Este trabalho foi financiado pela NSF concessão IOS 1121692, e pela Fundação Universidade de Washington Research.

1. Follection volátil <ol><li> Neste exemplo, nós usamos amostras voláteis de M. lewisii flores - um alpino nativa wildflower para a Califórnia. Os voláteis são recolhidos através de métodos de adsorção de acordo com a dinâmica Riffell et al. 14. Resumidamente, este método utiliza um sistema de retenção de ciclo fechado onde as flores são colocados num saco de Teflon. Utilizando uma bomba de vácuo inerte, o ar em torno das flores é aspirado através de uma &quot;armadilha&quot; composta de uma pipeta de Pasteur cheia com Porapak Q matriz. O ar de retorno a partir da bomba é filtrada através de carvão activado. Depois de um período de tempo prescrito, no nosso caso, de 24 h, o Porapak Q matriz é eluída com um solvente não-polar, tipicamente hexano, para recolher o extracto concentrado. O extracto é em seguida armazenado a -80 ° C até à análise. Se necessário, as amostras podem ser concentradas antes da análise, sob uma corrente de azoto gasoso. A não ser que a amostra já está bem caracterizado, executar-se uma alíquota da mesma através de umcromatografia gasosa-espectrometria de massa (GC-MS) para identificar os componentes voláteis, antes de utilizar a amostra. </li></ol> 2. Preparação eletrofisiológico <ol><li> Cortar cerca de 1 cm a partir da extremidade da ponta de uma pipeta de 1000 ul. Coloque uma abelha (impatiens do Bombus) na base da ponta de pipeta e empurre para a extremidade oposta da ponta até que apenas a cabeça é exposta. </li><li> Derreter a cera dental e moldá-lo ao redor da cabeça exposta, certificando-se que a parte de cera para os olhos compostos para a segurança e para fazer a cabeça da abelha completamente imóvel. Certifique-se de não obter qualquer cera na antena da abelha. </li><li> Uma vez que a cabeça é seguro, fazer um quadrado, janela como incisão na cápsula cabeça usando uma lâmina de barbear disjuntor ou o tamanho adequado de bisturi para cortar a cutícula. Com a lâmina de disjuntor, que começa no lado dorsal da cápsula cabeça, imediatamente atrás da antena e ao lado dos olhos compostos. Cortaruma linha reta, a partir de um olho composto para o olho composto contralateral. Depois de cortar uma linha reta para o olho composto oposto, começar a fazer uma incisão dorsal até que as curvas de cabeça cápsula e termina perto de seu tórax. Neste ponto, começam a cortar para a extremidade oposta da cápsula cabeça. Finalmente, uma vez que uma linha foi cortada para a extremidade oposta, uma linha de corte de volta para cima para a posição de partida da incisão inicial. É importante remover a cutícula, que fica ao lado da antena, pois isso irá impedir a inserção do eléctrodo. </li><li> Uma vez que a cutícula é cortado, usar um par de fórceps para remover a cutícula de abelha, que deve, em seguida, expor o cérebro de abelha, e, mais importante ainda, os lóbulos antenais. Começar imediatamente superfusing o cérebro com salino de insectos, de modo a que o cérebro não se torna dessicated. Após o cérebro é exposto, cuidadosamente utilizar uma pinça muito finas para remover a bainha perineural imediatamente acima dos lóbulos antenais. Tenha muito cuidado para nãopunção cérebro da abelha com a pinça. </li></ol> 3. Cromatografia Gasosa com multi-canal de gravação <ol><li> A abelha &quot;preparação&quot; - fixado em um tubo com seu cérebro exposto - está agora pronto para a gravação eletrofisiológico. Coloque a abelha em uma braçadeira, fixado a uma base magnética que se encontra sobre uma mesa de ar. </li><li> Providenciar um saco IV, o controlador de fluxo, e tubagem (cheio com solução salina de insectos), de modo que a solução salina continuamente superfuses cérebro. </li><li> Usando um micromanipulador, eletrodo insertareference, feito de fio de tungstênio, no olho da abelha. </li><li> Utilizando um micromanipulador separado, inserir o eléctrodo de canais múltiplos, como por exemplo um fio enrolado tetrode, ou de silício de canal multi-eléctrodo (NeuroNexus), em lobos antenal da abelha. Este eléctrodo está ligado a um pré-amplificador, tais como o sistema de TDT de S-3 série Z para o processador bioamp Z-bus do sistema TDT. A saída do Chro Gásdetector matogram via um cabo blindado BNC pode ser interligado com o sistema de aquisição de dados de amplificação e de tal forma que tanto o sinal neural e GC está sincronizado. </li><li> Espere cerca de 30-60 minutos para as gravações neurais para estabilizar. Uma vez que a actividade espontânea e da forma de onda de unidades nos canais de gravação tornaram-se consistentes, utilizar uma seringa de odor para estimular a abelha e observar a resposta dos canais de gravação para o odor. </li><li> Gravar picos extracelulares de neurónios por auto-thresholding os canais de gravação por 3,5-5 sigma do sinal dos canais individuais de gravação. Limiarização manual pode ser necessária para alguns canais para evitar a contaminação de ruído elétrico. Os potenciais de ação de neurônios aparecerá como picos de tensão no canal de gravação. Quando a tensão do canal exceder o limite, o sistema armazena e salva os poucos milissegundos antes e depois da passagem do limiar, assim, tomandoga instantâneo da forma de onda, ou espiga. </li><li> Imediatamente ao lado da mesa de ar é o GC. Antes de injectar o extracto floral no GC, certifique-se de que o método para a rampa de temperatura de funcionamento do GC está correcta. No nosso exemplo, iremos utilizar um método de temperatura a partir de 50 ° C durante 4 min, seguido por um aumento da temperatura a uma taxa de 10 ° C / min. a 220 ° C, altura em que se mantenha o GC para um min 6 adicional. Usamos uma coluna DB-5 GC (J &amp; W Scientific, Folsom, CA, EUA), com hélio como gás de transporte. A entrada é splitless, com uma temperatura programada de 200 ° C. A temperatura da chama detector de ionização é ajustado a 230 ° C. </li><li> Injectar a amostra de extracto de headspace floral na porta de injecção aquecida do GC para liberar os voláteis adsorvidos na coluna de GC. O efluente da coluna é dividido 1:1 entre o Detector de Ionização de Chama (FID) e a antena de abelha usando um copo conector &quot;Y&quot; (J &amp; W Scientific). Comece recording do eletrodo como você injetar uma amostra para o GC. </li><li> Após o ciclo de GC ter terminado, deixar o resto preparação de 5 a 15 min. Em seguida, injectar-se outra amostra no GC ou estimular a preparação de compostos voláteis usando individuais ou misturas de compostos. Neste último método de estimular a preparação, os impulsos de ar a partir de um fluxo de ar constante são desviadas através de uma seringa de vidro contendo um pedaço de papel de filtro em que os compostos tenham sido depositados. O estímulo odor foi pulsada por meio de uma válvula de solenóide activado controlada pelo software. </li><li> Se a atividade da unidade pára de repente ou alterações, verifique o gotejamento de solução salina e deixar o resto preparação para 15 min. Se a atividade espontânea não recuperar o seu nível anterior, então a preparação deve ser descartada e outra abelha usado se disponível. </li><li> Após a experiência, o cérebro fixar com 5% de formalina durante 20 minutos com a sonda ainda no tecido. Em seguida, o cérebro do consumoe colocá-lo em glutaraldeído a 2%, durante 4 horas, e, subsequentemente, fazer uma série graduada de etanol e desidratação limpar o cérebro com o salicilato de metilo. Com base na fixação e compensação do tecido, os locais na AL onde os eletrodos perfurado o tecido deve ser claramente visível por microscopia confocal. </li></ol> 4. Análise de Dados <ol><li> Analisar os dados coletados após o experimento para separar e identificar as unidades gravadas neurais. Usar programas de software típicos (Sorter Offline, MClust, e SClust) para formas de onda em separado, ou &quot;picos&quot;, baseada em formas de espiga, como pico ou amplitude vale, o pico de meia largura, etc, ou medidas reduzidas (componentes principais) 17 , 18 (Figura 2). Utilizar apenas os grupos de picos separados que no espaço tridimensional (PC1-PC3) e são estatisticamente diferentes um do outro (ANOVA multivariada, p &lt;0,05) (Figura 2) para análise posterior. Por favor, consultecitação # s 17-19 para a descrição completa de tetrode gravação e ponto-ordenação metodologias. </li><li> Carimbo de tempo picos de cada cluster e exportar esses dados para análise usando MATLAB ou Neuroexplorer (Nex Technologies, Winston-Salem, Carolina do Norte) para criar gráficos de varredura e respostas da taxa de queima (Figuras 2, 3A). </li><li> Identificar os tempos de retenção de compostos voláteis usando os dados registados simultaneamente GC. Utilizar os tempos de retenção dos compostos voláteis, determinado pelo vértice do pico do cromatograma, para analisar as respostas de unidade a esses pontos de tempo. </li><li> Para avaliar as respostas de cada unidade através da execução de GC, bin o número de espigas em intervalos de 100 ms e examinar o curso temporal das respostas da frequência de disparo, com referência ao tempo de retenção de voláteis de eluição. O binning de picos de 100 intervalos de mseg fornece detalhes suficientes, ou de sinal, sobre o curso temporal da resposta neuronal a um odorante por eluição a partir da GC. </li><li> Para examinar as respostas da população para os voláteis eluição diferentes, integrar as respostas da frequência de disparo das unidades individuais ao longo de uma janela de amostragem de 3 seg, 1,5 seg antes, e depois de 1,5 seg, o tempo de retenção do volátil (Figura 3). Este período de tempo é típico da duração de uma eluição volátil da GC. Mostramos disparando respostas da frequência das unidades de codificação de cores a eles (vermelho é uma resposta elevada taxa de queima; azul é uma resposta baixa) e dispondo-os como uma matriz de actividade com cada linha que representa a resposta do conjunto para o efluente GC (colunas) ( Figura 3). </li></ol>

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Insetos olfativo mediadas comportamentos conduzir muitos processos diferentes, incluindo a reprodução, host local, seleção e identificação de recursos alimentares adequados. O estudo destes processos requer a capacidade de identificar os voláteis emitidos a partir da fonte, bem como a capacidade de identificar esses compostos, que são mediadoras dos comportamentos. Para complicar as coisas é que os odores são compostas de dezenas a centenas de compostos individuais que, juntos, criam um aroma único que é per...

<table border="1"><tbody><tr><td> Nome do item </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Porapak Tipo Q 80-100 mesh </td><td> Waters </td><td> WAT027060 </td><td></td></tr><tr><td> Forno Sacos Reynolds </td><td> Reynolds </td><td></td><td></td></tr><tr><td> GC </td><td> Agilent </td><td> 7820A </td><td></td></tr><tr><td> Coluna GC </td><td> J &amp; W Scientific, Folsom, CA, EUA </td><td> DB-5 (30 m, 0,25 mm, 0,25 um) </td><td></td></tr><tr><td> Gás transportador hélio analítico </td><td> Praxair </td><td> HE K </td><td> 1 cc / min </td></tr><tr><td> 16-canal de eletrodos de silicone </td><td> NeuroNexus Techngias </td><td> a4x4-3mm50-177 </td><td></td></tr><tr><td> NiCr fio fino, 0,012 mm de diâmetro) </td><td> Sandvik Kanthal HP Reid </td><td> PX000004 </td><td> Para fazer tetrodes personalizados e stereotrodes </td></tr><tr><td> Pré-amplificador </td><td> Tucker-Davis Sistema </td><td> PZ-2 </td><td></td></tr><tr><td> Amplificador </td><td> Tucker-Davis Sistema </td><td> RZ-2 </td><td></td></tr><tr><td> Aquisição de dados do sistema - OpenEx suíte </td><td> Tucker-Davis Sistema </td><td></td><td></td></tr><tr><td> Software online pico de classificação - SpikePac </td><td> Tucker-Davis Sistema </td><td></td><td></td></tr><tr><td> Software ponto-classificação Offline - Mclust caixa de ferramentas Spike-ordenação </td><td> David Redish, do Departamento de Neurociência da Universidadede Minnesota </td><td> Download gratuito em <a href="http://redishlab.neuroscience.umn.edu/MClust/MClust.html" target="_blank">http://redishlab.neuroscience.umn.edu/MClust/MClust.html</a> </td><td> MATLAB caixa de ferramentas </td></tr></tbody></table>

identification of olfactory volatiles using gas chromatography-multi-unit recordings (gcmr) in the insect antennal lobe

Todos os organismos habitam um mundo cheio de estímulos sensoriais que determinam a sua resposta comportamental e fisiológica ao seu ambiente. O olfato é especialmente importante em insetos, que utilizam seus sistemas olfativos para responder, e discriminar entre estímulos, odor complexos. Estes odores provocam comportamentos que medeiam processos como a reprodução e seleção de habitat 1-3. Além disso, sensores químicos por comportamentos insetos media que são altamente significativa para a agricultura ea saúde humana, incluindo a polinização 4-6, herbivoria de culturas alimentares 7, e transmissão da doença de 8,9. Identificação de sinais olfativos e seu papel no comportamento dos insetos é, portanto, importante para a compreensão tanto dos processos ecológicos e os recursos alimentares humanos e bem-estar. Até à data, a identificação de compostos voláteis que dirigem o comportamento de insectos tem sido difícil e tedioso. As técnicas atuais incluemgás de gravação eletroantenogramas cromatografia acoplada (GC-EAG), e cromatografia em fase gasosa acoplada gravações individuais sensillum (GC-SSR) 10-12. Estas técnicas provaram ser vital para a identificação de compostos bioactivos. Nós desenvolvemos um método que usa cromatografia gasosa acoplada a multi-canal registros eletrofisiológicos (denominado «GCMR &#39;) de neurônios no lóbulo antenal (AL; principal centro do inseto olfativas) 13,14. Esta técnica estado-da-arte nos permite investigar como as informações odor é representado no cérebro do inseto. Além disso, porque as respostas neurais aos odores, a este nível de processamento olfactivo são altamente sensíveis devido ao grau de convergência de neurónios da antena do receptor em neurónios AL, AL gravações irá permitir a detecção de constituintes activos de odores naturais de forma eficiente e com elevada sensibilidade. Aqui descrevemos GCMR e dar um exemplo de seu uso. Várias etapas gerais são envolved na detecção de compostos voláteis bioactivos e da resposta de insectos. Voláteis primeiro precisam ser coletados a partir de fontes de interesse (neste exemplo, usar flores da Mimulus gênero (Phyrmaceae)) e caracteriza-se como necessário, utilizando padrão de GC-MS técnicas 14-16. Os insectos são preparados para o estudo utilizando dissecção mínima, após o qual um eléctrodo de gravação é inserida no lobo antenal e gravação multi-canal neural começa. Pós-processamento dos dados neurais, então, revela que odores particulares causar significativas respostas neurais do sistema nervoso dos insetos. Embora o exemplo que apresentamos aqui é específico para os estudos de polinização, GCMR pode ser expandida para uma ampla gama de organismos de estudo e de fontes voláteis. Por exemplo, este método pode ser utilizado na identificação de odores para atrair ou repelir insectos vectores e pragas de culturas. Além disso, GCMR também pode ser usado para identificar atractivos para os insectos benéficos, tais como pollinators. A técnica pode ser expandida para indivíduos não-insectos, bem.

No ensaio utilizando o GCMR M. lewisii aroma floral, injetamos 3 ul do extracto no GC. O número total de voláteis de eluição através da GC é tipicamente 60-70 voláteis. O cheiro de M. lewisii é predominantemente composto por monoterpenóides, incluindo β-mirceno (acíclico) e α-pineno, sendo o restante do aroma composto de seis carbonos, tais como compostos voláteis de 2-hexanol, e sesquiterpenos que compreendem &lt;1% do espaço livre. GCMR apr...

Watch this Scientific Journal Video about Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe at JoVE.com

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Pistas olfativas mediar muitos comportamentos diferentes em insetos, e muitas vezes são misturas complexas compostas de dezenas a centenas de compostos voláteis. Utilizando cromatografia em fase gasosa com multi-canais de gravação no lobo antenal inseto, descreve-se um método para a identificação de compostos bioactivos.

Identificação de voláteis olfativas utilizando cromatografia gasosa-Multi-unidade Gravações (GCMR) no lobo antenal Inseto

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

identification-olfactory-volatiles-using-gas-chromatography-multi

Research

JoVE Journal

Neuroscience

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

14.2K visualizações.  University of Washington. Pistas olfativas mediar muitos comportamentos diferentes em insetos, e muitas vezes são misturas complexas compostas de dezenas a centenas de compostos voláteis. Utilizando cromatografia em fase gasosa com multi-canais de gravação no lobo antenal inseto, descreve-se um método para a identificação de compostos bioactivos.

Vídeo: Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12.
Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane13. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field2. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor. 
Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.

The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.

Respostas análise de rato Olfactory neurônios sensoriais Usando a gravação Electroolfactogram Air fase

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

Neurociência

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

Imagens de cálcio de odor-provocou respostas no Drosophila Lobo antenal

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

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 ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Dois registros eletrofisiológicos de sinapticamente evocadas Respostas astroglial e neuronal em fatias hipocampais agudas

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

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 ...

Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects foraging for food or searching for mates3: the olfactory search is constrained by the physics of turbulent transport4. The concentration landscape of wind borne odors is discontinuous and consists of sporadically located patches. A pre-requisite to olfactory search is that intermittent odor patches are detected. Because of its high speed and sensitivity5-6, the olfactory organ of insects provides a unique opportunity for detection. Insect antennae have been used in the past to detect not only sex pheromones7 but also chemicals that are relevant to humans, e.g., volatile compounds emanating from cancer cells8 or toxic and illicit substances9-11. We describe here a protocol for using insect antennae on autonomous robots and present a proof of concept for tracking odor plumes to their source. The global response of olfactory neurons is recorded in situ in the form of electroantennograms (EAGs). Our experimental design, based on a whole insect preparation, allows stable recordings within a working day. In comparison, EAGs on excised antennae have a lifetime of 2 hr. A custom hardware/software interface was developed between the EAG electrodes and a robot. The measurement system resolves individual odor patches up to 10 Hz, which exceeds the time scale of artificial chemical sensors12. The efficiency of EAG sensors for olfactory searches is further demonstrated in driving the robot toward a source of pheromone. By using identical olfactory stimuli and sensors as in real animals, our robotic platform provides a direct means for testing biological hypotheses about olfactory coding and search strategies13. It may also prove beneficial for detecting other odorants of interests by combining EAGs from different insect species in a bioelectronic nose configuration14 or using nanostructured gas sensors that mimic insect antennae15.

We describe a protocol for using insect antennae in the form of electroantennograms (EAGs) on autonomous robots. Our experimental design allows stable recordings within a day and resolves individual odor patches up to 10 Hz. The efficiency of EAG sensors for olfactory searches is demonstrated in driving a robot toward an odor source.

Usando insetos eletroantenogramas Sensores em robôs autônomos para Pesquisas olfativas

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

Ex vivo preparações da Intacta Vomeronasal Órgão e acessórios bulbo olfatório

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular recording has historically been a common technique used to study sensitivities of individual photoreceptor cells to different light stimuli that is still being used today. However, there remains a dearth of detailed methodology in the literature for researchers wishing to replicate intracellular recording experiments in the eye. Here we present the insect as a model for examining eye physiology more generally. Insect photoreceptor cells are located near the surface of the eye and are therefore easy to reach, and many of the mechanisms involved in vision are conserved across animal phyla. We describe the basic procedure for in vivo intracellular recording of photoreceptor cells in the eye of a butterfly, with the goal of making this technique more accessible to researchers with little prior experience in electrophysiology. We introduce the basic equipment needed, how to prepare a live butterfly for recording, how to insert a glass microelectrode into a single cell, and finally the recording procedure itself. We also explain the basic analysis of raw response data for determining spectral sensitivity of individual cell types. Although our protocol focuses on determining spectral sensitivity, other stimuli (e.g., polarized light) and variations of the method are applicable to this setup.

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

The electrophysiological technique of intracellular recording is demonstrated and used to determine spectral sensitivities of single photoreceptor cells in the compound eye of a butterfly.

Determinação dos fotorreceptores celular Sensibilidade espectral em um inseto modelo de<em&gt; In Vivo</em&gt; Gravações intracelulares

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular ...

Insect-controlled Robot: A Mobile Robot Platform to Evaluate the Odor-tracking Capability of an Insect

Robotic odor source localization has been a challenging area and one to which biological knowledge has been expected to contribute, as finding odor sources is an essential task for organism survival. Insects are well-studied organisms with regard to odor tracking, and their behavioral strategies have been applied to mobile robots for evaluation. This "bottom-up" approach is a fundamental way to develop biomimetic robots; however, the biological analyses and the modeling of behavioral mechanisms are still ongoing. Therefore, it is still unknown how such a biological system actually works as the controller of a robotic platform. To answer this question, we have developed an insect-controlled robot in which a male adult silkmoth (Bombyx mori) drives a robot car in response to odor stimuli; this can be regarded as a prototype of a future insect-mimetic robot. In the cockpit of the robot, a tethered silkmoth walked on an air-supported ball and an optical sensor measured the ball rotations. These rotations were translated into the movement of the two-wheeled robot. The advantage of this "hybrid" approach is that experimenters can manipulate any parameter of the robot, which enables the evaluation of the odor-tracking capability of insects and provides useful suggestions for robotic odor-tracking. Furthermore, these manipulations are non-invasive ways to alter the sensory-motor relationship of a pilot insect and will be a useful technique for understanding adaptive behaviors.

The capability to localize an odor source is necessary for insect survival and is expected to be applicable to artificial odor-tracking. The insect-controlled robot is driven by an actual silkmoth and enables us to evaluate the odor-tracking capability of insects through a robotic platform.

Robô controlado por insetos: uma plataforma móvel Robot avaliar a capacidade Odor-tracking de um inseto

Robotic odor source localization has been a challenging area and one to which biological knowledge has been expected to contribute, as finding odor ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

Imunossinção Quadruple da Lâmpada Olfativa para Visualização de Códigos de Identidade Molecular de Axon Sensorial Olfativo

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...