Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

Claire  A. de March; Yosuke Fukutani; Aashutosh Vihani; Hitoshi Kida; Hiroaki Matsunami

doi:10.3791/59446

En temps réel en Vitro suivi d’Activation des récepteurs de l’odeur par une substance odorante en...

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JoVE Journal Neuroscience

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

En temps réel en Vitro suivi d’Activation des récepteurs de l’odeur par une substance odorante en Phase vapeur

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09:53 min

April 23, 2019

DOI: 10.3791/59446-v

Claire A. de March¹, Yosuke Fukutani^1,2, Aashutosh Vihani^1,3, Hitoshi Kida^1,4, Hiroaki Matsunami^1,3,5,6

¹Department of Molecular Genetics and Microbiology,Duke University Medical Center, ²Department of Biotechnology and Life Science,Tokyo University of Agriculture and Technology, ³Department of Neurobiology,Duke University Medical Center, ⁴Department of Mechanical Systems, Engineering,Tokyo University of Agriculture and Technology, ⁵Institute of Global Innovation Research,Tokyo University of Agriculture and Technology, ⁶Duke Institute for Brain Sciences,Duke University

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Overview

This study presents a novel method for real-time in vitro monitoring of odorant receptor activation using vapor phase odorant stimulation. Previous in vitro systems have largely relied on liquid phase stimulation, potentially limiting physiological relevance. The method provides insights into the activation kinetics of odorant receptors and their role in odor perception.

Key Study Components

Area of Science

Neuroscience
Olfactory biology
Cell culture techniques

Background

Odorant receptors respond to volatile odorant molecules, crucial for the sense of smell.
Existing in vitro methods primarily use liquid phase odorants, which may not accurately mimic natural conditions.
This research addresses gaps in current methodologies by allowing for vapor phase stimulation.

Purpose of Study

To develop a method that facilitates the detection and discrimination of volatile odors in vitro.
To enable real-time monitoring of odorant receptor activation.
To bridge the gap between in vitro and in vivo approaches in olfactory research.

Methods Used

Utilized cell culture techniques to grow cells in M10 PSF medium at physiological conditions.
Involves transfection of cells with specific odorant receptor plasmids to study their activation.
Key steps include careful cell preparation, including trypsinization and centrifugation.
Measurements of luminescence provide data on receptor activation upon exposure to odorants.

Main Results

The method allows for precise monitoring of odorant receptor responses to specific odorants like acetophenone.
Findings illustrate the kinetics of odor perception and potential roles of mucosal metabolic enzymes.
Successfully demonstrated the capability to detect and discriminate odors in a controlled in vitro setting.

Conclusions

This innovative approach enhances the understanding of olfactory receptor dynamics in a more physiologically relevant context.
The findings enable further research into the development of odorant receptor-based biosensors.
Implications extend to understanding mechanisms of odor detection, which could inform broader olfactory studies.

Frequently Asked Questions

What are the advantages of using vapor phase stimulation?

Vapor phase stimulation mimics natural conditions, providing a more physiologically relevant method to study odorant receptors compared to liquid phase approaches.

How is the biological model implemented in this study?

Cells are cultured in specific media and transfected with plasmids encoding odorant receptors to investigate their responses to volatile stimuli.

What types of data are obtained from this method?

The methodology provides luminescence data indicating the activation levels of odorant receptors in response to odorant exposure.

How can this method be applied to other research areas?

This technique can potentially be adapted for various studies involving sensory receptor activation and biosensor development across biological systems.

What are the key limitations or considerations for this study?

Care must be taken to ensure that experimental conditions, such as temperature and CO2 levels, remain constant to obtain accurate results in receptor activation studies.

Physiologiquement, les odorants récepteurs sont activés par des molécules odorantes inhalés en phase vapeur. Cependant, la plupart des systèmes in vitro utilisent une stimulation de la phase liquide odorant. Nous présentons ici une méthode qui permet un suivi en temps réel in vitro de l’activation des récepteurs odorants lors de la stimulation de la substance odorante en phase gazeuse.

Notre protocole permet de mesurer la détection et la discrimination des odeurs volatiles avec un panneau de récepteurs odorants. Cette méthode ouvre la possibilité de développer des biocapteurs à base de récepteurs odorants. Cette technique permet une surveillance in vitro en temps réel de l’activation des récepteurs odorants sur les molécules d’odorant en phase de vapeur.

Il comble l’écart entre les approches in vitro et in vivo. Cette méthode peut être utilisée pour comprendre la cinétique des événements qui conduisent à la perception des odeurs, y compris le rôle des enzymes métaboliques muqueales. Pour commencer, préparez M10 et M10 PSF selon le manuscrit.

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