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Neuroscience
Indicatori di sinapsi singola di Soldamento Release e Uptake in Acute Brain Slices da Topi Normal...
Indicatori di sinapsi singola di Soldamento Release e Uptake in Acute Brain Slices da Topi Normal...
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

Indicatori di sinapsi singola di Soldamento Release e Uptake in Acute Brain Slices da Topi Normali e Huntington

Full Text
6,652 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.

Presentiamo un protocollo per valutare l'equilibrio tra il rilascio del glutammato e l'autorizzazione alle singole sinapsi glutamatergiche corticosterogiche in fette acute di topi adulti. Questo protocollo utilizza il sensore fluorescente iGluu per il rilevamento del glutammato, una telecamera sCMOS per l'acquisizione del segnale e un dispositivo per l'illuminazione laser focale.

L'imaging ad alta risoluzione di singole sinapsi che esprimono un sensore di glutammato veloce consente di rilevare la mancata corrispondenza locale tra il rilascio del trasmettitore e l'assorbimento. Nel caso della malattia, questo metodo può essere utilizzato per identificare sinapsi disfunzionali. Per la correzione dell'autofluorescenza, in primo luogo, posizionare una fetta cerebrale dal mouse di interesse nella camera di registrazione di un microscopio con un fotone.

Immergere la fetta in liquido cerebrospinale ossigenato e artificiale e utilizzare l'obiettivo 20x di immersione in acqua per localizzare lo striato dorsale. Fissare le fette con una griglia di nylon su un'arpa di platino per ridurre al minimo il movimento del tessuto e passare all'obiettivo 63x di immersione in acqua. Utilizzando un filtro passa alto a 510 nanometri, acquisire un'immagine delle strutture positive del sensore autofluorescente e glutammato insieme.

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