The overall goal of this procedure is to be able to record very stable and reproducible long-term potentiation in acute hippocampal slices. This is accomplished by first extracting the mouse brain and dissecting the hippocampus in cold A CSF under the microscope. The second step is to cut transverse hippocampal slices with a tissue chopper.
Next, the slice is placed in the interface recording chamber, which will be perfused with oxygenated artificial cerebral spinal fluid. The final step is to place the electrodes and record the synaptic activity in the CA one region of the hippocampus. Ultimately, a protocol of high frequency stimulation is used to show the induction of a very stable long-term potentiation.
Through this method can provide insight into the mechanism underlying synaptic plasticity. It can also be applied to other system such as animal models of neurodegenerative or nervous system, or to immune disease. Generally, individuals new to this method will struggle because all the manipulations need great skill and lot of practice.
Begin this procedure by rinsing the perfusion circuit with dis distilled water for a minimum of 20 minutes. Then turn on the heating systems. Start the carbogen bubbling in the circuit.
The carbogen is delivered to the water bath below the recording chamber through air diffusers. Next, control the flow rate in the water bath by a flow meter Afterward, drain the circuit and fill it up with filtered A CSF tapping to remove the bubbles. Then place the ring, which will support the slice in the holding chamber.
Carefully remove all the air bubbles from the circuit. Then adjust the A CSF level in the recording chamber with the screw of the suction needle, the speed of the inlet pump is adjusted to one milliliter per minute, and the outlet pump is adjusted to five milliliters per minute. Before the dissection, prepare all the surgical instruments.
To remove the brain of a sacrificed mouse. Hold its head with your index finger and thumb, and make an incision with the dissecting scissors along the middle of the top of the head, starting at the edge of the guillotine, cut and running roly to the frontal bone. Next, cut through the cutaneous muscle on each side of the head to fully expose the skull plates.
Then remove the muscles at the coddle side of the head. Subsequently cut the temporalis muscle on each side along the temporalis plate. Then cut the frontal plates in the middle transversely.
Now make a little cut on the occipital bone between the two plates. Cut at the coddle base of the occipital plates at each side. Then with the spring scissors, cut along the sagittal suture.
Finally, remove the skull by spreading the halves away from each other with forceps. Now with the scalpel cut just before the cerebellum and just after the olfactory bulb, transversely then transfer the extracted brain to the dissecting dish with cold A CSF. Once the brain is emerged in the dissecting dish, sever the two hemispheres from each other with a scalpel in inserted in the middle under a binocular surgical microscope, carefully spread the structure of one hemisphere to reveal the lateral ventricle.
Remove the brainstem and cephalon by putting one spatula on the frontal cortex and the other spatula on the cephalon. Be careful not to touch the hippocampus with the spatula and not to stretch it during tissue sectioning. After that, sever the fornix.
Then gently push the hippocampus out of the cortex by inserting a spatula in the ventricle. When the hippocampus is extracted, remove excess cortical tissue and remaining blood vessels with a wide mouth plastic pasture pipette. Transfer the hippocampus in a spoon with its alvi surface upwards to the platform of the chopper.
Remove excess fluid from the spoon with a standard plastic pasture pipette. After that, tip the spoon up vertically and nearly touching the filter paper on the chopper. Drop the hippocampus off by rapidly touching the filter paper.
Then withdraw the spoon. Next, orientate the hippocampus and slice it. Transversely to 400 microns with the chopper.
Perform the procedure as quickly as possible. Subsequently, remove the filter paper with the hippocampal slices and wrap it around the metallic cylinder in order to spread the slices a little. Then free the slices with sprays of A CSF using a standard plastic pastoral pipette and collect them in a Petri dish filled with cold.
A CSF. Transfer the selected slice to the recording chamber with a standard plastic past pipette. Allow the slice to recover from the dissection trauma in the recording interface chamber at 28 degrees Celsius.
If the slice sinks lower the A CSF level to the slice level, then raise the A CSF level again to make the slice float. After that, orient the slice to the position that would facilitate electrodes positioning in the CA one region. Stop lowering the A CSF level when it is at the interface.
The meniscus of the media around the slice is indicative of a sufficient level of A CSF. The mesh must be saturated with media but not completely submerged. Then keep the chamber covered with filter papers over the perforated lid.
Let the slice rest for at least one hour and 30 minutes at 28 degrees Celsius before recording. Here is a sketch which shows two independent synaptic inputs as one and S two to the same neuronal population. S one pathway was used to induce LTP while S two pathway acted as a control.
These are the sample F-E-P-S-P traces recorded just before the LTP induction as indicated by the red traces and one hour after LDP induction indicated by the blue traces. Here are the F EPSPs from a perfectly healthy slice, and here are the F EPSPs from a slice presenting a high level of excitability when the slices are not perfectly healthy. Polys synaptic responses are observed, and F-E-P-S-P slope potentiation is reduced.
Here is a comparison of the time courses of the F-E-P-S-P slope after LTP induced by a single train of high frequency stimulation recorded in 2005, 2010, and 2011 in our laboratory. First experiments were recorded in 2005 indicated by filled circles. Subsequent recordings indicated by filled squares benefited from an improved dissection procedure.
Current results arise from the optimization of interface oxygenation and temperature control, along with electrode standardization as indicated by open circles. Here we show that increasing oxygen flow rate in the water bath beneath the recording chamber from 0.15 to 0.25 liters per minute as indicated by the blue curve, induces a decrease in F-E-P-S-P slope by 20%increasing the temperature from 28 to 29 degrees Celsius. As indicated, the green curve induces a more than 50%increase in F-E-P-S-P slope.
While attempting this procedure, it's important to remember that every step is crucial and errors in the protocol can lead to different results. After watching this video, you will have a good understanding of how to obtain very stable long-term potentiation. Recording in acute hippocampal slices.
