Este trabajo es apoyado por una beca de la Fundación Nacional de Ciencias naturales de China (No.31472037). Cualquier mención de nombres comerciales o productos comerciales en este artículo es únicamente con el propósito de proporcionar información específica y no implica una recomendación.

1. preparación de instrumentos y del insecto
<ol><li>
Preparación de tungsteno electrodos y estímulos soluciones
	<ol>
<li>Fijar un nuevo alambre de tungsteno (0,125 mm de diámetro, longitud de 75 mm) en un micromanipulador y afilar en una solución de nitrito (NaNO2) de sodio 10% (p/v) en una jeringa de 10 V proporcionado por una fuente de alimentación para 1 min. bajo un estereomicroscopio (aumento de X 40).</li>
		<li>Sumerja el alambre de tungsteno afilada repetidamente en la NaNO2 solución al 10%, aproximadamente de 4 mm a 5 V en < 1 minuto (figura 1A).</li>
		<li>Examinar el diámetro de la punta de tungsteno afilada con frecuencia bajo el estereomicroscopio hasta que esté lo suficientemente fina para penetrar la cutícula de un sensillum olfativa del palpo de langostas (figura 1B).</li>
		<li>Preparar las soluciones de estímulo. Diluir cada uno de la sustancia de estímulo químico en aceite mineral. Diluir 1-nonanol y ácido nonanoico en diluciones de 10%. Diluir E-2-hexenal y hexanal en 10-2, 10-3, 10-4y 10-5.</li>
		<li>Preparar Pasteur los tubos que llevan los estímulos: Inserte las tiras de papel de filtro (de 2 cm de largo, de 0,5 cm de ancho) en los tubos de Pasteur, agregar las soluciones diluidas de estímulo (cada 10 μl) a las tiras de papel de filtro y luego conecte los tubos de Pasteur con pipetas (1 ml).</li>
	</ol>
</li>
	<li>
Preparar el insecto
	<ol>
<li>Posterior las langostas (Locusta migratoria) con plántulas de trigo fresco bajo condiciones de hacinamiento en una humedad relativa de 60%, una temperatura de 28-30 ° C y un fotoperíodo de 18:6 h (luz: oscuridad). Elija de 1 a 3 días de edad: 5º instar ninfas de langosta y quitar las antenas con tijeras finas para evitar cualquier interferencia durante la grabación.</li>
	</ol>
</li>
	<li>
Preparar el soporte del palpo maxilar langosta
	<ol>
<li>Utilizar un portaobjeto (25 x 75 mm) como la base del soporte del palpo maxilar (MPH). Póngale un trozo de plástico (1 mm de altura, 10 mm de ancho, 35 mm de longitud) en una esquina de la diapositiva de cristal con cinta adhesiva de doble cara y finalmente fijar un cubierta de vidrio (18 mm x 18 mm) encima de la pieza de plástico con cinta adhesiva de doble cara. Coloque un pedazo pequeño de cinta de goma roja en la cubierta de vidrio como una capa de antideslizante. La pieza de plástico y el vidrio de la cubierta constituyen la plataforma para el palpo de langosta. La altura de la plataforma es de aproximadamente 1,5 mm.</li>
		<li>Instalar un alambre de tungsteno (diámetro de 0,125 mm, 36 mm de longitud) a una distancia de 1,5 mm paralelo al interior borde de la plataforma. Fijar los dos extremos del cable a la plataforma con cinta adhesiva de doble cara.</li>
	</ol>
</li>
</ol>2. preparación de palpos maxilares de langosta
<ol><li>Cortar un tubo de centrífuga (1.5 ml) verticalmente por la mitad y cortar la parte inferior. Ponga la langosta en el tubo preparado. Salir de la región ventral y la cabeza de la langosta expuesta. Fijar el conjunto a la diapositiva de cristal con cinta adhesiva de doble cara (figura 2A).</li>
	<li>Tire el palpo maxilar derecho en la plataforma.</li>
	<li>Poner el alambre de tungsteno en el cuarto segmento de la palpo. Coloque masilla adhesiva a cada lado del alambre de tungsteno, unos 2 mm de la palpo maxilar (figura 2A y 2B).</li>
</ol>3. solo Sensillum grabaciones
<ol><li>Colocar la preparación de langosta palpo maxilar bajo un microscopio a bajo aumento (100 X). Ajuste la posición de la preparación hasta que el palpo es perpendicular al electrodo de la grabación (Figura 3A).</li>
	<li>Insertar el electrodo de referencia (electrodo de tungsteno) en el ojo de la langosta mediante un micromanipulador. Mover el electrodo de la grabación (electrodo de tungsteno) cerca el palpo maxilar con el instrumental quirúrgico (figura 3B y 3C).</li>
	<li>Ajuste el dispositivo de entrega de olor a aproximadamente 1 cm desde el palpo maxilar (figura 3B).</li>
	<li>Abra el software de grabación Auto punto 32. Configurar los parámetros de grabación como sigue: la escala de grabación 500 μV; el alta corte del filtro de 300 Hz, el límite baja a 200 Hz; y pretrigger en 10 s.</li>
	<li>Conectar el electrodo de la grabación a un 10 x amplificador de AC/DC universal.</li>
	<li>Interruptor del microscopio a un aumento alto de (500 X). Insertar el electrodo de registro en la base de un sensillum de basiconic en el palpo maxilar y delicadamente ajustar el electrodo de la grabación para obtener buena picos espontáneos (figura 3D).</li>
	<li>Abra el controlador de estímulo para entregar una corriente de aire continua a 20 ml/s. establecer el tiempo de estimulación 1 señales de registro s. para 10 s, a partir de 10 s antes de la aparición del pulso del estímulo.</li>
	<li>Utilice un amplificador de AC/DC universal de 10 x para amplificar las señales. Alimentan las señales de los 4 de IDAC. Analizar las señales con el software Auto punto 32. Señales de CA son band-pass filtrada entre 200 a 300 Hz. uso Auto punto 32 para distinguir las amplitudes pico a través de ruidos. Calcular las respuestas de las neuronas como los aumentos en la frecuencia del potencial de acción (espigas por segundo) sobre las frecuencias espontáneas. Realizar un análisis estadístico utilizando GraphPad Prisma 7.</li>
</ol>

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</ol>

Los autores no tienen nada que revelar.

Insectos dependen de palpos para detectar olores de alimentos y los palpos se creen que juegan un papel importante en la especiación13,27. Los palpos son órganos olfativos simples y están recibiendo una atención creciente como un modelo atractivo para la exploración de la neuromolecular redes subyacentes chemosensation28.
Insectos labellar y palpo SSRs se han realizado con éxito en Drosophila melanogaster, gambiae de los Anophelesy Culex quinquefasciatus13,14,15,16 , 17 pero se han divulgado raramente en la forma de un vídeo de presentación16,29. Por el contrario, hay datos videos en SSRs antenales de Drosophila, la polilla del orangeworm del ombligo (Amyeloistransitella), Schistocerca Americanay la chinche (Cimex lectularius)16, 30 , 31 , 32 , 33.
Locust palpo sensilla basiconica tienen una estructura particular que difiere de la de sensillo antenal de langosta y muchos otros insectos sensilla. Utilizando el método descrito aquí, potenciales de acción generados por langosta palpo sensilla basiconica subtipos pb1 y pb2 pudo ser grabado y discriminadas (figura 4 y figura 5).
El paso crítico es la penetración de los electrodos de la grabación. El electrodo de la grabación debe ser insertado en la base de la sensillum y avanzado hasta que se adquieren buenas señales. Además, es importante evitar que la cúpula de la palpo que se derrumban cuando el electrodo de registro se inserta en la base de la sensillum. Para ello, establecer una plataforma que incluye un sostenedor del palpo maxilar langosta especial (MPH) y utiliza un alambre de tungsteno para comprimir el cuarto segmento de la palpo. Muchas repeticiones de este procedimiento muestran que es efectiva. Basado en los patrones de respuesta de las neuronas en la sensilla a varios odorantes, hemos, por primera vez, identificado dos subtipos de basiconica sensillo en el palpo maxilar de la langosta, es decir, pb1 y pb2.
La limitación de la técnica se describe en esta publicación es que podría ser utilizado para grabar grandes insectos (e.g., polillas, escarabajos y langostas) mientras no para grabar pequeños insectos (por ejemplo, moscas y mosquitos), que tienen sus propias plataformas y técnicas13,14,15,16,17. Esta técnica es complementaria a los métodos existentes.
En conclusión, un protocolo muy eficaz de un SSR de insectos palpo sensilla basiconica se describe en detalle. Este protocolo podría proporcionar los investigadores con una técnica útil en el estudio de mecanismos moleculares y celulares de insectos del olfato en el mouthpart. Este método con cromatografía de gases puede utilizarse para identificar ligandos naturales electrophysiologically activo en extractos de recursos alimenticios favorables.

Los animales han desarrollado una serie de órganos quimiosensoriales que detecta señales químicas exógenas. En los insectos, los órganos quimiosensoriales más importantes son las antenas y los palpos. En estos órganos, varios tipos de pelos quimiosensoriales, llamados chemosensory sensilla, están inervados por neuronas quimiosensoriales (CSNs) dentro de los pelos. CSNs en sensilla chemosensory reconocen señales químicas específicas a través de la transducción de estímulos químicos a potenciales eléctricos que posteriormente se transfieren hasta el sistema nervioso central1,2,3 .
CSNs expresan receptores quimiosensoriales varios [p. ej., receptores de olor (ORs)], ionotrópicos (IRs) de los receptores y los receptores gustativos (GRs) en sus membranas, que codifican señales químicos exógenos asociados con diferentes tipos de chemosensation 4,5,6. La caracterización de CSNs es clave para el esclarecimiento de los mecanismos celulares y moleculares de insectos quimiorrecepción. Ahora solo sensillum grabación (SSR) es una técnica ampliamente utilizada para la caracterización de insecto CSNs en la sensilla antenal de muchos insectos, incluyendo moscas7,8de polillas, escarabajos de9,10de áfidos, langostas11, y las hormigas12. Sin embargo, pocos estudios han aplicado un SSR a palpos insectos13,14,15,16,17, ya que las estructuras particulares de su sensillo un grabación electrofisiológica difícil18.
Enjambres de langostas (Orthoptera) a menudo causan graves daños y pérdidas económicas19. Los palpos se creen que juegan un papel importante en la selección del alimento de langosta20,21,22,23,24. Dos tipos de chemosensory sensilla son investigados por un microscopio electrónico de barrido (SEM). Generalmente, se observan en cada cúpula de langosta palpos18350 sensilla chaetica y sensilla basiconica de 7-8. Chaetica sensillo son sensillo gustativo que detecta señales químicas no volátil, mientras que sensillo basiconica tienen una función olfativa, detección de señales químicas volátiles.
En palpos de la langosta, los diámetros de las tomas del pelo de la basiconica de la sensilla (ca. 12 μm), son mucho mayores que los de sensilla chaetica (ca. 8 μm)18,25. La pared cuticular de la basiconica sensilla en los palpos es mucho más gruesa que el de sensillo antenal18. Además, la cúpula de la palpo tiene líquido contenido dentro de una cutícula flexible. Estas características significan que una penetración con un microelectrodo y adquisición de señales electrofisiológicas buena es más difícil que para sensillo antenal. En este documento, un protocolo SSR detallado y altamente eficaz para langosta palpo sensilla basiconica se presenta con un video.

<table><tbody><tr><td>Tungsten wire</td><td>ADVENT</td><td>W559504</td><td>Used for making the electrode and fixing the palp</td></tr><tr><td>NaNO&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-aldrich</td><td>563218-25G</td><td>Used for sharpening the tungsten wire</td></tr><tr><td>AC Power Supply</td><td>Syntech</td><td>A2-70</td><td>Providing the voltage in sharpening the tungsten wire</td></tr><tr><td>Stereoscope</td><td>Motic</td><td>SMZ-163</td><td>Used for observing the sharpening of tungsten wire</td></tr><tr><td>Microscope</td><td>Olympus</td><td>W-51</td><td>Used for observing the sensilla on locust maxillary palp</td></tr><tr><td>Intelligent Data Acquisition Controller</td><td>Syntech</td><td>IDAC-4</td><td>Real-time on screen display of all signals before and during recording</td></tr><tr><td>Stimulus controller</td><td>Syntech</td><td>CS-55</td><td>Used for controlling the stimulus application</td></tr><tr><td>Electronic micromanipulator</td><td>C.M.D.T</td><td>CFT-8301D</td><td>Used for minor movement of the recording electrode</td></tr><tr><td>Micromanipulator</td><td>Narishige</td><td>MN-151</td><td>Used for minor movement of the reference electrode</td></tr><tr><td>Speaker</td><td>EDIFIER</td><td>R101T06</td><td>Connected with IDAC-4 and providing sound for the signal</td></tr><tr><td>Magnetic base</td><td>PDOK</td><td>PD-101</td><td>Used to hold the electrode, and stimulus delivery tube</td></tr><tr><td>Vibration Isolation Table</td><td>TianHe</td><td>HAP-100-1208</td><td>Used for isolating the vibration from the equipment</td></tr><tr><td>Glass slide</td><td>CITOGLAS</td><td>ZBP-407</td><td>Used for making the base for the MPH</td></tr><tr><td>Blu-tack</td><td>Bostik</td><td>Blu-tack-45g</td><td>Fixing the tungsten wire</td></tr><tr><td>Pasteur tube</td><td>YARE</td><td>WITEG</td><td>Placing the filter paper containing stimuli stimulus solutions</td></tr></tbody></table>

single sensillum recordings for locust palp sensilla basiconica

Los palpos de piezas bucales de la langosta se consideran órganos gustativos convencionales que juegan un papel importante en la selección de alimentos de una langosta, especialmente para la detección de señales químicas no volátil por sensilla chaetica (anteriormente llamado terminal sensilla o sensillo crested). Hay creciente evidencia de que estos palpos tienen también una función olfatoria. Un receptor odorante (LmigOR2) y una proteína odorante (LmigOBP1) han sido localizados en las neuronas y células de accesorio, respectivamente, en el basiconica sensilla de los palpos. Sensillum sola grabación (SSR) se utiliza para registrar las respuestas de las neuronas receptoras del olor, que es un método eficaz para la detección de ligandos activos sobre los receptores de olor específico. SSR se utiliza en estudios funcionales de receptores odorantes en sensillo palp. La estructura de la basiconica sensilla situado en la cúpula de los palpos difiere un poco de la estructura de los de las antenas. Por lo tanto, cuando se realiza un SSR provocada por olores, algunos consejos específicos pueden ser útil para la obtención de resultados óptimos. En este papel, se presenta un protocolo detallado y altamente eficaz para un SSR de insectos palpo sensilla basiconica.

Se identifican dos subtipos sensilla (pb1 y pb2) en el palpo maxilar de langosta basado en dinámica de respuesta a olores químicos (10% 1-nonanol y 10% de ácido nonanoico). Las neuronas en pb1 producen significativamente más picos 1-nonanol de ácido nonanoico mientras las neuronas en pb2 son significativamente que menos activa 1-nonanol comparado con ácido nonanoico (figura 4). Hexanal y E-2-Hexenal pueden evocar un palpo langosta apertura respuesta (POR)26. Hexanal es una anfitrión abundante verde de la planta hoja volátil que puede contribuir a una confirmación adicional a la fuente de alimentos26. Los puntos sacados en las neuronas de pb1 último más de largo que los de pb2 cuando son estimulados por E-2-hexenal (figura 4). Las neuronas en pb1 y pb2 exhiben igualmente sólidas respuestas a hexanal (figura 4). Comparar los cambios promedios de todos los puntos entre los períodos 5 antes y 5 s después de que el estímulo indica que la respuesta a 1-nonanol es significativamente superior al ácido nonanoico en pb1, sino por el contrario en pb2 (figura 5). Las neuronas en estos dos subtipos de sensilla respondan dosis dependiente a E-2-hexenal y hexanal y sus patrones de respuesta a estas dos aldehídos diferencian (figura 6A y 6B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57863/57863fig1.jpg"> Figura 1. Preparación del electrodo. (A) este panel muestra un general ve del aparato de afilado del electrodo. La jeringa que contiene 10% NaNO2 (izquierda) se utiliza para afilar el electrodo (derecha). (B) este panel muestra una vista cercana de la punta del electrodo (a: adecuado, b: inadecuados). <a href="/files/ftp_upload/57863/57863fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57863/57863fig2.jpg"> Figura 2. Sostenedor del palpo maxilar langosta (MPH). (A) el MPH y una langosta están montados sobre el portaobjetos de vidrio antes de colocar bajo el microscopio. (B) este panel muestra un primer plano de la palpo maxilar de langosta, fijada por el alambre de tungsteno en la plataforma. <a href="/files/ftp_upload/57863/57863fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57863/57863fig3.jpg"> Figura 3. Solo grabaciones de sensillum. (A) este panel muestra una vista de la configuración de la electrofisiología. (B) este panel muestra una vista cercana de la preparación de langosta en el microscopio. (C) esta imagen muestra el palpo maxilar de langosta con 100 aumentos. (D) esta imagen muestra el palpo con 500 aumentos. La flecha indica un sensillum de basiconic. <a href="/files/ftp_upload/57863/57863fig3large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57863/57863fig4.jpg"> Figura 4. Rastros de la respuesta de grabaciones solo sensillum del palpo maxilar langosta. En este panel, pb1 está parado para el subtipo 1 de la basiconica sensilla de palpo; PB2 está parado para el subtipo 2 de la palpo sensilla basiconica. Las barras sobre las huellas indican la duración del estímulo (1 s). Para estas grabaciones, en diluciones de 10% excepto E-2-hexenal y hexanal, que son diluidas al 1% se utilizan todos los olores. Esta figura ha sido modificada de Zhang et al. 26. <a href="/files/ftp_upload/57863/57863fig4large.jpg" target="_blank">por favor haga clic aquí para ver una versión más grande de esta figura.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57863/57863fig5v2.jpg"> Figura 5. Comparación de medios números de espigas en las neuronas pb1 y pb2 estimulada por ácido nonanoico y 1-nonanol. El número promedio de los picos se calcula en los períodos 5 s antes y después del estímulo. En pb1, la cantidad media de los picos en las neuronas en respuesta a 1-nonanol aumenta significativamente más altos que los de las puntas de las neuronas en respuesta a ácido nonanoico (n = 11 palpos; ANOVA con post hoc prueba t; p < 0.0001), en contraste con pb2 (n = 10 palpos; ANOVA con post hoc prueba t; p = 0.0110). La barra de error representa SEM. Esta figura ha sido modificada de Zhang et al. 26. <a href="/files/ftp_upload/57863/57863fig5v2large.jpg" target="_blank">por favor haga clic aquí para ver una versión más grande de esta figura.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57863/57863fig6v3.jpg"> Figura 6. Los patrones de las neuronas en pb1 y pb2 dosis dependiente responde a E-2-hexenal y hexanal. (A) este panel muestra los patrones de las neuronas en pb1 (± SEM; n = 12 palpos). (B) este panel muestra los patrones de las neuronas en pb2 (± SEM; n = 10 palpos). Esta figura ha sido modificada de Zhang et al. 26. <a href="/files/ftp_upload/57863/57863fig6large.jpg" target="_blank">por favor haga clic aquí para ver una versión más grande de esta figura.</a>

Watch this Scientific Journal Video about Single Sensillum Recordings for Locust Palp Sensilla Basiconica at JoVE.com

Single Sensillum Recordings for Locust Palp Sensilla Basiconica

Este papel describe un protocolo detallado y muy eficaz para las grabaciones de solo sensillum de la basiconica sensilla en los palpos de partes bucales de insectos.

Grabaciones de Sensillum solo para langosta palpo Sensilla Basiconica

The palps of locust mouthparts are considered to be conventional gustatory organs that play an important role in a locust&#39;s food selection, especially ...

single-sensillum-recordings-for-locust-palp-sensilla-basiconica

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.2K vistas.  China Agricultural University. Este papel describe un protocolo detallado y muy eficaz para las grabaciones de solo sensillum de la basiconica sensilla en los palpos de partes bucales de insectos.

Video: Single Sensillum Recordings for Locust Palp Sensilla Basiconica

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

Grabaciones solo Sensillum en los insectos Drosophila melanogaster Y Anopheles gambiae</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

Investigación

Neurociencias

Electrophysiological Measurements from a Moth Olfactory System

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively. These approaches have been exploited for getting a better understanding of insect chemical communication. The identified stimuli can then be used as either attractants or repellents in management strategies for insect pests.

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively.

Las mediciones electrofisiológicas de un sistema olfativo Polilla

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single ...

Electrophysiology of Scorpion Peg Sensilla

We describe a modification of an existing tip-recording technique1,2 for electrophysiologically investigating short, peg-like sensory sensilla3,4. On the mid-ventral surface of all scorpions are two appendages called pectines, which have dense fields of mechano- and chemosensory peg sensilla5,6. One method for assessing chemoresponsiveness of these sensilla uses a tungsten electrode for extracellularly recording neural activity within a sensillum as a volatile odorant is introduced to the sensory field5,7. The limitations of this method include slow data collection and uncontrolled stimulant introduction to, and removal from, the peg field. To overcome these limitations, we developed a new tip-recording technique that uses nonpolar mineral oil as a medium through which to deliver water-based tastants to individual peg sensilla8,9. We have successfully applied this method to obtain sensillar chemoresponses to citric acid, ethanol, and salt. Here we describe the experimental protocol for such a study9. We think this new method may be useful for studying the response properties of other arthropod chemosensory systems, including those of insects10, 11 and crustaceans12.

This article describes an electrophysiological method for isolating chemical stimulation to individual sensilla via extracellular, tip-recordings under mineral oil.

Electrofisiología del Escorpión Peg sensilla

We describe a modification of an existing tip-recording technique1,2 for electrophysiologically investigating short, peg-like sensory sensilla3,4. On the ...

Sample Preparation Method of Scanning and Transmission Electron Microscope for the Appendages of Woodboring Beetle

This report described sample preparation methods that scanning and transmission electron microscope observations, demonstrated by preparing appendages of the woodboring beetle, Chlorophorus caragana Xie &#38; Wang (2012), for both types of electron microscopy. The scanning electron microscopy (SEM) sample preparation protocol was based on sample chemical fixation, dehydration in a series of ethanol baths, drying, and sputter-coating. By adding Tween 20 (Polyoxyethylene sorbitan laurate) to the fixative and the wash solution, the insect body surface of woodboring beetle was washed more cleanly in SEM. This study&#39;s transmission electron microscopy (TEM) sample preparation involved a series of steps including fixation, ethanol dehydration, embedding in resin, positioning using fluorescence microscopy, sectioning, and staining. Fixative with Tween 20 enabled penetrate the insect body wall of woodboring beetle more easily than it would had been without Tween 20, and subsequently better fixed tissues and organs in the body, thus yielded clear transmission electron microscope observations of insect sensilla ultrastructures. The next step of this preparation was determining the positions of insect sensilla in the sample embedded in the resin block by using fluorescence microscopy to increase the precision of target sensilla positioning. This improved slicing accuracy.

In order to observe ultrastructure of insect sensilla, scanning and transmission electron microscopy (SEM and TEM, respectively) sample preparation protocol were presented in the study. Tween 20 was added into the fixative to avoid sample deformation in SEM. Fluorescence microscopy was helpful for improving slicing accuracy in TEM.

Método de preparación de muestras de microscopio electrónico de escaneo y transmisión para los apéndices del escarabajo carpintero

This report described sample preparation methods that scanning and transmission electron microscope observations, demonstrated by preparing appendages of ...

Medio ambiente

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

Varias unidades Métodos de grabación para caracterizar la actividad neuronal en el Locust (<em&gt; Schistocerca americana</em&gt;) Circuitos olfativas

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

Electrofisiológico Grabación desde<em&gt; Drosophila</em&gt; Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house single or multiple neurons inside, are considered to make the major contribution in responding to the chemical stimuli. By directly recording action potential in the olfactory sensillum after exposure to stimuli, single sensillum recording (SSR) technique provides a powerful approach for investigating the neural responses of insects to chemical stimuli. For the bed bug, which is a notorious human parasite, multiple types of olfactory sensillum have been characterized. In this study, we demonstrated neural responses of bed bug olfactory sensilla to two chemical stimuli and the dose-dependent responses to one of them using the SSR method. This approach enables researchers to conduct early screening for individual chemical stimuli on the bed bug olfactory sensilla, which would provide valuable information for the development of new bed bug attractants or repellents and benefits the bed bug control efforts.

Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

Uso Individual sensillum grabación para detectar respuestas olfativo neurona de chinches de cama a Semioquímicos

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

Localización de Genes Receptor Odorante en Antenas Locustinas por RNA<em&gt; In Situ</em&gt; Hibridación

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, oviposition, and mating. Myriad chemicals of varying volatilities have been identified as natural odorants that activate insect Olfactory Receptor Neurons (ORNs). However, studying the olfactory responses to low-volatility odorants has been hampered by an inability to effectively present such stimuli using conventional odor-delivery methods. Here, we describe a procedure that permits the effective presentation of low-volatility odorants for in vivo Single-Sensillum Recording (SSR). By minimizing the distance between the odor source and the target tissue, this method allows for the application of biologically salient but hitherto inaccessible odorants, including palmitoleic acid, a stimulatory pheromone with a demonstrated effect on ORNs involved in courtship and mating behavior1. Our procedure thus affords a new avenue to assay a host of low-volatility odorants for the study of insect olfaction and pheromone communication.

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular pheromones.

Registro electrofisiológico de<em&gt; Drosophila</em&gt; Tricoide Sensilla en Respuesta a Odorantes de Baja Volatilidad

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, ...

Un método novedoso para mejorar el análisis de la actividad de las neuronas gustativas de los insectos

Base Recording: A Technique for Analyzing Responses of Taste Neurons in Drosophila

Insects taste the external world through taste hairs, or sensilla, that have pores at their tips. When a sensillum comes into contact with a potential food source, compounds from the food source enter through the pore and activate neurons within. For over 50 years, these responses have been recorded using a technique called tip recording. However, this method has major limitations, including the inability to measure neural activity before or after stimulus contact and the requirement for tastants to be soluble in aqueous solutions. We describe here a technique that we call base recording, which overcomes these limitations. Base recording allows the measurement of taste neuron activity before, during, and after the stimulus. Thus, it allows extensive analysis of OFF responses that occur after a taste stimulus. It can be used to study hydrophobic compounds such as long-chain pheromones that have very low solubility in water. In summary, base recording offers the advantages of single-sensillum electrophysiology as a means of measuring neuronal activity - high spatial and temporal resolution, without the need for genetic tools - and overcomes key limitations of the traditional tip recording technique.

Autor destacado: Avances en quimiorrecepción: de los receptores de olor de insectos a los ARN no codificantes

A rarely used method of electrophysiological recording, base recording, allows analysis of features of taste coding that cannot be examined by conventional recording methods. Base recording also allows the analysis of taste responses to hydrophobic stimuli that cannot be studied using traditional electrophysiological methods.

Registro de bases: una técnica para analizar las respuestas de las neuronas gustativas en Drosophila

Insects taste the external world through taste hairs, or sensilla, that have pores at their tips. When a sensillum comes into contact with a potential ...

Grabaciones de Sensillum solo para langosta palpo Sensilla Basiconica

Resumen

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