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Neuroscience
Indicateurs synapses uniques de la libération et de l’absorption des glutamates dans les tranches...
Indicateurs synapses uniques de la libération et de l’absorption des glutamates dans les tranches...
JoVE Journal
Neuroscience
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JoVE Journal Neuroscience
Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Indicateurs synapses uniques de la libération et de l’absorption des glutamates dans les tranches cérébrales aigues des souris normales et huntingtones

Full Text
6,650 Views
08:27 min
March 11, 2020

DOI: 10.3791/60113-v

Anton Dvorzhak1, Rosemarie Grantyn1

1Synaptic Dysfunction Group, Neuroscience Research Center,Charité - University Medicine

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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 evaluating the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses using acute slices from adult mice. By employing the fluorescent sensor iGlu u for glutamate detection, researchers can identify local mismatches in transmitter dynamics, aiding in the investigation of dysfunctional synapses in disease contexts.

Key Study Components

Area of Science

  • Neuroscience
  • Neuropharmacology
  • Synaptic physiology

Background

  • Release and clearance of glutamate are crucial for neurotransmission.
  • Imaging techniques can identify disruptions in neurotransmitter dynamics.
  • Pathological conditions may alter synaptic function.
  • Assessing these changes can provide insights into diseases such as Huntington's disease.

Purpose of Study

  • To develop a method for assessing synaptic glutamate dynamics.
  • To investigate how synapses respond to stimulation under different conditions.
  • To identify functional impairments in synapses associated with neurological disorders.

Methods Used

  • Ex vivo brain slices from adult mice were used for imaging studies.
  • Single corticostriatal glutamatergic synapses were the primary focus of investigation.
  • The fluorescence sensor iGlu u was employed for glutamate detection.
  • A two-photon microscope setup enabled high-resolution imaging and stimulation.
  • Custom protocols were followed for precise experimental conditions including electrical stimulation and fluorescence measurement.

Main Results

  • The protocol allowed for detection of glutamate release and clearance at the level of single synapses.
  • Differences in glutamate dynamics were observed between wild-type and mutant mice.
  • Specific decay parameters provided insights into synaptic function and potential dysfunction in Huntington's disease models.
  • Characterization of synaptic responses revealed differential behaviors in glutamate release.

Conclusions

  • This study enables detailed assessment of neurotransmitter dynamics at individual synapses, contributing to our understanding of synaptic dysfunction in neurological diseases.
  • The findings may aid in the identification of targets for therapeutic interventions.
  • Overall, the method enhances our understanding of glutamatergic transmission mechanisms and their plasticity in health and disease.

Frequently Asked Questions

What are the advantages of using acute brain slices?
Acute brain slices preserve the native environment of synapses, allowing for more accurate assessment of synaptic function and dynamics.
How is the glutamate sensor utilized in this method?
The fluorescent sensor iGlu u specifically detects glutamate release, enabling researchers to visualize and quantify neurotransmitter levels at synapses.
What types of outcomes can be measured with this protocol?
Outcomes include real-time measurements of glutamate release and clearance, as well as insights into synaptic dynamics and potential dysfunction in pathological conditions.
How can this method be adapted for other neurotransmitters?
This imaging protocol could potentially be modified to incorporate sensors specific to other neurotransmitters, allowing for broader applications in synaptic research.
What are the limitations of this imaging technique?
One limitation is the requirement for precise experimental conditions, which can be challenging to maintain, particularly in live slice preparations.
How can findings from this study be applied to neurological disorders?
By identifying dysfunction in glutamate dynamics, this research could lead to the development of targeted treatments for disorders such as Huntington's disease.

Nous présentons un protocole pour évaluer l’équilibre entre la libération de glutamate et le dégagement aux synapses glutamatergics corticostriatal simples dans des tranches aigues des souris adultes. Ce protocole utilise le capteur fluorescent iGluu pour la détection de glutamate, une caméra sCMOS pour l’acquisition du signal et un dispositif pour l’éclairage laser focal.

L’imagerie haute résolution de synapses simples exprimant un capteur de glutamate rapide permet de détecter l’inadéquation locale entre la libération de l’émetteur et l’absorption. Dans le cas de la maladie, cette méthode peut être utilisée pour identifier les synapses dysfonctionnelles. Pour la correction de l’autofluorescence, d’abord, placez une tranche de cerveau de la souris d’intérêt dans la chambre d’enregistrement d’un microscope à un photon.

Submergez la tranche dans du liquide céphalo-rachidien artificiel oxygéné et utilisez l’objectif d’immersion d’eau 20x pour localiser le striatum dorsal. Fixez les tranches avec une grille en nylon sur une harpe platine pour minimiser le mouvement des tissus et passez à l’objectif d’immersion d’eau 63x. À l’aide d’un filtre à passage élevé à 510 nanomètres, acquérir ensemble une image des structures positives du capteur autofluorescent et glutamate.

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Neurosciences Issue 157 glutamate release glutamate clearance glutamate spread presynaptic terminal astrocyte glutamate uptake decay kinetics spill-over optogenetics iGluu sCMOS

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