The overall goal of this procedure is to quantify the volume fraction and tortuosity of the extracellular space in brain tissue. This method answers key questions regarding the relationship between the extracellular space and the physiology and disease. The main advantage of this technique is that it allows the real-time characterization of two important parameters of the extracellular space.
Generally, individuals new to this method will struggle because the iontophoretic microelectrode exhibits a tendency to change its properties during experiments and frequently requires troubleshooting during experiments. Visual demonstration of this method is critical as the precise manipulation of an ion-selective microelectrode, an iontophoretic electrode over multiple steps is required to obtain a successful recording. To begin this procedure, run ACSF containing tetramethylammonium, or TMA, through the submersion chamber at two milliliters per minute.
Set the temperature controller to a desired temperature and bubble the ACSF with 95%oxygen, 5%carbon dioxide for the duration of the experiment. Next, mount the ground electrode on a holder and submerge its tip into the ACSF running through the submersion chamber. Then, fill the porous cup with 0.3%agarose containing TMA prepared previously and place it in the submersion chamber.
Now, secure an iontophoresis microelectrode to the pipette holder of one micromanipulator. Connect the iontophoresis microelectrode wire to the head stage of the iontophoresis unit. Next, secure a calibrated ISM to the second micromanipulator and connect the ISM to its head stages.
Afterwards, turn on the computer containing the Wanda and Walter programs. In the Wanda GUI, click Calibrate. Then, in the Calibration box, fill in the voltages measured during the ISM calibration and click Fit Data.
Following that, click Accept in the Calibrate box to automatically transfer the slope and interference generated to the main GUI. On the left side of the GUI, ensure that all the experimental parameters are set in the corresponding entries. Next, place a temperature probe in the agar cup.
Record the measured temperature in the Temperature entry in the Measuring Electrode box of the GUI. Then, lower the microelectrodes at least 1, 000 micrometers deep into the agarose and center them in the cup. Visualize them under the microscope using a 10X objective.
On the two-channel amplifier, manually move the ISM channel connector to the voltage subtraction output to set the subtraction on between the reference and ISM channels. Center the tips on each other in all three directional axes. Next, zero the relative positions of both microelectrodes on the micromanipulator control boxes and ensure that the microelectrodes are centered accurately and precisely.
Then, move the ISM 120 micrometers away from the iontophoresis microelectrode in one axis. Subsequently, start a recording by clicking on Acquire in the GUI and allow the program to run for a full recording. In this procedure, open the Walter program on the computer.
In the Zero Records From menu, click the Wanda Voltoro button to read the records generated by Wanda. In the next popup window, select one or more records to be read and click Open. In the One Write Excel menu, click Sheet 1.3 and follow prompts until the records are automatically graphed.
To begin the fitting procedure, in the Two Options menu, click on the select rec button. Then, in the figure two popup window, use the mouse to move the cross hairs over the first record to be processed and press either mouse button to choose the record. After that, click on fit curve in the menu and use at least 20 iterations of fitting to obtain an accurate fit of the data.
Next, select all to fit all data points and select continue. The program will fit the displayed curve. Then, select the option to write the result to the appropriate spreadsheet program by clicking Excel in the seven results menu.
Name the Excel file. Note and record the data generated which will be used to determine the functionality of the iontophoresis microelectrode. If the iontophoresis microelectrode is deemed suitable for the experiment, record the average transport number from all trials in the TransportNum end field in the Wanda GUI.
In this procedure, place a 400-micrometer-thick brain slice in the recording chamber ensuring that it is fully submerged in the flowing ACSF. Next, move both the iontophoresis microelectrode and the ISM above the field of interest on the brain slice. Submerge both electrodes in the flowing ACSF, but above the slice.
Then, offset the voltage for both the reference and the ion-sensing channels to zero millivolts. Wait for the voltage in both channels to stabilize. On the chart recorder, mark the voltage measured on the ion-sensing channel of the ISM.
Next, place the ISM and iontophoresis microelectrodes 200 micrometers deep in the slice and 120 micrometers away from each other. Calculate the voltage difference between the TMA signals measured in the flowing ACSF and in the brain and input this value into the Baseline Volt Millivolt field in the Measuring Electrode box of the Wanda GUI. On the left side of the GUI, ensure that all the experimental parameters are correctly entered.
Start the recording by clicking Acquire and allow it to take a full recording. After moving both microelectrodes out of the slice, use the chart recorder to determine any change between the voltage measured now and its measurement from before. To analyze the recordings from the brain, repeat the procedures for agarose data analysis using the Walter program.
Write the data onto the spreadsheet program by clicking Excel in the Walter menu. Then, record the volume fraction of the brain ECS, tortuosity of the brain ECS and the non-specific clearance. To check the transport number, take new recordings in agarose and repeat the procedures in the Walter program to obtain the transport number.
Inspect the spreadsheet and check if the transport number has changed by more than 10%from the transport number obtained prior to the brain measurements. If so, the data obtained with the iontophoretic microelectrode are not reliable. The transport number measured in this step must closely match that obtained prior to brain measurements, otherwise the volume fraction cannot be considered accurate.
Use newly obtained ISM calibration data as the input in the Wanda Calibrate box and check that the slope value differs by less than 10%from the previous calibration. Shown here is the representative data from a single trial obtained in agar demonstrating the concentration curve of TMA. And this is a fitted curve obtained from data processing in Walter.
The overlap of the data in the fitted curve demonstrates that the curve fitting done by Walter accurately models diffusion in this trial. Here is the table of agar measurements before experimentation in the brain. Here is a summary of measurements taken in a mouse brain slice under the control and experimental conditions.
Alpha decreased during a hypoosmolar challenge while tortuosity increased as expected. Alpha overshot the control values in the recovery conditions. And here is the table of agra measurements after experimentation in the brain.
The average transport numbers before and after the experiment were within 10%of each other which indicate that the values recorded in the brain were reliable. Once mastered, this procedure can be performed in 14 hours over two days. On day one, ISMs are fabricated and calibrated.
On day two, ISMs are re-calibrated and real-time iontophoresis is performed. While attempting this procedure, it's important to first be precise in the spacing of your electrodes. Secondly, it's important to only accept an experiment if the transport number of the iontophoretic microelectrode remains stable throughout the course of the experiment.
Following this procedure, which characterizes the diffusion of small cations in the extracellular space, you can go onto to perform other techniques like the integrative optical imaging method that allows you to answer the question of how large micromolecules move in the extracellular space. After watching this video, you should have a good understanding of how to perform the real-time iontophoresis method to quantify brain extracellular space volume and tortuosity in living brain in health and disease. Thank you for watching and good luck with your experiments.
