Los autores desean agradecer a Anni Lee y Maureen Byrne por su ayuda en el establecimiento del protocolo de preferencia un lugar acondicionado extendida.

Todos los procedimientos se llevaron a cabo de acuerdo con el Weill Cornell Medical College Institucional Cuidado de Animales y el empleo Comisión reglas. 1. Preparación y configuración de la administración estereotáxica de vectores virales <ol><li> Si un instrumento, hisopo estéril, o la mano usando guantes estériles están contaminados por el contacto con una superficie no estéril, deseche o volver a esterilizar el instrumento con un esterilizador de cuentas caliente, deseche el hisopo o cambiar a los nuevos guantes estériles. </li><li> Coloque una jaula del ratón con ropa de cama en un bloque de calor para calentar para la recuperación post-operatorio. </li><li> Configure afeitadora eléctrica para afeitarse la cabeza y el etanol y el yodo para esterilizar el cuero cabelludo. </li><li> Limpie los oídos bares y bodegas de la boca del stereotax con etanol al 70%. </li><li> Usando una jeringa de 5 l Hamilton, elaborará 5 l de vector viral (1 x 10 6 partículas virales / l) y creada en el soporte de jeringa stereotax. </li><li> Microondas almohadilla térmica o gire la almohadilla eléctrica sobre unº lugar en stereotax. Cubrir con una almohadilla absorbente limpio para reducir el riesgo de quemaduras. Monitor de temperatura del cuerpo del ratón (entre 97 a 99,5 ° F) con frecuencia durante todo el procedimiento. </li><li> Inyectar ratón (con un peso entre 22-35 g) con una mezcla de ketamina (100 mg / kg) y xilazina (20 mg / kg). Una vez que el ratón está anestesiado, pizca punta de la cola para comprobar los reflejos del ratón y tenga en cuenta cualquier movimiento o respuesta para asegurar que el ratón está adecuadamente anestesiado. </li><li> Afeitarse la cabeza entre las orejas y los ojos. </li><li> Limpie la cabeza rapada con etanol y yodo aplicadores de algodón estériles alterna. </li><li> Traslado mouse pad de calefacción en stereotax. </li></ol> 2. La microinyección estereotáxica de vectores virales <ol><li> Use guantes estériles durante toda la cirugía. Si los guantes se contaminan en cualquier momento al tocar superficies no estériles, cambiar los guantes. </li><li> Zero barra dentada y las escalas de barras para los oídos. </li><li> Establecer los dientes en la barra de diente. </li><li> Atornille hocico. Si se mueve o bigote movimientoción se observó en cualquier momento, administrar una dosis de refuerzo IP (~ 0,05 cc) de una mezcla de ketamina (100 mg / kg) y xilazina (20 mg / kg). </li><li> Atornille las barras de oído. </li><li> Lubricar los ojos con PuraLube. </li><li> Cortar el cuero cabelludo abierto a apenas detrás de las orejas con un bisturí. </li><li> Pinzas para sujetar papeles a cabo en las esquinas del cuero cabelludo a pelar la piel lejos de cráneo. Bregma y lambda debe ser fácilmente visible. </li><li> Asegúrese de sutura a bregma lambda se alinea directamente. Si la sutura es el posicionamiento reajuste torcida de la cabeza. </li><li> Asegúrese de que todos los ajustes del stereotax se ponen a cero. </li><li> Coloque la punta de aguja en su lugar a bregma. </li><li> Mida las coordenadas tanto para bregma y lambda. Esto incluye coordenadas dorsal / ventral, medial / lateral y anterior / posterior. </li><li> Ajuste de la cabeza hasta que las coordenadas ventrales para bregma y lambda son dentro de 0,02 mm el uno del otro. </li><li> Una vez alineados, desde el bregma ir medialmente a las coordenadas medial / lateral (M / L). Cerciorarselos lados izquierdo y derecho están alineados dentro de 0,02 mm el uno del otro. </li><li> Re-centro de la punta de la aguja en bregma y a partir de ahí se mueve en la dirección anterior / posterior (A / P) para la coordenada anterior / posterior deseada. </li><li> Si es necesario la aguja, el ángulo en una dirección y asegúrese de que el stereotax está bloqueado. Ir a la izquierda M / L de coordenadas (+ / - 1,52) </li><li> Medir la posición ventral de la parte superior del cráneo para usar como una referencia después se perfora el pozo de sondeo. </li><li> Perforar un pozo con una broca de estériles en la coordenada deseada. Compruebe que la aguja puede entrar sin interrupción a través del cráneo. </li><li> Baje la aguja hasta la cota deseada ventral. </li><li> Una vez alcanzada la cota, dip 0.015 mm por debajo de ~ 10 segundos para crear un pequeño espacio para el vector viral. </li><li> Inyectar el volumen deseado de vector viral a una velocidad de ~ 0,1 l / min. Espere 3 minutos para el vector viral para difundir completamente. Levante la punta de la aguja de 0,015 mm y esperar otros 2 min. </li><li>Sumergir la aguja (si haciendo inyecciones en ángulo) en la dirección opuesta. Una vez más, registrar la posición ventral como una referencia para la parte superior del cráneo. </li><li> Repita los mismos pasos que en el primer lado para inyectar vector viral en el lado opuesto (en caso de hacer inyecciones bilaterales). </li><li> Aplicar cera de hueso a ambos agujeros para sellarlos con el final de madera de un extremo del aplicador de algodón estéril. </li><li> Suturar el cuero cabelludo. </li><li> Aplicar el bloqueo del nervio a la zona de la herida. </li><li> Retire ratón desde stereotax y colocar en la jaula calentado en el bloque de calor para recuperar durante 45 min. </li><li> Vigilar los signos de dolor que incluyen: disminución de la actividad en general, inquietud, disminución de alimentos y el consumo de agua o el aumento de la vocalización. La buprenorfina (0,05-0,2 mg / kg) se puede administrar dos veces al día durante una semana si es necesario para el dolor. Alternativas aceptables incluyen: meperidina (20-60 mg / kg), pentazocina (10 mg / kg), nalbufina (4-8 mg / kg). </li></ol> 3. Preferencia de lugar condicionado: acquisition, extinción y reintegro <ol><li> Coloque los ratones en la sala central de un aparato de preferencia lugar de tres cámaras (Med Associates Inc., St. Albans, VT, EE.UU.) durante un periodo de habituación 60 segundos con puertas de guillotina cerrados. </li><li> Después de habituación, puertas de guillotina abierta y permitir que los ratones sin la exploración de las 3 cámaras para 1200 seg, con el tiempo transcurrido en cada cámara está grabando con el software MedPC IV (Med Associates, Inc.). </li><li> Asignar ratones para cámaras de acondicionamiento en base a su desempeño durante el pre-test. El uso de un diseño, administración arbitraria par medicamento durante los siguientes 3 días con el compartimiento que era menos preferido durante el pre-test basal. </li><li> Durante los próximos 3 días acondicionado, se inyectan ratones con la cocaína (10 mg / kg, ip) durante la sesión de la mañana y confinarlos en su cámara asignada para 1200 segundos inmediatamente después de la inyección. Ratones Volver a sus jaulas después de la sesión. Después de 4 horas, se inyectan ratones con solución salina (0,01 ml / gpeso corporal) y las confinan a la cámara opuesta para la sesión de la tarde 1200 seg. </li><li> Para la prueba de adquisición, lugar ratones en la cámara central con puertas de guillotina cerrados por un periodo de habituación 60 seg. Puertas de guillotina abierta y permitir la libre exploración de 1.200 seg, con el tiempo pasado en todas las cámaras grabadas por software MedPC IV (Med Associates, Inc.). Calcular preferencia restando la cantidad de tiempo gastado en la cámara de solución salina-emparejado de la cantidad de tiempo gastado en la cámara de cocaína-dos a dos. </li><li> Para apagar la preferencia de lugar, exponer ratones a dos sesiones de 20 min de la exploración libre de diarios en el mismo aparato, separados por al menos 2 horas, a partir de las 24 horas después de la prueba para la adquisición. El tiempo pasado en todas las cámaras de nuevo debe ser registrada y la magnitud de la CPP inicial debe ser comparado con el de cada sesión de extinción. </li><li> Determinar si los ratones han apagado la preferencia inducida por medicamentos, evaluando si la preferencia por un parparticular a través de la cámara de dos días consecutivos de extinción es significativamente menor en comparación con la puntuación de preferencia de adquisición. </li><li> Restablecer los ratones con una dosis de cebado de la cocaína (5 ó 10 mg / kg, ip) y volver a exponer al aparato que permite la exploración libre y registrar el tiempo empleado en cada compartimiento. </li><li> Administrar solución de la eutanasia Euthasol (150 mg / kg). Transcardially perfundir ratones con 4% de paraformaldehído (PFA) y le realizará un examen histológico para validar la colocación correcta microinyección. </li></ol>

<ol>
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Los autores no tienen nada que revelar.

Regional-y la ablación génica temporal específica mediante microinyección estereotáxica de vectores virales combinadas con CPP, que incluye las fases de extinción y reintegro posibilite la investigación de las contribuciones específicas de genes en tres fases distintas de comportamiento adictivo similar. Mientras knockout condicional que se logra utilizando el sistema Cre-loxP tradicional prevé espacio-temporalmente la ablación génica restringidos, estereotáxicamente microinyección Cre-recombinasa en áreas...

La cocaína es un psicoestimulante altamente de refuerzo. Después de la exposición repetida, varias adaptaciones moleculares y celulares que ocurren en la recompensa correspondiente circuitos del cerebro que se cree que resulta en compulsiva búsqueda de drogas, lo que provocó altas tasas de recaída que representan un grave problema clínico 1. La cocaína ejerce estos efectos en el comportamiento de larga duración mediante la regulación de la expresión génica. Para estudiar las adaptaciones que surgen del uso crónico de la cocaína, modelos de roedores preclínicos se han utilizado ampliamente. Uno de estos modelos es el lugar de preferencia paradigma condicionado (CPP). Este modelo implica el desarrollo de una asociación entre un medio ambiente aprendido previamente neutro y las propiedades de recompensa de la cocaína. Después de varias parejas de cocaína con una cámara en particular, los animales se les permite explorar libremente los entornos de cocaína apareadas y no apareadas cocaína y si prefieren el compartimiento de las drogas de pares, que se dice que han adquirido una plac inducida por cocaínapreferencia de correo. Por otra parte, después de un período de entrenamiento de extinción, este paradigma se puede utilizar para estudiar el restablecimiento contexto específico de la cocaína de la conducta de búsqueda. En comparación con otros modelos de comportamiento de comportamiento adictivo-como, tales como la sensibilización psicomotora, que se utiliza para estudiar a largo plazo inducida por la cocaína y la plasticidad de comportamiento molecular de 2,3 y la auto-administración (SA), que se cree que con más precisión imitan adictivo un comportamiento similar se encuentra en los seres humanos, el paradigma CPP es un procedimiento simple para estudiar la cocaína aprendizaje contextual 4. Protocolos de CPP pueden ser convenientemente ampliarse para incluir la extinción y fases de reintegro, al igual que SA, que permiten la investigación de los mecanismos que subyacen ansia por la droga y la recaída 5-7 y que se cree que recapitular los aspectos de lo que ocurre en el hombre la búsqueda de drogas y el narcotráfico - y cue-inducida recaída 8-10. Un mecanismo que subyace a lainducido por cocaína plasticidad de comportamiento que es característico de cada una de las distintas fases de la CPP, incluyendo la adquisición, la extinción, y el restablecimiento, es la activación de firmas únicas de la expresión de genes dentro de las diferentes regiones del cerebro. Para probar directamente que los genes dentro de las regiones cerebrales relevantes para mediar recompensa inducido por cocaína cambios de comportamiento, es útil ser capaz de manipular de forma selectiva de una manera regionalmente específica. Una forma de lograr esto es a microinject recombinantes vectores virales adenoasociados (rAAV) que expresan la recombinasa Cre utilizando la cirugía estereotáxica, en distintas áreas del cerebro de ratones que tienen genes diana flanqueado por sitios LoxP (ratones floxed). Este método permite un control temporal y regional muy preciso sobre el momento y donde los genes se realiza la ablación, tanto en neuronas que dividen y que no se dividen, sin inducir respuestas inmunes 11-13. Este nivel de control representa una importante ventaja sobre la tecnología tradicional Cre-loxP de criar ratones floxed with ratones que expresan la recombinasa Cre bajo el control de un promotor de gen endógeno, en el que el tiempo y distribución de supresión de genes pueden ser reguladas con más fuerza. Además, viral génica mediada por vectores evita posibles efectos compensatorios de desarrollo que pueden producirse utilizando estrategias knockout tradicionales. En adición a la cocaína CPP, que se describe aquí, la microinyección de rAAV-Cre en el cerebro de los ratones floxed para evaluar el papel de los genes específicos se puede aplicar ubicua a paradigmas de comportamiento que implican fases separadas, incluyendo la auto-administración y la sensibilización psicomotora. Por ejemplo, nuestro laboratorio ha utilizado la tecnología de rAAV-Cre para estudiar el papel de Ca v 1.2 2 canales tipo L de Ca + en cocaína sensibilización psicomotora 14. Específicamente, rAAV-Cre se microinyectó en el núcleo accumbens (NAc) de ratones con el gen que codifica Ca v 1.2 floxed, para demostrar que el Ca v </sub&gt; 1,2 actuando en esta región interviene en la fase de expresión de la sensibilización psicomotora 14,15. Sin embargo, la estrategia de rAAV-Cre no se puede utilizar si no existen ratones con un gen floxed de interés, como fue nuestra experiencia cuando la evaluación de la función de Ca v 1.3 2 canales de tipo L Ca + en la sensibilización psicomotora. Por lo tanto, una limitación de la utilización de rAAV-Cre se presenta si los ratones condicionales no existen para un gen particular de interés. Sin embargo, rAAVs que expresan siRNA se puede utilizar para los genes diana desmontables, como lo hemos hecho para examinar el papel de Ca v 1.3 canales 14,15. Microinyección de rAAV-Cre en regiones discretas del cerebro de ratones floxed y luego ponerlos a prueba en el paradigma de la CPP permite la investigación de los genes específicos que median en las diversas fases de comportamiento adictivo-como y donde están actuando. El uso de este paradigma ha ayudado en nuestra comprensión de cómo la administración repetida de cocaína, en esencia, secuestra el brains circuito de recompensa causando cambios inadaptadas en las vías de transducción de señales moleculares y la expresión génica que conducen al estado de adicción 1,16,17.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Conditioned place preference activity chambers</td>
			<td>Med Associates, Inc., St. Albans, VT, USA</td>
			<td>MED-CPP-MS</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stereotaxic alignment system for mouse</td>
			<td>David Kopf Instruments, Tujunga, CA, USA</td>
			<td>model 900</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hamilton syringes</td>
			<td>Hamilton Company, Reno, Nevada, USA</td>
			<td>7634-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>rAAV2-Cre-GFP</td>
			<td>Vector BioLabs, Philadelphia, PA, USA</td>
			<td>7016</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>rAAV2-GFP</td>
			<td>Vector BioLabs, Philadelphia, PA, USA</td>
			<td>7004</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
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stereotaxic microinjection of viral vectors expressing cre recombinase to study the role of target genes in cocaine conditioned place preference

Microinyección recombinante vectores virales adenoasociados (rAAV) que expresan la recombinasa Cre en distintas regiones del cerebro del ratón se convierte selectivamente genes knockout de interés permite un mejor control temporal y regionalmente específica de supresión de genes, en comparación con los métodos existentes. Mientras que la supresión condicional también se puede lograr por los ratones de apareamiento que expresan la recombinasa Cre bajo el control de promotores de genes específicos con ratones que portan un gen floxed, microinyección estereotáxica permite la selección de las zonas discretas del cerebro en puntos de tiempo experimentador-determinadas de interés. En el contexto de la cocaína lugar acondicionado preferencia, y otros paradigmas de comportamiento cocaína tales como la auto-administración o sensibilización psicomotora que puede implicar la retirada, extinción y / o fases de reinstalación, esta técnica es particularmente útil en la exploración de la contribución única de los genes diana a estos distintos fases de los modelos de comportamiento de plasticidad inducida por la cocaína. Específicamente,esta técnica permite la ablación selectiva de los genes diana durante las fases discretas de un comportamiento a prueba su contribución al comportamiento a través del tiempo. En última instancia, este conocimiento permite terapias más específicas que están en mejores condiciones para hacer frente a los factores de riesgo más potentes que se presentan durante cada fase de la conducta adictiva.

CPP Después de realizar CPP en ratones microinyectados, se debe verificar que la cohorte de control de inyección de (rAAV-GFP) ratones han adquirido normalmente preferencia por la cámara de drogas emparejado (Figura 1A, 1B). Los ratones se considera que han adquirido preferencia por una cámara en particular cuando preferencia cocaína (tiempo de permanencia en la cámara de la cocaína de pares de menos tiempo de permanencia en la cámara de solución salina-emparejado) es ...

Watch this Scientific Journal Video about Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference at JoVE.com

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

En este artículo se describe cómo microinject vectores virales en el cerebro del ratón y luego probar en un paradigma de preferencia un lugar acondicionado que incluye una fase de adquisición, extinción y reintegro.

La microinyección estereotáxica de vectores virales que expresan Cre recombinasa para estudiar el papel de los genes diana de cocaína acondicionado Place Preferencia

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes ...

stereotaxic-microinjection-viral-vectors-expressing-cre-recombinase

Research

JoVE Journal

Behavior

18.5K vistas.  Weill Cornell Graduate School of Biomedical Sciences. En este artículo se describe cómo microinject vectores virales en el cerebro del ratón y luego probar en un paradigma de preferencia un lugar acondicionado que incluye una fase de adquisición, extinción y reintegro.

Video: Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with na&iuml;ve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

Inyección estereotáctica de microARN que expresan lentivirus al ratón hipocampo CA1 Región y evaluación de los resultados del comportamiento

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

Investigación

Comportamiento

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

Un procedimiento para estudiar el efecto de la prolongada restricción de alimentos en heroína Buscando en ratas abstinentes

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

Fat Preference: A Novel Model of Eating Behavior in Rats

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to this multifactorial disorder is the consumption of a high fat diet, a behavior that has been shown to increase both caloric intake and body fat content. However, the elements regulating preference for high fat food over other foods remain understudied.
To overcome this deficit, a model to quickly and easily test changes in the preference for dietary fat was developed. The Fat Preference model presents rats with a series of choices between foods with differing fat content. Like humans, rats have a natural bias toward consuming high fat food, making the rat model ideal for translational studies. Changes in preference can be ascribed to the effect of either genetic differences or pharmacological interventions. This model allows for the exploration of determinates of fat preference and screening pharmacotherapeutic agents that influence acquisition of obesity.

Dietary fat content influences both energy intake and body fat composition in mammals. By examining rats&#8217; preference for high fat food in a series of choice experiments, it is possible to test genetic differences and pharmacological interventions on their preference for high fat food.

Preferencia grasa: Un Modelo de Novela de la conducta alimentaria en ratas

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to ...

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

Un procedimiento para observar inducida Contexto-Renovación-pavloviano acondicionado Alcohol comportamiento de búsqueda en ratas

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as appropriate models, partly because the response to behaviorally relevant stimuli and the circuitry parsing stimulus-action outcomes is astonishingly similar between humans and rodents. In studying molecular substrates of complex behaviors, the microinjection of reagents that modify, augment, or silence specific systems is an invaluable technique. However, it is crucial that the microinjection site is precisely targeted in order to aid interpretation of the results. We present a method for the manufacture of surgical implements and microinjection needles that enables accurate microinjection and unlimited customizability with minimal cost. Importantly, this technique can be successfully completed in awake rodents if conducted in conjunction with other JoVE articles that covered requisite surgical procedures. Additionally, there are many behavioral paradigms that are well suited for measuring motivation. The progressive ratio is a commonly used method that quantifies the efficacy of a reinforcer to maintain responding despite an (often exponentially) increasing work requirement. This assay is sensitive to reinforcer magnitude and pharmacological manipulations, which allows reinforcing efficacy and/ or motivation to be determined. We also present a straightforward approach to program operant software to accommodate a progressive ratio reinforcement schedule.

Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented.

Roedor cerebro microinyección para el Estudio de los sustratos moleculares de Comportamiento Motivado

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as ...

Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to drugs of abuse make lasting alterations in the brain function of vulnerable individuals, and these persistent alterations likely serve to maintain the maladaptive drug seeking and taking behaviors characteristic of addiction/dependence2. In rodents, reward experience and contextual associations are frequently measured using the conditioned place preference assay, or CPP, wherein preference for a previously drug-paired context is measured. Behavioral sensitization, on the other hand, is an increase in a drug-induced behavior that develops progressively over repeated exposures. Since sensitized behaviors can often be measured after several months of drug abstinence, depending on the dose and length of initial exposure, they are considered observable correlates of lasting drug-induced plasticity. Researchers have found these assays useful in determining the neurobiological substrates mediating aspects of addiction as well as assessing the potential of different interventions in disrupting these behaviors. This manuscript describes basic, effective protocols for mouse CPP and locomotor behavioral sensitization to cocaine.

This protocol is intended to enable researchers to conduct experiments designed to test these aspects of addiction using the conditioned place preference and locomotor behavioral sensitization assays.

Evaluación del comportamiento de sensibilización inducida por la cocaína y la preferencia de lugar condicionado en ratones

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to ...

Place and Response Learning in the Open-field Tower Maze

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for examining how rats learn to use a place- or response-learning to successfully navigate in an open-field arena. Additionally, this protocol describes how the OFTM differs from other behavioral maze paradigms that are commonly used to study spatial learning in rodents. The OFTM described in this article was adapted from the one previously described by Cole, Clipperton, and Walt (2007). Specifically, the OFTM was created to test spatial learning in rodents without the experimenter having to consider how &#8220;stress&#8221; might play a role as a confounding variable. Experiments have shown that stress-alone can significantly affect cognitive function1. The representative results section contains data from an experiment that used the OFTM to examine the effects of estradiol treatment on place- and response-learning in adult female Sprague Dawley rats2. Future studies will be designed to examine the role of the hippocampus and striatum in place- and response-learning in the OFTM.

The Open-field Tower Maze (OFTM) is a maze created to study the behavioral and neural mechanisms of spatial learning (e.g., place- or response-learning) in rats. This maze is especially useful for experimenters who want to use a non-stressful maze paradigm to investigate spatial learning in their research.

Coloque y Aprendizaje de respuesta en el Abierto de campo Torre Maze

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for ...

Reinstatement of Drug-seeking in Mice Using the Conditioned Place Preference Paradigm

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to acquire a conditioned place preference in a drug-paired compartment, and after the post-conditioning test, they perform several sessions to extinguish the established preference. The CPP permits the evaluation of the conditioned rewarding effects of drugs related to environmental cues. Then, the extinguished CPP can be robustly reinstated by the non-contingent administration of a priming dose of the drug, and by exposure to stressful stimuli. Both methods will be explained here. When the animal reinitiates the behavioral response, a reinstatement of the conditioned reward is considered to have taken place.
The main advantages of this protocol are that it is non-invasive, inexpensive, and simple with good validity criteria. In addition, it allows the study of different environmental manipulations, such as stress or diet, which can modulate relapse into drug seeking behaviors. However, one limitation is that if the researcher aims to explore the motivation and primary reinforcing effects of the drug, it should be complemented with self-administration procedures, as they involve operant responses of animals.

This protocol describes the Conditioned Place Preference (CPP) as a model of relapse. This procedure permits the measurement of relapse in laboratory animals, considering the impact of drug-associated environmental cues as craving and relapse in abstaining addicts is currently the focus of drug-abuse treatment programs.

Reintegro de la búsqueda de drogas en ratones utilizando el paradigma de preferencia condicionada del lugar

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to ...

A Conditioned Place Preference Protocol for Measuring Incubation of Craving in Rats

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed incubation of drug craving. Here, we describe a morphine conditioned place preference (CPP) protocol for measuring the incubation of craving in rats. In this protocol, a CPP paradigm mainly employing somatosensory cues is used to establish a long-term reward memory of morphine. A three-chamber CPP box that differs in the texture of the chamber floor is constructed. First, the animals are tested for their baseline preference to the two side chambers for three consecutive days. Then, they are injected intraperitoneally with morphine/saline and put into their non-preferred/preferred chamber for 45 min. After 6 days of conditioning, their preference to the side chambers is tested for 15 min at different time points after the last conditioning session. With this paradigm, the reward memory of morphine could last for at least 18 days. To test whether the above-mentioned protocol can model increased craving, the number of entrances into the two side chambers are counted during the abstinence period. The results show that the entrances increased, suggesting that the CPP paradigm could mimic the incubation of craving. Future studies can employ this model to study neural mechanisms underlying long-term memory and incubation of craving.

Here, a morphine conditioned place preference (CPP) protocol is described to measure incubation of craving in rats.

Un protocolo de preferencia de lugar condicionada para medir la incubación de antojo en ratas

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed ...

Assessing the Coherence of Parents' Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with parents. This paper focuses on such a method, namely Five-Minute Speech Sample-Coherence (FMSS-Coherence). In this method, a parent is asked to speak for five uninterrupted minutes about her/his child and their relationship. The resulted narrative is coded for coherence, namely the extent to which the parent provides&#160;in the narrative&#160;a clear, consistent, multidimensional and well-supported portrayal of the child. FMSS-Coherence is based on attachment research that shows that the coherence of parents&#8217; narratives is indicative of the quality of the parent-child relationship and child adjustment. It overcomes the limitations of attachment narrative measures which are typically labor intensive. FMSS-Coherence is less nuanced than extant attachment narrative measures. Yet, studies of families from different cultural backgrounds, across different child ages and in the context of typically developing children as well as children with special needs suggest that coherence can be reliably evaluated using the FMSS procedure. Furthermore, parents&#8217; FMSS-Coherence is associated with parenting quality and children&#8217;s socio-emotional adjustment. Thus, it holds promise for researchers and practitioners who seek a relatively time- and cost-effective method for assessing the coherence of parents&#8217; narratives regarding their child.

Assessing the Coherence of Parents&#039; Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

This paper introduces a method for assessing the coherence of parents' short narratives regarding their child and their relationship using the five-minute speech sample procedure.

Evaluar la coherencia de las narrativas cortas de los padres con respecto a su hijo utilizando el procedimiento de muestra de habla de cinco minutos

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with ...