This work was supported in part by Japan Society for the Promotion of Science Overseas Research Fellowships (HO), Endowed Scholar Program (TK), Human Frontier Science Program (TK), Brain Research Foundation (TK), Faculty Science and Technology Acquisition and Retention Program (TK), Brain &#38; Behavior Research Foundation (TK), and by The Sumitomo Foundation Research Grant (JY), NARSAD Young Investigator Research Grant (JY). We thank W. Marks for valuable comments and suggestions during the preparation of the manuscript.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas Southwestern Medical Center.
1. Preparations of microdrive array parts
<ol>
	<li>Print the microdrive array parts using a 3D printer using dental model resin (Figure 1A,B). Ensure that the thickness of individual 3D printed layers is less than 50 &#181;m to keep the small holes on the printed parts clear and viable. 
	NOTE: The microdrive array consists of five parts (Figure 1C): (1) the main body of the microdrive array, which includes nine microdrive-screws for tetrodes and one screw for a silicon-probe (Figure 1Ca-d). The coordination of the tetrode bundle and hole for the opto-silicon probe at the bottom depends on the target brain area&#8217;s coordinates (Figure 1Cd); (2) a shuttle to attach a silicon-probe or optrode (Figure 1Ce); (3) a probe electrical connecter mount to hold the silicon probe connecter (Figure 1Cf); (4) a fiber ferrule holder that clamps to the center part of the body to prevent undesired movements of the implanted opto-silicon probe when plugging/unplugging an optical fiber connector (Figure 1Cg); and (5) a shielding cone that provides physical and electrical shielding to the microdrive array for stable recording (Figure 1Ch). The total weight of the microdrive array is 5.9 g, including the shielding cone (Table 1). If holes are clogged in the printed parts, drill out the holes using drill bits: #76 for the inner holes and #68 for the outer holes for tetrode-microdrive screws, #71 for tetrode microdrive-screw supporter hole, and #77 for the holes for the guide-posts at the bottom of the body.</li>
	<li>Insertion of guide posts into the microdrive array body.
	<ol>
		<li>Cut two 16 mm lengths of 26-Ga stainless steel wire. Gently sharpen the wire tips using a rotary grinder.</li>
		<li>Insert the wires into the bottom holes of the body (Figure 2A). Apply a small amount of cyanoacrylate glue at the bottom of the body to secure the guide posts.</li>
	</ol>
	</li>
</ol>
2. Opto-silicon probe preparation
<ol>
	<li>Prepare the microdrive screw for a silicon-probe. 
	NOTE: The microdrive screw for the silicon probe consists of a custom screw (300 &#181;m pitch), supporting a support tube, and an L-shape tube (Figure 2B).
	<ol>
		<li>Prepare the mold for the microdrive head (Figure 2C). To construct the mold, prepare the 3D-printed plastic pattern of the microdrive (Figure 2Ca). Then, pour liquid silicone gel after making a temporal wall by putting tapes around the pattern. Remove air bubbles by shaking gently, wait until it is cured, then remove the silicone-gel mold from the pattern (Figure 2Cb).</li>
		<li>Cut 18 mm and 9.5 mm lengths of 23 G stainless wire using a rotary grinder. Roughen the top 2&#8211;3 mm of the wires with a rotary grinder to enhance adhesion of the dental acrylic.</li>
		<li>Take one custom screw and apply small amount of silicon oil to reduce the friction with the dental acrylic. Set the wires and a custom-screw to the mold.</li>
		<li>Pour dental acrylic into the mold using a syringe to eliminate air bubbles around the wires and the screws. Air bubble contamination will make the microdrive fragile. Wait until the dental acrylic is fully cured, then take off the microdrive screws from the mold. Bend 6 mm of the longer wire tip to a 60&#176; angle using pliers.</li>
		<li>Check the quality of the microdrive screws (e.g., cracks, air-bubbles, and friction) to rotate the screw. If there is high friction, rotate the screw until they become smooth using an electric screw driver with a customized driver tip, which couples with the microdrive screw.</li>
		<li>Install the microdrive screw into the microdrive array body to check whether it moves up and down smoothly by turning the screw. Threads for the screw are automatically created when inserting the screw into the hole of the body.</li>
	</ol>
	</li>
	<li>Prepare the shuttle (Figure 3Aa).
	<ol>
		<li>Cut two 5 mm lengths of polyetheretherketone (PEEK) tubing using sharp scissors. Align the tubes at both sides of the shuttle. Glue the tubes and shuttle using epoxy.</li>
		<li>Apply small amount of silicon oil on the guide posts. Check the quality of the shuttle by inserting onto the guide posts of the microdrive array body. Make sure the shuttle moves smoothly without excessive friction.</li>
	</ol>
	</li>
	<li>Prepare an optorode (Figure 3Ab). This step can be skipped if an optogenetic experiment is not required.
	<ol>
		<li>Cleave the optical fiber to 21 mm in length using a ruby cutter. Grind the fiber tip to make the tip flat and shiny.</li>
		<li>Gently place the optical fiber on the front side of the silicon-probe. The fiber tip is positioned 200&#8211;300 &#181;m above the top of the electrode sites. Hold the fiber temporarily with transparent tape.</li>
		<li>Glue the optical fiber to the base of the silicon-probe using small amount of epoxy. Wait for at least 5 h until the epoxy is fully cured. 
		NOTE: It is recommended to attach the optical fiber on the same side as the electrode sites. Attaching the fiber at the backside may prevent light from properly illuminating the recording sites.</li>
	</ol>
	</li>
	<li>Attach the shuttle to the silicon probe (Figure 3Ac): apply a small amount of epoxy at the back of the silicon-probe&#8217;s base. Attach the bottom part of the shuttle to the silicon-probe&#8217;s base, and gently hold in position for 2&#8211;3 min to avoid formation of a gap between the shuttle and silicon-probe base during initial cure of the epoxy. Wait for at least 5 h until the epoxy is completely cured.</li>
	<li>Carefully insert the shuttle tubes onto the guide posts of the main body under the microscope (Figure 3B). During this procedure, hold the groove of the shuttle with fine tweezers.</li>
	<li>Insert the microdrive-screw into the screw hole by turning the screw. Engage the silicon probe and microdrive-screw by inserting the tip of the L-shape wire into the groove of the shuttle head (Figure 3C).</li>
	<li>Attach the probe electrical connecter holder to the microdrive array body (Figure 3D).
	<ol>
		<li>Cut two #0 screws to 3.5 mm thread length. Grind the tips to remove burrs.</li>
		<li>Place the probe connecter holder on the body. Place the silicon probe electrical connector into the holder.</li>
		<li>Secure the silicon probe connector in the holder using epoxy, and be sure to not glue it to the microdrive array body to allow for the recovery procedure of the silicon probe. Insert the screws to hold the probe connecter holder.</li>
	</ol>
	</li>
	<li>Attach the ferrule-holder to the opto-silicon probe and microdrive array body (Figure 3D).
	<ol>
		<li>Cut two #0 screws to 6 mm thread length. Grind the tips to remove burrs.</li>
		<li>Grind the outside of two #0 machine screw nuts to make small hex nuts with 2.5&#8211;3.0 mm outer diameter to reduce the weight and space.</li>
		<li>Insert the screws into component A of the holder. Glue the screw heads using epoxy.</li>
		<li>Apply small amount of silicon grease to component A and B to reduce friction with the body. Insert component A into the body, then temporally hold using inverse tweezers.</li>
		<li>Place component B onto component A&#8217;s screws. Thread the customized nuts into the screws. Use pliers to tighten the nuts to secure the ferrule holder on the body.</li>
		<li>Insert the fiber ferrule into the groove of the fiber ferrule holder (component B). Ensure that the fiber ferrule is sticking 4&#8211;5 mm out from the holder.</li>
		<li>Apply small amount of epoxy between the ferrule and the holder groove. Wait until the epoxy is fully cured and check that the ferrule does not move. Check the shuttle and ferrule holder for smooth motion by loosening the nuts before turning the microdrive-screw.</li>
		<li>Check the working distance of the probe. Ensure that the probe tip completely retracts into the body when the ferrule-holder is at the top position while the shuttle tubes are still associated with the guide-posts. The maximum working distance is determined by the length of the silicone probe and the target brain region.</li>
		<li>If the microdrive-screw is loose, apply small amount of dental acrylic around the screw to add more threads for support. When it is cured, rotate the screw to check tightness and stability.</li>
	</ol>
	</li>
</ol>
3. Tetrode preparation
NOTE: This procedure is similar to previously published articles8,19,20,39.
<ol>
	<li>Prepare the microdrive screws for the tetrode. The microdrive for a tetrode consists of a custom-machined screw and a 23 G tubing (Figure 2B). This procedure is similar to section 2.1.</li>
	<li>Make a bundle of 30 G stainless steel tubing that has a 5.5 mil wire inside. In this case, a total of nine 30 G tubing (eight recording tetrodes and one reference electrode) were used.</li>
	<li>Thread the 30 G bundle from the bottom of the drive body, and secure them with 20 G thin-walled tubing to the main body. Trim the bottom of the bundle with a rotary grinder to make the tip even and flush. Trim top part of the 30 G tubes with a rotary grinder so that the 30 G tube sticks out about 0.5 mm from the main body.</li>
	<li>Load 5.5 mil polyimide insulating tubes into the 30 G tubing. Prepare tetrode wires and load them into a 32-channel electric interface board (EIB). Check electrical connection with the impedance tester before final precision cut.</li>
	<li>Lower electrode tip impedance to 250&#8211;350 k&#937; with gold plating solution. Fix all tetrodes with superglue.</li>
	<li>Fill excessive gap between polyimide tube and tetrode with mineral oil for sealing and lubrication. Route the ground wire to the EIB. 
	NOTE: If necessary, the optical fiber can be integrated along tetrode wires12.</li>
</ol>
4. Attaching the shielding cone
<ol>
	<li>Paint silver conductive shielding paint on the inside of the printed cone. Place the microdrive array inside of the cone (Figure 3E).</li>
	<li>Cut two #0 screws to 3.5 mm thread length. Fasten the screws from the outside of the cone to hold the microdrive array in place.</li>
	<li>Apply silver paint around the screw head to electrically connect the shielding cone with electrical ground. Check the electrical connectivity between the ground wire and cone. Apply a small amount of epoxy between the microdrive array body and shielding cone to securely attach the body. 
	NOTE: Another way to prepare the shielding cone is to use aluminum tape40 (Figure 3F). First, prepare the pattern paper for the shielding cone after sticking aluminum foil to the paper (Figure 3Fa). Then, roll the paper and attach it to the microdrive body using a small amount of cyanoacrylate glue (Figure 3Fb). The weight of this cone is 0.72 g and total weight of the microdrive array is reduced to 4.7 g (Table 1).</li>
</ol>
5. Implant surgery
NOTE: This procedure is modified from previously published articles18,39,41 for dual-site implantation. Ensure that the weight of the animal is over 25 g for the microdrive implant for faster recovery after the surgery.
<ol>
	<li>Preparation
	<ol>
		<li>To prepare a ground screw, attach the silver wire to a skull screw and apply silver paint. Then, attach a gold pin to the opposite side of the wire using silver paint.</li>
		<li>Prepare the drive holding adapter to hold the microdrive array to a stereotactic device. Attach a male connecter to a stainless handle using epoxy. Make sure that alignment of the connecter and stainless handle is straight.</li>
		<li>In the case that histological confirmation is needed after recording, apply Di-I to the tetrodes or backside of the silicon-probe38.</li>
		<li>Lower the silicon-probe down to be desired depth. Loosen the nuts of the ferrule holder using pliers, lower the silicon-probe (opto-silicon probe) by turning the silicon-probe&#8217;s microdrive-screw, then fasten the nuts to secure the ferrule holder. When implanting tetrodes in hippocampal area CA1 and a silicon-probe in MEC, the distance between the tetrode cannula and silicon-probe&#8217;s tip is 3&#8211;4 mm.</li>
	</ol>
	</li>
	<li>Set the anesthetized mouse (0.8%&#8211;1.5% isoflurane) in a stereotaxic device. The anesthetic condition of the mouse is confirmed by absence of the toe-pinch reflex. Apply clear ointment to the eyes to prevent drying. Cover the eyes with a piece of foil to protect from strong surgical light exposure.</li>
	<li>Disinfect the mouse&#8217;s scalp with iodine and isopropanol after shaving the fur. Make a 1.5&#8211;2.0 cm incision at the scalp using standard surgical scissors, and remove the tissue over the skull using cotton swabs after subcutaneously applying lidocaine.</li>
	<li>Align the mouse head with the stereotaxic tool. Ensure that the height difference between bregma and lambda is less than 100 &#956;m. Determine the craniotomy location using an atlas and mark these locations with a sterilized pencil.</li>
	<li>Anchor the skull screws (0.8 mm diameter, 0.200 mm thread pitch) by rotating them 1.5 turns (0.3 mm) on the skull, using surgical tweezers and a screwdriver after drilling 8&#8211;11 holes in the skull using 0.5 mm drill bit. 
	NOTE: 2&#8211;4 holes in the frontal skull, 2&#8211;3 holes in each side of the parietal skull, and 1&#8211;2 holes in the interparietal skull are suggested.</li>
	<li>Attach the ground screw to the hole by rotating it one turn (0.2 mm) after drilling a hole in the interparietal bone. Ensure that this hole does not penetrate through the bone into the brain case; otherwise, cerebellar signals will contaminate the recording. Check that the impedance is less than 20 k&#8486; at 1 kHz between the ground screw and skull screws using an impedance meter. 
	NOTE: Larger impedance will cause the introduction of motion artifacts during recording.</li>
	<li>Perform the craniotomy at the marked locations. The dura can be left intact in mice.</li>
	<li>Connect the male pin of the ground screw and the microdrive array&#8217;s ground connector. Check connectivity using the impedance meter by measuring between the ground screw and shielding.</li>
	<li>Set the microdrive array to the adapter, set it to the stereotaxic device, and slowly lower the silicon probe until the desired depth. Ensure that the tetrode bundles are placed above the brain surface but still inside of the microdrive array when the silicon probe is inserted into the brain (Figure 4A).</li>
	<li>Carefully apply the silicon grease to seal the area of the silicon probe and the tetrode bundle (Figure 4B). Put a small amount of the silicon grease at the tip of a 20 G needle and apply the grease around the probes using the needle. Repeat until silicon grease completely covers the area around the probe so that dental acrylic does not flow onto or underneath the electrodes/probes. Be careful not to let the grease touch the electrode sites, otherwise it will dramatically increase the impedance of the recording sites.</li>
	<li>Apply dental acrylic to fix the microdrive array to the anchoring screws in the skull. 
	NOTE: It is recommended to apply dental acrylic in three layers to avoid the excessive heat produced during curing of the acrylic.</li>
	<li>Remove the adapter from the microdrive array carefully. Inject 1 mL of PBS subcutaneously to prevent dehydration. Inject 5 mg/kg meloxicam subcutaneously as an analgesic treatment.</li>
	<li>Cover the silicon-probe connector by a piece of tape to prevent any dirt from getting inside of the electrical connections. Cover the microdrive array using a plastic paraffin film and tape it in place.</li>
	<li>Administer appropriate analgesic treatment for 3 days (e.g., subcutaneous injections of 2 mg/kg meloxicam once per day). Allow 3&#8211;5 days for recovery before starting the tetrode adjustment. The implanted mouse after the recovery period is shown in Figure 4C.</li>
</ol>
6. Recovering the silicon-probe (Figure 4D)
<ol>
	<li>Inject ketamine (75 mg/kg) and dexmedetomidine (1 mg/kg) anesthetics intraperitoneally and confirmed absence of the toe-pinch reflex. Fix the anesthetized mouse by directly perfusing 4% paraformaldehyde through the heart using a hood. Surgical methods for rodents are described previously42.</li>
	<li>Loosen the nuts of the ferrule holder using a plier. Then, carefully move it to the top of the body by turning the adjusting screw to fully retract the silicon-probe towards inside of the microdrive array body. Fasten the nuts to hold the probe at the top position.</li>
	<li>Take the mouse brain out from the bottom by cracking the skull from the side. The microdrive array is now separated from the animal.</li>
	<li>Completely remove the L-shaped microdrive-screw that drives the silicon-probe. Loosen and take out the nuts of the ferrule holder using pliers. Take out the component A of the ferrule holder.</li>
	<li>Unscrew the probe connector mount and detach from the drive body. Check that the probe connector mount can come off from the microdrive array body.</li>
	<li>Hold the top part of the shuttle with tweezers, then carefully slide the silicon-probe assembly out from the microdrive array.</li>
	<li>Clean the probe tip with contact lens cleaner (first with enzyme, then 3% hydrogen peroxide) for at least 1 day. Carefully wipe the electrode tip using isopropanol pads under the microscope. Keep the probe in a static-free storage box. 
	NOTE: The shuttle and probe connector mount remain attached to the silicon probe and can be reused in the next implantation. 
	NOTE: Some silicon probes are not tolerable with hydrogen peroxide. In this case, use the contact lens solution containing proteolytic enzyme only.</li>
	<li>To reuse the microdrive array body for the next surgery, remove the dental acrylic using a combination of fine-tip drills and nippers. Then, recover the skull-screws by immersing the removed dental acrylic into acetone. Note that the acetone will dissolve plastic parts of the microdrive array.</li>
	<li>Remove the epoxy between the microdrive body and shielding cone using a scalpel. 
	NOTE: No additional parts need to be printed again for the next surgery if the microdrive is not broken.</li>
</ol>

The authors have nothing to disclose.

The protocol demonstrates how to construct and implant a hybrid microdrive array that allows recording of neural activities from two brain areas using independent adjustable tetrodes and a silicon-probe in freely behaving mice. It also demonstrates optogenetic experiments and the recovery of the silicon probe after experiments. While adjustable silicon probe33 or opto-silicon probe36 implantation are previously demonstrated in mice, this protocol has clear advantages in the...

It is crucial to understand how neuronal activity supports cognitive process, such as learning and memory, by investigating how different brain regions dynamically interact with each other. To elucidate dynamics of the neural activity underlying cognitive tasks, large-scale extracellular electrophysiology has been conducted in freely moving animals with the aid of microdrive arrays1,2,3,4. In the past two decades, several types of microdrive array have been developed to implant electrodes into multiple brain regions for rats5,6,7,8 and mice9,10,11,12. Nonetheless, current microdrive designs generally do not allow for the use of multiple probe types, forcing researchers to choose a single electrode type with specific benefits and limitations. For example, tetrode arrays work well for densely populated brain regions such as the dorsal hippocampus CA11,13, while silicon probes give a better geometrical profile for studying anatomical connections14,15.
Tetrodes and silicon probes are often used for in vivo chronic recording, and each has its own advantages and disadvantages. Tetrodes have been proven to have significant advantages in better single unit isolation than single electrodes16,17, in addition to cost effectiveness and mechanical rigidity. They also provide higher yields of single unit activities when combined with microdrives8,18,19,20. It is essential to increase the number of simultaneously recorded neurons for understanding the function of neural circuits21. For example, large numbers of cells are needed to investigate small populations of functionally heterogeneous cell types such as time-related22 or reward coding23 cells. Much higher cell numbers are required to improve the decoding quality of spike sequences13,24,25.
Tetrodes, however, have a disadvantage in recording spatially distributed cells, such as in the cortex or thalamus. In contrast to tetrodes, silicon probes can provide spatial distribution and interaction of local field potentials (LFPs) and spiking activities within a local structure14,26. Multi-shank silicon probes further increase the number of recording sites and allow recording across single or neighboring structures27. However, such arrays are less flexible in the positioning of electrode sites compared to tetrodes. In addition, complex spike sorting algorithms are required in high-density probes to extract information about action potentials of neighboring channels to mirror the data acquired by tetrodes28,29,30. Hence, the overall yield of single units is often less than tetrodes. Moreover, silicon probes are disadvantageous due to their fragility and high cost. Thus, the choice of tetrodes vs. silicon probes depends on the aim of the recording, which is a question of whether obtaining a high yield of single-units or spatial profiling at the recording sites is prioritized.
In addition to recording neural activity, optogenetic manipulation has become one of the more powerful tools in neuroscience to examine how specific cell types and/or pathways contribute to neural circuit functions13,31,32,33. However, optogenetic experiments require additional consideration in microdrive array design to attach the fiber connector to stimulation light sources34,35,36. Often, connecting fiber-optics requires a relatively large force, which may lead to a mechanical shift of the probe in the brain. Therefore, it is not a trivial task to combine an implantable optical fiber to conventional microdrive arrays.
For the above reasons, researchers are required to optimize the selection of the type of electrode or to implant an optical fiber depending on the aim of the recording. For example tetrodes are used to achieve higher unit yield in hippocampus1,13, while silicon probes are used to investigate the laminar depth profile of cortical areas, such as the medial entorhinal cortex (MEC)37. Currently, microdrives for simultaneous implantation of tetrodes and silicon probes had been reported for rats5,11. However, it is extremely challenging to implant multiple tetrodes and silicon probes in mice because of the weight of the microdrives, limited space on the mouse head, and spatial requirements for designing the microdrive to employ different probes. Although it is possible to implant silicon probes without a microdrive, this procedure does not allow for adjustment of the probe and lowers the success rate of silicon-probe recovery12,38. Furthermore, optogenetic experiments require additional considerations in microdrive array design. This protocol demonstrates how to construct and implant a microdrive array for chronic recording in freely moving mice, which allows implantation of nine independently adjustable tetrodes and one adjustable opto-silicon probe. This microdrive array also facilitates optogenetic experiments and recovery of the silicon probe.

<table><tbody><tr><td>#00-90 screw</td><td>J.I. Morris</td><td>#00-90-1/8</td><td>EIB screws</td></tr><tr><td>#0-80 nut</td><td>Small Parts</td><td>B00DGB7CT2</td><td>brass nut for holding fiber ferrule holder</td></tr><tr><td>#0-80 screw</td><td>Small Parts</td><td>B000FMZ57G</td><td>brass machine screw for probe connector mount, fiber ferrule holder, and shielding cone</td></tr><tr><td>22 Ga polyetheretherketone tubes</td><td>Small Parts</td><td>SLPT-22-24</td><td>for attaching to the shuttle, 0.025 inches inner diameter</td></tr><tr><td>23 Ga stainless tubing</td><td>Small Parts</td><td>HTX-23R</td><td>for tetrode</td></tr><tr><td>23 Ga stainless wire</td><td>Small Parts</td><td>HTX-23R-24-10</td><td>for L-shape/support wire</td></tr><tr><td>26 Ga stainless wire</td><td>Small Parts</td><td>GWX-0200</td><td>for guide-posts</td></tr><tr><td>30 Ga stainless wire</td><td>Small Parts</td><td>HTX-30R</td><td>for tetrode</td></tr><tr><td>3-D CAD software package</td><td>Dassault Syst&amp;egrave;mes</td><td>SolidWorks 2003</td><td></td></tr><tr><td>3D printer</td><td>FormLab</td><td>Form2</td><td></td></tr><tr><td>5.5mil polyimide insulating tubes</td><td>HPC Medical</td><td>72113900001-012</td><td></td></tr><tr><td>aluminum foil tape</td><td>Tyco</td><td>Tyco Adhesives 617022 Aluminum Foil Tape</td><td>for the alternative shielding cone</td></tr><tr><td>conductive paste</td><td>YSHIELD</td><td>HSF54</td><td>for shielding cone</td></tr><tr><td>customized screws for silicon-probe microdrive</td><td>AMT</td><td>UNM1.25-HalfMoon</td><td>half-moon stainless screw, 1.5 mm diameter, 300 &amp;micro;m thread pitch</td></tr><tr><td>customized screws for tetrode microdrive</td><td>AMT</td><td>Yamamoto_0000-160_9mm</td><td>slotted stainless screw, 0.5 mm diameter, 160 &amp;micro;m thread pitch, custom-made to order for our design</td></tr><tr><td>dental acrylic</td><td>Stoelting</td><td>51459</td><td></td></tr><tr><td>dental model resin</td><td>FormLab</td><td>RS-F2-DMBE-02</td><td></td></tr><tr><td>Dremel rotary tool</td><td>Dremel</td><td>model 800</td><td>a grinder</td></tr><tr><td>drill bit</td><td>Fine Science Tool</td><td>19007-05</td><td></td></tr><tr><td>electric interface board</td><td>Neuralynx</td><td>EIB-36-Narrow</td><td></td></tr><tr><td>epoxy</td><td>Devcon</td><td>GLU-735.90</td><td>5 minutes epoxy</td></tr><tr><td>eye ointment</td><td>Dechra</td><td>Puralube Ophthalmic Ointment</td><td>to prevent mice eyes from drying during surgery</td></tr><tr><td>fiber polishing sheet</td><td>Thorlabs</td><td>LFG5P</td><td>for polishing the optical fiber</td></tr><tr><td>fine tweezers</td><td>Protech International</td><td>15-368</td><td>for loading/recovering the silicon probe</td></tr><tr><td>gold pins</td><td>Neuralynx</td><td>EIB Pins Small</td><td></td></tr><tr><td>ground wire</td><td>A-M Systems</td><td>781500</td><td>0.010 inch bare silver wire</td></tr><tr><td>headstage preamp</td><td>Neuralynx</td><td>HS-36</td><td></td></tr><tr><td>impedance meter</td><td>BAK electronics</td><td>Model IMP-2</td><td>1 kHz testing frequency</td></tr><tr><td>mineral oil</td><td>ZONA</td><td>36-105</td><td>for lubricating screws and wires</td></tr><tr><td>optical fiber</td><td>Doric</td><td>MFC_200/260-0.22_50mm_ZF1.25(G)_FLT</td><td></td></tr><tr><td>Recording system</td><td>Neuralynx</td><td>Digital Lynx 4SX</td><td></td></tr><tr><td>ruby fiber scribe</td><td>Thorlabs</td><td>S90R</td><td>for cleaving the optical fiber</td></tr><tr><td>silicon grease</td><td>Fine Science Tool</td><td>29051-45</td><td></td></tr><tr><td>silicon probe</td><td>Neuronexus</td><td>A1x32-Edge-5mm-20-177</td><td>Fig. 3, 4A, 4B, 5</td></tr><tr><td>silicon probe</td><td>Neuronexus</td><td>A1x32-6mm-50-177</td><td>Fig. 4C</td></tr><tr><td>silicon probe washing solution</td><td>Alcon</td><td>AL10078844</td><td>contact lens cleaner</td></tr><tr><td>silicone lubber</td><td>Smooth-On</td><td>Dragon Skin 10 FAST</td><td>for preparation of microdrive mold</td></tr><tr><td>silver paint</td><td>GC electronic</td><td>22-023</td><td>silver print II coating, used for ground wires</td></tr><tr><td>skull screw</td><td>Otto Frei</td><td>2647-10AC</td><td>0.8 mm diameter, 0.200 mm thread pitch</td></tr><tr><td>standard surgical scissors</td><td>ROBOZ</td><td>RS-5880</td><td></td></tr><tr><td>stereotaxic apparatus</td><td>Kopf</td><td>Model 942</td><td></td></tr><tr><td>super glue</td><td>Loctite</td><td>LOC230992</td><td>for applying to guide-posts</td></tr><tr><td>surgical tweezers</td><td>ROBOZ</td><td>RS-5135</td><td></td></tr><tr><td>Tetrode Twister</td><td>Jun Yamamoto</td><td>TT-01</td><td></td></tr><tr><td>tetrode wires</td><td>Sandvik</td><td>PX000004</td><td></td></tr></tbody></table>

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.

The microdrive array was constructed within 5 days. The timeline of microdrive preparation is described in Table 2. Using this microdrive, nine tetrodes and one silicon probe were implanted into the hippocampal CA1 and MEC of the mouse [21 week old/29 g body weight male pOxr1-Cre (C57BL/6 background)], respectively. This transgenic mouse expresses Cre in MEC layer III pyramidal neurons. The mouse was injected with 200 nL of AAV5-DIO-ChR2-YFP (titer: 7.7 x 1012 gc/mL) into the MEC 10 weeks befo...

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Hybrid Microdrive System with Recoverable Opto-Silicon Probe and Tetrode for Dual-Site High Density Recording 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, ...

hybrid-microdrive-system-with-recoverable-opto-silicon-probe-tetrode

Research

JoVE Journal

Neuroscience

11.0K Views.  University of Texas Southwestern Medical Center. 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.

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

Construction of Microdrive Arrays for Chronic Neural Recordings in Awake Behaving Mice

State-of-the-art electrophysiological recordings from the brains of freely behaving animals allow researchers to simultaneously examine local field potentials (LFPs) from populations of neurons and action potentials from individual cells, as the animal engages in experimentally relevant tasks. Chronically implanted microdrives allow for brain recordings to last over periods of several weeks. Miniaturized drives and lightweight components allow for these long-term recordings to occur in small mammals, such as mice. By using tetrodes, which consist of tightly braided bundles of four electrodes in which each wire has a diameter of 12.5 &mu;m, it is possible to isolate physiologically active neurons in superficial brain regions such as the cerebral cortex, dorsal hippocampus, and subiculum, as well as deeper regions such as the striatum and the amygdala. Moreover, this technique insures stable, high-fidelity neural recordings as the animal is challenged with a variety of behavioral tasks. This manuscript describes several techniques that have been optimized to record from the mouse brain. First, we show how to fabricate tetrodes, load them into driveable tubes, and gold-plate their tips in order to reduce their impedance from M&Omega; to K&Omega; range. Second, we show how to construct a custom microdrive assembly for carrying and moving the tetrodes vertically, with the use of inexpensive materials. Third, we show the steps for assembling a commercially available microdrive (Neuralynx VersaDrive) that is designed to carry independently movable tetrodes. Finally, we present representative results of local field potentials and single-unit signals obtained in the dorsal subiculum of mice. These techniques can be easily modified to accommodate different types of electrode arrays and recording schemes in the mouse brain.

The design and assembly of microdrives for in vivo electrophysiological recordings of brain signals from the mouse is described. By attaching microelectrode bundles to sturdy driveable carriers, these techniques allow for long-term and stable neural recordings. The lightweight design allows for unrestricted behavioral performance by the animal following drive implantation.

State-of-the-art electrophysiological recordings from the brains of freely behaving animals allow researchers to simultaneously examine local field ...

Behavior

High-density EEG Recordings of the Freely Moving Mice using Polyimide-based Microelectrode

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a noninvasive brain monitoring tool in cognitive neuroscience as well as a diagnostic tool for epilepsy and sleep disorders in neurology. However, the underlying mechanism of EEG rhythm generation is still under the veil. Recently introduced polyimide-based microelectrode (PBM-array) for high resolution mouse EEG1 is one of the trials to answer the neurophysiological questions on EEG signals based on a rich genetic resource that the mouse model contains for the analysis of complex EEG generation process. This application of nanofabricated PBM-array to mouse skull is an efficient tool for collecting large-scale brain activity of transgenic mice and accommodates to identify the neural correlates to certain EEG rhythms in conjunction with behavior. However its ultra-thin thickness and bifurcated structure cause a trouble in handling and implantation of PBM-array. In the presented video, the preparation and surgery steps for the implantation of PBM-array on a mouse skull are described step by step. Handling and surgery tips to help researchers succeed in implantation are also provided.

In this article, we described the surgery procedure and handling tips for implantation of ultra-thin polyimide-based microelectrode array (PBM-array) on the mouse skull for acquisition of high-density encephalography (EEG) in a mouse model.

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a ...

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 ...

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. Lightweight recording devices carrying a high density array of tetrodes allow for the simultaneous monitoring of the activity of tens to hundreds of individual neurons. Here we describe a protocol for the fabrication of a micro-drive array with twenty one independently movable micro-drives. This device has been used successfully to record from hippocampal and cortical neurons in our lab. We show how to prepare a custom designed, 3-D printed plastic base that will hold the micro-drives. We demonstrate how to construct the individual micro-drives and how to assemble the complete micro-drive array. Further preparation of the drive array for surgical implantation, such as the fabrication of tetrodes, loading of tetrodes into the drive array and gold-plating, is covered in a subsequent video article.

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

In this protocol we demonstrate how to fabricate a micro-drive array for chronic electrophysiological recordings in rats.

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. ...

Biology

Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

The tetrode, a bundle of four electrodes, has proven to be a valuable tool for the simultaneous recording of multiple neurons in-vivo.  The differential amplitude of action potential signatures over the channels of a tetrode allows for the isolation of single-unit activity from multi-unit signals.  The ability to precisely control the stereotaxic location and depth of the tetrode is critical for studying coordinated neural activity across brain regions.  In combination with a micro-drive array, it is possible to achieve precise placement and stable control of many tetrodes over the course of days to weeks.  In this protocol, we demonstrate how to fabricate and condition tetrodes using basic tools and materials, install the tetrodes into a multi-drive tetrode array for chronic in-vivo recording in the rat, make ground wire connections to the micro-drive array, and attach a protective cone onto the micro-drive array in order to protect the tetrodes from physical contact with the environment.



Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

In this protocol we demonstrate how to fabricate and condition tetrodes for use with a micro-drive array, which was designed for chronic electrophysiological recordings in rats. In addition, we illustrate the final stages of micro-drive array construction, which includes installing ground wires and a protective cone.

The tetrode, a bundle of four electrodes, has proven to be a valuable tool for the simultaneous recording of multiple neurons in-vivo.  The differential ...

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic targeting for microcircuit dissection and disease modeling. The introduction of optogenetics, for example, has allowed for bidirectional manipulation of genetically-identified neurons, at an unprecedented temporal resolution. To capitalize on these tools and gain insight into dynamic interactions among brain microcircuits, it is essential that one has the ability to record from ensembles of neurons deep within the brain of this small rodent, in both head-fixed and freely behaving preparations. To record from deep structures and distinct cell layers requires a preparation that allows precise advancement of electrodes towards desired brain regions. To record neural ensembles, it is necessary that each electrode be independently movable, allowing the experimenter to resolve individual cells while leaving neighboring electrodes undisturbed. To do both in a freely behaving mouse requires an electrode drive that is lightweight, resilient, and highly customizable for targeting specific brain structures.
A technique for designing and fabricating miniature, ultralight weight, microdrive electrode arrays that are individually customizable and easily assembled from commercially available parts is presented. These devices are easily scalable and can be customized to the structure being targeted; it has been used successfully to record from thalamic and cortical regions in a freely behaving animal during natural behavior.

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic ...

Construction of an Improved Multi-Tetrode Hyperdrive for Large-Scale Neural Recording in Behaving Rats

Monitoring the activity patterns of a large population of neurons over many days in awake animals is a valuable technique in the field of systems neuroscience. One key component of this technique consists of the precise placement of multiple electrodes into desired brain regions and the maintenance of their stability. Here, we describe a protocol for the construction of a 3D-printable hyperdrive, which includes eighteen independently adjustable tetrodes, and is specifically designed for in vivo extracellular neural recording in freely behaving rats. The tetrodes attached to the microdrives can either be individually advanced into multiple brain regions along the track, or can be used to place an array of electrodes into a smaller area. The multiple tetrodes allow for simultaneous examination of action potentials from dozens of individual neurons, as well as local field potentials from populations of neurons in the brain during active behavior. In addition, the design provides for simpler 3D drafting software that can easily be modified for differing experimental needs.

We present the construction of a 3D-printable hyperdrive with eighteen independently adjustable tetrodes. The hyperdrive is designed to record brain activity in freely behaving rats over a period of several weeks.

Monitoring the activity patterns of a large population of neurons over many days in awake animals is a valuable technique in the field of systems ...

Extracellular Electrophysiological Recording in the Motor Cortex of Mice

Multichannel Extracellular Recording in Freely Moving Mice

The protocol aims to uncover the properties of neuronal firing and network local field potentials (LFPs) in behaving mice carrying out specific tasks by correlating the electrophysiological signals with spontaneous and/or specific behavior. This technique represents a valuable tool in studying the neuronal network activity underlying these behaviors. The article provides a detailed and complete procedure for electrode implantation and consequent extracellular recording in free-moving conscious mice. The study includes a detailed method for implanting the microelectrode arrays, capturing the LFP and neuronal spiking signals in the motor cortex (MC) using a multichannel system, and the subsequent offline data analysis. The advantage of multichannel recording in conscious animals is that a greater number of spiking neurons and neuronal subtypes can be obtained and compared, which allows the evaluation of the relationship between a specific behavior and the associated electrophysiological signals. Notably, the multichannel extracellular recording technique and the data analysis procedure described in the present study can be applied to other brain areas when conducting experiments in behaving mice.

Author Spotlight: Advancements in Multichannel Extracellular Recording for Studying Neuronal Activity in Freely Moving Mice 

The protocol describes the methodology of extracellular recording in the motor cortex (MC) to reveal extracellular electrophysiological properties in freely moving conscious mice, as well as the data analysis of local field potentials (LFPs) and spikes, which is useful for evaluating the network neural activity underlying behaviors of interest.

The protocol aims to uncover the properties of neuronal firing and network local field potentials (LFPs) in behaving mice carrying out specific tasks by ...

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 ...

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. ...

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

Summary

Explore More Videos

Construction of Microdrive Arrays for Chronic Neural Recordings in Awake Behaving Mice

High-density EEG Recordings of the Freely Moving Mice using Polyimide-based Microelectrode

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

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

Construction of an Improved Multi-Tetrode Hyperdrive for Large-Scale Neural Recording in Behaving Rats

Multichannel Extracellular Recording in Freely Moving Mice

A Lightweight Drive Implant for Chronic Tetrode Recordings in Juvenile Mice

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