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JoVE Journal
Neuroscience
Indicadores de sinapsis únicas de liberación y aceptación de glutamato en rebanadas cerebrales ag...
Indicadores de sinapsis únicas de liberación y aceptación de glutamato en rebanadas cerebrales ag...
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

Indicadores de sinapsis únicas de liberación y aceptación de glutamato en rebanadas cerebrales agudas de ratones normales y Huntington

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.

Presentamos un protocolo para evaluar el equilibrio entre la liberación de glutamato y el aclaramiento en sinapsis glutamatérgicas corticostriatales en rodajas agudas de ratones adultos. Este protocolo utiliza el sensor fluorescente iGluu para la detección de glutamato, una cámara sCMOS para la adquisición de señal y un dispositivo para la iluminación láser focal.

La imagen de alta resolución de una sola sinapsis que expresa un sensor de glutamato rápido permite una detección de discordancia local entre la liberación y la absorción del transmisor. En el caso de la enfermedad, este método se puede utilizar para identificar sinapsis disfuncionales. Para la corrección de autofluorescencia, primero, coloque una rebanada cerebral del ratón de interés en la cámara de grabación de un microscopio de un solo fotón.

Sumerja la rebanada en líquido cefalorraquídeo artificial oxigenado y utilice el objetivo de inmersión en agua de 20x para localizar el estriado dorsal. Fije las rodajas con una rejilla de nylon en un arpa de platino para minimizar el movimiento del tejido, y cambie al objetivo de inmersión en agua 63x. Usando un filtro de paso alto a 510 nanómetros, adquiera una imagen de las estructuras positivas del sensor autofluorescente y glutamato juntos.

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