Name: using single sensillum recording to detect olfactory neuron responses of bed bugs to semiochemicals
Uploaded: 2016-01-18T15:00:00.000+00:00
Duration: 6 min 55 s
Description: Watch this Scientific Journal Video about Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals at JoVE.com

The project was supported by Award AAES 461Hatch/Multistate Grants ALA08-045 and ALA015-1-10026 to N.L.

1. Preparação de Instrumentos, Soluções de estímulos, e Percevejos <ol><li> Prepara-se uma solução de KNO 2 50% (w / v) em um frasco de 20 ml. </li><li> Afie dois microeléctrodos de tungsténio em solução KNO 2 a 5 V mergulhando repetidamente os eletrodos de tungstênio dentro e fora da solução. <ol><li> Cerca de afiar o fio de tungsténio por imersão a cerca de 10 mm de fio de tungsténio a dentro e para fora da solução de KNO 2 a uma velocidade de 2 imersões / seg durante cerca de 5 min, o que pode consumir muito, a extremidade dianteira do fio de tungsténio. </li><li> Delicadamente afiar o eléctrodo por meio de imersão de cerca de 1 mm da ponta do fio dentro e para fora da solução a uma velocidade de 2 imersões / seg durante pelo menos 1 minuto, de modo a fazer um ponto de o eléctrodo fino e afiado. Verifique o diâmetro ponta do eletrodo sob o microscópio com freqüência até que ele atinja 0,2-0,5 mm, que deve estar bem o suficiente para perfurar a cutícula percevejos sensillum olfativo. Nota: Enquanto manualmente afiadaening o eléctrodo, a velocidade de imersão de o fio de tungsténio na solução de KNO 2 não é constante durante todo o tempo. Com mais prática, é muito mais fácil de manter uma velocidade relativamente constante no afiar do eléctrodo. O tempo de nitidez também é incerto, dependendo de como o eléctrodo fino deve ser. Aqui, uma ponta de eléctrodo com o diâmetro de 0,2 m ~ é suficiente para perfurar através do sensillum olfactiva. </li></ol></li><li> Dilui-se cada um dos estímulos químicos em sulfóxido de dimetilo (DMSO) a partir do composto puro a uma concentração inicial de 1:10 v / v como uma solução estoque. Criar uma série de diluições Decádico dependendo do número de doses necessárias na experiência, novamente com o DMSO, a partir de cada uma das soluções de reserva para cada químico. Aqui, usa 10% (+) - β-pineno e eucaliptol. </li><li> Coloque os dias em jejum ou sete de pós-alimentação percevejos adultos (macho ou fêmea) do Ft. Dix colónia (uma oferta do Dr. Haynes na Universidade de Kentucky) para ser usado em tele experimentar numa placa de Petri. Nota: Não há um número exato para percevejos colocados na placa de Petri. Ele pode ser um pouco ou muito. </li></ol> 2. Bed Bug Antena Preparação <ol><li> Anestesiar os percevejos no gelo (2-3 min). </li><li> Corrigir tanto as antenas eo corpo inseto em uma lamela de microscópio com fita dupla face e remover as pernas com uma tesoura fina. </li><li> Use um pequeno pino para tocar suavemente as antenas de modo a colocá-los na fita de forma constante. </li><li> Descansar a lamela contra uma esfera pequena (~ 1 cm de diâmetro) de cera dental para facilitar a manipulação e ajustá-lo a um ângulo apropriado (~ 90 °) para o eléctrodo de registo (Figura 1). </li><li> Uma vez garantido, coloque o bug cama sob um microscópio estéreo, ligue a fonte de luz fria e ajustar a intensidade da iluminação até a antena está claramente apresentada, e concentrar-se o microscópio sobre a segunda flagelo da antena de percevejos em alta ampliação (720x) . Nota: A intensidade de iluminação utilizado na experiência não é quantificada, que realmente depende de como os olhos do experimentador sentir a intensidade de iluminação. </li></ol> 3. Único Sensillum Recording <ol><li> Ligue o pré-amplificador (10X) com o controlador de aquisição de sinal, o qual está ligado com o computador para o registo e visualização de sinais. Ligue o computador e iniciar o software, por exemplo, AutoSpike32 e clique no modo &quot;Record&quot; na barra de menu. Em seguida, escolha a &quot;onda&quot; de modo a começar a gravar os sinais de onda. Nota: Uma linha reta em execução a partir da esquerda para a direita do monitor repetidamente deverão ser agora visíveis. Aqui, a janela de gravação dura 40 seg. Gravação de onda máximo é de 10 segundos. Taxa de amostragem selecionada é 96.000 e taxa de amostragem digital é 240. Há 0 deslocamento% e sem filtragem, nenhuma retificação para os sinais de gravação. Todas estas definições de parâmetros do software pode ser modificado conforme necessário. </li> &lt;li&gt; Ligue o alto-falante ligado ao pré-amplif icador, que é usado para apresentar o modo de tonificação para as respostas neuronais a partir de sensillum antenal. </li><li> Insira o eletrodo de referência para o abdome do percevejo estabilizado. Nota: O eletrodo de referência foi realizada por um metal estande fixada magneticamente à mesa do ar. </li><li> Depois de o eléctrodo de referência tenha sido ligado ao abdómen da percevejo, mover o eléctrodo de registo, que está conectado ao pré-amplificador e manipulado por um micromanipulador, no sentido da extremidade posterior da antena do percevejo. </li><li> Quando o eléctrodo de gravação está em contacto com a ponta direita da antena, ligar o microscópio e localizar o eléctrodo a baixa ampliação. </li><li> Ajustar o eléctrodo de registo enquanto aumenta gradualmente até que a ampliação tanto o eléctrodo e o sensillum antenal estão no mesmo plano e claramente visíveis ao microscópio. Nota: Por esta altura, o microscópio é habitually com a maior ampliação. </li><li> Insira o eletrodo de registro no eixo da sensillum usando o micromanipulador e ir um pouco mais fundo, se o ruído de fundo é elevado em comparação com o potencial de ação. </li><li> Uma vez que os potenciais de acção claros são observados a partir do sensillum gravado, preencher uma micropipeta com 10% de (+) - β-pineno. Utilizar a micropipeta para depositar 10 ul aliquota de 10% (+) - β-pineno em uma tira de papel de filtro (~ 3 x 15 mm) colocado dentro de uma pipeta de Pasteur de vidro. <ol><li> Ligar a pipeta carregado para o tubo de saída do fluxo de impulsos de estímulo e o controlador de colocar a ponta da pipeta no pequeno orifício no tubo orientado para a antena. </li></ol></li><li> Quando todas essas ligações tenham estabilizado, pressionar o pedal do controlador de estímulo para fornecer um sopro de 0,5 seg estímulo (0,5 L / min) para o fluxo de ar humidificado contínua. A gravação do potencial de ação será iniciada simultaneamente quando o footswitch é deprimido. O processo de gravação será passado para 10 segundos a partir de 1 segundo antes da estimulação. </li><li> Conte os potenciais de ação off-line para dois períodos 500 ms, uma antes e outra após a estimulação. Subtrair qualquer alteração na taxa de pico durante a 500 mseg após a estimulação da actividade espontânea registados durante os últimos 500 mseg e converter as contagens na escala convencional de espigas / s por sua multiplicação por 2. </li></ol> 4. Estímulo substituição na SSR <ol><li> Uma vez que o interruptor de pé foi desencadeada, entregar a 10% (+) - β-pineno na pipeta para o percevejo antenas e gravar a resposta a este odorante específico durante 10 seg, após o que a pipeta é removido. </li><li> Rotular outra nova pipeta com o eucaliptol 0,001% a ser testado. Coloque um pequeno pedaço de papel de filtro sobre a qual 10 ul do estímulo foi aplicado, na nova pipeta. </li><li> Espere por 2-5 min até que o estímulo é completamente vaporized na pipeta de vidro. Fixar a pipeta para o tubo de saída do fluxo de impulsos. </li><li> Insira a ponta da pipeta no pequeno orifício do tubo orientado para a antena. Pressionar o pedal e iniciar a gravação de 10 segundos. </li><li> Desligue a pipeta e preparar outra pipeta com 0,01% de eucaliptol. </li><li> Teste todo o resto das doses de eucaliptol (de 0,001% a 10%) sobre o sensilla antennal para observar as respostas dependentes da dose. Teste a partir do mais diluída para as doses menos diluir. </li></ol>

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The authors have nothing to disclose.

A técnica standard sensillum gravação tem sido extensivamente utilizado para testar as respostas neurais de insectos, tais como moscas de fruta, mosquitos e percevejos a diferentes estímulos químicos no ambiente. Estes estímulos químicos são frequentemente dissolvidos e diluídos num solvente comum, a fim de preparar a diferentes doses de tratamentos. No entanto, diferentes solventes pode produzir diferentes taxas de libertação bastante para os estímulos. Estudos anteriores sobre alguns insetos amplamente est...

O percevejo comum Cimex lectularius L (Hemiptera: Cimicidae), como um ectoparasita temporária, é um inseto sugador de sangue obrigado, o que significa a sua sobrevivência, desenvolvimento e reprodução necessitam de fontes de sangue de hospedeiros, incluindo seres humanos e animais 1,2. Embora a transmissão do vírus raramente tem sido relatada devido a C. lectularius, o incômodo gerado por morder uma infestação afeta seriamente anfitriões tanto física como psicologicamente 3. A introdução e uso generalizado de inseticidas químicos, especialmente o DDT, reduziu o risco de infestações e até o final de 1950 as infestações estavam em um nível tão baixo que eles já não eram uma preocupação pública grave. No entanto, uma série de possíveis fatores levaram a ressurgimento em populações de percevejos em todo o mundo, tais como a redução do uso de inseticidas, um declínio de sensibilização da opinião pública, o aumento da atividade viajar, eo desenvolvimento de resistência a inseticidas 4-9. &lt;/ p&gt; Sinais químicos no ambiente são detectados e reconhecidos por insetos através de órgãos olfativos, como antenas e palpos maxilares. O sensilla olfativo sobre o inseto antenas desempenham um papel crucial na detecção desses sinais químicos. As moléculas químicas entrar na cutícula antenal através de poros na superfície da cutícula. Proteínas de ligação odorante no ligamento linfa antennal a estas moléculas químicas e transportá-los para os receptores olfativos 10. Os receptores olfactivos e os seus co-receptores do canal de iões catiónica não-selectivos na membrana neuronal, que será despolarizada uma vez que estas moléculas químicas são reconhecidas pelos receptores olfactivos 11. Sensillum gravação único (SSR) foi desenvolvido para detectar a alteração extracelular no potencial de acção causada pela aplicação de um destes estímulos químicos ou não químicos. Através da inserção de um eletrodo de registro na linfa sensillum e um eléctrodo de referênciaem alguma outra parte do corpo do insecto (geralmente se os olhos compostos ou do abdómen), a taxa de disparo dos neurónios em resposta a estímulos podem ser gravados 12. As alterações no número de picos representam a sensibilidade do insecto a estímulos específicos. Estímulos químicos de diferentes identidades e concentração irá eliciar respostas neurais diferentes, com diferentes taxas de combustão e estruturas temporais, e pode assim ser utilizado para investigar o processo de codificação de o insecto a substâncias químicas específicas. Para o percevejo comum, ambas as formas sexuais compartilhar o mesmo padrão de sensilla olfativa nas antenas: nove sulcado sensilla peg C, 29 cabelo-como E (E1 e E2) sensilla, e um par de cada um dos Dα, Dβ, Dγ peg suave sensilla 13,14. Como vários neurónios foram identificadas em cada tipo de sensillum, não é fácil de distinguir os potenciais de acção de diferentes neurónios alojados no mesmo sensillum, assim, para esta experiência a total números de potenciais de ação foram contadas off-line por um período de 500 ms antes e após a estimulação. O número de potenciais de ação após a estimulação foi então subtraído do número de potenciais de ação antes do estímulo e multiplicado por dois, a fim de quantificar as mudanças na taxa de disparo em cada sensillum indivíduo em picos por segundo 15.

<table><tbody><tr><td>Tungsten wire</td><td>A-M SYSTEMS</td><td>#716500</td><td>Used for preparing the electrode</td></tr><tr><td>KNO2</td><td>Sigma</td><td>#310484</td><td>Used for sharpening the tungsten wire</td></tr><tr><td>AC Power Supply</td><td>BK Precision</td><td>1653A</td><td>Providing the voltage in sharpening the tungsten wire</td></tr><tr><td>Leica Z6 APO Microscope</td><td>Leica</td><td>10447424</td><td>Used for observing the sensilla on antennae</td></tr><tr><td>Simulus controller</td><td>Syntech</td><td>CS-55</td><td>Used for controlling the stimulus application</td></tr><tr><td>4-Channel USB 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>Light Source</td><td>SCHOTT</td><td>A20500</td><td>Providing light sources for observation</td></tr><tr><td>Micromanupulator</td><td>Leica</td><td>115378</td><td>Used for minor movement of electrode</td></tr><tr><td>Speaker</td><td>Juster</td><td>95a</td><td>Connected with Acquisition Controller IDAC-4 and providing sound for the signal</td></tr><tr><td>Magnetic stand</td><td>Narishige</td><td>GJ-1</td><td>Used to hold the reference electrode, stablized bed bug and stimulus delivery tube</td></tr><tr><td>TMC Vibration Isolation Table</td><td>TMC</td><td>63-500</td><td>Used for isolating the vibration from the equipments</td></tr><tr><td>Coverslip</td><td>Tedpella</td><td>2225-1</td><td>Used for holding the bed bug</td></tr><tr><td>Double-sided Tape</td><td>3M</td><td>XT6110</td><td>Used for stablizing the bed bug on the coverclip</td></tr><tr><td>Dental Wax</td><td>Dentakit</td><td>DK-R012</td><td>Used for supporting the coverclip where bed bug is stablized&amp;nbsp;</td></tr></tbody></table>

using single sensillum recording to detect olfactory neuron responses of bed bugs to semiochemicals

O sistema olfactivo inseto desempenha um papel importante na detecção de semioquímicos no ambiente. Em particular, o sensilas antenal que abrigam neurónios simples ou múltiplas, para dentro, são considerados para tornar a maior contribuição para responder aos estímulos químicos. Ao gravar diretamente potencial de ação no sensillum olfativa após a exposição a estímulos, a gravação sensillum único (SSR) técnica fornece uma abordagem poderosa para investigar as respostas neurais dos insetos a estímulos químicos. Para o bug cama, que é um parasita humano notório, vários tipos de sensillum olfatório foram caracterizados. Neste estudo, demonstramos respostas neurais de cama bug sensilla olfativo a dois estímulos químicos e as respostas dependentes da dose para um deles usando o método SSR. Esta abordagem permite que pesquisadores para realizar rastreio precoce de estímulos químicos individuais na olfativo sensilla cama bug, que iria fornecer informações valiosas para o desenvolvimento de novos atrativos ou repelentes de insetos de cama e benefícios dos esforços de controle de cama bug.

Sensillum gravação única é uma técnica de investigação poderoso utilizado em estudos de insetos ecologia química e fisiologia neural. Investigando as respostas neurais dos insetos aos compostos voláteis diferentes, especialmente aqueles pensado para ser ecologicamente relacionado com a sobrevivência eo desenvolvimento dos insetos, não só nos dá insights valiosos no processo de olfato do inseto, mas também abre novos caminhos promissores que poderiam, potencialmente, levar p...

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

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.

Usando Individual Sensillum Gravação para detectar respostas Olfativo Neurônio de percevejos para Semioquímicos

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

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Research

JoVE Journal

Neuroscience

9.8K visualizações.  Auburn University. 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.

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

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).

Único Recordings Sensillum na Insetos Drosophila melanogaster E 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 ...

Neurociência

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.

Medidas eletrofisiológicas partir de um sistema olfativo Traça

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.

Eletrofisiologia da 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 ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Odorante induzidas respostas registradas a partir de neurônios receptores olfativos utilizando a técnica de sucção da pipeta

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

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.

Multi-unidade Métodos de gravação para caracterizar a atividade neural no Locust (<em&gt; Schistocerca Americana</em&gt;) Circuitos olfatórias

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

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Perfurado gravação patch-clamp de rato Olfactory neurônios sensoriais na Intact neuroepitélio: Análise Funcional do Neurônios Expressando uma Odorant Receptor Identificados

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of an adapted &#34;olfactory chip&#34; for the efficient calcium imaging of C. elegans males is described here. Studies of male exposure to glycerol and a pheromone are also shown.

Usando um Chip olfativo Microfluidic adaptado para a imagem da atividade Neuronal em resposta aos Pheromones em neurônios de cabeça masculino C. Elegans

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...

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.

Gravação eletrofisiológica de<em&gt; Drosophila</em&gt; Sensilla Tricoide em resposta aos odorantes de baixa volatilidade

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, ...

Single Sensillum Recordings for Locust Palp 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 for the detection of non-volatile chemical cues through sensilla chaetica (previously named terminal sensilla or crested sensilla). There is now increasing evidence that these palps also have an olfactory function. An odorant receptor (LmigOR2) and an odorant-binding protein (LmigOBP1) have been localized in the neurons and accessory cells, respectively, in the sensilla basiconica of the palps. Single sensillum recording (SSR) is used for recording the responses of odorant receptor neurons, which is an effective method for screening active ligands on specific odorant receptors. SSR is used in functional studies of odorant receptors in palp sensilla. The structure of the sensilla basiconica located on the dome of the palps differs somewhat from the structure of those on the antennae. Therefore, when performing an SSR elicited by odorants, some specific advice may be helpful for obtaining optimum results. In this paper, a detailed and highly effective protocol for an SSR from insect palp sensilla basiconica is introduced.

This paper describes a detailed and highly effective protocol for single sensillum recordings from the sensilla basiconica on the palps of insect mouthparts.

Único Sensillum gravações para Locust palpo sensilas 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 ...

Um novo método para análise aprimorada da atividade dos neurônios gustativos do inseto

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 em destaque: Avanços na quimiorrecepção - de receptores de odor de insetos a RNAs não 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.

Gravação de base: uma técnica para analisar as respostas dos neurônios gustativos em 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 ...

Usando Individual Sensillum Gravação para detectar respostas Olfativo Neurônio de percevejos para Semioquímicos

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Único Recordings Sensillum na Insetos Drosophila melanogaster E Anopheles gambiae