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Neuroscience
전 비보 내 분 비 신호 초파리 에 두뇌 응답을 시각화 칼슘 이미징
전 비보 내 분 비 신호 초파리 에 두뇌 응답을 시각화 칼슘 이미징
JoVE Journal
Neuroscience
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JoVE Journal Neuroscience
Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

전 비보 내 분 비 신호 초파리 에 두뇌 응답을 시각화 칼슘 이미징

Full Text
9,859 Views
06:49 min
June 2, 2018

DOI: 10.3791/57701-v

Hiroshi Ishimoto1, Hiroko Sano2

1Division of Biological Science, Graduate School of Science,Nagoya University, 2Department of Molecular Genetics, Institute of Life Science,Kurume University

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Overview

This study presents a protocol for ex vivo calcium imaging of the Drosophila brain, aiming to explore neuronal responses to endocrine signals. The method allows for the testing of natural or synthetic compounds to activate specific neurons, providing insights into the intersections of endocrinology and neuroscience.

Key Study Components

Area of Science

  • Neuroscience
  • Endocrinology
  • Neurobiology of Drosophila

Background

  • The study investigates the direct effects of endocrine signals on the brain.
  • Utilizes Drosophila to provide a model for understanding hormonal networks.
  • Focuses on how specific peptides can influence neuronal activation.
  • Established the groundwork for further research on receptor specificity.

Purpose of Study

  • To develop a protocol that allows for the assessment of brain responses to endocrine signals.
  • To enable researchers to examine neuronal activity separate from other tissues.
  • To facilitate screening of compounds that may influence neuronal function.

Methods Used

  • The method involves dissection and imaging of the Drosophila larval brain using calcium indicators.
  • Larvae are collected and prepared for imaging approximately 90-120 hours after egg laying.
  • Calcium fluorescence imaging parameters are established, allowing for detailed observation of neuronal activity.
  • Application of test peptides enables measurement of the brain's responsive signals over time.

Main Results

  • Successful imaging of GCaMP6 signals reveals neuronal activation in response to various peptides.
  • Statistical analysis incorporates baseline signal intensity to evaluate neuronal responses post-peptide application.
  • The findings confirm the usefulness of this technique for exploring hormonal control of brain function.

Conclusions

  • This study demonstrates a valuable protocol for investigating endocrine influence on neuronal activity in Drosophila.
  • It enables exploration of receptor specificity and hormonal networks in neuroscience research.
  • The implications extend to understanding plasticity and mechanisms underlying brain function and responses.

Frequently Asked Questions

What are the advantages of using Drosophila for calcium imaging?
Drosophila offers a simplified model system where genetic modifications can easily be made, allowing for targeted studies on neuronal functions and responses to hormones.
How is the larval brain prepared for imaging?
The larval brain is dissected using fine forceps to separate it from other tissues, then mounted in an imaging chamber for calcium fluorescence assessment.
What outcomes can be obtained from this imaging method?
The method provides real-time data on neuronal calcium signals, enabling insights into neuronal activation and response to hormonal signals.
How long does it take to perform the entire procedure?
With proper preparation, the technique can be completed in about one hour, making it efficient for screening experiments.
What considerations should be taken into account when preparing the brain sample?
It is crucial to prepare the brain sample quickly and handle it gently to avoid any damage during dissection, which can affect imaging results.
Can this protocol be adapted for other types of experiments?
Yes, the protocol can be combined with genetic manipulations to explore different receptor types and their specific roles in neuronal signaling.

이 종이 초파리 뇌의 칼슘 이미징 비보 전 프로토콜을 설명합니다. 이 방법에서는, 천연 또는 합성 화합물은 뇌의 특정 신경 세포를 활성화 하는 능력을 테스트 하는 버퍼에 적용할 수 있습니다.

이 방법은 내분비 신호에 대한 뇌 반응을 검토하는 연구와 같은 신경 과학 분야뿐만 아니라 내분비학 분야의 주요 질문에 답하는 데 도움이 될 수 있습니다. 이 기술의 가장 큰 장점은 다른 조직과 분리된 뇌에 대한 내분비 신호의 직접적인 영향을 조사할 수 있다는 것입니다. 이 절차를 시작하려면 집게를 사용하여 플라스틱 배양 접시의 하단 중앙을 긁어 해부 후 유충 뇌의 복부 신경절을 쉽게 장착할 수 있도록 움푹 들어간 곳을 만듭니다.

이쑤시개로 움푹 들어간 곳의 양쪽에 초강력 접착제를 한 방울 떨어뜨리고 접착제에 텅스텐 막대를 부착합니다. 그런 다음 퍼티와 같은 재사용 가능한 작은 접착제 조각을 사용하여 움푹 들어간 곳을 둘러싼 원형 벽을 만듭니다. 유충의 뇌를 해부하기 위해 알을 낳은 후 90-120시간 후에 유충을 채취하여 증류수로 최소 3회 세척하여 부착된 음식물 찌꺼기를 제거합니다.

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