This work was supported by the Korea Institute of Science and Technology Institutional Program (Projects No. 2E26190 and 2E26170) and the Human Frontier Science Program (RGY0089/2012).

All methods described have been approved by the Animal Care and Use Committee of the Korea Institute of Science and Technology.
1. Preparing the Micro-drive and Electrode
<ol>
	<li>Assembling the micro-drive.
	<ol>
		<li>Print the parts of the micro-drive (slider, body, and shell)14 using a high-resolution 3D printer. Make sure the parts have no defects.</li>
		<li>Fix the slider into the micro-drive body with a screw (size 000-120x1/4).</li>
		<li>So...

<ol>
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Chronic recording of neuronal activity is critical for understanding neural processes such as hippocampal place fields. Our approach to perform chronic silicon probe implantantation distinguishes itself from other methods7,18,19,20 by the fact that it is relatively simple to recover the electrode package at the end of the experiment. While place cells are typically studied in freely-moving cond...

Silicon probes present several advantages for electrophysiological recordings, including the fact that they are designed with sharp profiles minimizing tissue damage and that they present a precise layout of densely packed recording sites1,2,3,4. They are used to study various systems in different species, including humans3,5,6, with diverse approaches1,7. Yet, their recurrent use is still relatively limited because of their cost, fragility, and the fact that convenient methods for chronic experiments are lacking8. Recent advances in 3D printing technology have made possible the custom designing of devices such as micro-drives and head-plates to allow an easier handling of these delicate electrodes. In a first step, we will describe how to build and use a set of tools that we have developed for the implantation of chronic silicon probes14.
While place cells are typically studied using freely-moving animals running in mazes, recently they were also investigated in virtual environments15 and in treadmill apparatii9 (Figure 1A). These experimental methods offer the advantage that animals can be head-restrained, making the use of 2-photon microscope15, patch-clamp16, and optrode9,10,11 techniques easier, in addition to providing enhanced control on animal behavior and environmental cues12. In a second step, we will present the procedures for training mice and recording place cell activity in a treadmill apparatus.

<table>
	<tbody>
		<tr>
			<td>Silicon Probe</td>
			<td>Neuronexus</td>
			<td>Buzsabi32</td>
			<td>Recording electrode</td>
		</tr>
		<tr>
			<td>Recording system</td>
			<td>Intantech</td>
			<td>RHD2132/RHD2000</td>
			<td></td>
		</tr>
		<tr>
			<td>3D printer</td>
			<td>Asiga</td>
			<td>Pico Plus 27</td>
			<td>High resolution printer for micro-drive</td>
		</tr>
		<tr>
			<td>3D printer</td>
			<td>Stratasys</td>
			<td>Mojo</td>
			<td>Lower resolution printer for hat components</td>
		</tr>
		<tr>
			<td>Stereotaxic apparatus</td>
			<td>Kopf</td>
			<td>Model 963</td>
			<td></td>
		</tr>
		<tr>
			<td>Binocular microscope</td>
			<td>Leica</td>
			<td>M60</td>
			<td></td>
		</tr>
		<tr>
			<td>Treadmill apparatus</td>
			<td></td>
			<td></td>
			<td>We build them</td>
		</tr>
	</tbody>
</table>

implantation of chronic silicon probes and recording of hippocampal place cells in an enriched treadmill apparatus

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced electrodes for large-scale electrophysiological recording of neuronal activity, but the procedures for their chronic implantation are still underdeveloped. The activity of hippocampal place cells is known to correlate with an animal&#39;s position in the environment, but the underlying mechanisms are still unclear. To investigate place cells, here we describe a set of techniques which range from the fabrication of devices for chronic silicon probe implants to the monitoring of place field activity in a cue-enriched treadmill apparatus. A micro-drive and a hat are built by fitting and fastening together 3D-printed plastic parts. A silicon probe is mounted on the micro-drive, cleaned, and coated with dye. A first surgery is performed to fix the hat on the skull of a mouse. Small landmarks are fabricated and attached to the belt of a treadmill. The mouse is trained to run head-fixed on the treadmill. A second surgery is performed to implant the silicon probe in the hippocampus, following which broadband electrophysiological signals are recorded. Finally, the silicon probe is recovered and cleaned for reuse. The analysis of place cell activity in the treadmill reveals a diversity of place field mechanisms, outlining the benefit of the approach.

A mouse was first trained to run on a two-meter long belt devoid of cues (Figure 1C). Following electrode implantation, a new belt of the same length but presenting 3 pairs of cues was installed on the treadmill, in order to generate allocentric spatial representations12,14. Broadband signals were recorded at a sampling rate of 30,000 Hz, using a 250-channel recording system (amplifier board with USB ...

Watch this Scientific Journal Video about Implantation of Chronic Silicon Probes and Recording of Hippocampal Place Cells in an Enriched Treadmill Apparatus at JoVE.com

Implantation of Chronic Silicon Probes and Recording of Hippocampal Place Cells in an Enriched Treadmill Apparatus

We describe the diverse steps to implant chronic silicon probes and to record place cells in mice that are running head-fixed on a cue-enriched treadmill apparatus.

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced ...

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.

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Neuroscience

12.5K 次观看.  Korea Institute of Science and Technology. 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 si...