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

곤충 맛 뉴런 활동의 향상된 분석을 위한 새로운 방법

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.

저자 스포트라이트: 화학수용의 발전 – 곤충 냄새 수용체에서 비코딩 RNA까지

염기 기록: 초파리에서 미각 뉴런의 반응을 분석하는 기법

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.

미각 뉴런의 반응을 분석하기 위한 기록 전극 및 초파리의 준비

10-15마리의 새로 출현한 초파리를 새로운 표준 배양 바이알에 넣는 것으로 시작합니다. 이 바이알은 섭씨 25도, 상대 습도 60%에서 12시간 명암 주기로 유지합니다. 자극을 유지하기 위한 유리 모세관을 준비하려면 붕규산 유리 모세관을 선택하고 피펫 풀러 기기를 사용하여 유리 모세관을 당깁니다.그런 다음 참조 전극과 기록 전극 모두에 텅스텐 막대를 사용하십시오. 이 전극을 10% 질산칼륨 용액 또는 수산화칼륨 용액에 몇 초 동안 반복적으로 담궈 1마이크로미터 팁 직경으로 날카롭게 합니다. 바이알에서 파리 한 마리를 흡인기로 끌어들이려면 흡인기를 조심스럽게 빼내고 흡인기 끝에 손가락을 올려 파리를 가둡니다.이제 흡입기의 끝을 200마이크로리터 피펫 팁에 유지하고 끝을 사용하여 플라이를 피펫 팁의 좁은 끝을 향해 머리부터 앞으로 밉니다. 면도날을 사용하여 피펫 팁의 양쪽 끝, 플라이의 앞쪽과 뒤쪽을 자릅니다. 점토나 작은 면봉을 사용하여 머리의 절반이 다듬어진 피펫 팁 끝에서 튀어나올 때까지 파리를 앞으로 더 밉니다.집게를 사용하여 머리 앞쪽의 라벨럼을 부드럽게 노출시킵니다. 실체 현미경 아래에서 라벨럼을 커버 슬립에 측면으로 배치하여 31개의 맛 감각이 있는 하나의 로브가 노출되도록 합니다.

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

Research

JoVE Journal

Neuroscience

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

62 조회수.  Yale University. 10-15마리의 새로 출현한 초파리를 새로운 표준 배양 바이알에 넣는 것으로 시작합니다. 이 바이알은 섭씨 25도, 상대 습도 60%에서 12시간 명암 주기로 유지합니다. 자극을 유지하기 위한 유리 모세관을 준비하려면 붕규산 유리 모세관을 선택하고 피펫 풀러 기기를 사용하여 유리 모세관을 당깁니다.그런 다음 참조 전극과 기록 전극 모두에 텅스텐 막대를 사용하십시오. ?...

비디오: 미각 뉴런의 반응을 분석하기 위한 기록 전극 및 초파리의 준비

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 ...