Generation of Local CA1 γ Oscillations by Tetanic Stimulation

The overall goal of this procedure is to prepare brain slices for recording extracellular gamma oscillations as a model of neuronal excitability. This is accomplished by first removing the brain from the skull. The second step is to rapidly cool the brain in an ice of A CSF and cut it into 450 micron slices.

Next, transfer a slice to the recording chamber and place the stimulating electrode in the stratum and a recording electrode in the ca one stratum parama doole. The final step is to elicit gamma oscillations and assess the impact of drugs on neuronal excitability. Ultimately, the characterization of hippocampal gamma oscillations is used to show the effects of drugs on network function or to understand the mechanisms of disease genesis in neurogenetic disease.

This method can be used to provide insights into disease mechanisms where increases in neuronal network excitability occur such as epilepsy, autism, and Alzheimer's, and can also be used to investigate the method of action for drugs. To begin this procedure, repair a holding chamber by inserting a raised nylon mesh in a 250 milliliter beaker. Next, fill the beaker with a CSF to cover the mesh by approximately two centimeters and bubble it with carbogen at room temperature.

Ensure that the bubbles do not directly disrupt the slice holding area. Then place the dissecting instruments, including a large pair of scissors, a small pair of scissors, a small and a large micros spatula, and a small and a large pair of forceps on ice. Place the cooled Vibram tissue cutting block onto an aluminum foil on ice.

After that repair, two pieces of six centimeter filter paper, a single edge razor blade, and a 25 milliliter beaker filled with a cutting solution slurry. Next, fill a second container with ice for brain dissection. Lay a piece of tissue paper on the ice and place a 12 centimeter culture dish on top.

Subsequently fill the culture dish with cutting solution ice slurry and bubble it with carbogen. In this procedure, heal the skin and connective tissue of the head towards the nose using the small scissors, cut the connective tissue to reveal the underlying skull. Then remove the muscles overlying the dorsal aspect of the skull and neck.

To remove the brain, secure the front of the skull with a pair of large forceps. Then make two lateral cuts through the bone on both sides of the rem and magnum. After that, make another cut between the eyes just anterior of the bgma.

Carefully cut along the sagittal suture and reflect the cut skull sections to reveal the brain. Next, use the small spatula to scoop out the brain and being careful to sever the cranial nerves. Place the brain in a cultured dish.

Then transfer the brain to a 25 milliliter beaker filled with cutting solution slurry. To prepare the brain hemisphere for slicing, place a piece of six centimeter filter paper in the bottom of a cultured dish. Then fill it with fresh cutting solution slurry and bubble with car.

In subsequently, place the brain ventral side down onto the filter paper. Next, remove the cerebellum with a new and cleaned single edge razor blade. Make a cut along the midline to separate the two hemispheres.

To prepare the Vibram tissue block, dry the surface of the chilled Vibram tissue cutting platform. Place a drop of Sano ACRL glue in the middle and spread it evenly to the approximate size of the brain. Use a small spatula to transfer one hemisphere onto a large micros spatula and position it so the medial side of the brain faces down.

Absorb the solution of the brain with a piece of filter paper as much as possible. Then slide the brain off the larger spatula and use the smaller spatula to guide it onto the glue. After that, secure the cutting platform to the Vibram chamber.

Build the chamber with cutting solution slurry to completely cover the brain and bubble the solution with carbogen. Then rotate the cutting platform so that the ventral side of the brain is facing the blade. Slice the brain into sections of 450 micrometers thick, cut through the brain from the ventral surface to the dorsal surface to produce whole brain sagittal slices for electrophysiological recordings.

If the base of the slice lifts, use a bent 27 gauge needle to gently press the slice down. Then use the pipette to transfer the slice to the holding chamber. Typically three to four hippo Campbell slices can be cut from each hemisphere to mount the slice In the recording chamber.

Place a brain slice into a submerged recording chamber perfused with a CSF flowing at one to two milliliters per minute and heated to 32 degrees celsius. The A CSF use for recordings differs from that in the holding chamber as the magnesium concentration is increased from two millimolar to four millimolar. Secure the slice by placing a semicircular stainless steel weight on it with its strands parallel to hippocampal ca one.

After that, increase the speed of perfusion to eight to 10 milliliters per minute at 32 degrees Celsius. Using a dissecting microscope, move the stimulating electrode to the middle of the stratum orients. Then move the recording electrode to the parameter layer as close to the stimulating electrode as possible.

Subsequently, lower both the stimulating and recording electrodes 50 to 100 micrometers into the slice. In order to generate the field EPSP of one millivolt in response to the 120 to 150 micro test pulse to generate gamma oscillations, stimulate the tissue with a train of 20 times 0.1 millisecond pulses delivered at 200 hertz. This Titanic stimulus can yield reproducible responses when delivered every five minutes.

This schematic shows the locations of the stimulating electrode in the stratum orients and the recording electrode in the stratum para of the ca one. And here's an example of the gamma oscillations induced by Titanic stimulation. This figure shows the pharmacological blockade of AMPA receptors with 20 micromolar CN QX gabaa receptors with 20 micromolar by Coline IH current with 20 micromolar peridium, chloride, and T type calcium channels.

With 100 micromolar nickel, each agent was able to independently reduce the number of spikes generated. This preparation is ideal for determining the network scale action of drugs. Tine is a clinically used anti-epileptic medication that opens potassium channels and reduces membrane excitability bath.

Application of tine produced a dose dependent reduction in the number of spikes in the duration of oscillation. Once mastered the preparation of brain slices and the dissection for electrophysiological recordings can be performed in about 10 minutes following this procedure. The addition of other methods such as whole cell patch clamp recordings can be added to understand the role of individual neurons within a network in diseases as well as following application of drugs.