocan la superficie cortical sin penetrar en él (Figura 1A ). Estos dos electrodos de tornillo sirven de suelo y de referencia. </li><li> Mount estimulantes y grabación electrodos en electroDe los titulares en los brazos estereotáxica izquierdo y derecho, respectivamente. </li><li> Conectar los terminales de electrodo de estimulación a un estimulador electrofisiológico con el aislamiento de corriente y el terminal de electrodo de registro a un amplificador electrofisiológico diferencial (ganancia = 1,000, filtro de paso de banda = 1 Hz-3 kHz) y luego a un osciloscopio digital para la inspección visual de las respuestas evocadas. Además, conecte GND y terminales de los electrodos REF al amplificador diferencial. </li><li> Suave y lentamente baja estimulante y electrodos de grabación en incrementos de 0,5 mm, mientras que el seguimiento de la respuesta evocada visualmente a un choque de estímulo de 600 a 800 uA. Continúe bajando cada electrodo hasta que llegan a su respectiva posición DV meta y se observa una señal estereotipada en el osciloscopio (Figura 1B). </li><li> Después, utilizar el cemento acrílico dental para hacer una tapa para mantener los terminales de los electrodos en su lugar, cualquier cemento dental que toca la piel debe limpiarseinmediatamente. Una vez que el cemento dental está completamente curado transferir al animal del marco estereotáxico a una jaula de roedores limpia. Utilice una lámpara de calor o una almohadilla para mantener la temperatura central. </li><li> Monitorear el animal cada hora hasta que se recupere la conciencia. Una inyección de flunixina se puede administrar como un analgésico. </li></ol> 2. La inducción de LTP <ol><li> Deje que los animales de 5-7 días para recuperarse de la cirugía. Colocar el animal en el entorno de grabación que consiste en una jaula de Faraday (122 cm x 43 cm x 43 cm) equipado con material de insonorización y un conmutador giratorio 5-canal que conecta el electrodo conduce a la configuración de estimulante / grabación. </li><li> Permita que el animal se aclimate durante 1-2 horas en el entorno de grabación antes de conectar los electrodos a los instrumentos de estimulación y registro (consulte el paso 1.17). </li><li> Ajuste los controles del estimulador de la producción de 400 uA y registrar la amplitud de un promedio de 10 respuestas evocadas asegurándose ªen al menos 10 segundos transcurrido entre los estímulos. Utilice el método que se muestra en la Figura 1C para cuantificar la respuesta evocada. Repita este procedimiento para 600, 800, 1.000, 1.200, y 1.400 uA. </li><li> Construir una curva de entrada / salida por el trazado de la amplitud media de la respuesta evocada frente a la intensidad del estímulo. De esta figura determinar la intensidad del estímulo que corresponde a 50% de la amplitud máxima medida. Utilice la intensidad de 50% para el resto del experimento. </li><li> Entonces, usando la intensidad del estímulo 50%, obtener una línea de base mediante el registro de la amplitud media de 5 respuestas evocadas cada minuto durante 15 minutos. Una vez más asegurarse de que al menos 10 segundos transcurrido entre estímulos. </li><li> Posteriormente entregar la estimulación tetánica que consiste de 10 ráfagas de 10 pulsos suministrados a 400 Hz con una velocidad de ráfaga de 5 Hz a la vía perforante medial. Monitorear el animal para detectar signos de convulsiones, incluyendo sacudidas de perro mojado. Si el animal tiene una convulsión del experiment debe ser terminada y todos los datos de ese animal debe ser excluido de los resultados del estudio. </li><li> Luego, continuará grabando la amplitud promedio de 5 respuestas evocadas cada minuto durante 30 minutos posttetanization. Después de eso, comparar esta amplitud a la amplitud de línea de base obtenida en la etapa 2.5 mediante el cálculo del porcentaje de cambio desde la línea base (Figura 2). </li><li> Tras la conclusión de los experimentos, el animal es sacrificado por medio de la inhalación de isoflurano-un método que sea consistente con las Directrices sobre la eutanasia de la Asociación Americana de Medicina Veterinaria. </li></ol>

<ol>
	<li>Paxinos, G., Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The mouse brain in stereotaxic coordinates. , (2004).</li><li>Bliss, T. V. P., Lomo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-lasting+potentiation+of+synaptic+transmission+in+the+dentate+area+of+the+anesthetized+rabbit+following+stimulation+of+the+perforant+path.">Long-lasting potentiation of synaptic transmission in the dentate area of the anesthetized rabbit following stimulation of the perforant path.</a> J. Physiol. 232, 331-356 (1973).</li><li>Blaise, J. H., Bronzino, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+stimulus+frequency+and+age+on+bidirectional+synaptic+plasticity+in+the+dentate+gyrus+of+freely+moving+rats.">Effects of stimulus frequency and age on bidirectional synaptic plasticity in the dentate gyrus of freely moving rats.</a> Exp. Neurol. 182, 497-506 (2003).</li><li>Blaise, J. H., Koranda, J. L., Chow, U., Haines, K. E., Dorward, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+isolation+stress+alters+bidirectional+long-term+synaptic+plasticity+in+amygdalo-hippocampal+synapses+in+freely+behaving+adult+rats.">Neonatal isolation stress alters bidirectional long-term synaptic plasticity in amygdalo-hippocampal synapses in freely behaving adult rats.</a> Brain Res. 1193, 25-33 (2008).</li><li>Koranda, J. L., Masino, S. A., Blaise, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+synaptic+plasticity+in+the+dentate+gyrus+of+the+awake+freely+behaving+mouse.">Bidirectional synaptic plasticity in the dentate gyrus of the awake freely behaving mouse.</a> J. Neurosci. Methods. 167, 160-166 (2008).</li><li>Cooke, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophosphorylation+of+alphaCaMKII+is+not+a+general+requirement+for+NMDA+receptor-dependent+LTP+in+the+adult+mouse.">Autophosphorylation of alphaCaMKII is not a general requirement for NMDA receptor-dependent LTP in the adult mouse.</a> J. Physiol. 574, 805-818 (2006).</li><li>Davis, S., Bliss, T. V., Dutrieux, G., Laroche, S., Errington, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+duration+of+long-term+potentiation+in+the+hippocampus+of+the+freely+moving+mouse.">Induction and duration of long-term potentiation in the hippocampus of the freely moving mouse.</a> J. Neurosci. Methods. 75, 75-80 (1997).</li><li>Jones, M. W., Peckham, H. M., Errington, M. L., Bliss, T. V., Routtenberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+plasticity+in+the+hippocampus+of+awake+C57BL/6+and+DBA/2+mice:+interstrain+differences+and+parallels+with+behavior.">Synaptic plasticity in the hippocampus of awake C57BL/6 and DBA/2 mice: interstrain differences and parallels with behavior.</a> Hippocampus. 11, 391-396 (2001).</li><li>Zhang, T. A., Tang, J., Pidoplichko, V. I., Dani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addictive+nicotine+alters+local+circuit+inhibition+during+the+induction+of+in+vivo+hippocampal+synaptic+potentiation.">Addictive nicotine alters local circuit inhibition during the induction of in vivo hippocampal synaptic potentiation.</a> J. Neurosci. 30, 6443-6453 (2010).</li><li>Tang, J., Dani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+enables+in+vivo+synaptic+plasticity+associated+with+the+addictive+drug+nicotine.">Dopamine enables in vivo synaptic plasticity associated with the addictive drug nicotine.</a> Neuron. 63, 673-682 (2009).</li></ol>

Los autores tienen nada que revelar.

En este protocolo, hemos demostrado un método fiable y sencillo para el estudio de LTP en la Dirección General en ratones comportarse libremente. Mientras que muchos estudios de LTP en ratas despiertas se han realizado 3,4, muy pocos se han realizado en ratones despiertos todo debido a la complejidad técnica que supone la inmobiliaria craneal limitada en ratones y el peso de Headstages electrodos en relación con el peso medio de 5 ratones. Los pocos estudios que han demostrado la PLP en la Dire...

El desarrollo de la tecnología para manipular genes ha producido transgénicos y knock-out modelos de ratón de casi todas las enfermedades neurodegenerativas y neurológicas. Esto ha requerido traducción de técnicas de investigación electrofisiológicos utilizados previamente en especies de roedores más grandes en el modelo animal de ratón. Una de tales técnicas de investigación neurofisiológica es el uso de la potenciación a largo plazo (LTP) para probar la eficacia de las conexiones sinápticas dentro de las redes neuronales implicados en diversos trastornos neuropatológicos. Este protocolo describe las técnicas para la investigación electrofisiológica fiable de LTP en comportarse libremente ratones. La ventaja de este protocolo sobre otros es que es simple y fácil de implementar, sino que también es bastante menos costoso, ya que no requiere ni el uso de costosos sistemas de microdrive controlados por el ordenador ni Headstages transistor de efecto de campo, y, a nuestro conocimiento, es el primer protocolo de video de grabaciones electrofisiológicas crónicas to estudiar la LTP en comportarse libremente ratones. Con este fin, se describe en este artículo metodologías sencillas para estudiar la potenciación a largo plazo en ratones comportarse libremente. Estas metodologías pueden ser fácilmente traducidos a modelos transgénicos y knockout de ratón de trastornos neuropatológicos.

<table border="0" cellpadding="0" cellspacing="0" width="514">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Ketamine (100 mg/ml)</td>
			<td style="width:104px;">Henry Schein</td>
			<td style="width:137px;">10177</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Xylazine (20 mg/ml)</td>
			<td style="width:104px;">Henry Schein</td>
			<td style="width:137px;">33197</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Acepromazine (10 mg/ml)</td>
			<td style="width:104px;">Henry Schein</td>
			<td style="width:137px;">2177</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">Dental acrylic powder</td>
			<td style="width:104px;">Lang Dental Manufacturing Co.</td>
			<td style="width:137px;">1330CLR</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">Dental acrylic liquid</td>
			<td style="width:104px;">Lang Dental Manufacturing Co.</td>
			<td style="width:137px;">1306CLR</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">Tungsten wire (0.127 mm)</td>
			<td style="width:104px;">World Precision Instruments</td>
			<td style="width:137px;">TGW0515</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">Stainless Steel Hypodermic Tubing (0.286 mm)</td>
			<td style="width:104px;">World Precision Instruments</td>
			<td style="width:137px;">832400</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Flunixin (50 mg/ml)</td>
			<td style="width:104px;">Henry Schein</td>
			<td style="width:137px;">14165</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Epoxilyte</td>
			<td style="width:104px;">Superior Essex</td>
			<td style="width:137px;">EP 6001-M</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">Stainless steel wire insert (0.2 mm)</td>
			<td style="width:104px;">World Precision Instruments</td>
			<td>792900</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Stereotaxic frame apparatus</td>
			<td style="width:95px;">Kopf Instruments</td>
			<td style="width:137px;">Model 902</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Ear cuffs (ear cups)</td>
			<td style="width:95px;">Kopf Instruments</td>
			<td style="width:137px;">Model 921&#160;</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Electrophysiological stimulator</td>
			<td style="width:95px;">Astro-Med, Inc.</td>
			<td style="width:137px;">S88</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">Digital oscilloscope</td>
			<td style="width:95px;">B &#38; K Precision Corp.</td>
			<td style="width:137px;">2542</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Current isolation unit</td>
			<td style="width:95px;">Astro-Med, Inc.</td>
			<td style="width:137px;">PSIU-6</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:195px;">Differential amplifier</td>
			<td style="width:95px;">World Precision Instruments, Inc.</td>
			<td style="width:137px;">DAM-50</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Commutator</td>
			<td style="width:95px;">Plastics One</td>
			<td style="width:137px;">SLC6</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Dental drill</td>
			<td style="width:95px;">Stoelting</td>
			<td style="width:137px;">58650</td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

long-term potentiation of perforant pathway-dentate gyrus synapse in freely behaving mice

Estudios de la potenciación a largo plazo de la eficacia sináptica, un fenómeno sináptica dependiente de la actividad que tiene propiedades que lo hacen atractivo como un potencial mecanismo celular de almacenamiento de aprendizaje y la información subyacente, han sido durante mucho tiempo utilizado para elucidar la fisiología de varios circuitos neuronales en el hipocampo, la amígdala , y otras estructuras límbicas y corticales. Con esto en mente, los modelos de ratones transgénicos de enfermedades neurológicas representan plataformas útiles para llevar a cabo la potenciación a largo plazo (LTP) estudios para desarrollar una mayor comprensión de la función de los genes en la comunicación sináptica normal y anormal en las redes neuronales implicadas en el aprendizaje, la emoción y la información procesamiento. En este artículo se describen las metodologías para la inducción de LTP en forma confiable el ratón libremente comportarse. Estas metodologías se pueden utilizar en estudios de transgénicos y Knockout comportarse libremente modelos de ratón de enfermedades neurodegenerativas.

La Tabla 1 muestra las coordenadas de la DG y mPP empleado en el presente protocolo Figura 1A muestra las marcas de las estructuras diana en el cráneo;.. También se muestran la ubicación de los electrodos de tierra y de referencia Figura 1B ilustra representante respuesta evocada traza tanto pre- y posttetanization en el mismo animal. Tenga en cuenta que la posttetanization respuesta evocada es mayor que la respuesta pretetanization que es indicativo de la inducción...

Watch this Scientific Journal Video about Long-term Potentiation of Perforant Pathway-dentate Gyrus Synapse in Freely Behaving Mice at JoVE.com

Long-term Potentiation of Perforant Pathway-dentate Gyrus Synapse in Freely Behaving Mice

Transgénicos y knockout modelos de ratón de enfermedades neurológicas son útiles para estudiar el papel de los genes en la neurofisiología normal y anormal. Este artículo describe metodologías que pueden utilizarse para estudiar la potenciación a largo plazo, un mecanismo celular que puede ser la base de aprendizaje y la memoria, en transgénicos y knockout libremente comportarse modelos de ratón de la neuropatología.

La potenciación a largo plazo de la Vía Perforante-giro dentado sinapsis en comportarse libremente Ratones

Studies of long-term potentiation of synaptic efficacy, an activity-dependent synaptic phenomenon having properties that make it attractive as a potential ...

long-term-potentiation-perforant-pathway-dentate-gyrus-synapse-freely

Research

JoVE Journal

Behavior

14.0K vistas.  Trinity College.  Transgénicos y knockout modelos de ratón de enfermedades neurológicas son útiles para estudiar el papel de los genes en la neurofisiología normal y anormal. Este artículo describe metodologías que pueden utilizarse para estudiar la potenciación a largo plazo, un mecanismo celular que puede ser la base de aprendizaje y la memoria, en transgénicos y knockout libremente comportarse modelos de ratón de la neuropatología. 

Video: Long-term Potentiation of Perforant Pathway-dentate Gyrus Synapse in Freely Behaving Mice

Mouse Short- and Long-term Locomotor Activity Analyzed by Video Tracking Software

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice and other rodents. Improvements in video tracking software (VTS) have allowed it to be coupled to LMA testing, dramatically improving specificity and sensitivity when compared to the line crossings method with manual scoring. In addition, VTS enables high-throughput experimentation. While similar to automated video tracking used for the open field test (OFT), LMA testing is unique in that it allows mice to remain in their home cage and does not utilize the anxiogenic stimulus of bright lighting during the active phase of the light-dark cycle. Traditionally, LMA has been used for short periods of time (mins), while longer movement studies (hrs-days) have often used implanted transmitters and biotelemetry. With the option of real-time tracking, long-, like short-term LMA testing, can now be conducted using videography. Long-term LMA testing requires a specialized, but easily constructed, cage so that food and water (which is usually positioned on the cage top) does not obstruct videography. Importantly, videography and VTS allows for the quantification of parameters, such as path of mouse movement, that are difficult or unfeasible to measure with line crossing and/or biotelemetry. In sum, LMA testing coupled to VTS affords a more complete description of mouse movement and the ability to examine locomotion over an extended period of time.

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice. Coupling of video tracking software (VTS) and LMA allows for the improvement of specificity and sensitivity, especially when compared with the manual, line crossing method of LMA analysis. Additionally VTS allows long-term tracking of mouse LMA.

Ratón de corto y largo plazo la actividad locomotriz analizados por el software de seguimiento de vídeo

Locomotor  activity (LMA) is a simple and easily performed measurement of behavior in mice  and other rodents. Improvements in video tracking software ...

Investigación

Comportamiento

Construction of Microdrive Arrays for Chronic Neural Recordings in Awake Behaving Mice

State-of-the-art electrophysiological recordings from the brains of freely behaving animals allow researchers to simultaneously examine local field potentials (LFPs) from populations of neurons and action potentials from individual cells, as the animal engages in experimentally relevant tasks. Chronically implanted microdrives allow for brain recordings to last over periods of several weeks. Miniaturized drives and lightweight components allow for these long-term recordings to occur in small mammals, such as mice. By using tetrodes, which consist of tightly braided bundles of four electrodes in which each wire has a diameter of 12.5 &mu;m, it is possible to isolate physiologically active neurons in superficial brain regions such as the cerebral cortex, dorsal hippocampus, and subiculum, as well as deeper regions such as the striatum and the amygdala. Moreover, this technique insures stable, high-fidelity neural recordings as the animal is challenged with a variety of behavioral tasks. This manuscript describes several techniques that have been optimized to record from the mouse brain. First, we show how to fabricate tetrodes, load them into driveable tubes, and gold-plate their tips in order to reduce their impedance from M&Omega; to K&Omega; range. Second, we show how to construct a custom microdrive assembly for carrying and moving the tetrodes vertically, with the use of inexpensive materials. Third, we show the steps for assembling a commercially available microdrive (Neuralynx VersaDrive) that is designed to carry independently movable tetrodes. Finally, we present representative results of local field potentials and single-unit signals obtained in the dorsal subiculum of mice. These techniques can be easily modified to accommodate different types of electrode arrays and recording schemes in the mouse brain.

The design and assembly of microdrives for in vivo electrophysiological recordings of brain signals from the mouse is described. By attaching microelectrode bundles to sturdy driveable carriers, these techniques allow for long-term and stable neural recordings. The lightweight design allows for unrestricted behavioral performance by the animal following drive implantation.

Construcción de Matrices Microdrive para grabaciones neuronales crónicas en Awake Ratones Behaving

State-of-the-art electrophysiological recordings from the brains of freely behaving animals allow researchers to simultaneously examine local field ...

Trace Fear Conditioning in Mice

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings between a neutral stimulus and an unconditioned stimulus. There is a 20 sec trace period that separates each conditioning trial. On the following day freezing is measured during presentation of the conditioned stimulus (CS) and trace period. On the third day there is an 8 min test to measure contextual memory. The representative results are from mice that were presented with the aversive unconditioned stimulus (shock) compared to mice that received the tone presentations without the unconditioned stimulus. Trace fear conditioning has been successfully used to detect subtle learning and memory deficits and enhancements in mice that are not found with other fear conditioning methods. This type of fear conditioning is believed to be dependent upon connections between the medial prefrontal cortex and the hippocampus. One current controversy is whether this method is believed to be amygdala-independent. Therefore, other fear conditioning testing is needed to examine amygdala-dependent learning and memory effects, such as through the delay fear conditioning.

In the following experiment we describe a protocol for trace fear conditioning in mice. This type of associative memory includes a trace period that separates the neutral stimulus and the unconditioned stimulus.

Traza el condicionamiento del miedo en ratones

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

La Junta Agujero Modificado - Medición de Comportamiento, Cognición e interacción social en ratones y ratas

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

A Novel Experimental and Analytical Approach to the Multimodal Neural Decoding of Intent During Social Interaction in Freely-behaving Human Infants

Understanding typical and atypical development remains one of the fundamental questions in developmental human neuroscience. Traditionally, experimental paradigms and analysis tools have been limited to constrained laboratory tasks and contexts due to technical limitations imposed by the available set of measuring and analysis techniques and the age of the subjects. These limitations severely limit the study of developmental neural dynamics and associated neural networks engaged in cognition, perception and action in infants performing &#8220;in action and in context&#8221;. This protocol presents a novel approach to study infants and young children as they freely organize their own behavior, and its consequences in a complex, partly unpredictable and highly dynamic environment. The proposed methodology integrates synchronized high-density active scalp electroencephalography (EEG), inertial measurement units (IMUs), video recording and behavioral analysis to capture brain activity and movement non-invasively in freely-behaving infants. This setup allows for the study of neural network dynamics in the developing brain, in action and context, as these networks are recruited during goal-oriented, exploration and social interaction tasks.

This protocol presents a novel methodology for the neural decoding of intent from freely-behaving infants during unscripted social interaction with an actor. Neural activity is acquired using non-invasive high-density active scalp electroencephalography (EEG). Kinematic data is collected with inertial measurement units and supplemented with synchronized video recording.

Un nuevo enfoque experimental y analítico al Multimodal Neural Decodificación de Intención Durante interacción social en Libremente-comportarse Infantes Humanos

Understanding typical and atypical development remains one of the fundamental questions in developmental human neuroscience. Traditionally, experimental ...

A Pressure Injection System for Investigating the Neuropharmacology of Information Processing in Awake Behaving Macaque Monkey Cortex

The top-down modulation of feed-forward cortical information processing is functionally important for many cognitive processes, including the modulation of sensory information processing by attention. However, little is known about which neurotransmitter systems are involved in such modulations. A practical way to address this question is to combine single-cell recording with local and temporary neuropharmacological manipulation in a suitable animal model. Here we demonstrate a technique combining acute single-cell recordings with the injection of neuropharmacological agents in the direct vicinity of the recording electrode. The video shows the preparation of the pressure injection/recording system, including preparation of the substance to be injected. We show a rhesus monkey performing a visual attention task and the procedure of single-unit recording with block-wise pharmacological manipulations.

Here, we show the pressure injection of neuropharmacological substances during single-cell recording in an awake, behaving macaque monkey. This procedure allows pharmacological manipulation in the direct vicinity of a cortical recording site.

Un sistema de inyección a presión de Investigación de la Neurofarmacología del procesamiento de la información en que se comporta Awake mono macaco de la corteza

The top-down modulation of feed-forward cortical information processing is functionally important for many cognitive processes, including the modulation ...

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical neurons with two-photon microscopy has provided unique insights into cortical structure, function and plasticity. However, these studies are limited to head fixed animals, greatly reducing the behavioral complexity available for study. In this paper, we describe a procedure for performing chronic fluorescence microscopy with cellular-resolution across multiple cortical layers in freely behaving mice. We used an integrated miniaturized fluorescence microscope paired with an implanted prism probe to simultaneously visualize and record the calcium dynamics of hundreds of neurons across multiple layers of the somatosensory cortex as the mouse engaged in a novel object exploration task, over several days. This technique can be adapted to other brain regions in different animal species for other behavioral paradigms.

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

Here, we present a procedure for performing large-scale Ca2+ imaging with cellular-resolution across multiple cortical layers in freely moving mice. Hundreds of active cells can be observed simultaneously using a miniature, head-mounted microscope coupled with an implanted prism probe.

Ca Cortical Multi-capa<sup&gt; 2+</sup&gt; Imágenes en ratones con movimientos libres con sondas de prisma y microscopía de fluorescencia miniaturizada

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical ...

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer scale. However, current methods mostly rely on tethered cable recordings or only use unidirectional systems, allowing either recording or stimulation of neural activity, but not at the same time or same target. Here, a new wireless, bidirectional device for simultaneous multichannel recording and stimulation of neural activity in freely behaving rats is described. The system operates through a single portable head stage that both transmits recorded activity and can be targeted in real-time for brain stimulation using a telemetry-based multichannel software. The head stage is equipped with a preamplifier and a rechargeable battery, allowing stable long-term recordings or stimulation for up to 1 h. Importantly, the head stage is compact, weighs 12 g (including battery) and thus has minimal impact on the animal&#180;s behavioral repertoire, making the method applicable to a broad set of behavioral tasks. Moreover, the method has the major advantage that the effect of brain stimulation on neural activity and behavior can be measured simultaneously, providing a tool to assess the causal relationships between specific brain activation patterns and behavior. This feature makes the method particularly valuable for the field of deep brain stimulation, allowing precise assessment, monitoring, and adjustment of stimulation parameters during long-term behavioral experiments. The applicability of the system has been validated using the inferior colliculus as a model structure.

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

A wireless, bidirectional system for multi-channel neural recordings and stimulation in freely behaving rats is introduced. The system is light and compact, thus having minimal impact on the animal&#180;s behavioral repertoire. Moreover, this bidirectional system provides a sophisticated tool to assess causal relationships between brain activation patterns and behavior.

Un inalámbrico, interfaz bidireccional para la grabación en Vivo y estimulación de la actividad Neural en comportarse libremente las ratas

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer ...

Automated, Long-term Behavioral Assay for Cognitive Functions in Multiple Genetic Models of Alzheimer's Disease, Using IntelliCage

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive decline, such as Alzheimer&#39;s disease (AD), have become a promising tool to elucidate the underlying mechanisms and promote the therapeutic advances. An important step is the validation and characterization of expected behavioral abnormality in the models, in the case of AD, cognitive decline. The long-term behavioral investigations of laboratory animals to study the effect of aging demand substantial efforts from researchers. The IntelliCage system is a high-throughput and cost-effective test battery for mice that eliminates the need for daily human handling. Here, we describe how the system is utilized in the long-term phenotyping of a genetic Alzheimer&#39;s disease model, specifically focusing on the cognitive functions. The experiment employs repeated battery of tests that assess spatial learning and executive functions. This cost-effective age-dependent phenotyping allows us to identify the transient and/or permanent effects of genes on various cognitive aspects.

Automated, Long-term Behavioral Assay for Cognitive Functions in Multiple Genetic Models of Alzheimer&#039;s Disease, Using IntelliCage

This paper describes a protocol for cognitive assessments for genetic models of the Alzheimer&#39;s disease using the IntelliCage system, which is a high throughput automated behavioral monitoring system with operant conditioning.

Automatizado, ensayo conductual a largo plazo para las funciones cognitivas en múltiples modelos genéticos de la enfermedad de Alzheimer, utilizando IntelliCage

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive ...

Simultaneous Recordings of Cortical Local Field Potentials and Electrocorticograms in Response to Nociceptive Laser Stimuli from Freely Moving Rats

Electrocortical responses, elicited by laser heat pulses that selectively activate nociceptive free nerve endings, are widely used in many animal and human studies to investigate the cortical processing of nociceptive information. These laser-evoked brain potentials (LEPs) consist of several transient responses that are time-locked to the onset of laser stimuli. However, the functional properties of the LEP responses are still largely unknown, due to the lack of a sampling technique that can simultaneously record neural activities at the surface of the cortex (i.e., electrocorticogram [ECoG] and scalp electroencephalogram [scalp EEG]) and inside the brain (i.e., local field potential [LFP]). To address this issue, we present here an animal protocol using freely moving rats. This protocol is composed of three main procedures: (1) animal preparation and surgical procedures, (2) a simultaneous recording of ECoG and LFP in response to nociceptive laser stimuli, and (3) data analysis and feature extraction. Specifically, with the help of a 3D-printed protective shell, both ECoG and LFP electrodes implanted on the rat&#39;s skull were securely held together. During data collection, laser pulses were delivered on the rat&#39;s forepaws through gaps in the bottom of the chamber when the animal was in spontaneous stillness. Ongoing white noise was played to avoid the activation of the auditory system by the laser-generated ultrasounds. As a consequence, only nociceptive responses were selectively recorded. Using the standard analytical procedures (e.g., band-pass filtering, epoch extraction, and baseline correction) to extract stimulus-related brain responses, we obtained results showing that LEPs with a high signal-to-noise ratio were simultaneously recorded from ECoG and LFP electrodes. This methodology makes the simultaneous recording of ECoG and LFP activities possible, which provides a bridge of electrocortical signals at the mesoscopic and macroscopic levels, thereby facilitating the investigation of nociceptive information processing in the brain.

We developed a technique that simultaneously records both electrocorticography and local field potentials in response to nociceptive laser stimuli from freely moving rats. This technique helps establish a direct relationship of electrocortical signals at the mesoscopic and macroscopic levels, which facilitates the investigation of nociceptive information processing in the brain.

Grabaciones simultáneas de los potenciales de campo Local Cortical y Electrocorticograms en respuesta a estímulos nociceptivos Laser se mueva libremente las ratas

Electrocortical responses, elicited by laser heat pulses that selectively activate nociceptive free nerve endings, are widely used in many animal and ...