The overall goal of this procedure is to illustrate a general protocol for slice fast scan cyclic V Telemetry. This is accomplished by first preparing the brain tissue for coronal slicing. Then electrodes are placed onto the discrete brain region of interest.
The next step is to deliver voltage as a triangular waveform through the electrode to detect dopamine. Finally, a pharmacological agent of interest is applied to the brain slice and its effect on presynaptic dopamine dynamics is observed. Ultimately, this protocol shows changes in presynaptic dopamine dynamics, such as stimulated dopamine release and dopamine uptake rates through fast scan cyclic vol Telemetry.
Though this method can provide insight into presynaptic dopamine dynamics, it can also be applied to other systems, including animal and neurotransmitters, such as rats and fruit flies, and serotonin and norepinephrine respectably demonstrating the procedure will be Francis Mena, MEA Khalid, and Erin oo, the graduate students from my laboratory To begin this procedure, according to the accompanied manuscript, prepare three types of artificial cerebral spinal fluid solutions, sucrose, A-C-S-F-A-C-S-F solution for Volta metric recordings and modified A CSF solution for electrode calibration. Next, transfer 10 milliliters of sucrose A CSF into a small beaker and place it in an ice bucket. Then place the instant Loctite adhesive in an ice bucket.
After that, prepare a razor blade and the necessary tools required for dissection, such as forceps, spatula, and scissors by wiping them with an alcohol pad. Then sacrifice a mouse by decapitation and quickly remove its brain. Place the brain in a beaker of ice cold sucrose, A CSF for approximately 10 minutes.
In the meantime, prepare the VIBRAM for slicing. Place some crushed ice in the specimen bath. Then place the specimen chamber in the Vibram and fix it firmly.
Add more ice around the specimen chamber to fill the gaps and make sure that no ice gets into the chamber. Afterward, place a cleaned razor blade into the blade holder. Then fill the specimen chamber with ice cold sucrose A CSF.
Subsequently, pour some cold sucrose A CSF on a piece of paper towel on an upturn Petri dish. Transfer the freshly dissected mouse brain onto it with a pair of forceps. Then remove the cerebellum by cutting along the medial lateral axis with a razor blade.
This creates a flat base that can be affixed to the vibram stage. Next, add a drop of the Loctite adhesive on the specimen stage. Affix the flat end of the prepared brain on the stage immediately, and keep it as upright as possible.
Then place the stage in the specimen chamber and tighten the screw. Ensure that the brain is completely immersed in the sucrose A CSF in the chamber. After that, adjust the stage with the control so that the razor blade lines up with the top of the brain.
Optimal parameters for the vibrator are obtained by setting the frequency and speed to low. Next, set the slice thickness to 400 micrometers. The first few slices usually do not contain the striatum.
Repeat the slicing until the slices containing the striatum are obtained. Once the stri AAL region is reached, use a paintbrush to lift the slice and place it in a beaker with oxygenated room temperature. A CSF.
Typically, one can obtain three to four slices containing thesal complex in which the caudate putamen and the nucleus accumbens are included. Allow the slices to acclimate in A CSF bubbling with 95%O2 5%CO2 at room temperature for at least one hour before proceeding with the experiment. To begin this procedure, connect the tubing to the submersion recording chamber.
Then pump the oxygenated room temperature A CSF at the rate of one milliliter per minute. Set the temperature controller to 32 degrees Celsius after A CSF has filled the custom-built slice holder. Remove any air bubbles under the mesh using a needle syringe.
Create negative pressure in the outflow tubing with a syringe in order to start the flow To control the buffer overflow. Place a Kim wipe on the edge of the slice holder to act as a wick. Then transfer a slice to the slice holder in the recording chamber, which is continuously perfused with oxygenated A CSF at room temperature.
Submerge the silver silver chloride reference electrode in the slice holder and connect it to the head stage with an alligator clip. Attach the carbon fiber micro electrode to the micro manipulator and connect it to the head stage with an alligator clip. Next, lower the tungsten stimulating electrode and the carbon fiber micro electrode to the surface of the sal brain slice.
The stimulating electrode will be used to deliver either a single pulse or multiple pulses of electric stimulations to the slice to evoke neurotransmitter release to measure the electrically evoked dopamine using fast scan cyclic vol telemetry or FSCV, apply voltage in a triangular waveform to the electrode. Hold the potential of the carbon fiber micro electrode at negative 0.4 volts. Ramp it to a positive limit of 1.2 volts.
Then bring it back down to negative 0.4 volts at a scan rate of 400 volts per second. Stimulate the slice every five minutes and make the vol metric measurement of the resulting dopamine FLX for 15 seconds each time after at least three. Stable electrically stimulated dopamine release recordings defined as less than 10%difference between peak heights.
Switch to perfusing the slice with a CSF containing pharmacological agent of interest at a flow rate of one milliliter per minute for 30 minutes to obtain maximal effect similar to baseline recording record. The electrically stimulated dopamine release every five minutes during the pharmacological perfusion. Figure A is an example of the electrically evoked dopamine release measurement using FSCV, following single pulse stimulation in the caudate putamen.
Figure B shows a three dimensional pseudo color plot that combines the information from both the current versus time traces and the cyclic volt Graham. Similarly, figure A and C show the dopamine release evoked by a single electrical stimulation pulse in the nucleus accumbens core and shell respectively, and the three dimensional pseudo color plots for the nucleus accumbens core and shell are indicated in figure B and D respectively. Shown here are the representative traces after the slice had been treated with a pharmacological agent for 30 minutes, followed by single pulse stimulations to evoke dopamine release in the caudate putamen.
The agents used in the experiments are quinol, methamphetamine, and brain derived neurotrophic factor. After watching this video, you should have a good understanding of how to prepare mouse coronal brain slices for obtaining and measuring presynaptic dopamine dynamics using fast and likely will telemetry.
