만성 실리콘 프로브 및 녹음 풍부한 디딜 방 아 장치에 Hippocampal 장소 세포의 이식

The overall goal of this procedure is to implant chronic silicon probes in the mouse hippocampus, and to record place cell activity in mice that are running head-fixed in a cue-enriched treadmill. This method can help answer key questions about the underlying mechanisms of place cell activity. However, it could be applied to a wide range of other systems.

The main advantage of silicon probes is that hundreds of place cells can be monitored simultaneously with temporal resolution of a single action potential, and with information about cells'especial configuration. Visual demonstration is critical, as it involves a series of manual steps that are delicate and need to be practiced. Begin probe preparation by carefully detaching the probe from the shipment enclosure.

While working under a binocular microscope, use a flat alligator clip with rubber padding to grab the flex cable of the silicon probe and lift it from the enclosure while holding the enclosure with forceps. Fix the alligator clip to a manipulator, then use the manipulator to lay the silicon probe on the micro drive slider parallel to the direction of movement. Apply a drop of room temperature curing acrylic dental repair material to fix the probe to the slider.

Correct the probe position if it is shifted. Fix the connector of the probe to the connector holder with dental cement. Then, fix the micro drive and probe assemblage on the probe cleaning device, which is equipped with two small rotating foam sponges.

Adjust the gap using the manipulator. Soak the sponges with detergent. Then, monitor the cleaning process under a microscope while slowly and gently moving the probe up and down between the sponges.

Begin by installing an anesthetized mouse in a stereotaxic apparatus. Place a nose cone to administer 1.5 to 2%isoflurane. Apply eye ointment to the eyes.

Then, after shaving the scalp, clean the head of the animal with antiseptic. Inject buprenorphine under the scalp for analgesia. Then, check for a surgical plane of anesthesia by the absence of a reaction to a paw pinch.

Next, cut and remove part of the parietal skin of the mouse head using fine scissors to expose the skull at its edges. Use saline and a hemostatic sponge to clean and control bleeding during the surgery. After removing the periosteum using a scraper tool, find bregma and lambda and the coronal and sagittal suture reference points on the skull.

Adjust the head's angle along the sagittal access such that the bregma and lambda points are at the same height. Drill two holes in the skull for the reference and ground electrodes. The hole should be approximately one milometer caudal and one millimeter lateral to lambda.

Insert the ground and reference electrodes into the holes. Then, apply ultraviolet light bunting dentin activator on the skull and expose to UV light for 45 to 60 seconds. Next, apply a layer of dental cement to the edges of the skull.

Then, fix the head plate to a stereotaxic manipulator and position it above the skull. Slowly lower the head plate until it slightly touches the skull and apply dental cement at the junction with the skull. Let the dental cement cure for 15 minutes.

After removing the nose cone, fix a connector holder and a cap to the head plate. Put the mouse in its cage after giving a subcutaneous injection of analgesic. Anesthetize the mouse as before and place it in the stereotaxic frame.

After opening the skin and cleaning the skull with saline, measure the distance to the point of insertion and mark it. Drill the bone carefully until it becomes thin and transparent. Moisten and clean with saline while drilling.

Use precision forceps to carefully remove the thinned bone and the dura mater. Keep the brain surface wet with saline all the time. Fix the micro drive and silicon probe assemblage to the stereotaxic manipulator.

Bring the silicon probe to just above the craniotomy, then screw the silicon probe connector holder to the head plate. Connect the recording amplifier and electrodes. Shield the mouse with aluminum foil to protect from electromagnetic noise.

Start the recording system to monitor the electrical activity of the brain. Slowly insert the silicon probe into the brain using the micromanipulator. Continuously check the electrical signals, the manipulator traveled distance, and the shanks of the probe to make sure they are penetrating in the brain.

Unit activity is visible in the cortex while the white matter underneath is relatively silent. Unit activity reappears when the shanks touch the pyramidal layer of the hippocampus. From this point, retract the silicon probe 200 microns.

Cover the surface of the brain with a mixture of bone wax and mineral oil, then fix the micro drive to the head plate using dental cement. After allowing the dental cement to cure for 15 minutes, detach the micro drive from the stereotaxic manipulator and put the head cap back on. Return the mouse to the cage after administering analgesia and monitor for any sign of pain.

As the silicon probe needs to stabilize in the brain, allow the mouse to recover for an entire day before recording when the mouse runs on the treadmill. Install the anesthetized mouse in the stereotaxis apparatus by fixing the head plate. Remove the head cap.

Bring the stereotaxic manipulator just above the micro drive. Fix the micro drive to the manipulator. Fix the micro drive to the micro drive handle by tightening the dedicated screws, and remove the screw connecting the micro drive to the shell part.

Remove the screw connecting the shell and body of the micro drive. Under binocular microscope supervision, slowly pull up the micro drive and silicon probe assemblage with the stereotaxic manipulator, leaving the shell part behind. This is a color coded representation of place fields for a cell recorded on day one of treadmill running after probe implantation.

Some cells showed repeated place fields correlated with the identity of the cues. This is the spike auto correlogram for the cell, and this image shows the spike wave forms for the cell example recorded on day one. This image shows the color coded representation of place fields for a cell recorded on day five after repeated exposure to the belt.

Cells here showed a unique place field. Once mastered, each surgical procedure takes about one hour. Special care should be given to the craniotomy and the remitter removal to avoid any brain damage.

Always keep the tools and surgical area clean and aseptic, and monitor the animal wellbeing during and after the surgeries. After watching this video, you should have a good understanding of how to implant chronic silicon probes in the hippocampus and how to record place cell activity.