Neurodegenerative and mental health disorders result from changes in synaptic communication. Multifactorial events change synaptic receptors in ways that we do not completely understand. Microtransplantation of synaptic membranes provides insight into these alterations.
The main advantage of this technique is the possibility to record the activity of native brain receptors working in human brain, and therefore, represent the diseases we are studying. Synaptic receptors are major targets for medicine regulating the electrical activity of the brain, but understanding how these receptors change, better therapies restoring their functionality can be developed. This method has been successfully applied to different species, from insects, fish, mammals, but can also be applied to membrane receptors outside of the brain, for example, muscles and tumors.
Possibilities are wide. Trimming injection needles is tricky. Too small can prevent protein injection, and two big can kill the cell.
It helps use a consistent measurement, like a razor blade's edge. Begin by sonicating the selected samples prepared from the human brain region of interest three times in a bath with floating wet ice. Use five second cycles each time, and wait for one minute on wet ice in between cycles.
Place one microliter of the sample onto the thermoplastic, and make an indentation on the surface, if needed, before sample placement to prevent movement of the sample. Use the knobs of the manipulator holding the nanoinjector to position the needle, and move it toward the sample to be filled. Once the needle tip is in the sample, press and hold the fill button until enough sample is taken up.
About 0.5 microliter sample is required for 10 oocytes. Use the knobs to move the nanoinjector's needle back into the highest position. Penetrate the oocyte just underneath the surface with a needle, no deeper, and use the foot pedal to inject the sample.
Wait for about two to three seconds. After successful injection, the cell will expand. Use the knobs on the manipulator to exit the cell.
Move the Petri dish so that the next oocyte is aligned with the needle. Repeat the process until the desired number of oocytes in the dish have been injected, then retract the nanoinjector to its original position and remove the needle. Ensure that one well of oocytes is left non-injected to be used as a control.
Turn on all the equipment used for the recording of ion currents, then turn on the desktop computer, and log into WinEDR version 3.9.1. Ensure that the program is running, and the record is set to Disc. After that, set the recording duration, and clear out the Simulator option.
For the voltage electrode section, turn off the negative capacity compensation. Set the gain selector switch to 10 for the bath electrode section, then set the three position toggle switch, which selects the gain multiplier, to x1. The LED lights will indicate the gain multiplier selection.
Turn off the clamp mode selector, put the DC gain control to in, and set the gain control to around half of the full bandwidth open-loop. Set commands to 40 millivolts, the hold controls to negative, and the scale multiplier to x2. For the current electrode, use the VE offset to establish a zero reference, before impaling the oocyte.
Next, open the WinEDR version 3.9.1 software, go to the top menu, and select File, followed by New. Create your own folder and save the file. In the main menu, select Record, followed by Record to Disk.
Place the agar bridges in the respective circular holes behind the recording chamber, connecting the wells for the electrodes used as a ground reference in the recording chamber. Add 1x Ringer's Working Solution to the corresponding perfusion valve. Open the valve until the recording chamber is filled with Ringer's Solution.
Using the right and left manipulators holding the electrodes, guide the electrode needles into the recording chamber that is filled with Ringer's Solution. Check the resistance of each electrode by zeroing out the VM and VE offset knobs, and then pressing the electrode test buttons. The resistance should be between 0.5 to three megaohm.
If the resistance is out of the range, replace it using new microelectrodes. Next, fill the recording chamber with fresh Ringer's Solution. Use a glass pipette to place a non-injected or a micro-transplanted oocyte in the center of the recording chamber.
Ensure that the oocyte is clearly visible under the microscope with the animal side up. Guide the electrode into the Ringer's Solution until they touch the oocyte membrane. Gently pierce the oocyte membrane on the animal side with both the electrodes, and record the resting membrane potential.
Turn on the Ringer's Solution flow. Using the Oocyte Clamp Amplifier, change the mode to voltage clamp slow, and set the holding voltage to 80 millivolts. The current on the monitor should be negative, usually between 0 and 0.4 microamperes.
Go to File, New, and then save the recording on the software to create a new file, and save the recording. Press Record followed by Record to Disc to start the recording. Add relevant information to the file using the Mark Chart dialogue.
Apply agonists for chemical stimulation by opening the valves, and perfusing them into the recording chamber for 15 seconds. Once the recording is completed, turn the voltage clamp to the off position, and turn the Ringer's Solution valve off. Remove the oocyte, and discard following institutional policy for human brain samples, then stop the recording and save the file.
Save a copy of the recordings to a USB drive. From there, open the desired file to be analyzed. To measure the AMPA maximum response, and the GABA A peak response, first, establish a 0 level or baseline by finding the red line, then drag it to the current generated by the Ringer application or baseline.
Once the 0 level is established, determine the response measurements by dragging the green vertical readout cursor to the desired part of the graph tracer. Ion currents from the oocytes injected with the membranes from the human synaptic receptors were recorded. AMPA receptors were activated with 100 micromolar kainate, and GABA A receptors with one millimolar GABA.
Co-injection of the filtered membranes from the electric organ of torpedo, rich in acetylcholine receptors, and cDNA coding for the GABA row one receptor into the animal pole produced mainly two groups of oocytes, based on their responses. One group of oocytes had large responses to acetylcholine, but no responses to one micromolar GABA. Oocytes in another group had null or low responses to acetylcholine, but large responses to GABA.
The graphical image shows the mean plus minus standard error of the mean of peak current in group one and group two. One oocyte in group two had large GABA and acetylcholine responses, suggesting the rupture of the nucleus. Consequently, the distribution of the responses was skewed to low values, as noted by the difference between the mean and the median of the distribution of membrane current.
One of the causes of the low response oocytes is that membranes are being injected, and trapped into the nucleus of the oocyte. GABA and kainate responses of oocytes injected into the animal or vegetal poles with unfiltered membranes obtained from a non-AD brain, and an AD brain are shown here. Oocytes injected near the animal pole without targeting the nucleus gave larger responses than oocytes injected into the vegetal pole, thus allowing the study of tissue samples with very low numbers of receptors.
While attempting this procedure, always monitor your Ringer level. It's always during the important recordings that you'll run out, and then it'll use your result, and death of the oocyte, and loss of very valuable data. Following this procedure, we can perform Western loads to validate the presence of the synaptic receptors, or proteins of interest.
It allows to measure global excitation to inhibition synaptic ratios in different brain regions, and different brain disorders, allowing to make direct test on theoretical prediction about the causes of disease.
