Analyzing the Glutamate Release and Clearance at a Single Neuron Synapse in a Mouse Brain Slice
Transcription
Begin with an electrophysiology setup containing a mouse forebrain slice submerged in an oxygenated aCSF.
aCSF contains an ion-channel blocker that selectively inhibits the sodium-ion channels, preventing sodium-induced neuronal excitation.
The cortical neurons express fluorescent glutamate-detection sensor proteins.
Locate a single presynaptic neuron axonal bouton capable of releasing glutamate, an excitatory neurotransmitter, at the synapse, a junction where it connects with postsynaptic neurons.
Position a stimulation electrode near this bouton and apply depolarizing currents.
This excites the presynaptic neuron, causing a calcium ion influx and facilitating glutamate release.
Start fluorescence intensity recording around the selected bouton.
The released glutamate interacts with the sensor proteins and receptors on post-synaptic neurons.
Meanwhile, illuminate the selected area with laser light, causing the glutamate-bound sensor proteins to fluoresce; an increasing fluorescence intensity indicates glutamate release.
Over time, glutamate dissociates from the sensor proteins and enters the astrocyte, causing a decrease in fluorescence intensity, indicating glutamate clearance.
