preparing the recording electrodes and drosophila for analyzing responses of taste neurons

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

Insects, including drosophilid flies, are endowed with a sophisticated taste system that enables them to extract complex chemical information from their surroundings. This system allows them to discern the chemical composition of various substances, distinguishing between those that are nutritious and those that are harmful1,2.
At the core of this system are specialized structures known as taste hairs or sensilla, strategically located on various body parts. In drosophilid flies, these sensilla are located on the labellum, which is the major taste organ of the fly head1,2,3,4, as well as on the legs and wings1,2,5,6. The labellum is located at the tip of the proboscis and contains two lobes4,7,8. Each lobe is covered with 31 taste sensilla categorized as short, long, and intermediate4,7,8. These sensilla each house 2-4 taste neurons1,2,9,10. These taste neurons express members of at least four different gene families, namely the Gustatory receptor (Gr), Ionotropic receptor (Ir), Pickpocket (Ppk), and Transient receptor potential (Trp) genes1,2,11,12,13. This diversity of receptors and channels equips insects with the ability to recognize a wide array of chemical compounds, including both nonvolatile and volatile cues1,2,14.
For over 50 years, scientists have quantified the response of taste neurons and their receptors using a technique called tip recording3,4,6,8,13,15,16,17,18,19,20,21,22,23,24,25,26,27,28, 
29,30,31,32,33,34,35. However, this method has major limitations. First, neural activity can be measured only during contact with the stimulus, and not before or after contact. This limitation precludes the measurement of spontaneous spiking activity and prevents the measurement of OFF responses. Second, only tastants that are soluble in aqueous solutions can be tested.
These limitations can be overcome by a rarely used alternative electrophysiological technique called &#34;base recording.&#34; Here we describe this technique, which we have adapted from a method used by Marion-Poll and colleagues24, and show the crucial taste coding features that it can now conveniently measure14.

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

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

Our lab investigates how animals detect and respond to odors, taste, and some pheromone. We've made significant strides in understanding the molecular basis of chemoreception. We've made several advances in the field, including the discovery of the first insect odor receptors, the discovery of the first insect taste receptors, and the elucidation of basic principles of the logic of odor and taste coding.This technique allows the measurement of test neuron activity before, during, and after the stimulus. Thus, it allows extensive analysis of OFF responses that occur after a taste stimulus. It also allows the measurement of taste neuron activity to the river of volatile compounds.The classical technique for taste electrophysiology was unable to measure taste responses to hydrophobic compounds such as long-chain pheromones that have very low solubility in water. However, with the help of this technique, we can overcome this limitation by separating the recording electrode from the glass stimulus capillary. Our research explores the functioning of the chemosensory systems in animals, focusing on their role in feeding and mating choices.We're currently examining non-coding RNAs and micro peptides in fruit flies'olfactory systems, and how mosquitoes and tsetse flies use these systems to find human hosts.

Preparando os eletrodos de gravação e a drosófila para analisar as respostas dos neurônios gustativos

Comece colocando de 10 a 15 moscas Drosophila recém-surgidas em frascos de cultura padrão frescos. Mantenha esses frascos a 25 graus Celsius e 60% de umidade relativa em um ciclo de luz e escuridão de 12 horas. Para preparar um capilar de vidro para reter o estímulo, selecione um capilar de vidro de borossilicato e use um instrumento extrator de pipeta para puxar o capilar de vidro.Em seguida, pegue hastes de tungstênio para eletrodos de referência e gravação. Mergulhe esses eletrodos repetidamente por vários segundos em uma solução de nitrato de potássio a 10% ou em uma solução de hidróxido de potássio para afiá-los até um diâmetro de ponta de um micrômetro. Para puxar uma única mosca do frasco para um aspirador, retire cuidadosamente o aspirador e coloque um dedo sobre a extremidade do aspirador para prender a mosca.Agora, mantenha a extremidade do aspirador na ponta da pipeta de 200 microlitros e use a extremidade para empurrar a mosca para frente de cabeça em direção à extremidade estreita da ponta da pipeta. Usando uma lâmina de barbear, apare ambas as extremidades da ponta da pipeta, anterior e posterior à mosca. Com a ajuda de argila ou um pequeno pedaço de algodão, empurre ainda mais a mosca para frente até que metade de sua cabeça se projete da extremidade da ponta da pipeta aparada.Empregue uma pinça para expor suavemente o labelo na frente da cabeça. Sob um microscópio estereoscópico, posicione o labelo lateralmente em uma lamínula, garantindo que um lóbulo com sua sensila gustativa 31 seja exposto.

preparing-recording-electrodes-drosophila-for-analyzing-responses

Research

JoVE Journal

Neuroscience

Preparing the Recording Electrodes and Drosophila for Analyzing Responses of Taste Neurons

62 visualizações.  Yale University. Comece colocando de 10 a 15 moscas Drosophila recém-surgidas em frascos de cultura padrão frescos. Mantenha esses frascos a 25 graus Celsius e 60% de umidade relativa em um ciclo de luz e escuridão de 12 horas. Para preparar um capilar de vidro para reter o estímulo, selecione um capilar de vidro de borossilicato e use um instrumento extrator de pipeta para puxar o capilar de vidro.Em seguida, pegue hastes de tungstênio para eletrodos de referência e gravação. Mergulhe esses el...

Vídeo: Preparando os eletrodos de gravação e a drosófila para analisar as respostas dos neurônios gustativos

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.

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.

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 ...