The overall goal of the experiment is to estimate the time course of glutamate clearance from astrocytic membranes following its release from excitatory synapses. The first step in this process is to record synaptically activated glutamate transport occurrence from astrocytes in acute brain slices in the absence and presence of a subs saturating concentration of the broad spectrum glutamate transporter antagonist, TBOA. The second step is to analyze the recordings to isolate the synaptically activated glutamate transporter currents from all other components of the evoked response.
Next, the filter time course is estimated by analyzing the transporter currents recorded in the presence of TBOA by de involving the filter from the transport occurrence recorded in controlled conditions. The time course of glutamate clearance from astrocytic membranes is obtained. The results show that in the mouse hippocampus astrocytes require only a few milliseconds to remove synaptically released glutamate from the extracellular space base.
This approach provides a sensitive tool to detect changes in astrocytic uptake capacity in different brain regions during physiological and pathological conditions. The main advantage of this approach over other methods based, for example, on displacement analysis of rapidly dissociating glutamate receptor antagonists on fluorescent glutamate indicators or on diffusion reaction computer simulations, is that it provides a high temporal resolution, direct experimental readout of glutamate clearance from astrocytes. This method allows us to understand the spatial and temporal dynamics of synaptic transmission in the brain.
It can help us to interpret pathophysiological changes in glutamate, diffusion, and uptake that may occur in certain neurological disorders such as Alzheimer's disease. Begin this procedure by making five cuts on a dissected mouse brain with a scalpel. First, remove the olfactory bulbs and the frontal cortex.
Second, remove the cerebellum. Next, remove the left and right temporal lobes. Then separate the two brain hemispheres with amid sagittal cut.
Next, glue the lateral surface of each brain section to the cold vibram base plate. The dorsal side of the brain should be facing the Vibram blade and the ventral side of the brain should be in contact with the agar block away from the vibrato blade. Then secure the vibrato base plate to the dissecting chamber.
Set the slice thickness to 250 micrometers and adjust the width of the blade before slicing. Once the blade has passed through the cortex and hippocampus, use a scalpel to cut the cortex hippocampus from the midbrain. Subsequently, place a slice in the submersion chamber at 34 degrees Celsius after keeping the slices at 34 degrees Celsius for 30 minutes.
Let them cool down to room temperature for 30 minutes before using them for the electrophysiology recordings in this procedure, transfer a slice to the recording chamber. Visually inspect the slice under the dot illumination or I-R-D-I-C. Astrocytes can be identified by their small cell body and prominent nucleus for synaptic stimulation.
Place a bipolar stainless steel electrode about 100 micrometers away from the astrocyte that you plan to patch. Patch the astrocyte and break in the whole cell configuration by applying a very gentle suction. Then alternate single and paired stimuli every 10 to 20 seconds to isolate the facilitated portions of synaptically activated transport occurrence.
First average at least 20 sweeps obtained with paired stimulations in controlled conditions, and in the presence of 10 micromolar broad spectrum glutamate transporter antagonist. TBOA then average an identical number of sweeps obtained with single stimulations in control conditions and in 10 micromolar TBOA subtract the average trace obtained with single stimulations from the average trace obtained with paired stimulations. This step allows isolating the STO geometric and the sustained potassium current evoked by the second stimulus.
Next shift, the average trace obtained with single stimulations by a time interval that matches the PUL interval used to deliver paired pulses subtract the time shifted, average response obtained with single stimulations from the average response to the second stimulus obtained previously. This step isolates a facilitated portion of the transporter current evoked by the second stimulus. In most cases, this step removes the sustained potassium current entirely.
If this is the case, the isolation of the FSTC is complete and you can proceed with the deconvolution analysis and arrive the time course of glutamate clearance from astrocytes. In some cases, however, a small sustained potassium current is still present and further analysis is required. Measure the amplitude of the sustained potassium current remaining after performing.
The analysis described previously scale the amplitude of the mono exponential function to the amplitude of the residual sustained potassium current by setting the amplitude of the measured sustained potassium current as a in this equation. But the rise time constant represents the average value of rise estimated in separate experiments in which a sustained potassium current is isolated. Pharmacologically using a high saturating concentration of TBOA then subtract the resulting mono exponential function from the FSTC and sustained potassium current.
To complete the isolation of the FSTC. To isolate the flash activated transport occurrence. Average at least 20 sweeps obtained with the light path open in control conditions and in 10 micromolar TBOA then average the same number of sweeps obtained with the light path closed in control conditions, and in 10 micromolar TBOA subtract the average trace obtained with the light path blocked from the average trace obtained with the light path open.
This step allows removing the stimulus artifact and isolating the FTCs in this step fit the transport current either FSTC or FTC recorded in control conditions and in 10 micromolar TBOA and fit them with a multi exponential function, create an instantaneously rising function that decays mono exponentially according to this function and that best describes the decaying phase of the transporter Current recorded at 10 micromolar TBOA. Next devolve the mono exponential function from the fit of the transporter current recorded in 10 micromolar TBOA to derive the filter. Then devolve the filter from the fit of the transporter current in controlled conditions.
To obtain a quantitative estimate of the overall time course of glutamate clearance, calculate the OID of the waveform While attempting this procedure. It's important to confirm that the transport occurrence are recorded under experimental conditions where the filter operates in a linear regime and introduces only linear distortions of the transport. Current wave from these can be done by checking the manipulations that change the size of the transport current.
Do not change its stamp course. After watching this video, you should have a good understanding of how to use astrocytic recordings to extract information about glutamate, diffusion and uptake in the brain.
