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>Solder a nut (size 000-120x5/64 Hex) onto the tip of the screw.</li>
		<li>Rotate the screw clockwise to move the slider up or counter-clockwise to move it down.</li>
		<li>Fix the shell to the body with a screw (size 000-120x1/4).</li>
	</ol>
	</li>
	<li>Mounting the probe on the micro-drive.
	<ol>
		<li>Fix the micro-drive in a horizontal position under a binocular microscope.</li>
		<li>Use a flat alligator clip with rubber padding to grab the flex cable of the silicon probe, while carefully detaching the probe from the shipment enclosure with forceps. Fix the alligator clip to a manipulator.</li>
		<li>Using the manipulator, lay the silicon probe on the micro-drive slider, parallel to the direction of movement.</li>
		<li>Apply a drop of dental cement (room temperature-curing acrylic dental repair material) to fix the probe to the slider. Correct the probe position if it has shifted. Glue is not recommended as it cures very fast and makes it difficult to readjust the electrode position.</li>
		<li>Fix the connector of the probe to the C-holder (connector holder) with dental cement.</li>
	</ol>
	</li>
	<li>Cleaning and putting dye on the probe.
	<ol>
		<li>Fix the Microdrive/probe assemblage on the probe-cleaning device. The device is equipped with 2 small rotating foam sponges (non-sterile swabs). Adjust the gap by the manipulator.</li>
		<li>Soak the sponges with detergent.</li>
		<li>Slowly move the probe up and down between the sponges, allowing a gentle touch. Monitor the cleaning process under a microscope.</li>
		<li>Rinse the probe with distilled water. Dip the probe in alcohol for sterilization.</li>
		<li>Apply Dil (lipophilic fluorescent dye) on the back of the shanks of the probe, using a foam swab. This will allow in a later stage the visualization of shank locations in the brain.</li>
	</ol>
	</li>
	<li>Assembling the hat
	<ol>
		<li>Print the parts of the hat (head-plate, connector holder, and cap)14 using a 3D-printer. The 3 parts fit together to form a sealed enclosure.</li>
		<li>Insert nuts (size M2 and 00-90x1/4) in the slots of the head-plate and fix with glue and dental cement.</li>
		<li>Insert a washer in the slot of the cap and fix with dental cement.</li>
	</ol>
	</li>
</ol>
2. Surgery for Fixation of the Hat on the Skull
All tools used for surgery are sterilized beforehand by autoclaving. A dry heat beads device is used forto sterilize tools that become contaminated and need to be sterilized during the surgery.
<ol>
	<li>Set the level of isoflurane to 4% for the initiation of anesthesia. Put the mouse in the anesthesia chamber for 5 min.</li>
	<li>Install the mouse in a stereotaxic apparatus.</li>
	<li>Reduce the isoflurane level to 1.5-2%. Adjust the level during the surgery according to animal state, breathing rate, and body temperature20.</li>
	<li>Apply eye ointment on the eyes.</li>
	<li>Shave the scalp and clean the head of the animal with antiseptic (iodopovidone). Maintain aseptic conditions during all steps of the surgery.</li>
	<li>Inject bupivacaine (1 mg/kg) under the scalp. 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 the bleeding during the surgery.</li>
	<li>Remove the periosteum using a scraper tool.</li>
	<li>Find the reference points on the skull21: bregma, lambda, coronal, and sagittal suture. Adjust the head&#39;s angle along the sagittal axis such that the Bregma and lambda points are at the same height.</li>
	<li>Drill two holes (~0.5 mm diameter) in the skull for the reference and ground electrodes. The holes should be approximately 1 mm caudal and 1 mm lateral of the Lambda.</li>
	<li>Insert the ground and reference electrodes (size 000-120x1/16 miniature stainless-steel screws wire-coupled to pin connectors).</li>
	<li>Apply ultraviolet (UV) light bonding dentin activator on the skull and then apply UV light for 45-60 s.</li>
	<li>Apply a layer of dental cement on the edges of the skull. Avoid spreading cement on the skin and hair of the mice.</li>
	<li>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 min.</li>
	<li>Remove the anesthesia. Fix a connector-holder and a cap to the head-plate. Put the mouse in its cage after giving a sub-cutaneous injection of ketaprofen 5 mg/kg.</li>
	<li>Give sub-cutaneous injections of ketaprofen 5 mg/kg for the two next days and monitor carefully for any sign of pain. Mice typically wake up from anesthesia within 20-40 min. The hat implant is relatively light (3.34g), such that mice have no trouble to lift their head, run in mazes and climb on the edges of their home cage.</li>
</ol>
3. Behavioral Training
<ol>
	<li>After a post-surgery recovery period of 7 days, start the restriction of water at 1 mL per day.</li>
	<li>To make the treadmill belt, cut a piece of velvet fabric (5 cm by 1-2 m) and fix small objects on it using hot glue. Attach erected objects on the edges of the belt in order not to interfere with the motion of the mouse.</li>
	<li>Fix the belt on the wheels of the treadmill by suturing together the two ends.</li>
	<li>Install the mouse head-fixed in the treadmill by inserting and tightening the two screws of the head-plate into the slots of the head-fixation plate.</li>
	<li>Start training the mouse to run head-restrained on the treadmill for the water reward. Deliver the water reward through a lick port. Initially, put the mouse on the treadmill for periods of 10 min, 3 times per day.</li>
	<li>As the mouse gets used to the head-fixation and starts to move the belt (typically after ~3 days), increase the training session duration to 30 min. After 2-3 weeks, some mice run more than 100 trials in 30 min (a trial being a full rotation of the belt).</li>
	<li>Choose the mice showing the best behavioral performances for the recording experiments.</li>
</ol>
4. Implantation of the Silicon Probe
<ol>
	<li>Put the mouse under anesthesia.</li>
	<li>Install the mouse in the stereotaxic apparatus by fixing the head-plate. Clean the skull surface with saline.</li>
	<li>Find stereotaxic markers: bregma, lambda, coronal, and sagittal suture. Measure the distance to the point of insertion and mark it.</li>
	<li>Drill the bone carefully until it becomes thin and transparent. Moisten and clean with saline while drilling.</li>
	<li>Carefully remove the thinned bone and the Dura matter using precision forceps. Keep the brain surface wet with saline all the time.</li>
	<li>Fix the Microdrive/silicon probe assemblage to a stereotaxic manipulator. Bring the silicon probe just above the craniotomy. Screw the silicon probe connector holder to the head-plate.</li>
	<li>Connect the recording amplifier and ground/reference electrodes. Shield the mouse with aluminum foil to protect from electro-magnetic noise. Start the recording system to monitor the electrical activity of the brain.</li>
	<li>Slowly insert the silicon probe into the brain using the micromanipulator. Continuously check the electrical signals, the manipulator travelled distance, and the shanks of the probe (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 &#181;m (use the micro-drive the next day to bring the shank back into the pyramidal layer).</li>
	<li>Cover the surface of the brain with a mixture of bone wax and mineral oil.</li>
	<li>Fix the micro-drive to the head-plate using dental cement. Let the dental cement cure for 15 min. Then detach the micro-drive from the stereotaxic manipulator and put the hat cap back on.</li>
	<li>Put the mouse back in its cage and give a sub-cutaneous injection of ketaprofen 5 mg/kg. Check for any sign of pain. This surgery is much less invasive than the first one and mice are typically active within 45 min after they wake-up from the anesthesia. However, let the mouse recover an entire day as the silicon probe needs to stabilize in the brain.</li>
</ol>
5. Recording
<ol>
	<li>Install the mouse head-fixed on the treadmill. Remove the hat cap.</li>
	<li>Connect the recording amplifier and start the recording.</li>
	<li>On the first day, use the micro-drive to move the silicon probe into the pyramidal layer of the hippocampus. Each counter-clockwise turn of the screw moves the shanks 200 &#181;m deeper. Adjust the shank position slowly (~20-50 &#181;m at a time) until ripple oscillations13 and unit activity appear.</li>
	<li>On the next days, tune the shank position and wait ~1 h before recording the hippocampal activity while the mouse runs on the belt. To maintain good recording signal quality for several days, remove hard objects and low ceiling from the mouse&#39;s cage to minimize the chance the hat bumps into hard surfaces.</li>
</ol>
6. Recovering the probe
<ol>
	<li>Put the mouse under anesthesia.</li>
	<li>Install the mouse in the stereotaxic apparatus by fixing the head-plate. Remove the hat cap.</li>
	<li>Bring the stereotaxic manipulator just above the micro-drive. Fix the micro-drive to the manipulator. Unscrew the connector holder from the head-plate. Remove the screw connecting the shell and body of the micro-drive.</li>
	<li>Under binocular microscope supervision, slowly pull up the micro-drive/silicon probe assemblage with the stereotaxic manipulator, leaving the shell part behind.</li>
	<li>Clean the silicon probe with the cleaning device.</li>
</ol>

<ol>
	<li>Schjetnan, A. G., Luczak, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+large-scale+neuronal+ensembles+with+silicon+probes+in+the+anesthetized+rat.">Recording large-scale neuronal ensembles with silicon probes in the anesthetized rat.</a> J Vis Exp. (56), (2011).</li><li>Buzsaki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+recording+of+neuronal+ensembles.">Large-scale recording of neuronal ensembles.</a> Nat Neurosci. 7 (5), 446-451 (2004).</li><li>Suner, S., Fellows, M. R., Vargas-Irwin, C., Nakata, G. K., Donoghue, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+of+signals+from+a+chronically+implanted,+silicon-based+electrode+array+in+non-human+primate+primary+motor+cortex.">Reliability of signals from a chronically implanted, silicon-based electrode array in non-human primate primary motor cortex.</a> IEEE Trans Neural Syst Rehabil Eng. 13 (4), 524-541 (2005).</li><li>Csicsvari, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massively+parallel+recording+of+unit+and+local+field+potentials+with+silicon-based+electrodes.">Massively parallel recording of unit and local field potentials with silicon-based electrodes.</a> J Neurophysiol. 90 (2), 1314-1323 (2003).</li><li>Hochberg, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+ensemble+control+of+prosthetic+devices+by+a+human+with+tetraplegia.">Neuronal ensemble control of prosthetic devices by a human with tetraplegia.</a> Nature. 442 (7099), 164-171 (2006).</li><li>Okun, M., Lak, A., Carandini, M., Harris, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+Term+Recordings+with+Immobile+Silicon+Probes+in+the+Mouse+Cortex.">Long Term Recordings with Immobile Silicon Probes in the Mouse Cortex.</a> PLoS One. 11 (3), e0151180 (2016).</li><li>Vandecasteele, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+recording+of+neurons+by+movable+silicon+probes+in+behaving+rodents.">Large-scale recording of neurons by movable silicon probes in behaving rodents.</a> J Vis Exp. (61), e3568 (2012).</li><li>Kipke, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+neurotechnologies+for+chronic+neural+interfaces:+new+horizons+and+clinical+opportunities.">Advanced neurotechnologies for chronic neural interfaces: new horizons and clinical opportunities.</a> J Neurosci. 28 (46), 11830-11838 (2008).</li><li>Royer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+timing,+rate+and+bursts+of+hippocampal+place+cells+by+dendritic+and+somatic+inhibition.">Control of timing, rate and bursts of hippocampal place cells by dendritic and somatic inhibition.</a> Nat Neurosci. 15 (5), 769-775 (2012).</li><li>Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond-timescale+genetically+targeted+optical+control+of+neural+activity.">Millisecond-timescale genetically targeted optical control of neural activity.</a> Nat Neurosci. 8 (9), 1263-1268 (2005).</li><li>Royer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-array+silicon+probes+with+integrated+optical+fibers:+light-assisted+perturbation+and+recording+of+local+neural+circuits+in+the+behaving+animal.">Multi-array silicon probes with integrated optical fibers: light-assisted perturbation and recording of local neural circuits in the behaving animal.</a> Eur J Neurosci. 31 (12), 2279-2291 (2010).</li><li>Geiller, T., Fattahi, M., Choi, J. S., Royer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Place+cells+are+more+strongly+tied+to+landmarks+in+deep+than+in+superficial+CA1.">Place cells are more strongly tied to landmarks in deep than in superficial CA1.</a> Nat Commun. 8, 14531 (2017).</li><li>Ylinen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sharp+wave-associated+high-frequency+oscillation+(200+Hz)+in+the+intact+hippocampus:+network+and+intracellular+mechanisms.">Sharp wave-associated high-frequency oscillation (200 Hz) in the intact hippocampus: network and intracellular mechanisms.</a> J Neurosci. 15 (1 Pt 1), 30-46 (1995).</li><li>Battaglia, F. P., Sutherland, G. R., McNaughton, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+sensory+cues+and+place+cell+directionality:+additional+evidence+of+prospective+coding+in+the+hippocampus.">Local sensory cues and place cell directionality: additional evidence of prospective coding in the hippocampus.</a> J Neurosci. 24 (19), 4541-4550 (2004).</li><li>Hazan, L., Zugaro, M., Buzsaki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Klusters,+NeuroScope,+NDManager:+a+free+software+suite+for+neurophysiological+data+processing+and+visualization.">Klusters, NeuroScope, NDManager: a free software suite for neurophysiological data processing and visualization.</a> J Neurosci Methods. 155 (2), 207-216 (2006).</li><li>Kadir, S. N., Goodman, D. F., Harris, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-dimensional+cluster+analysis+with+the+masked+EM+algorithm.">High-dimensional cluster analysis with the masked EM algorithm.</a> Neural Comput. 26 (11), 2379-2394 (2014).</li><li>Lewicki, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+methods+for+spike+sorting:+the+detection+and+classification+of+neural+action+potentials.">A review of methods for spike sorting: the detection and classification of neural action potentials.</a> Network. 9 (4), R53-R78 (1998).</li><li>Battaglia, F. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Lantern:+an+ultra-light+micro-drive+for+multi-tetrode+recordings+in+mice+and+other+small+animals.">The Lantern: an ultra-light micro-drive for multi-tetrode recordings in mice and other small animals.</a> J Neurosci Methods. 178 (2), 291-300 (2009).</li><li>Blumberg, M. S., Sokoloff, G., Tiriac, A., Del Rio-Bermudez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+valuable+and+promising+method+for+recording+brain+activity+in+behaving+newborn+rodents.">A valuable and promising method for recording brain activity in behaving newborn rodents.</a> Dev Psychobiol. 57 (4), 506-517 (2015).</li><li>Haiss, F., Butovas, S., Schwarz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+miniaturized+chronic+microelectrode+drive+for+awake+behaving+head+restrained+mice+and+rats.">A miniaturized chronic microelectrode drive for awake behaving head restrained mice and rats.</a> J Neurosci Methods. 187 (1), 67-72 (2010).</li><li>Dombeck, D. A., Khabbaz, A. N., Collman, F., Adelman, T. L., Tank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+large-scale+neural+activity+with+cellular+resolution+in+awake,+mobile+mice.">Imaging large-scale neural activity with cellular resolution in awake, mobile mice.</a> Neuron. 56 (1), 43-57 (2007).</li><li>Villette, V., Malvache, A., Tressard, T., Dupuy, N., Cossart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internally+Recurring+Hippocampal+Sequences+as+a+Population+Template+of+Spatiotemporal+Information.">Internally Recurring Hippocampal Sequences as a Population Template of Spatiotemporal Information.</a> Neuron. 88 (2), 357-366 (2015).</li><li>Ziv, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+dynamics+of+CA1+hippocampal+place+codes.">Long-term dynamics of CA1 hippocampal place codes.</a> Nat Neurosci. 16 (3), 264-266 (2013).</li><li>Danielson, N. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+Contribution+of+Adult-Born+Hippocampal+Granule+Cells+to+Context+Encoding.">Distinct Contribution of Adult-Born Hippocampal Granule Cells to Context Encoding.</a> Neuron. 90 (1), 101-112 (2016).</li><li>Stark, E., Koos, T., Buzsaki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diode+probes+for+spatiotemporal+optical+control+of+multiple+neurons+in+freely+moving+animals.">Diode probes for spatiotemporal optical control of multiple neurons in freely moving animals.</a> J Neurophysiol. 108 (1), 349-363 (2012).</li><li>Wu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+implantable+neural+probe+with+monolithically+integrated+dielectric+waveguide+and+recording+electrodes+for+optogenetics+applications.">An implantable neural probe with monolithically integrated dielectric waveguide and recording electrodes for optogenetics applications.</a> J Neural Eng. 10 (5), 056012 (2013).</li></ol>

The authors have nothing to disclose.

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 conditions, the treadmill apparatus not only significantly simplifies experimental design and data analysis, it also allows researchers to scrutinize place field mechanisms in minimal contexts during many repetitions of stereotyped animal trajectories12. It is also much simpler to build than virtual reality systems, since it only requires 3D-printed wheels, one-dimensional motion sensors, and a microcontroller.
Stable place field activity could be recorded over many days, an important requisite for investigating the long-term network dynamics associated with learning. In this respect, some issues should be considered. First, the processes of brain tissue degeneration and scar formation around the silicone probes might themselves generate long-term variations in activity patterns. It is therefore important for all surgical procedures to be done carefully to cause minimal damage to brain tissues and blood vessels. Second, it is relatively difficult to track individual cells over consecutive days, because of electrode drifts in the brain14. In this respect, imaging techniques such as two-photon microscopy and micro-endoscopy might be better suited, as cell identity can be traced from the morphology and spatial configuration of cells21,22,23. They also allow a larger yield in term of number of cells recorded and provide direct information about cell gene expressions24. However, imaging techniques are quite invasive when deep brain regions are concerned and cannot resolve single action potentials as they mainly rely on calcium signals. Hence, an optimal solution for longitudinal studies might be to combine both optical and electrophysiological approaches.
Finally, it is worth noting that the set of techniques we have described might be easily adaptable for other types of electrode, including Neuropixels (https://www.janelia.org/lab/harris-lab-apig) and optrodes11,25,26, and can be used in a range of experimental models other than hippocampal place cells. With the fast progress in 3D-printing technology, the further improvement and customization of recording and behavioral devices should become more and more straightforward and accessible to the labs.

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 interface board and custom-made Labview interface) and two silicon probes (Figure 1A, 4 shanks, 8 sites per shanks) implanted in CA1 and CA3 (Figure 1B). Units were detected using a threshold function on the high-pass filtered signals (0.8-5 kHz). Cell isolation was performed using a semi-automatic spike sorting method15,16,17. The forward/backward motion of the treadmill was monitored using LED and photo-sensor couples and recorded by digital input channels of the recording system.
An average of 148&#177;35 cells (mean&#177;s.e.m) could be isolated in each session, among which 38.4&#177;3.5% showed clear place field activity (Figure 2). The place fields were relatively stable across many trials, in either the initial day of recording (Figure 2A) or after several days of exposure to the cued belt (Figure 2B and Figure 2C). Different types of spatial representations could be discerned. Some cells showed repeated place fields correlated with the identity of the cues (Figure 2A), while other cells showed a unique place field (Figure 2C)12. Hence, we could record hippocampal place cells over several days and identify a diversity of place field mechanisms, two important requisites for studying the mechanisms and dynamics associated with spatial navigation, learning, and memory.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56438/56438fig1.jpg" /> 
Figure 1: Silicon probe and treadmill apparatus. (A) Shank and site layout of silicon probe. (B) Implantation site. (C) Treadmill apparatus and layout of cues on the belt. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56438/56438fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56438/56438fig2v2.jpg" /> 
Figure 2: Chronic recording of place cells. (A) Color coded representation of place fields (top), spike auto-correlogram (bottom left) and spike waveforms (bottom right) for a cell example recorded on day 1. (B-C) Same as (A) for cells examples recorded on day 3 (B) and day 5 (C). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56438/56438fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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 ...

implantation-chronic-silicon-probes-recording-hippocampal-place-cells

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.6K Views.  Korea Institute of Science and Technology. We beschrijven de diverse stappen implantaat chronische silicon sondes en het optekenen van plaats cellen in muizen die running hoofd-gefixeerd op een loopband cue-verrijkt-apparaat zijn.

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

Recording Large-scale Neuronal Ensembles with Silicon Probes in the Anesthetized Rat

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of behaviors from the spiking activity of its neurons. One of the most effective methods to monitor spiking activity from a large number of neurons in multiple local neuronal circuits simultaneously is by using silicon electrode arrays1-3.
Action potentials produce large transmembrane voltage changes in the vicinity of cell somata. These output signals can be measured by placing a conductor in close proximity of a neuron. If there are many active (spiking) neurons in the vicinity of the tip, the electrode records combined signal from all of them, where contribution of a single neuron is weighted by its 'electrical distance'. Silicon probes are ideal recording electrodes to monitor multiple neurons because of a large number of recording sites (+64) and a small volume. Furthermore, multiple sites can be arranged over a distance of millimeters, thus allowing for the simultaneous recordings of neuronal activity in the various cortical layers or in multiple cortical columns (Fig. 1). Importantly, the geometrically precise distribution of the recording sites also allows for the determination of the spatial relationship of the isolated single neurons4. Here, we describe an acute, large-scale neuronal recording from the left and right forelimb somatosensory cortex simultaneously in an anesthetized rat with silicon probes (Fig. 2).

Extracellular recordings of neuronal activity using silicon probes in the anesthetized rat will be described. This technique allows information to be obtained across multiple brain areas from more than 100 neurons simultaneously. It provides information with single cell resolution about neuronal ensembles dynamics in multiple local circuits.

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

Simultaneous Monitoring of Wireless Electrophysiology and Memory Behavioral Test as a Tool to Study Hippocampal Neurogenesis

Brainwaves amplitude obtained from electroencephalography (EEG) has been well-recognized as a basis for cognitive capacity, memory, and learning on animals and humans. Adult neurogenesis mechanism is also linked to memory and learning improvement. Traditionally, researchers used to assess learning and memory parameters in rodent models by behavioral tasks. Therefore, the simultaneous monitoring of behavioral changes and EEG is particularly interesting in correlating data between brain activity and task-related behaviors. However, most of the equipment required to perform both studies are either complex, expensive, or uses a wired setup network that hinders the natural animals&#8217; movement. In this study, EEG was recorded with a wireless electrophysiology device along with the execution of a novel object recognition task (NORT). The animal&#8217;s behavior was monitored simultaneously by a video tracking system. Both recordings were analyzed offline by their timestamps which were synched to link EEG signals with the animal&#8217;s actions. Subjects consist of adult Wistar rats after medium-term environmental enrichment treatment. Six skull screw electrodes were fixed in pairs on both hemispheres over frontal, central, and parietal regions and were referenced to an electrode located posterior of the nasal bone. NORT protocol consists of exposing the animal to two identical objects for 10 min. After 2 h and 24 h, one of the objects was replaced with a novel one. Exploration time for each object was monitored by a behavioral tracking software (BTS) and EEG data recording. The analysis of the EEG synced with behavioral data consists of estimations of alpha and beta relative band power and comparisons between novel object recognition versus familiar object exploration, between three experimental stages. In this manuscript, we have discussed electrodes manufacturing process, epidural electrodes implantation surgery, environmental enrichment protocol, NORT protocol, BTS setup, EEG &#8211; BTS coupling for simultaneous monitoring in real-time, and EEG data analysis based on automatic events detection.

The protocol presented here provides information on the simultaneous electroencephalography (EEG) and behavioral assessment in real-time. We have discussed all steps involved in this protocol as an attractive solution for researchers in many fields of neuroscience, particularly in learning and memory areas.

Brainwaves amplitude obtained from electroencephalography (EEG) has been well-recognized as a basis for cognitive capacity, memory, and learning on ...

Behavior

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of scientific and clinical development. The use of flexible polymer electrode arrays can support long-lasting recording, but the same mechanical properties that allow for longevity of recording make multiple insertions and integration into a chronic implant a challenge. Here is a methodology by which multiple polymer electrode arrays can be targeted to a relatively spatially unconstrained set of brain areas.
The method utilizes thin-film polymer devices, selected for their biocompatibility and capability to achieve long-term and stable&#160;electrophysiologic recording interfaces. The resultant implant allows accurate and flexible targeting of anatomically distant regions, physical stability for months, and robustness to electrical noise. The methodology supports up to sixteen serially inserted devices across eight different anatomic targets. As previously demonstrated, the methodology is capable of recording from 1024 channels. Of these, the 512 channels in this demonstration used for single neuron recording yielded 375 single units distributed across six recording sites. Importantly, this method also can record single units for at least 160 days.
This implantation strategy, including temporarily bracing each device with a retractable silicon insertion shuttle, involves tethering of devices at their target depths to a skull-adhered plastic base piece that is custom-designed for each set of recording targets, and stabilization/protection of the devices within a silicone-filled, custom-designed plastic case. Also covered is the preparation of devices for implantation, and design principles that should guide adaptation to different combinations of brain areas or array designs.

Described below is a method for implantation of multiple polymer electrode arrays across anatomically distant brain regions for chronic electrophysiological recording in freely moving rats. Preparation and surgical implantation are described in detail, with emphasis on design principles to guide adaptation of these methods for use in other species.

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of ...

Hybrid Microdrive System with Recoverable Opto-Silicon Probe and Tetrode for Dual-Site High Density Recording in Freely Moving Mice

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, conventional microdrive designs often only allow use of one type of electrode to record from single or multiple regions, limiting the yield of single-unit or depth profile recordings. It also often limits the ability to combine electrode recordings with optogenetic tools to target pathway and/or cell type specific activity. Presented here is a hybrid microdrive array for freely moving mice to optimize yield and a description of its fabrication and reuse of the microdrive array. The current design employs nine tetrodes and one opto-silicon probe implanted in two different brain areas simultaneously in freely moving mice. The tetrodes and the opto-silicon probe are independently adjustable along the dorsoventral axis in the brain to maximize the yield of unit and oscillatory activities. This microdrive array also incorporates a set-up for light, mediating optogenetic manipulation to investigate the regional- or cell type-specific responses and functions of long-range neural circuits. In addition, the opto-silicon probe can be safely recovered and reused after each experiment. Because the microdrive array consists of 3D-printed parts, the design of microdrives can be easily modified to accommodate various settings. First described is the design of the microdrive array and how to attach the optical fiber to a silicon probe for optogenetics experiments, followed by fabrication of the tetrode bundle and implantation of the array into a mouse brain. The recording of local field potentials and unit spiking combined with optogenetic stimulation also demonstrate feasibility of the microdrive array system in freely moving mice.

This protocol describes the construction of a hybrid microdrive array that allows implantation of nine independently adjustable tetrodes and one adjustable opto-silicon probe in two brain regions in freely moving mice. Also demonstrated is a method for safely recovering and reusing the opto-silicon probe for multiple purposes.

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, ...

Micro-Drive Implant for Recording Daily Neural Activity in Juvenile Mice

A Lightweight Drive Implant for Chronic Tetrode Recordings in Juvenile Mice

In vivo electrophysiology provides unparalleled insight into the sub-second-level circuit dynamics of the intact brain and represents a method of particular importance for studying mouse models of human neuropsychiatric disorders. However, such methods often require large cranial implants, which cannot be used in mice at early developmental time points. As such, virtually no studies of in vivo physiology have been performed in freely behaving infant or juvenile mice, despite the fact that a better understanding of neurological development in this critical window would likely provide unique insights into age-dependent developmental disorders such as autism or schizophrenia. Here, a micro-drive design, surgical implantation procedure, and post-surgery recovery strategy are described that allow for chronic field and single-unit recordings from multiple brain regions simultaneously in mice as they age from postnatal day 20 (p20) to postnatal day 60 (p60) and beyond, a time window roughly corresponding to the human ages of 2 years old through to adulthood. The number of recording electrodes and final recording sites can be easily modified and expanded, thus allowing flexible experimental control of the in vivo monitoring of behavior- or disease-relevant brain regions across development.

Author Spotlight: A Lightweight Drive Implant for Chronic Tetrode Recordings in Juvenile Mice  

Here, we describe a micro-drive design, surgical implantation procedure, and post-surgery recovery strategy that allow for chronic field and single-unit recordings from multiple brain regions simultaneously in juvenile and adolescent mice across a critical developmental window from postnatal day 20 (p20) to postnatal day 60 (p60) and beyond.

In vivo electrophysiology provides unparalleled insight into the sub-second-level circuit dynamics of the intact brain and represents a method of ...

Development of an X-Y Electrode Stage for Precise Neural Recording in Marmosets

Electrophysiology of Laminar Cortical Activity in the Common Marmoset

The marmoset monkey provides an ideal model for examining laminar cortical circuits due to its smooth cortical surface, which facilitates recordings with linear arrays. The marmoset has recently grown in popularity due to its similar neural functional organization to other primates and its technical advantages for recording and imaging. However, neurophysiology in this model poses some unique challenges due to the small size and lack of gyri as anatomical landmarks. Using custom-built micro-drives, researchers can manipulate linear array placement to sub-millimeter precision and reliably record at the same retinotopically targeted location across recording days. This protocol describes the step-by-step construction of the micro-drive positioning system and the neurophysiological recording technique with silicon linear electrode arrays. With precise control of electrode placement across recording sessions, researchers can easily traverse the cortex to identify areas of interest based on their retinotopic organization and the tuning properties of the recorded neurons. Further, using this laminar array electrode system, it is possible to apply a current source density analysis (CSD) to determine the recording depth of individual neurons. This protocol also demonstrates examples of laminar recordings, including spike waveforms isolated in Kilosort, which span multiple channels on the arrays.

Author Spotlight: Optimizing Micro-Drive Systems for Precise Electrode Positioning in Marmoset

Custom-built micro-drives enable the sub-millimeter targeting of cortical recording sites with linear silicon arrays.

The marmoset monkey provides an ideal model for examining laminar cortical circuits due to its smooth cortical surface, which facilitates recordings with ...

A Cost-Effective, Versatile Probe Implant System for Electrophysiology in Mice

The DREAM Implant: A Lightweight, Modular, and Cost-Effective Implant System for Chronic Electrophysiology in Head-Fixed and Freely Behaving Mice

Chronic electrophysiological recordings in rodents have significantly improved our understanding of neuronal dynamics and their behavioral relevance. However, current methods for chronically implanting probes present steep trade-offs between cost, ease of use, size, adaptability, and long-term stability.
This protocol introduces a novel chronic probe implant system for mice called the DREAM (Dynamic, Recoverable, Economical, Adaptable, and Modular), designed to overcome the trade-offs associated with currently available options. The system provides a lightweight, modular and cost-effective solution with standardized hardware elements that can be combined and implanted in straightforward steps and explanted safely for recovery and multiple reuse of probes, significantly reducing experimental costs.
The DREAM implant system integrates three hardware modules: (1) a microdrive that can carry all standard silicon probes, allowing experimenters to adjust recording depth across a travel distance of up to 7 mm; (2) a three-dimensional (3D)-printable, open-source design for a wearable Faraday cage covered in copper mesh for electrical shielding, impact protection, and connector placement, and (3) a miniaturized head-fixation system for improved animal welfare and ease of use. The corresponding surgery protocol was optimized for speed (total duration: 2 h), probe safety, and animal welfare.
The implants had minimal impact on animals&#39; behavioral repertoire, were easily applicable in freely moving and head-fixed contexts, and delivered clearly identifiable spike waveforms and healthy neuronal responses for weeks of post-implant data collection. Infections and other surgery complications were extremely rare.
As such, the DREAM implant system is a versatile, cost-effective solution for chronic electrophysiology in mice, enhancing animal well-being, and enabling more ethologically sound experiments. Its design simplifies experimental procedures across various research needs, increasing accessibility of chronic electrophysiology in rodents to a wide range of research labs.

Here, we introduce a lightweight, cost-effective probe implant system for chronic electrophysiology in rodents optimized for ease of use, probe recovery, experimental versatility, and compatibility with behavior.

Chronic electrophysiological recordings in rodents have significantly improved our understanding of neuronal dynamics and their behavioral relevance. ...

Implanting an Electrode Array and Optic Fiber into the Mouse Hippocampus

Source: Bender, F., et. al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57349">Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice.</a> J. Vis. Exp. (2018)
This video demonstrates the stereotactic implantation of a wire electrode array and an optic fiber in the hippocampus of a mouse model.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Implantation of Chronic Silicon Probes and Recording of Hippocampal Place Cells in an Enriched Treadmill Apparatus

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