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&#232;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 &#181;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 &#181;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...

Watch this Scientific Journal Video about Hybrid Microdrive System with Recoverable Opto-Silicon Probe and Tetrode for Dual-Site High Density Recording in Freely Moving Mice at JoVE.com

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

10.9K 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

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 ...

Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed single-neuron activity while animals are acquiring an operantly conditioned response, or when that response is extinguished. But even in these cases, observation periods usually encompass only a single stage of learning, i.e. acquisition or extinction, but not both (exceptions include protocols employing reversal learning; see Bingman&#160;et al.1 for an example). However, acquisition and extinction entail different learning mechanisms and are therefore expected to be accompanied by different types and/or loci of neural plasticity.
Accordingly, we developed a behavioral paradigm which institutes three stages of learning in a single behavioral session and which is well suited for the simultaneous recording of single neurons&#39; action potentials. Animals are trained on a single-interval forced choice task which requires mapping each of two possible choice responses to the presentation of different novel visual stimuli (acquisition). After having reached a predefined performance criterion, one of the two choice responses is no longer reinforced (extinction). Following a certain decrement in performance level, correct responses are reinforced again (reacquisition). By using a new set of stimuli in every session, animals can undergo the acquisition-extinction-reacquisition process repeatedly. Because all three stages of learning occur in a single behavioral session, the paradigm is ideal for the simultaneous observation of the spiking output of multiple single neurons. We use pigeons as model systems, but the task can easily be adapted to any other species capable of conditioned discrimination learning.

Recording Single Neurons&#039; Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

Learning new stimulus-response associations engages a wide range of neural processes which are ultimately reflected in changing spike output of individual neurons. Here we describe a behavioral protocol allowing for the continuous registration of single-neuron activity while animals acquire, extinguish, and reacquire a conditioned response within a single experimental session.

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed ...

Extracellular Wire Tetrode Recording in Brain of Freely Walking Insects

Increasing interest in the role of brain activity in insect motor control requires that we be able to monitor neural activity while insects perform natural behavior. We previously developed a technique for implanting tetrode wires into the central complex of cockroach brains that allowed us to record activity from multiple neurons simultaneously while a tethered cockroach turned or altered walking speed. While a major advance, tethered preparations provide access to limited behaviors and often lack feedback processes that occur in freely moving animals. We now present a modified version of that technique that allows us to record from the central complex of freely moving cockroaches as they walk in an arena and deal with barriers by turning, climbing or tunneling. Coupled with high speed video and cluster cutting, we can now relate brain activity to various parameters of the movement of freely behaving insects.

We previously developed a technique for implanting tetrode wires into the central complex of cockroach brains that allows us to monitor activity in individual units of tethered cockroaches. Here we present a modified version of that technique that allows us to also record brain activity in freely moving insects.

Increasing interest in the role of brain activity in insect motor control requires that we be able to monitor neural activity while insects perform ...

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle (Mercynorrhina torquata)

The rise of radio-enabled digital electronic devices has prompted the use of small wireless neuromuscular recorders and stimulators for studying in-flight insect behavior. This technology enables the development of an insect-machine hybrid system using a living insect platform described in this protocol. Moreover, this protocol presents the system configuration and free flight experimental procedures for evaluating the function of the flight muscles in an untethered insect. For demonstration, we targeted the third axillary sclerite (3Ax) muscle to control and achieve left or right turning of a flying beetle. A thin silver wire electrode was implanted on the 3Ax muscle on each side of the beetle. These were connected to the outputs of a wireless backpack (i.e., a neuromuscular electrical stimulator) mounted on the pronotum of the beetle. The muscle was stimulated in free flight by alternating the stimulation side (left or right) or varying the stimulation frequency. The beetle turned to the ipsilateral side when the muscle was stimulated and exhibited a graded response to an increasing frequency. The implantation process and volume calibration of the 3 dimensional motion capture camera system need to be carried out with care to avoid damaging the muscle and losing track of the marker, respectively. This method is highly beneficial to study insect flight, as it helps to reveal the functions of the flight muscle of interest in free flight.

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle Mercynorrhina torquata

This protocol describes the process of constructing an insect-machine hybrid system and carrying out wireless electrical stimulation of the flight muscles required to control the turning motion of a flying insect.

The rise of radio-enabled digital electronic devices has prompted the use of small wireless neuromuscular recorders and stimulators for studying in-flight ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and smaller functional subgroups, it becomes paramount to record from projections and/or genetically-defined subpopulations of neurons. Fiber photometry is an accessible and powerful approach that can overcome these challenges. By combining optical and genetic methodologies, neural activity can be measured in deep brain structures by expressing genetically-encoded calcium indicators, which translate neural activity into an optical signal that can be easily measured. The current protocol details the components of a multi-fiber photometry system, how to access deep brain structures to deliver and collect light, a method to account for motion artifacts, and how to process and analyze fluorescent signals. The protocol details experimental considerations when performing single and dual color imaging, from either single or multiple implanted optic fibers.

This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and ...

Intracerebroventricular Delivery of Gut-Derived Microbial Metabolites in Freely Moving Mice

The impact of gut microbiota and their metabolites on host physiology and behavior has been extensively investigated in this decade. Numerous studies have revealed that gut microbiota-derived metabolites modulate brain-mediated physiological functions through intricate gut-brain pathways in the host. Short-chain fatty acids (SCFAs) are the major bacteria-derived metabolites produced during dietary fiber fermentation by the gut microbiome. Secreted SCFAs from the gut can act at multiple sites in the periphery, affecting the immune, endocrine, and neural responses due to the vast distribution of SCFAs receptors. Therefore, it is challenging to differentiate the central and peripheral effects of SCFAs through oral and intraperitoneal administration of SCFAs. This paper presents a video-based method to interrogate the functional role of SCFAs in the brain via a guide cannula in freely moving mice. The amount and type of SCFAs in the brain can be adjusted by controlling the infusion volume and rate. This method can provide scientists with a way to appreciate the role of gut-derived metabolites in the brain.

Gut-derived microbial metabolites have multifaceted effects leading to complex behavior in animals. We aim to provide a step-by-step method to delineate the effects of gut-derived microbial metabolites in the brain via intracerebroventricular delivery via a guide cannula.

The impact of gut microbiota and their metabolites on host physiology and behavior has been extensively investigated in this decade. Numerous studies have ...

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 ...

Multichannel Recording in the Bilateral MC in Free-Moving Mice and Neurophysiological Data Analysis

Multilayer Imaging with Microprism in Head-Fixed and Freely Moving Animals

Long-Term Imaging of Identified Neural Populations using Microprisms in Freely Moving and Head-Fixed Animals

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for studying the structure, function, and plasticity of living brain tissues. In comparison to conventional electrophysiology, fluorescence microscopy can capture the neural activity as well as the morphology of the cells, enabling long-term recordings of the identified neuron populations at single-cell or subcellular resolution. However, high-resolution imaging typically requires a stable, head-fixed setup that restricts the movement of the animal, and the preparation of a flat surface of transparent glass allows visualization of neurons at one or more horizontal planes but is limited in studying the vertical processes running across different depths. Here, we describe a procedure to combine a head plate fixation and a microprism that gives multilayer and multimodal imaging. This surgical preparation not only gives access to the entire column of the mouse visual cortex but allows two-photon imaging in a head-fixed position and one-photon imaging in a freely moving paradigm. Using this approach, one can sample identified cell populations across different cortical layers, register their responses under head-fixed and freely moving states, and track the long-term changes over months. Thus, this method provides a comprehensive assay of the microcircuits, enabling direct comparison of neural activities evoked by well-controlled stimuli and under a natural behavioral paradigm.

Author Spotlight: Comparative Imaging of Neural Activity in Awake and Freely Moving States

When integrated with a head-plate and an optical design compatible with both single- and two-photon microscopes, the microprism lens presents a significant advantage in measuring neural responses in a vertical column under diverse conditions, including well-controlled experiments in head-fixed states or natural behavioral tasks in freely moving animals.

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for ...