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JoVE Journal
Neuroscience
In vivo Optical cálcio Imaging de aprendizagem induzida por plasticidade sináptica em Drosoph...
In vivo Optical cálcio Imaging de aprendizagem induzida por plasticidade sináptica em Drosoph...
JoVE Journal
Neuroscience
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JoVE Journal Neuroscience
In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

In vivo Optical cálcio Imaging de aprendizagem induzida por plasticidade sináptica em Drosophila melanogaster

Full Text
9,801 Views
06:35 min
October 8, 2019

DOI: 10.3791/60288-v

Clare E. Hancock1, Florian Bilz1, André Fiala1

1Department of Molecular Neurobiology of Behavior,University of Göttingen

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Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This study presents a protocol for visualizing pre- and postsynaptic calcium in Drosophila to investigate learning and memory. Employing in vivo calcium imaging with synaptically localized sensors, the research combines this with classical olfactory conditioning to explore synaptic plasticity associated with associative learning.

Key Study Components

Area of Science

  • Neuroscience
  • Behavioral Biology
  • Calcium Imaging

Background

  • Drosophila melanogaster serves as a model for studying learning and memory.
  • Understanding synaptic calcium activity can illuminate the physiological processes behind memory formation.
  • Associative learning is fundamental to how memories are encoded and retrieved.
  • The mushroom body in Drosophila is crucial for learning and memory functionalities.

Purpose of Study

  • To visualize calcium activity at synapses during learning processes.
  • To correlate synaptic activity with memory trace formation.
  • To utilize a classical conditioning paradigm to examine synaptic changes.

Methods Used

  • Calcium imaging is performed using genetically encoded calcium indicators in a custom imaging chamber.
  • Drosophila are prepared for imaging following surgical procedures to expose the brain region of interest.
  • The experiment involves a pre-conditioning baseline measurement followed by odor stimulus delivery and post-conditioning measurements.
  • The imaging system includes a multi-photon microscope tuned for specific excitation wavelengths and scanning protocols.
  • Data collection involves measuring calcium transients linked with odor stimuli before and after associative training.

Main Results

  • The study provides insights into synaptic changes associated with olfactory learning.
  • Differences in calcium responses were observed between various genetically altered flies, demonstrating the impact of specific indicators on synaptic activity.
  • Visualizations reveal how olfactory memory is stored and modulated at the cellular level.
  • Results underscore the utility of this method in understanding complex brain structures.

Conclusions

  • This protocol enables real-time visualization of calcium dynamics, contributing to our comprehension of neuronal mechanisms in memory formation.
  • Insights gained through this method can illuminate synaptic plasticity principles and enhance our understanding of learning processes.
  • The results have broad implications for neuroscience, particularly in the context of associative memory storage.

Frequently Asked Questions

What advantages does this imaging method offer?
This method provides real-time observation of calcium activity, allowing for direct correlational studies between synaptic responses and learning.
How are Drosophila prepared for imaging?
Drosophila undergo surgical procedures to expose brain areas and are fixed in an imaging chamber for optimal visualization.
What outcomes can be expected from this protocol?
Outcomes include detailed imaging of calcium transients and insights into synaptic plasticity during learning events.
Can this method be adapted for other models?
While designed for Drosophila, the principles of this imaging technique may be applicable to other model organisms with adaptations.
What are the limitations of this study?
Limitations may include the specificity of the calcium indicators used and potential variations in individual animal responses.

Aqui nós apresentamos um protocolo com que o cálcio pre-e/ou pós-sináptica pode ser visualizado no contexto da aprendizagem e da memória de Drosophila . A imagem latente in vivo do cálcio que usa sensores synaptically localizados do cálcio é combinada com um paradigma olfactory clássico do condicionamento tal que a plasticidade sináptica subjacente este tipo de aprendizagem associativa possa ser determinada.

Esta técnica facilita a observação em tempo real da atividade sináptica de cálcio como parâmetro fisiológico para os processos celulares subjacentes à aprendizagem e formação da memória em drosophila melanogaster. Comparando respostas neuronais com odores antes e depois do treinamento associativo, podemos desenhar correlações diretas entre a atividade sináptica e a formação do traço de memória em moscas de frutas individuais. Para produzir moscas de frutas transgênicas em que neurônios específicos de interesse expressam um indicador de cálcio geneticamente codificado cruzam moscas fêmeas e machos carregando as construções GAL4 e UAS desejadas, e envelhecem a progêneria feminina até três a seis dias após a eclosão.

Para preparar uma mosca para a imagem, use fórceps finos para colocar uma mosca anesthetizada de gelo em uma câmara de imagem personalizada. Usando um microscópio dissecando para posicionar o tórax e as pernas em contato com os fios elétricos na parte inferior da câmara, e com a cabeça deitada plana. Fixar a mosca no lugar com fita adesiva clara, e use uma lâmina de bisturi cirúrgica para cortar uma janela na fita ao redor da cabeça, deixando a antena coberta e apenas a parte anterior da parte mais do tórax exposta.

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