We thank the Moser Lab at the Kavli Institute for Systems Neuroscience and Centre for Neural Computation, Norwegian University of Science and Technology, for the chronic neural recording procedures in rats. This work was supported by NIH grant R21 NS098146, and Human Frontier Science Program Long-Term Fellowship LT000211/2016-L to L. Lu.

1. Stereolithography of 3D Models
<ol>
	<li>Use stereolithographic techniques to print the hyperdrive parts and accessories. Each hyperdrive is comprised of eighteen shuttles, eighteen shuttle bolts, and one each of all other plastic pieces (Figure 1). 
	NOTE: The accessories are not part of the hyperdrive but are necessary for hyperdrive construction.</li>
</ol>
2. Preparation of Accessories (Figure 2).
<ol>
	<li>Preparation of the microdrive rack (Figure 2C).
	<ol>
		<li>Clean and expand the smaller through-holes and the larger blind-holes in the rack with a &#248; 0.71 mm (0.028&#34;) drill bit and a &#248; 0.84 mm (0.033&#34;) drill bit, respectively.</li>
		<li>Cut a &#248; 0.89 mm (0.035&#34;) welding rod into 17 mm long segments, round both ends, and insert each guide rod into the &#248; 0.84 mm (0.033&#34;) holes on the rack, leaving 11.5 mm outside (flush with the threaded rods).</li>
		<li>Fully insert six 0-80 threaded, 15.88 mm (5/8&#34;) long flat head screws down into the slots in the rack. Ensure that the guide rods and threaded rods are straight and parallel to one another. Fill the remaining space in the slots with dilute dental cement. Air dry on a benchtop for 15 min.</li>
		<li>Glue the welding rods and screws into the rack with thin super glue and allow to air dry for 15 min.</li>
	</ol>
	</li>
	<li>Preparation of the core station (Figure 2E) .
	<ol>
		<li>Thread the four holes with a 2-56 tap, and use 2-56, 4.76 mm (3/16&#34;) long nylon screws to secure the core in the station, if necessary.</li>
	</ol>
	</li>
	<li>Preparation of the turning tool (Figure 2F) .
	<ol>
		<li>Thread the hole on the handle with a 4-40 tap. Insert the machined tip into the slot in the handle and secure with a 4-40, 4.76 mm (3/16&#34;) long cup screw.</li>
	</ol>
	</li>
	<li>Preparation of the hyperdrive holder (Figure 2G) .
	<ol>
		<li>Thread the screw hole with an 8-32 tap. Use an 8-32, 9.52 mm (3/8&#34;) long nylon thumb screw to secure the hyperdrive when in use.</li>
	</ol>
	</li>
	<li>Preparation of the rod positioning complex (Figure 2H).
	<ol>
		<li>Thread the stem from the side with the larger hole (top) with an 8-32 tap to a depth of about 7 mm. Thread the smaller holes (six in the top, eighteen in the bottom) with a 0-80 tap. Expand the central hole in the top with a &#248; 4.76 mm (3/16&#34;) drill bit, if necessary.</li>
		<li>Assemble the stem to the top, using an 8-32, &#248; 4.76 mm (3/16&#34;), 6.35 mm (1/4&#34;) long shoulder screw. Secure the bottom to the top with 0-80, 6.35 mm (1/4&#34;) long screws when in use.</li>
	</ol>
	</li>
</ol>
3. Preparation of the Hyperdrive Components (Figure 3).
<ol>
	<li>Preparation of the hyperdrive nut (Figure 3A).
	<ol>
		<li>Using the nut holder (Figure 2D), thread the nut with a 3/8-24 bottoming tap until smooth.</li>
	</ol>
	</li>
	<li>Assembly of the hyperdrive core (Figure 3B).
	<ol>
		<li>Clean and expand the holes in the core using different sized drill bits (twelve ground wire through-holes (inner ring):&#248;&#160;0.61 mm (0.024&#34;); the eighteen tetrode through-holes (middle ring): &#248; 0.66 mm (0.026&#34;) first, and then &#248; 0.71 mm (0.028&#34;); the eighteen guide rod blind-holes (outer ring): &#248; 0.84 mm (0.033&#34;)).</li>
		<li>Thread the two through-holes on top of the core and the remaining eight blind-holes (four on the side, four near the bottom) with a 0-80 tap. Use a bottoming tap for the blind-holes.</li>
		<li>Create external threads at the base of the core using a 3/8-24 die. Adjust the die properly so the hyperdrive nut will fit over the new threads.</li>
		<li>Depending on the number of ground wires desired, insert multiple 6 mm long segments of 23-gauge metal tubing (cannulas) into the ground wire holes in the core, gluing them if necessary. File the&#160;ends of the ground wire cannulas until flush with the outside of the core, and clean the cannulas with a &#248; 0.30 mm (0.012&#34;) steel wire.</li>
		<li>Fully insert eighteen 0-80, 15.88 mm (5/8&#34;) long flat head screws head down into the slots in the core. Do not bend the screws or damage the threads during this process.</li>
		<li>Using the rod positioning complex and the core station, position eighteen 17 mm segments of &#248; 0.89 mm (0.035&#34;) welding rod over the guide rod holes in the core and hammer them down to be flush with the screws (about 5 mm).</li>
		<li>Correct the positions of the welding rods and screws if necessary, then tighten the central shoulder screw and the surrounding six screws in the rod positioning complex to secure the outward directions of the rods in the core. Screw the nut onto the core (with the rod positioning complex) and fit the core into the hyperdrive holder to allow easier positioning under a stereoscope.</li>
		<li>Fill the slots with dilute dental cement to secure the screws to the core and allow air drying for 15 min. Fill 2-3 slots at a time before the dental cement gets too thick. Scrape away any excess dental cement on the core to maintain a proper fit with the shield.</li>
		<li>Glue the screws and rods into the core with thin super glue, allow air drying for 15 min.</li>
	</ol>
	</li>
	<li>Assembly of the microdrive (Figure 3C) .
	<ol>
		<li>Clean and expand the two outer holes in the shuttle with drill bits (smaller hole: &#248; 0.61 mm (0.024&#34;) drill bit; larger hole: &#248; 0.89 mm (0.035&#34;) drill bit).</li>
		<li>Insert the shuttle bolt into the bolt holder base. Pay attention to the orientation. Close the bolt holder lid, hold tightly, and thread slowly through the hole in the lid with a 0-80 tap. Tap 2-3 times until smooth.</li>
		<li>Insert the shuttle bolt into the shuttle from the side with the smaller opening. Place the shuttle-shuttle bolt complex upside-down in the microdrive assembly station base.</li>
		<li>Cut a 15 mm segment of 23 gauge metal tubing and smooth both ends, then position the tubing over the &#248; 0.61 mm (0.024&#34;) hole, guided by the slot on the station Lid. Hammer the cannula into the hole until the upper end is flush with the station Lid.</li>
		<li>Remove the outer half of the upper tip of the cannula with a sanding wheel. Clean the cannula with a &#248; 0.30 mm (0.012&#34;) metal wire. Glue the cannula onto the shuttle using thin super glue, making sure not to glue the shuttle bolt to the shuttle, and air dry for 15 min.</li>
		<li>Prepare at least eighteen microdrives, test the microdrive on the microdrive rack. Make sure that the shuttle bolt can rotate smoothly in the shuttle and that the entire microdrive moves freely along the length of the threaded rod.</li>
	</ol>
	</li>
	<li>Preparation of the central column (Figure 3D).
	<ol>
		<li>Sand the top and bottom of the central column until flat, if needed. Thread the two holes in the central column with a 0-80 tap. Insert a 0-80 hex nut (3.18 mm (1/8&#34;) wide, 1.19 mm (3/64&#34;) high) into each slot.</li>
	</ol>
	</li>
	<li>Preparation of the hyperdrive cap (Figure 3E).
	<ol>
		<li>Using non-magnetic forceps, glue four magnets (3 mm in diameter, 1 mm thick) into the four wells, matching them to the N and S poles on the electrode interface board.</li>
	</ol>
	</li>
	<li>Assembly of the guide cannulas into a bundle (Figure 3F).
	<ol>
		<li>Place eighteen 30 gauge, thin wall cannulas (ID 0.19 mm,0.0075&#34;) into &#248; 2.29 mm (0.09&#34;) heat-shrink tubes (3-5 mm long, spaced apart along the bundle by 5-10 mm). Make all cannulas flush with one another on one end of the bundle.</li>
		<li>Shrink the heat-shrink tubes using a heat gun until the bundle is tight. Squeeze the bundle gently to shape it as desired (round or oval). Confirm that all cannulas are in the correct positions with no twisting, crossing, or bending.</li>
		<li>Mark the area(s) for soldering on the cannulas. The unsoldered portion should be 26 mm in length, while the soldered portion should be 5-10 mm. Move the shrinking tubes to the soldering marks to prevent spreading.</li>
		<li>Apply flux to one soldering area and solder while rotating the bundle. Cool at room temperature for at least 1 min. Repeat this step to solder the same area two more times. Smooth out the soldered portion by soldering without applying flux and filler material. Cool at room temperature for at least 1 min.</li>
		<li>Cut the bundle to the proper length with a diamond wheel at the highest speed, polish both ends to adjust the length (unsoldered part: 26 mm, soldered part: 5-10 mm as desired). Clean the guide cannulas with a &#248; 0.18 mm (0.007&#34;) metal wire under a stereoscope.</li>
	</ol>
	</li>
	<li>Preparing the tetrodes. Similar procedures have been described8,16,17 .
	<ol>
		<li>Adjust the height of the horizontal T bar and the position of the magnetic stirrer, so that the horizontal arm at the cross of the T bar is directly above the center of the magnetic stirrer. Hook one end of an S-hook to the center of a small magnetic stir bar, then glue them together. Clean the tetrode&#160;making space with compressed air and ethanol wipes.</li>
		<li>Circle the two ends of a piece of single tetrode wire around 40 cm in length together, then secure with a piece of copper tape.</li>
		<li>Lift the wire circle by holding the copper tape. Place the end opposite to the copper tape onto the horizontal arm of the T bar. Lower the copper tape gently (while the other end is still on the T bar), twist once, and place the copper tape onto the T&#160;bar. The tetrode circle is now in a figure eight (&#34;&#8734;&#34;) configuration with the copper tape&#160;sitting on top of the cross of the horizontal bar.</li>
		<li>Hold the copper tape on the T bar with one hand gently. With your other hand, hook the free end of the S-hook (with a magnetic stir attached to the other end) through the bottom of the tetrode wire circle, release the S-hook gently and let it straighten the four wires by the weight of the S hook.</li>
		<li>Adjust the height of the horizontal bar until the bottom of the S-hook is about 1 cm above the center of the magnetic stirrer plate.</li>
		<li>Bend the edge of the copper tape down to secure it to the horizontal bar. Examine the four straight tetrode wires by eye, then remove any debris.</li>
		<li>Turn on the stirrer twisting the four wires at a speed around 60 rpm, until the angle between the two opposite untwisted wires is about 60&#176;.</li>
		<li>Set the heat gun to 210 &#176;C, and heat the twisted wires by sweeping the gun along the straight length of the wires from different angles for 2 min to fuse them together by melting the VG bond coat.</li>
		<li>Lift the S-hook with stir gently and cut the lower end of the tetrode with fine scissors.</li>
		<li>Hold the copper tape on the horizontal bar with a finger, cut the wires from both edges of the copper tape with scissors, and remove the copper tape. Cut the remaining wire on the horizontal bar to release the tetrode.</li>
		<li>Place the completed tetrode in a dust-free box for storage. Prepare at least twenty-five tetrodes.</li>
	</ol>
	</li>
</ol>
4. Assembly of the hyperdrive (Figure 4).
<ol>
	<li>Inserting the guide cannulas into the hyperdrive core (Figure 4A).
	<ol>
		<li>Remove the heat-shrink tubes and slide a 4 mm segment of silicon tubing (ID 1.02 mm (0.04&#34;), OD 2.16 mm (0.085&#34;)) along the bundle to the soldered/unsoldered border. Wedge the slit in the hyperdrive spacer to widen the central hole, allowing the spacer to slip around the silicon tube. Remove the wedge when the spacer sits at the center of the silicon tube.</li>
		<li>Organize the positions of the guide cannulas in the bundle by placing long segments (10 cm) of the &#248; 0.18mm (0.007&#34;) metal wire through each cannula into a specific tetrode hole in the hyperdrive core, preventing any crossover of the wires or cannulas in the process. Bend the ends of the wires to hold them in place.</li>
		<li>Push the cannulas through their respective holes in the core, being careful to avoid bending or crossing between them, until the free end of each cannula is at least 2 mm outside the upper end of the tetrode hole. Secure the spacer by screwing the nut onto the core, being careful to prevent the spacer from rotating. Apply a drop of very dilute dental cement from the top of the core onto the junction of the cannulas to secure their relative positions.</li>
		<li>Cut the guide wires from the soldered end of the bundle, and remove them from the cannulas by retracting from the free end.</li>
	</ol>
	</li>
	<li>Assembly of the microdrives onto the hyperdrive Core (Figure 4B). A detailed spatial arrangement of the microdrives in the hyperdrive has been previously described11,13.
	<ol>
		<li>Load the microdrives slowly and carefully onto each threaded rod of the core. Confirm that (1) the 23 gauge microdrive cannula goes into the tetrode hole smoothly, (2) the 30 gauge guide cannula goes into the 23 gauge microdrive cannula smoothly, and (3) the shuttle bolt turns smoothly along the threaded rod. Screw the microdrives down to 1.0-1.5 mm above the lower end of the threaded rods.</li>
		<li>Cut eighteen pieces of polyimide tubing (ID 0.11 mm (0.0045&#34;), OD 0.14 mm (0.0055&#34;)) into 38-43 mm segments (length of the guide cannula bundle plus 7 mm). Clean each tube with a &#248; 0.08 mm (0.003&#34;) steel wire.</li>
		<li>Invert the core, insert the polyimide tubes carefully into the guide cannulas from the soldered end, and push them all the way in under a stereoscope. Flip the core upright and glue the upper end of the polyimide tube onto the microdrive cannula with thick super glue. Place the core upside-down and let the glue dry for 15 min.</li>
		<li>Cut the extra polyimide tubing at the upper end, leaving 0.5-1.0 mm outside of the microdrive cannula.</li>
	</ol>
	</li>
	<li>Assembly of the ground wires (Figure 4C).
	<ol>
		<li>Cut the number of ground wires necessary to lengths of 25-30 mm from coated steel wire (coated &#248; 0.20 mm (0.008&#34;), bare &#248; 0.13 mm (0.005&#34;)). Strip 2 mm of the plastic insulation from both tips of the wires and insert one end of each into the ends of&#160;6-8&#160;mm long 30 gauge cannulas. Flatten the&#160;ends of the cannulas to secure the connection to their respective wires.</li>
		<li>Use a Dremel tool to cut the cannulas in half to create two complete ground wires from each.</li>
		<li>Insert the round end of the 30 gauge cannula into the upper end of the ground wire cannula in the core and press to make the insertion tight.</li>
	</ol>
	</li>
	<li>Assembly of the electrode interface board (Figure 4D).
	<ol>
		<li>Insert the central column into the core and secure with two 0-80, 7.94 mm (5/16&#34;) long socket head screws. Glue if necessary to make the central column steady in the core.</li>
		<li>Expand the portions of the slots in the EIB-72-QC-Large board that correspond to the two tapped holes in the central column with a &#248; 1.2 mm tap. Attach the electrode interface board to the central column with two 0-80, 3.97 mm (5/32&#34;) long pan head screws. Make sure the board is situated in the center and is secure.</li>
	</ol>
	</li>
	<li>Connecting the ground wires (Figure 4E).
	<ol>
		<li>Route each ground wire around the central column and connect the exposed free end to the electrode interface board with a gold pin at the designated ground hole.</li>
	</ol>
	</li>
	<li>Loading the tetrodes into the hyperdrive, as previously described16,17 .
	<ol>
		<li>Load each tetrode carefully into the polyimide tubes of the microdrives, being careful not to bend them during the process.</li>
		<li>Gently feed the free end wires into their designated holes in the electrode interface board and electrically connect them using gold pins.</li>
		<li>Cut the tetrodes individually to a proper length. Confirm that the portion of tetrodes protruding from the lower ends of the polyimide tubes after cutting is straight, otherwise replace the entire tetrode and recut.</li>
	</ol>
	</li>
	<li>Attaching the shield.
	<ol>
		<li>Attach the shield to the core using four 0-80, 3.97 mm (5/32&#34;) pan head screws. The numbers on the shield should match with the numbers on the electrode interface board.</li>
	</ol>
	</li>
	<li>Plating the tetrode tips.
	<ol>
		<li>Plate the tips of the tetrodes using the NanoZ plating device equipped with an ADPT-NZ-EIB-36 connector and an ADPT-EIB-72-QC-HS-36 adaptor17. Alternatively, plate them manually one by one as described elsewhere16. Plate the tetrode tips prior to use (e.g., one day before implantation), as impedance will gradually increase over time after plating. Replace the tetrodes that are shorted or obstructed during the process of plating, cut them to a proper length, and re-plate.</li>
	</ol>
	</li>
	<li>Finalizing the hyperdrive (Figure 4F).
	<ol>
		<li>Glue the tetrodes to their polyimide tubes as previously described16. Retract all of them back into their guide cannulas so the plated tips are not exposed.</li>
		<li>Screw four 0-80, 6.35 mm (1/4&#34;) long socket head screws into the four holes near the bottom of the hyperdrive core.</li>
		<li>Using a stereoscope, lower each tetrode slowly until the tip of the tetrode is just above the edge of the guide cannula. Meanwhile, locate the position of each tetrode in the guide cannula bundle. The map of the tetrode&#39;s position is critical for reconstruction of recording sites.</li>
		<li>Attach the cap to the drive and store the hyperdrive properly for implantation.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>O'Keefe, J., Recce, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+relationship+between+hippocampal+place+units+and+the+EEG+theta+rhythm.">Phase relationship between hippocampal place units and the EEG theta rhythm.</a> Hippocampus. 3 (3), 317-330 (1993).</li><li>Wilson, M. A., McNaughton, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+hippocampal+ensemble+code+for+space.">Dynamics of the hippocampal ensemble code for space.</a> Science. 261 (5124), 1055-1058 (1993).</li><li>Gothard, K. M., Skaggs, W. E., Moore, K. M., McNaughton, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+hippocampal+CA1+neural+activity+to+multiple+reference+frames+in+a+landmark-based+navigation+task.">Binding of hippocampal CA1 neural activity to multiple reference frames in a landmark-based navigation task.</a> J Neurosci. 16 (2), 823-835 (1996).</li><li>Gray, C. M., Maldonado, P. E., Wilson, M., McNaughton, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrodes+markedly+improve+the+reliability+and+yield+of+multiple+single-unit+isolation+from+multi-unit+recordings+in+cat+striate+cortex.">Tetrodes markedly improve the reliability and yield of multiple single-unit isolation from multi-unit recordings in cat striate cortex.</a> J Neurosci Methods. 63 (1-2), 43-54 (1995).</li><li>Buzsaki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+recording+of+neuronal+ensembles.">Large-scale recording of neuronal ensembles.</a> Nat Neurosci. 7 (5), 446-451 (2004).</li><li>Fyhn, M., Hafting, T., Witter, M. P., Moser, E. I., Moser, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grid+cells+in+mice.">Grid cells in mice.</a> Hippocampus. 18 (12), 1230-1238 (2008).</li><li>Skaggs, W. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EEG+sharp+waves+and+sparse+ensemble+unit+activity+in+the+macaque+hippocampus.">EEG sharp waves and sparse ensemble unit activity in the macaque hippocampus.</a> J Neurophysiol. 98 (2), 898-910 (2007).</li><li>Guo, P., Pollack, A. J., Varga, A. G., Martin, J. P., Ritzmann, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+wire+tetrode+recording+in+brain+of+freely+walking+insects.">Extracellular wire tetrode recording in brain of freely walking insects.</a> J Vis Exp. (86), (2014).</li><li>Knierim, J. J., McNaughton, B. L., Poe, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+spatial+selectivity+of+hippocampal+neurons+during+space+flight.">Three-dimensional spatial selectivity of hippocampal neurons during space flight.</a> Nat Neurosci. 3 (3), 209-210 (2000).</li><li>Leutgeb, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+codes+for+spatial+and+episodic+memory+in+hippocampal+neuronal+ensembles.">Independent codes for spatial and episodic memory in hippocampal neuronal ensembles.</a> Science. 309 (5734), 619-623 (2005).</li><li>Lansink, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+split+microdrive+for+simultaneous+multi-electrode+recordings+from+two+brain+areas+in+awake+small+animals.">A split microdrive for simultaneous multi-electrode recordings from two brain areas in awake small animals.</a> J Neurosci Methods. 162 (1-2), 129-138 (2007).</li><li>Kloosterman, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-drive+array+for+chronic+in+vivo+recording:+drive+fabrication.">Micro-drive array for chronic in vivo recording: drive fabrication.</a> J Vis Exp. (26), (2009).</li><li>. Google Patents Available from: <a target="_blank" href="https://www.google.com/patents/US5928143">https://www.google.com/patents/US5928143</a> (1999)</li><li>Redish, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independence+of+firing+correlates+of+anatomically+proximate+hippocampal+pyramidal+cells.">Independence of firing correlates of anatomically proximate hippocampal pyramidal cells.</a> J Neurosci. 21 (5), RC134 (2001).</li><li>Schmitzer-Torbert, N., Redish, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+activity+in+the+rodent+dorsal+striatum+in+sequential+navigation:+separation+of+spatial+and+reward+responses+on+the+multiple+T+task.">Neuronal activity in the rodent dorsal striatum in sequential navigation: separation of spatial and reward responses on the multiple T task.</a> J Neurophysiol. 91 (5), 2259-2272 (2004).</li><li>Nguyen, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-drive+array+for+chronic+in+vivo+recording:+tetrode+assembly.">Micro-drive array for chronic in vivo recording: tetrode assembly.</a> J Vis Exp. (26), (2009).</li><li>Chang, E. H., Frattini, S. A., Robbiati, S., Huerta, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+microdrive+arrays+for+chronic+neural+recordings+in+awake+behaving+mice.">Construction of microdrive arrays for chronic neural recordings in awake behaving mice.</a> J Vis Exp. (77), e50470 (2013).</li><li>Vandecasteele, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+recording+of+neurons+by+movable+silicon+probes+in+behaving+rodents.">Large-scale recording of neurons by movable silicon probes in behaving rodents.</a> J Vis Exp. (61), e3568 (2012).</li><li>Siegle, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronically+implanted+hyperdrive+for+cortical+recording+and+optogenetic+control+in+behaving+mice.">Chronically implanted hyperdrive for cortical recording and optogenetic control in behaving mice.</a> Conf Proc IEEE Eng Med Biol Soc. 2011, 7529-7532 (2011).</li><li>Brunetti, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+fabrication+of+ultralight+weight,+adjustable+multi-electrode+probes+for+electrophysiological+recordings+in+mice.">Design and fabrication of ultralight weight, adjustable multi-electrode probes for electrophysiological recordings in mice.</a> J Vis Exp. (91), e51675 (2014).</li><li>. Google Patents Available from: <a target="_blank" href="https://www.google.com/patents/US4575330">https://www.google.com/patents/US4575330</a> (1986)</li><li>Ludvig, N., Potter, P. E., Fox, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+single-cell+recording+and+microdialysis+within+the+same+brain+site+in+freely+behaving+rats:+a+novel+neurobiological+method.">Simultaneous single-cell recording and microdialysis within the same brain site in freely behaving rats: a novel neurobiological method.</a> J Neurosci Methods. 55 (1), 31-40 (1994).</li></ol>

The authors have nothing to disclose.

Here, we describe the process of constructing a newly developed hyperdrive comprised of eighteen independently movable tetrodes. The drive can be constructed from affordable parts purchased at many available hardware stores, combined with components created by stereolithographic printing. The hyperdrive can be chronically implanted onto a rat&#39;s skull using standard surgical procedures and is capable of recording extracellular neural activity while the animal performs various behavioral tasks.
<p class="jove_conte...

In the field of systems neuroscience, scientists study the neural correlates underlying cognitive processes such as spatial navigation, memory, and decision-making. For these types of studies, it is critical to monitor the activity of many individual neurons during animal behavior. Over the past decades, two important advances have been made to meet the experimental needs for extracellular neural recording in small animals1,2,3. First was the development of the tetrode, a bundle of four microwires used to record neural activity of neurons simultaneously1,2,4. The differential signal amplitudes of activity across the four channels of a tetrode allows for the isolation of individual neuron activity from many simultaneously recorded cells5. In addition, the flexible nature of the microwires allows greater stability of the tetrode minimizing the relative displacement between the tetrode and the target cell population. Tetrodes are now widely used instead of a single electrode for many brain studies in various species, including rodents1,2,6, primates7, and insects8. Second was the development of a hyperdrive carrying multiple independently movable tetrodes, which allows for the simultaneous monitoring of neural activity from larger populations of neurons from multiple recording locations3,9,10,11,12.
The availability of a reliable and affordable multi-tetrode recording device for small animals is limited. The classic hyperdrive, initially developed by Bruce McNaughton13, has been successfully used for neural recordings in freely behaving rats in many labs in the past two decades9,10,14,15. However, for technical reasons, the original components needed to build the McNaughton drive are now very difficult to obtain and are not compatible with recently improved data acquisition interfaces. The other well accepted design of hyperdrive requires the microdrives to be individually handcrafted, which could yield inconsistent results and consume substantial time12. In order to record neural activity from various brain regions in behaving rats, we developed a new hyperdrive using stereolithographic technology. We sought to satisfy the following requirements: (1) the new hyperdrive must allow precise displacement of tetrodes in the brain and provide stable recording from multiple target regions; (2) the new hyperdrive must be compatible with the magnetic quickclip system recently developed to allow easy connection; and (3) the new hyperdrive can be accurately reproduced with materials easily available. Here, we provide a technique for building the 3D-printable hyperdrive containing eighteen independently movable tetrodes, based upon the McNaughton design. In the protocol, we describe the details of the fabrication process of the new hyperdrive, which we have used successfully to record single-neuron action potentials and local field potentials from the postrhinal and medial entorhinal cortices over weeks in a freely behaving rat during natural foraging tasks.

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			<td>New England Small Tube</td>
			<td>Custom-made</td>
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			<td>A-M Systems</td>
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			<td>A-M Systems</td>
			<td>791500</td>
			<td>0.005&#34; bare, 0.008&#34; coated, half hard</td>
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			<td>California Fine Wire</td>
			<td>Custom-made</td>
			<td>0.0007&#34; in diameter, platinum-iridium (90%-10%), HML and VG coating</td>
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			<td>Tap</td>
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			<td>McMaster</td>
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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 used a newly built hyperdrive to obtain trial results. The drive was equipped with tetrodes constructed from &#248; 17 &#181;m (0.0007&#34;), polyimide-coated platinum-iridium (90%-10%) wire. The tips of the tetrodes were plated in platinum black solution to reduce electrode impedances to between 100 and 200 k&#937; at 1 kHz. The hyperdrive was implanted 4.6 mm left of the midline and 0.5 mm anterior to the transverse sinus on the skull of a 550 g, male Long-Evans rat. Additional groun...

Watch this Scientific Journal Video about Construction of an Improved Multi-Tetrode Hyperdrive for Large-Scale Neural Recording in Behaving Rats at JoVE.com

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

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 ...

construction-an-improved-multi-tetrode-hyperdrive-for-large-scale

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JoVE Journal

Neuroscience

11.2K Views.  Baylor College of Medicine. 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.

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

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

Design and Construction of a Cost Effective Headstage for Simultaneous Neural Stimulation and Recording in the Water Maze

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial products are often expensive, not easily customized, and not submersible. Here we describe a method to design and build a customized, integrated circuit headstage for simultaneous 4-channel neural recording and 2-channel simulation in awake, behaving animals. The headstage is designed using a free, commercially available CAD-type design package, and can be modified easily to accommodate different scales (e.g. to add channels). A customized printed circuit board is built using surface mount resistors, capacitors and operational amplifiers to construct the unity gain source follower circuit. The headstage is made water-proof with a combination of epoxy, parafilm and a synthetic rubber putty. We have successfully used this device to record local field potentials and stimulate different brain regions simultaneously via independent channels in rats swimming in a water maze. The total cost is &lt; $30/unit and can be manufactured readily.

We present a low-cost method to design and construct a light headstage pre-amplifier system with simultaneous neural recording and stimulation capability. This device can be waterproofed for use in swimming animals.

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial ...

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

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

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

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

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

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

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

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

Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers

It is well known that anesthesia alters neural response properties in various regions of the brain.13. In the auditory system, fundamental response properties of brainstem neurons including threshold, frequency specificity, and inhibitory sidebands are altered in significant ways under anesthesia1-2. These observations prompted physiologists to seek ways to record from single neurons without the contaminating effects of anesthesia. One result was a decerebrate preparation, where the brainstem was completely transected at the level of the midbrain4. The drawbacks of this preparation are a formidable surgery, the elimination of descending projections from the forebrain, and an inability to use sensory stimulation to examine structures above the midbrain. A different strategy has been to implant electrode arrays chronically to record from single neurons and multiunit clusters while the animal is awake and/or behaving5,6. These techniques however are not compatible with injecting tracer dyes after first electrophysiologically characterizing a brain structure. To avoid altering neural response properties with anesthetics while recording electrophysiological response properties from single neurons, we have adapted a head restraint technique long used in bats7-9 to mouse10-12. Using this method, we are able to conduct electrophysiological recordings over several days in the unanesthetized mouse. At the end of the recording sessions, we can then inject a dye to reconstruct electrode positions and recording sites or inject a tracer so that pathways to and from the recording loci can be determined. This method allows for well isolated single neuron recordings over multiple days without the use anesthetics.

Electrophysiological characterization of neuronal responses is important for understanding brain function and for guiding the placement of dyes for pathway tracing. However, many studies are performed in anesthetized animals. To understand brain function without anesthetics, we developed a method to record neuronal response properties and inject dyes in awake mouse.

It is well known that anesthesia alters neural response properties in various regions of the brain.13.  In the auditory system, fundamental response ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Neural Circuit Recording from an Intact Cockroach Nervous System

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.

This article describes the cockroach ventral nerve cord dissection and extracellular recordings from the cercal nerve and connectives. Evoked responses are generated by electrical stimulation of the cercal nerve or direct mechanical stimulation of the cerci.

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

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

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

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

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.