Este trabalho é apoiado por uma concessão da Fundação Nacional de ciências naturais da China (No.31472037). Qualquer menção de nomes comerciais ou produtos comerciais neste artigo é exclusivamente com o propósito de fornecer informações específicas e não implica uma recomendação.

1. preparação dos instrumentos e inseto
<ol><li>
Preparando o tungstênio soluções eletrodos e estímulos
	<ol>
<li>Fixar um novo fio de tungstênio (diâmetro de 0,125 mm, comprimento de 75 mm) em um micromanipulador e apontá-lo em uma solução de nitrito (NaNO2) de sódio 10% (p/v) em uma seringa de 10 V fornecida por uma fonte de alimentação por cerca de 1 min sob um estereomicroscópio (ampliação de 40 X).</li>
		<li>Mergulhe o fio de tungstênio afiada repetidamente em solução 10% NaNO2 , cerca de 4 mm em 5 V em < 1 min (figura 1A).</li>
		<li>Examine o diâmetro da ponta afiada tungstênio frequentemente sob o microscópio estereoscópico até é fino o suficiente penetrar a cutícula de um sensillum de olfativo locust ao toque (figura 1B).</li>
		<li>Prepare as soluções de estímulo. Dilua cada um da substância química estímulo em óleo mineral. Dilua 1-nonanol e ácido nonanoico em diluições de 10%. Dilua E-2-hexenal e hexanal em 10-2, 10-3, 10-4e 10-5.</li>
		<li>Preparar Pasteur tubos carregando os estímulos: inserir as tiras de papel de filtro (de 2 cm de comprimento, largura de 0,5 cm) para os tubos de Pasteur, adicione as soluções diluídas de estímulo (cada 10 µ l) para as tiras de papel de filtro e em seguida, conecte os tubos de Pasteur com pontas de pipetas (1 ml).</li>
	</ol>
</li>
	<li>
Preparar o inseto
	<ol>
<li>Gafanhotos traseiros (Locusta migratoria) com mudas de trigo fresco sob condições de superlotação em um fotoperíodo de 18:6 h (luz: escuro), uma temperatura de 28-30 ° C e uma humidade relativa de 60%. Escolha 1 de 3-dias de idade 5th ínstar ninfas de gafanhoto e remover as antenas com uma tesoura bem para evitar qualquer interferência durante a gravação.</li>
	</ol>
</li>
	<li>
Preparando o titular do palpo maxilar locust
	<ol>
<li>Use uma lâmina de vidro (25 x 75 mm) como a base do titular do palpo maxilar (MPH). Prenda um pedaço de plástico (1 mm de altura, 10 mm de largura, 35 mm de comprimento) para um canto do vidro slide com fita adesiva dupla-face e finalmente corrigir um tampa de vidro (18 x 18 mm) em cima do pedaço de plástico com fita adesiva dupla-face. Coloque um pequeno pedaço de fita vermelha de borracha sobre o vidro de cobertura como uma camada anti-derrapante. A peça de plástico e o vidro de cobertura constituem a plataforma para o palpo locust. A altura da plataforma é de aproximadamente 1,5 mm.</li>
		<li>Instalar um fio de tungstênio (diâmetro de 0,125 mm, comprimento de 36 mm) a uma distância de 1,5 mm paralelo ao interior borda da plataforma. Fixe as duas extremidades do fio até a plataforma com fita adesiva dupla-face.</li>
	</ol>
</li>
</ol>2. preparação de Locust palpos maxilares
<ol><li>Corte um tubo de centrifugação (1,5 ml) verticalmente ao meio e corte o fundo. Coloque o gafanhoto dentro do tubo preparado. Deixe a região ventral e a cabeça dos locust exposto. Corrigi o assembly para a lâmina de vidro com fita adesiva de dupla face (Figura 2A).</li>
	<li>Puxe o maxilar bem palpável para a plataforma.</li>
	<li>Coloque o fio de tungstênio no segmento quarto do palpo. Coloque a massa adesiva em cada lado do fio de tungstênio, cerca de 2 mm do palpo maxilar (Figura 2A e 2B).</li>
</ol>3. único Sensillum gravações
<ol><li>Coloque a preparação de palpo maxilar locust sob um microscópio em um pequeno aumento (100 X). Ajuste a posição da preparação até o palpo é perpendicular ao eléctrodo de gravação (Figura 3A).</li>
	<li>Introduza o eléctrodo de referência (eletrodo de tungstênio) o olho de gafanhoto usando um micromanipulador. Mova o eletrodo de gravação (eletrodo de tungstênio) perto o palpo maxilar com o micromanipulador (Figura 3B e 3C).</li>
	<li>Ajuste o dispositivo de entrega de odor para cerca de 1 cm do palpo maxilar (Figura 3B).</li>
	<li>Abra o software de gravação automática Spike 32. Defina os parâmetros de gravação como segue: a escala de gravação em 500 mV; o alto corte do filtro em 300 Hz, o corte baixo em 200 Hz; e o pretrigger em 10 s.</li>
	<li>Conecte o eletrodo gravação um 10 x universal AC/DC amplificador.</li>
	<li>Alterne o microscópio para uma alta ampliação (500 X). Insira o eletrodo gravação na base de um sensillum de basiconic sobre o palpo maxilar e ajustar delicadamente o eléctrodo de gravação para obter boas picos espontâneos (Figura 3D).</li>
	<li>Abra o controlador de estímulo para fornecer um fluxo contínuo de ar a 20 ml/s. o tempo de estimulação para 1 s. sinais de registro por 10 s, começando 10 s antes do início do pulso estímulo.</li>
	<li>Use um amplificador de AC/DC 10 x universal para amplificar os sinais. Alimente os sinais em 4 o IDAC. Analise os sinais com o software Auto Spike 32. Sinais de AC são band-pass filtrado entre 200 a 300 Hz. uso Auto Spike 32 para distinguir amplitudes pico-de-cocho de ruídos. Calcule as respostas dos neurônios como os aumentos nas frequências do potencial de ação (picos por segundo) sobre as frequências espontâneas. Realizar uma análise estatística utilizando GraphPad Prism 7.</li>
</ol>

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Os autores não têm nada para divulgar.

Insetos dependem de palpos para detectar odores de comida, e seus palpos são acreditados para jogar um papel importante na especiação13,27. Os palpos são simples órgãos olfativos e estão recebendo crescente atenção como um atraente modelo de exploração da neuromolecular redes subjacente chemosensation28.
Inseto labellar e palpo SSRs foram realizados com sucesso em Drosophila melanogaster, Anopheles gambiaee Culex quinquefasciatus13,14,15,16 , 17 mas raramente têm sido relatados na forma de um vídeo de apresentação16,29. Em contrapartida, dados de vídeo em SSRs antenais estão disponíveis para a Drosophila, a traça do orangeworm umbigo (Amyeloistransitella), Schistocerca Americanae o percevejo (Cimex lectularius)16, 30 , 31 , 32 , 33.
Locust palpo sensilas basiconica têm uma estrutura especial que difere das sensilas antenais locust e muitos outras insetos sensilas. Usando o método descrito aqui, potenciais de ação gerados pelo gafanhoto palpo sensilas basiconica subtipos pb1 e pb2, pode ser gravado e discriminados (Figura 4 e Figura 5).
O passo crítico é a penetração do eletrodo gravação. O eletrodo de gravação deve ser inserido na base do sensillum e avançado até bons sinais são adquiridos. Além disso, é importante evitar que a cúpula do palpo em colapso quando o eletrodo de gravação é inserido na base do sensillum. Para conseguir isso, montamos uma plataforma incluindo um titular de palpo maxilar locust especial (MPH) e usado um fio de tungstênio para comprimir o quarto segmento do palpo. Muitas repetições deste procedimento demonstram que este é eficaz. Com base nos padrões de resposta dos neurônios nos sensilas para várias odorantes, nós, pela primeira vez, identificamos dois subtipos de sensilas basiconica sobre o palpo maxilar locust, nomeadamente pb1 e pb2.
A limitação da técnica descrita nesta publicação é que poderia ser usado para gravar os insetos grandes (por exemplo, mariposas, besouros e gafanhotos) tempo para não gravar pequenos insetos (por exemplo, moscas e mosquitos), que têm suas próprias plataformas e técnicas13,14,15,16,17. Esta técnica é complementar aos métodos existentes.
Em conclusão, um protocolo altamente eficaz de um SSR de inseto palpo sensilas basiconica é descrito em detalhes. Este protocolo poderia fornecer pesquisadores com uma técnica útil no estudo dos mecanismos moleculares e celulares da olfação inseto sobre o mouthpart. Esse método vinculado com cromatografia gasosa poderia ser usado para identificar os ligands naturais electrophysiologically-ativo em extratos de recursos alimentares favoráveis.

Animais têm evoluído de uma gama de órgãos chemosensory que detetam sugestões químicas exógenas. Em insetos, os mais importantes órgãos de chemosensory são as antenas e os palpos. Nestes órgãos, diversos tipos de cabelos chemosensory, chamados chemosensory sensilas, são inervados pelos neurônios chemosensory (CSNs) dentro os pelos. CSNs em chemosensory sensilas reconhecem sugestões químicas específicas através de transdução de sinal de estímulos químicos para potenciais eléctricos que posteriormente são transferidos até o sistema nervoso central1,2,3 .
CSNs expressam vários receptores chemosensory [por exemplo, receptores de odorante (ORs)], geleificação receptores (IRs) e receptores gustativos (GRs) em suas membranas, que codificam exógenas sugestões químicas associadas com diferentes tipos de chemosensation 4,5,6. A caracterização da CSNs é a chave para a elucidação dos mecanismos celulares e moleculares de inseto quimiorecepção. Agora gravação única sensillum (SSR) é uma técnica amplamente utilizada para a caracterização de insecto CSNs nos sensilas antenais de muitos insetos, incluindo moscas7, traça8, besouros9, pulgões10, gafanhotos,11, e formigas,12. No entanto, poucos estudos têm aplicado um SSR a inseto palpos13,14,15,16,17, porque as estruturas particulares da suas sensilas fazem um gravação eletrofisiológicas difícil18.
Enxames de gafanhotos (Orthoptera) muitas vezes causam danos à lavoura grave e perda econômica19. Os palpos são acreditados para jogar um papel importante na seleção alimentar de gafanhotos20,21,22,23,24. Dois tipos de sensilas chemosensory são investigados por um microscópio eletrônico de varredura (MEV). Geralmente, chaetica 350 sensilas e 7-8 sensilas basiconica são observados em cada cúpula dos locust palpos18. Chaetica de sensilas são sensilas gustativas que detetam sugestões químicas não-volátil, enquanto sensilas basiconica têm uma função olfativa, sensoriamento sugestões químicas voláteis.
Em palpos de locust, os diâmetros das tomadas de cabelo da basiconica de sensilas (ca. 12 µm), são muito maiores do que aqueles de sensilas chaetica (ca. 8 µm)18,25. A parede cuticular do basiconica de sensilas sobre os palpos é muito mais espessa do que a de sensilas antenais18. Além disso, a cúpula do palpo tem conteúdo líquido dentro de uma cutícula altamente flexível. Estas características significam que uma penetração com um microeléctrodo e uma aquisição de sinais eletrofisiológicos boas é mais difícil do que para sensilas antenais. Neste trabalho, um protocolo SSR detalhado e altamente eficaz para locust palpo sensilas basiconica é apresentado com um vídeo.

<table border="0" cellpadding="0" cellspacing="0" style="width:1145px;" width="1145">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">Tungsten wire</td>
			<td style="width:140px;">ADVENT</td>
			<td style="width:217px;">W559504</td>
			<td style="width:485px;">Used for making the electrode and fixing the palp</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NaNO2</td>
			<td>Sigma-aldrich</td>
			<td>563218-25G</td>
			<td>Used for sharpening the tungsten wire</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AC Power Supply</td>
			<td>Syntech</td>
			<td>A2-70</td>
			<td>Providing the voltage in sharpening the tungsten wire</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Stereoscope</td>
			<td>Motic</td>
			<td>SMZ-163</td>
			<td>Used for observing the sharpening of tungsten wire</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microscope</td>
			<td>Olympus</td>
			<td>W-51</td>
			<td>Used for observing the sensilla on locust maxillary palp</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">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 height="40">
			<td height="40" style="height:40px;">Stimulus controller</td>
			<td>Syntech</td>
			<td>CS-55</td>
			<td>Used for controlling the stimulus application</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">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 height="40">
			<td height="40" style="height:40px;">Micromanipulator</td>
			<td>Narishige</td>
			<td>MN-151</td>
			<td>Used for minor movement of the reference electrode</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Speaker</td>
			<td>EDIFIER</td>
			<td>R101T06</td>
			<td>Connected with IDAC-4 and providing sound for the signal</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Magnetic base</td>
			<td>PDOK</td>
			<td>PD-101</td>
			<td>Used to hold the electrode, and stimulus delivery tube</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Vibration Isolation Table</td>
			<td>TianHe</td>
			<td>HAP-100-1208</td>
			<td>Used for isolating the vibration from the equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass slide</td>
			<td>CITOGLAS</td>
			<td>ZBP-407</td>
			<td>Used for making the base for the MPH</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Blu-tack</td>
			<td>Bostik</td>
			<td>Blu-tack-45g</td>
			<td>Fixing the tungsten wire</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Pasteur tube</td>
			<td>YARE</td>
			<td>WITEG</td>
			<td>Placing the filter paper containing stimuli solutions</td>
		</tr>
	</tbody>
</table>

single sensillum recordings for locust palp sensilla basiconica

Os palpos das peças bucais locust são considerados órgãos gustativos convencionais que desempenham um papel importante na seleção de alimentos de um gafanhoto, especialmente para a detecção de não-volátil sugestões químicas através de sensilas chaetica (anteriormente denominado terminais sensilas ou crista sensilas). Há crescente evidência de que estes palpos também têm uma função olfativa. Um receptor de odorante (LmigOR2) e uma proteína ligadora de odorante (LmigOBP1) foram localizadas nos neurônios e células de acessório, respectivamente, na basiconica de sensilas dos palpos. Sensillum única gravação (SSR) é usado para gravar as respostas dos neurônios receptores de odorante, que é um método eficaz para ligantes ativos sobre os receptores específicos odorante de triagem. SSR é usado em estudos funcionais de receptores de odorante no sensilas palpo. A estrutura do basiconica de sensilas localizado na cúpula dos palpos difere um pouco da estrutura das pessoas sobre as antenas. Portanto, ao realizar um SSR eliciado odorantes, alguns conselhos específicos podem ser útil para a obtenção de melhores resultados. Neste trabalho, é apresentado um protocolo detalhado e altamente eficaz para um SSR de inseto palpo sensilas basiconica.

Dois subtipos de sensilas (pb1 e pb2) sobre o palpo maxilar locust são identificados com base em dinâmica de resposta diferente de químicos odorantes (10% 1-nonanol e ácido nonanoico de 10%). Os neurônios no pb1 produzem significativamente mais picos de até 1-nonanol do ácido nonanoico enquanto os neurônios em pb2 são significativamente que menos ativado por 1-nonanol comparado com ácido nonanoico (Figura 4). Hexanal e E-2-Hexenal podem evocar um palpo locust abrindo a resposta (POR)26. Hexanal é uma planta verde folha anfitrião abundante voláteis que possam contribuir para uma mais uma confirmação para a fonte de alimento26. Os picos que suscitou nos neurônios pb1 última mais do que aqueles de pb2 quando eles são estimulados por E-2-hexenal (Figura 4). Os neurônios no pb1 e pb2 similarmente apresentam respostas robustas para hexanal (Figura 4). Comparando as alterações médios de todas as pontas entre os períodos 5 s antes e 5 s após a estimulação indica que a resposta a 1-nonanol é significativamente superior ao ácido nonanoico pb1, mas contrariamente em pb2 (Figura 5). Os neurônios nestes dois subtipos de sensilas respondem dose-dependente E-2-hexenal e hexanal, e seus padrões de resposta para estes dois aldeídos são diferentes (figura 6A e 6B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57863/57863fig1.jpg"> Figura 1. Preparação do eléctrodo. (A), este painel mostra um general Ver os do aparato afiação do eletrodo. A seringa contendo 10% NaNO2 (à esquerda) é usada para afiar o eletrodo (à direita). (B), este painel mostra uma visão estreita da ponta do eletrodo (r: adequado; b: impróprios). <a href="/files/ftp_upload/57863/57863fig1large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57863/57863fig2.jpg"> Figura 2. Titular de Locust palpo maxilar (MPH). (A) The MPH e um gafanhoto são montados sobre a lâmina de vidro antes de posicioná-lo no microscópio. (B) este painel mostra um close-up do palpo maxilar locust, fixado por fio de tungstênio na plataforma. <a href="/files/ftp_upload/57863/57863fig2large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57863/57863fig3.jpg"> Figura 3. Único gravações sensillum. (A), este painel mostra uma vista sobre a instalação de eletrofisiologia. (B) este painel mostra uma visão estreita da preparação gafanhoto montada no microscópio. (C), esta imagem mostra o palpo maxilar locust ampliação de 100 X. (D), esta imagem mostra o palpo ampliação de 500 X. A seta indica um sensillum basiconic. <a href="/files/ftp_upload/57863/57863fig3large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57863/57863fig4.jpg"> Figura 4. Vestígios de resposta de gravações de sensillum único do palpo maxilar locust. Neste painel, pb1 defende subtipo 1 do basiconica de sensilas palpável; PB2 defende subtipo 2 de basiconica de sensilas o palpo. As barras acima os vestígios indicam a duração do estímulo (1 s). Para estas gravações, todos os odores são usados em diluições de 10% exceto para E-2-hexenal e hexanal, que são diluídas a 1%. Esta figura foi modificada de Zhang et al . 26. <a href="/files/ftp_upload/57863/57863fig4large.jpg" target="_blank">clique aqui para ver uma versão maior desta figura.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57863/57863fig5v2.jpg"> Figura 5. Comparação de números médios de picos nos neurônios pb1 e pb2 estimulada pelo ácido nonanoico e 1-nonanol. Os números médios de espinhos são calculados no s períodos 5 antes e após a estimulação. No pb1, os números médios de espinhos nos neurônios respondendo a 1-nonanol aumentam significativamente mais altamente do que aqueles de espinhos nos neurônios respondendo ao ácido nonanoico (n = 11 palpos; ANOVA com t-testes post hoc ; p < 0,0001), em contraste com pb2 (n = 10 palpos; ANOVA com t-testes post hoc ; p = 0.0110). A barra de erro representa SEM. Esta figura foi modificada de Zhang et al . 26. <a href="/files/ftp_upload/57863/57863fig5v2large.jpg" target="_blank">clique aqui para ver uma versão maior desta figura.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57863/57863fig6v3.jpg"> Figura 6. Os padrões de neurônios no pb1 e pb2 resposta dose-dependente para E-2-hexenal e hexanal. (A), este painel mostra os padrões dos neurônios no pb1 (± SEM; n = 12 palpos). (B), este painel mostra os padrões dos neurônios em pb2 (± SEM; n = 10 palpos). Esta figura foi modificada de Zhang et al . 26. <a href="/files/ftp_upload/57863/57863fig6large.jpg" target="_blank">clique aqui para ver uma versão maior desta 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 artigo descreve um protocolo detalhado e altamente eficaz para gravações de único sensillum do basiconica de sensilas sobre os palpos de peças bucais de insetos.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.1K visualizações.  China Agricultural University. Este artigo descreve um protocolo detalhado e altamente eficaz para gravações de único sensillum do basiconica de sensilas sobre os palpos de peças bucais de insetos.

Vídeo: 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).

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

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

Única unidade de In vivo Gravações do quiasma óptico de rato

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

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 ...

Single Drosophila Ommatidium Dissection and Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative diseases because of its powerful genetics1. The fly eye in particular has been the organ of choice for neurodegeneration research, being the most accessible and life-dispensable part of the Drosophila nervous system. However the major caveat of intact eyes is the difficulty, because of the intense autofluorescence of the pigment, in imaging intracellular events, such as autophagy dynamics2, which are paramount to understanding of neurodegeneration.
We have recently used the dissection and culture of single ommatidia3 that has been essential for our understanding of autophagic dysfunctions in a fly model of Dentatorubro-Pallidoluysian Atrophy (DRPLA)3, 4.
We now report a comprehensive description of this technique (Fig. 1), adapted from electrophysiological studies5, which is likely to expand dramatically the possibility of fly models for neurodegeneration. This method can be adapted to image live subcellular events and to monitor effective drug administration onto photoreceptor cells (Fig. 2). If used in combination with mosaic techniques6-8, the responses of genetically different cells can be assayed in parallel (Fig. 2).

Single Drosophila Ommatidium Dissection and Imaging

The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.

Único Drosophila Dissection omatídeo e Imagem

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

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 ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

Um Procedimento para Implantação de Arrays organizado de microfios para gravações a única unidade em Awake, comportando-se Animais

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

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.

Eletrofisiológica Gravação De<em&gt; Drosophila</em&gt; Labellar Prove sensilla

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

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Emparelhados Recordings células inteiras em organotï¿½icas fatias do hipocampo

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

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.

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 ...

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.

Localização de Genes de Receptor de Odorantes em Antenas de Locustídeo por RNA<em&gt; In Situ</em&gt; Hibridação

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