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

A Novel Method for Enhanced Analysis of Insect Taste Neuron Activity

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.

Author Spotlight: Advances in Chemoreception &#8211; From Insect Odor Receptors to Non-Coding RNAs

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

Preparación de los electrodos de registro y Drosophila para analizar las respuestas de las neuronas gustativas

Comience colocando de 10 a 15 moscas Drosophila recién emergidas en viales de cultivo estándar frescos. Mantenga estos viales a 25 grados centígrados y 60% de humedad relativa en un ciclo de luz y oscuridad de 12 horas. Para preparar un capilar de vidrio para sostener el estímulo, seleccione un capilar de vidrio de borosilicato y use un instrumento extractor de pipetas para tirar del capilar de vidrio.Luego, tome varillas de tungsteno como electrodos de referencia y de registro. Sumerja estos electrodos repetidamente durante varios segundos en una solución de nitrato de potasio al 10% o en una solución de hidróxido de potasio para afilarlos hasta un diámetro de punta de un micrómetro. Para extraer una sola mosca del vial y colocarla en un aspirador, retire con cuidado el aspirador y coloque un dedo sobre el extremo del aspirador para atrapar la mosca.Ahora, mantenga el extremo del aspirador en la punta de la pipeta de 200 microlitros y utilice el extremo para empujar la mosca hacia adelante de cabeza, hacia el extremo estrecho de la punta de la pipeta. Con una cuchilla de afeitar, recorte ambos extremos de la punta de la pipeta, anterior y posterior a la mosca. Con la ayuda de arcilla o un pequeño trozo de algodón, empuje aún más la mosca hacia adelante hasta que la mitad de su cabeza sobresalga del extremo de la punta de la pipeta recortada.Emplea fórceps para exponer suavemente el labelo en la parte delantera de la cabeza. Bajo un microscopio estereoscópico, coloque el labelo lateralmente en un cubreobjetos, asegurándose de que un lóbulo con sus 31 sensillas gustativas quede expuesto.

Research

JoVE Journal

Neuroscience

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

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 ...

Investigación

Neurociencias

Base Recording from Taste Sensilla of Drosophila for Analyzing Responses of Taste Neurons

Registro de la base de la sensilla gustativa de Drosophila para analizar las respuestas de las neuronas gustativas