This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.
The overall goal of this procedure is to provide a method to ensure simplified means of carbogenation of small-volume recycling ACSF and to achieve long-term recordings of field potentials using a recording chamber mounted on a fluorescence microscope. This method can help us answer the key questions in the field of neuroplasticities relating to the underlying lignin pathways that regulates the synaptic transmission and formation of memory in a cellular mode of learning and memories. The main advantage of this technique is that it helps to stabilize the oxygen level of a small bio reservoir to enhance the cost efficiency of the experiments using specific antagonists or agonists.
The preparation of the acute hippocampus slices is demonstrated by the PhD students, Xia and Weiguang of our laboratory. To begin this procedure, use a cold surgical blade to dissect a piece of the dorsal cortex at a 70 degree angle along the dorsal edge of each hemisphere. With a cold dental cement spatula, transfer each hemisphere to a piece of filter paper to briefly dry the surface.
Mount the two hemispheres with freshly cut surfaces on the ice cold slicing platform using fast-acting adhesive glue so the caudal end of the hemispheres face the razor blade. Next, place the slicing platform into the cooling chamber of a vibratome. Fill the chamber with the slicing buffer at two to four degrees Celsius and carbogenate it.
After that, cut 350 micrometer slices using adequate vibrotome settings. Isolate the hippocampal formation from the subiculum and the entorhinal cortices using two injection cannulas as scissors. Remove the bubbles from the slice-holding mesh of the previously prepared incubation chamber using a pasteur pipette.
Then, transfer the slices that have some transparency at the stratum pyramidal from the slicing platform on the mesh of the incubation chamber with a large-mouth pasteur pipette. In this step, add a three-way tube connector to the outflow of the recycling system. Connect the middle arm of the three-way tube connector to a carbogen supply tube and regulate the flow rate with an airflow meter.
After that, add a second three-way tube connector to the inflow of the recycling system. Connect the middle arm to the tube facing the inline heater, the upper arm to a thin silicone tube, and the lower arm to the inflow tube from the reservoir. Then, place a tube squeezer on a thin silicone tube at a position along the tube where the pulsation of the ACSF in the slice chamber generated by the peristolic pump is at its minimum.
Now, presoak a small piece of lens-cleaning paper and a nylon mesh fixed on a U-shaped platinum wire in the submersion slice chamber for a few minutes. After that, remove the U-shaped platinum wire. Switch off the recycling and place a slice on the lens-cleaning paper.
Then, immediately place the slice-holding mesh on top of the slice. Switch on the pump and let the slice equilibrate for 30 minutes without dipping the objective into ACSF. In the meantime, fill the recording electrode with ACSF and mount it into the pipette holder.
Check the insulation of the metal stimulation electrode by placing it into sodium bicarbonate solution. Connect the electrode to the negative pole of a DC voltage generator and observe the formation of bubbles at the tip under a dissection microscope. Next, place the tested apoxy-insulated tungsten stimulation electrode in the manipulator holder and the reference wire in the slice chamber.
Add a second reference electrode to the chamber and connect it to the reference socket of the head stage of the recording electrode. In the amplifier control software, click on the zero clamp mode box, then double click the output gain list box. Choose a gain of 100, double click the high pass filter box vessel, choose three kilohertz, and double click the low pass filter list box AC to choose 0.1 hertz.
In the recording software, click on acquire, open protocol. Choose a protocol that has the settings allowing for episodic stimulations and the digitalization of amplified potentials at 10 to 20 kilohertz for 50 to 100 milliseconds and that automatically triggers a stimulus isolator 10 milliseconds after the start of an episodic recording. Place the stimulation and recording electrodes in line and parallel to the stratum pyramidal.
Subsequently, click acquire, edit protocol, and choose the trigger tab in the popup window. Click on the trigger source box and choose space bar as the trigger source. Then, click the okay button and click the record button to start the acquisition.
In the stimulus isolator software, click on the voltage control box and enter zero. Click the download button. Following that, click on the recording software window and press the space bar.
Repeat this cycle by sequentially entering values from 1000 to 8000. Correlate the stimulation strength with FEPSP slope values and determine the stimulation strength required to get 40%of the FEPSP slope maximum. Click the voltage control box and enter the determined value.
Then, click the download button. In the recording software, click the stop button and then the acquire menu. Click on edit protocol and choose the trigger tab in the popup window.
Afterward, click on the trigger source box and choose the internal timer as the trigger source. Click the okay button, followed by the record button that starts the automatic recording of field potentials every 60 seconds. Click the stop button after 30 to 60 minutes.
In the recording software, click acquire, open protocol, and choose the same protocol as the baseline recording. Click the okay button and then click record. Keep automatically running the field potential recordings for two to four hours.
When it is finished, click the stop button to terminate the recording. This figure shows the representative recordings of activity-dependent potentiation of synaptic transmission using outflow carbogenation and reservoir carbogenation with the recycling of a small ACSF reservoir. The potentiation was induced after time-point zero by three times 100 hertz per second trains.
This figure shows the outflow carbogenation with recycling of a small ACSF reservoir improved the LTD induction in comparison to experiments with reservoir carbogenation only. Once mastered, the slice preparation can be done in five to 10 minutes and a stable field potential recording can also be achieved for many hours if it is performed properly. While attempting this procedure, it is important to adjust your carbogenation level, perfusion speed, the position of the electrodes, and the stimulation stress of your specific equivalence.
Following this procedure, other method like the patch clamp, the forensics microscopy can be performed in order to answer the additional questions like the virilization of sap cellular localization of ravin probings in response to the induction of activity-dependent synaptic plasticity. After its development, this technique paves the way for researchers in the field of neuroplasticity to explore activity-dependent synaptic plasticity as a cellular mode of learning and memory in in vitro hippocampal slices of well tab and transgenic mouse genes. After watching this video, you should have a good understanding of how to perform acute slice preparation from the temporal hippocampus and how to obtain stable conditions for long-term recording of synaptic transmissions using a small volume of recycled ACSF.
Don't forget that working with animals and chemical compounds can be hazardous and precautions such as wearing gloves and the proper disinfection should always be taken while performing this procedure.
