J. S. Pezaris, M. Sahani, and R. A. AndersenNew and Improved Tetrodes for MonkeysCalifornia Institute of TechnologyComputation and Neural Systems Technical Report TR-98-03December, 1998

NEW AND IMPROVED

TETRODES FOR MONKEYS*

John S. Pezaris

Maneesh Sahani

Richard A. Andersen

Computation and Neural Systems

California Institute of Technology

Mail Code 216-76

Pasadena, CA 91125, U.S.A.

Abstract

We have adapted the Recce-O’Keefe tetrode for use in monkey cortex. Our designhas been in use for three years in our laboratory, and has proven capable and robust.Details on construction of tetrodes, carrier tubes, and jigs are given, including sources formaterials. Example recordings show excellent isolation and histologically verified tracksare straight at up to 12 millimeters of tetrode extension.

Introduction

Here we report on adapting Recce-O’Keefe tetrodes (Recce and O’Keefe, 1989) to recordfrom monkey cortex, and to detail the parameters and techniques used in that process.The design of our tetrodes is presented, including specific construction details, followed bythe description of experiments performed to measure their characteristics, and discussionof the results.

Tetrodes and Spheres of Sensitivity

A tetrode is a bundle of four individually insulated fine wire electrodes whose tips liecloser together than their respective spheres of sensitivity. Neural recordings made with

∗ A preliminary version of this paper can be found in Computational Neuroscience: Trends in Research1997, J. M. Bower (ed), Plenum Press, New York, 1997, pp. 937–942.

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Figure 1: Tetrode Mk II Rev F, photographAn assembled and loaded tetrode carrier.

traditional (single-wire) electrodes are similar to monophonic musical recordings, just astetrode recordings are similar to quadraphonic ones: the multi-channel recordings con-tain spatial information that the single-channel recordings lack. A neuron that lies inthe overlap of two or more of these spheres is detected by two or more electrodes. As-symetries, whether in electrode construction or relative geometry, insure that differentelectrodes record this neural signal through slightly different filters. The cross-channelcomparison allows the disambiguation of signals that appear identical to a single elec-trode.

It can be argued that since four non-coplanar voltage measurements are sufficient touniquely locate sources in 3-space, four wires are therefore sufficient for a multiwireneural electrode to uniquely identify neurons in the brain. This, however, relies uponthe tacit assumption that the brain is a noiseless, homogeneous (or at least isotropic),linear, non-dissipative medium populated with point sources; while there is some evidencefor linearity (Wehr, et al., 1998), it is certainly not noiseless, homogeneous, nor non-dissipative, and neurons are not point sources. Four wires have been used in our multiwireelectrodes merely as a matter of tradition, and we therefore adhere to the establishednomenclature and call them tetrodes.

Animal Preparation

Previous reports describing tetrodes have been concerned with rat (Wilson and Mc-Naughton, 1993) or cat (Gray, et al, 1994) preparations. Two features of our awakemonkey preparation constrained our design. First, while a recording chamber is chron-ically implanted, the intracranial surface is exposed and electrodes are inserted andremoved during each recording session. Thus, the dura mater must remain intact and issubject to toughening. Second, the brain of the macaque is larger than those of eitherthe rat or the cat and profoundly gyrated in comparison. Thus even cortical areas canlie quite far from the exposed surface. A mechanism is therefore required to penetratethe dura and deliver the delicate tetrode wires to relatively deep neural structures, whileminimizing tissue insult.

Our laboratory currently uses commercially available traditional electrodes and hydraulicmicrodrives (FHC, Brunswick, Maine), and we have designed our tetrodes to use the same

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equipment wherever possible. Specifically, our design will mount directly into an FHCmicrodrive, and is manipulated much like a traditional electrode would be.

Methods

Introduction

Tetrodes consist of two parts, the bundle of four wires, often referred to as the tetrodeitself, and a telescoping guide tube through which the tetrode bundle is advanced. Con-struction of the two will be described in the following sections, including details for thejigs used in assembly. We will first discuss construction of the special tetrode guidetubes, followed by construction of the machine to wind wire into tetrodes, and finallyinstructions on winding and preparation of a tetrode.

Construction of Tetrode Carrier, Mk II Rev F

To protect the tetrode when not in use, to penetrate the dura, and to prevent bucklingas the bundle is advanced, a stainless-steel assembly called a carrier tube is used. Thisuber-guidetube, seen in Figures 1 and 2, is constructed from readily available parts.

Carrier tubes are difficult to build as the parts as small and lengths and positions mustbe kept accurate to ±0.2 mm. A parts list is found in Table 1. The following detailedinstructions assume that parts cut and machined to specification have been made oracquired. Exterior surfaces of lengths which will be glued (see Figure 2) should beroughed with 400 grit sandpaper. All lengths should be thoroughly cleaned (preferablyultrasonically) and dried.

Take a 10 mm length of 22.5◦ beveled 32 gauge cannula (C5) and slip the chamferedend into a 34 mm length of 26 gauge cannula (C4) to a depth of about 1 mm. Place asmall amount of freshly mixed epoxy at the junction and slide into place until 5 mm ofthe finer cannula remains visible. Additional epoxy may be required as the two are puttogether, however, be sure to remove any excess. Call this assembly A1. Leave to dry.

Take a 5 mm length of 26 gauge cannula (C3) and similarly glue into place 18 mmfrom the blunt end of the 54 mm length of 32 gauge cannula (C6). Bring the smallerlength almost into place before applying the epoxy to minimize any excess which mustbe removed. Call this assembly A2. Leave to dry.

Once A1 is dry, take a 49 mm length of 21 gauge cannula (C2) and glue into place 15 mmfrom the sharp end of A1. Call this new assembly A1′. Leave to dry.

Once A1′ is dry, insert A2 into A1′, beveled end outwards. Then, take a 5 mm length of26 gauge cannula (C3) and slide over the free end of A2 and glue to the outer cannula ofA1′ (the length of 21 gauge) so that the ends are flush. Leave to dry.

Once the epoxy has set, the carrier tube should telescope smoothly approximately 15 mmfrom stop to stop. The two parts should also rotate freely against each other. Theremaining step is to glue the free end of A2 to the clamping plate. To prevent gluecreeping into the hole during this process, seal it with a bit of bone wax or sealingwax. To align the carrier tube to the clamping plate, this last step is best done using amicrodrive as a jig. Insert the carrier into the guide tube hole and fix in place by lightly

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34

18105

5 520

50

55

Figure 2: Tetrode Carrier Mk II Rev F, sectionThe carrier tube punctures the dura directly, but is not advancedinto the brain. The inner assembly is advanced into the carrier,forcing the tetrode bundle, which has been clamped to it whenmounted in a microdrive, into the brain. All dimensions in mil-limeters. Red hatching is glue. Heavy orange lines are the tetrodebundle. The carrier is shown 1 mm from the closed position. Draw-ing not to scale (vertical dimensions exaggerated in portions).

item quantity description source part number

C1 1 clamping plate handmadeC2 1 21 ga, 50 mm longa Small Parts O-HTX-21-12e

C3 2 26 ga, 5 mm long Small Parts O-HTX-26-12e

C4 1 26 ga, 34 mm longa Small Parts O-HTX-25-12e

C5 1 32 ga, 20 mm longb Small Parts O-HTX-32-12e

C6 1 32 ga, 55 mm longc Small Parts O-HTX-32-12e

C7 1 connectord DigiKey S2231-36C8 misc quick-setting epoxyf Devcon S-208C9 misc soft waxC10 misc cellophane cooking wrap

Table 1: Tetrode Carrier Parts ListaOD chamfer one end. bID chamfer one end, 22.5◦ bevel cut other end. c45◦ bevel cut oneend. dCut 4 pins (2×2) out of overall length. eCannulae part numbers are for uncut 12-inchlengths; Small Parts will provide lengths cut and machined to specification at additional cost.fTrade name 5-Minute Epoxy.

tightening the microdrive set screw. Slide A2 until it extends 5 mm over the edge of themicrodrive clamping block, rotating it so that the bevel is towards the block. Slide abit of cooking wrap (cellophane) under the end of A2. Apply a small amount of glueto the 32 gauge cannula and screw the clamping plate down. Glue the 4-pin connectorto the facing side of the clamping plate in a transverse orientation (it may be necessaryto put a very small section of circuit board underneath the connector as a spacer) usingquick-setting epoxy. Leave to dry.

At removal, carefully peel off the plastic sheet, insert a reaming wire from the penetrating

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item quantity description source part number

T1 1 chemistry standT2 2 clamp (one machined)T3 1 68 RPM reversible AC motor C&H Sales 54191814001T4 1 7.5 µF 440 VAC capacitora C&H Sales CAP0835T5 1 DPDT center off 10 A switch C&H Sales SW9603T6 1 three-pronged power cord C&H Sales PFC8300T7 1 shaft couplingb

T8 1 5 cm × 5 cm blank PC boardT9 2 glass hooksc

T10 2 plastic tubingd

T11 misc wire, solder, electrical tape

Table 2: Tetrode Twister Parts ListaStarter capacitor; adjust to motor specifications. bTo fit output shaft of motor. cBend fromglass rod using a Bunsen burner. dSized to fit glass rod; 5–10 cm long.

end and work out the wax sealing the opposite end. Thoroughly clean and lubricatebefore use (see below).

Note that because the glue used in construction is non-conductive, the outermost sectionof the carrier (the 21 gauge cannula of A1) may not be in electrical contact with theinnermost (the 32 gauge cannula of A1), and therefore may not be in contact with tissuewhen in use. It is advantageous to ground this cannula from a shielding standpoint, butit should not be used as a source of ground unless additional steps are taken, such asusing a conductive epoxy for assembly or filling the recording chamber with saline duringuse. Our initial experience with conductive epoxies has been disappointing, and giventheir excessive cost, it seems prudent to use non-conductive glue and assume the lengthsof cannulae are all isolated.

Carrier Tube Cleaning and Lubrication

Carrier tubes are cleaned in four steps by repeated reaming first with music wire wettedwith acetone, then dry wire, then wire wetted with ispropyl, and again dry wire (this isdone with the same length of wire). They are then lubricated by reaming with wire wettedwith silicone oil. This should be done before the first use, and every time the tetrodebundle is replaced. Oil-filling the carrier tube is important to increase construction yield,reduce the chances of electrode kinking during use, and prevent wicking of physiologicalfluid up the carrier.

Tetrode Twister

Tetrodes are built by winding fine wire on a motorized jig called a tetrode twister. Ourtwister was constructed out of readily-available inexpensive parts and provides sufficientflexibility for nearly any taste. The twister (see Figure 3) has two glass hooks, onesuspended some 15 cm above the other, arranged on a chemistry stand. The upper hookis clamped to the stand, and therefore adjustable in height but normally remains fixed;the lower hook is attached to the output shaft of an electrical motor, and can be rotatedabout the vertical axis in either direction. Parts required for construction are listed inTable 1. Some machining of the lower clamp is required, and the capacitor and switchmust be glued to the motor along with a guard.

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Figure 3: Tetrode TwisterThis jig is used to twist tungsten wire into tetrodes. Wire is loopedbetween the two glass hooks, held in place with adhesive tape, andthen twisted when the motor is engaged. Forward and reversedirections are by convention only.

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Twisting a Tetrode

Twisting a tetrode is a straightforward but delicate operation. It must be done with cleanhands. First, place a 5 cm strip of adhesive tape on the motor from the rear, leaving theends free. Draw a 60 cm length of tungsten wire and cut with fine surgical scissors (usean inverted glass funnel as a low-friction bearing for the spool of wire). Place one endof the wire against the adhesive of one end of the tape and press the tape against themotor shaft. Carefully wrap the wire from the lower hook to the upper hook, makingtwo loops. Bring the wire just barely taught, and wind 4 or 5 times just above the lowerhook before fixing it to the shaft with the other end of the tape. Engage the motor inthe forward direction for approximately 65 turns. After winding, evenly pass a heat gun(FIT GUN 3, Alpha Wire; set to high) over the wire at a distance of 5–10 cm taking 5seconds for each of two passes. Carefully cut the tetrode free from the bottom (it mayhelp to hold the bundle just above the cut point with a pair of plastic-coated forceps,releasing the tension slowly after the cut). A properly twisted tetrode is dead straight.Once wound, take care not to bend or kink the tetrode bundle; a number 10 envelopemakes a nice storage container.

Stripping

Electrical connections are made to the looped end of the bundle (as it comes from thetwister). With a pair of very sharp scissors, cut the loops and trim the wires even. Placethe tetrode on a pad of paper, and cover the final 3–5 mm of wire at the (now cut)loop ends with chemical strip (Strip-X, GC Electronics) for 20–30 minutes. Gently pullthe wire out of the chemical strip, discard the upper sheet of paper and lay the tetrodebundle back on the pad. Take one cotton swab and use it to hold the bundle against thepad (press the cotton bud quite hard against the wire about 1 cm below the strippedarea); take another cotton swab, dip it in isopropyl and wipe any remaining chemicalstrip off the wire. Finally, check the bundle under a low-power microscope to insure theinsulation has been properly stripped.

Loading

Loading a tetrode bundle into the carrier should be done under low-power magnifica-tion (4× to 8×) by someone with steady hands. Caffeine use is discouraged. Grasp therecording end of the bundle with a pair of plastic-coated forceps and approach the clamp-ing plate end of the carrier. Carefully, and repeatedly, attempt insertion until the wirehas been threaded. If the wire gets kinked, trim the damaged part off before continuingfurther attempts. Once the bundle has been threaded, use the forceps to slide it into thecarrier, a few millimeters at a time. Be careful not to bend or kink the wire during thisprocess. Eventually, the bundle will encounter the lower end of the innermost cannula,and it may prove difficult to thread. Patience! Try extending the carrier to differentlengths, holding the bundle against the clamping plate with the forceps, and using acombined sliding and rotating motion on the lower part of the carrier to thread the wire,watching for bends where it exits the carrier at the top. It may become necessary toremove the bundle and trim the end if it proves impossible to thread through the lastsection. Once the recording tip appears at the sharp end of the carrier, pull the bundlethrough until 2–3 cm remain at the other end and proceede to the next step. Be carefulnot to bend or kink the wire.

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Figure 4: Tetrode ToolsThe set of hand tools used to create tetrode bundles and carriers.

item quantity description source part number

T1 1 Dumont Pattern 1a BRI 10-1400T2 1 Dumont Pattern 5 BRI 10-1425T3 1 Dumont Pattern 5b BRI 10-1425T4 1 micro dissecting scissorsc BRI 11-2030T5 1 lamp with magnifier glassd Luxo 17253BKT6 1 sharp wire cuttersd Xcelite MS54T7 1 heat gun Newark Electronics 93F1914T8 misc conductive painte GC Electronics 22-201T9 misc chemical stripf GC Electronics 10-2602T10 misc tungsten wire California Fine Wire CFW211-0005-HMLT11 misc acetoneT12 misc isopropyl alcoholT13 misc reaming wireg

Table 3: Tetrode Tools and Supplies

List of tools and supplies used to twist bundles, construct carriers, and to load bundles intocarriers. aBlunt tips slightly with a file or grinding wheel. bCover tips with fine surgical tubing.cLabel TETRODE WIRE ONLY and separate from other tools. dAvailable through DigiKey.eTrade name Silver Print, available through electronics distributors and retailers. fTrade nameStrip-X, available through electronics distributors and retailers. gThis should be music or springwire no larger than 0.003 inches in diameter.

Making Electrical Connection

The stripped wire is physically and electrically attached to the connector with conductivepaint. The connector should first be cleaned, by dipping the head of the carrier inisopropyl alcohol, and drying. Then, each wire (previously stripped) is attached to the

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0

1

2

3

10ms

b c dad dc bbbbb a a

Figure 5: Example Tetrode RecordingOn the left is a short stretch of the four channels of data. Heaviersections represent detected events. Letters signify events fromfour presumably different units. On the right are the averagesof all events from each of the identified cells. Signals have beenhigh-pass filtered at 300 Hz.

connector by placing a drop of conductive paint on a connector pin, and, again undermagnification, one of the four wires brought into the paint and held in place until thepaint dries. Clean uncoated blunted forceps should be used for this, and again, patienceis necessary to hold the wire as the paint dries (10–15 seconds is usually sufficient to makea physical connection; full electrical connection takes several minutes). An articulated-arm incandescent lamp with conical shade makes an excellent low-temperature hood tospeed drying. See Figure 1 for an assembled and loaded carrier.

Tools and Supplies

Recommended tools and supplies to construct tetrode bundles, carriers, loading andcleaning can be found in Table 3. Most should be readily available in biology andelectronics stock rooms, however, sources for nearly all are listed at the end of the paper.

Reserve the fine surgical scissors for cutting tetrode wire only. Do not use them for anyother purpose, as they must be extremely sharp to cleanly cut the fine tungsten. A pairof scissors lasts approximately one year in our laboratory; once they become too dull forthis task, they remain quite sharp and can be used elsewhere.

Equipment

Our experimental setup is as follows. A hydraulic microdrive (Fred Haer Corp, Brunswick,Maine) is used to position the tetrodes. Tetrode signals are amplified by a customfour-channel headstage amplifier (A = 100) feeding a custom four-channel variable-gainpreamplifier (A = 1 to 5000, nominally set to 200). The preamplifier feeds anti-aliasfilters (fc = 10 kHz, Tucker-Davis Technologies, Gainsville, Florida) and four-channelinstrumentation-grade A/D (fs = 20 kHz, also TDT). Data are streamed to disk, eventu-ally written to CD-ROM, and analyzed using in-house Matlab code. Our spike sortingalgorithms can have been described elsewhere (Sahani, et al, 1998a, b).

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Results

An example tetrode recording is shown in Figure 5. This is of particular interest becauseit clearly shows one unit (a) detected on channel 3, a second unit (b) on channel 2, andboth on channel 1 at approximately equal amplitude, illustrating the monophonic-versus-quadraphonic analogy suggested earlier. Two additional units (c, d) were detected in thisrecording. Notice how similar three of the four waveforms are on channel 1, making itvery difficult to disambiguate between them given only the information from that trace.

Penetration Straightness and Tissue insult

We have histologically identified all marked penetrations and have determined that thetetrode bundles ran straight and the tracks are not unusual. The deepest tetrode penetra-tion, some 12 mm, included 11 lesions which lie in a straight line, even at the maximumextension. Informal comparison of tracks between the control penetrations and the twowire diameters show that all three are comparable. That is, the tetrode bundle causedno additional tissue insult.

Summary and Conclusions

We have described the design and construction of a tetrode design appropriate for use inthe chronic monkey preparation. Experiments were performed to verify the mechanicaland electrical performance of the tetrodes which showed them to be functional andadequate for simultaneously detecting multiple neighboring cells in monkey cortex.

Sources

Microdrives

Frederick Haer & CoBrunswick, ME 04011, USA800.326.2905207.729.1601http://www.fh-co.com

Tetrode Wire

California Fine Wire338 South Fourth StreetGrover Beach, CA 93433, USA805.489.5144http://www.calfinewire.com

0.0005 inch tungsten wire, HML insulated, 0.0007–0.0008 inch nominal OD (part numberCFW211-0005-HML).

Cannulae

Small Parts13980 N.W. 58th Court

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Figure 6: Series of Lesions11 lesions left along a tetrode penetration. Black ticks are 500 µm apart.

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Miami Lakes, FL 33014800.220.4242http://www.smallparts.com

Stainless steel cannula, uncut, 21 gauge (part number O-HTX-21-12); 26 gauge (O-HTX-26-12); 32 gauge (O-HTX-32-12). These part numbers are for uncut 12-inch lengths ofcannula; Small Parts will cut and machine to specification for additional cost.

Tetrode Twister Parts

C&H Sales2176 E. Colorado Blvd.Pasadena, CA 91107800.325.9465http://www.candhsales.com

Reversible AC gearhead motor (part number 54191814001), switch (SW9603), capacitor(CAP0835), power cord (PFC8300).

Epoxy

Devcon Consumer ProductsITW Devcon30 Endicott StreetDanvers, MA 01923, USA800-933-8266

5 Minute Fast Drying Epoxy (product number S-208). Sold through distributors andretailers.

Heat Gun

Newark Electronics(Many locations in the US)800-463-9275http://www.newark.com

Alpha Wire Heat Gun FIT GUN 3 (order number 93F1914).

Reaming Wire

InterWire GroupCorporate Headquarters355 Main Street, Armonk, NY 10504914.273.6633

phosphate coated music wire 0.0025 inch diameter.

Chemicals

GC Electronics1801 Morgan StreetRockford, IL 61102

Strip-X (product number 10-2602), Silver Print (22-201). Sold through distributors andretailers.

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Forceps and Scissors

Biomedical Research Instruments, Inc.12264 Wilkins AvenueRockville, MD 20852800.327.9498301.881.7911

Dumont Pattern Number 1 (order number 10-1400); Dumont Pattern Number 5 (10-1425, need 2); BRI Micro Dissecting Scissors (11-2030)

Connector and Tools

DigiKey701 Brooks Avenue SouthThief River Falls, MN 56701-0677800.344.4539http://www.digikey.com

Dual row straight 72-pin connector with gold plated pins (part number S2231-36); Luxo17253BK lamp with magnifying glass; Xcelite MS54 diagonal cutters.

References

M. L. Recce, J. O’Keefe, The Tetrode: An Improved Technique for Multi-Unit Extracel-

lular Recording, Soc. for Neurosci. Abstr., 15(2) p. 1250, 1989.

C. M. Gray, et al, “Tetrodes markedly improve the yield and reliability of multiple singleunit isolation from multiunit recordings in cat striate cortex,” Society for Neuroscience

Abstracts, 20(1) p. 625 (1994).

M. Sahani, J. S. Pezaris, R. A. Andersen, “On the Separation of Signals from NeighboringCells in Tetrode Recordings,” Advances in Neural Information Processing Systems 10,M. I. Jordan, M. J. Kearns, S. A. Solla (eds.), MIT Press, Cambridge, MA (1998).

M. Sahani, J. S. Pezaris, R. A. Andersen, “Extracellular Recording from Multiple Neigh-boring Cells: A Maximum-Likelihood Solution to the Spike-Separation Problem,” Com-

putational Neuroscience, J. M. Bower (Ed.), Plenum Press, New York (1998).

M. Wehr, J. S. Pezaris, M. Sahani, “Simultaneous Paired Intracellular and TetrodeRecordings for Evaluating the Performance of Spike Sorting Algorithms,” to appear inNeurocomputing (1998).

M. A. Wilson, and B. L. McNaughton, “Dynamics of the Hippocampal Ensemble Codefor Space,” Science, 261(5124) pp. 1055-1058 (1993).