IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. BME-33, NO. 2, FEBRUARY 1986

Recording from the Aplysia Abdominal Ganglion with

a Planar Microelectrode Array

JAMES L. NOVAK, STUDENT MEMBER, IEEE, AND BRUCE C. WHEELER, MEMBER, IEEE

Abstract-A passive multimicroelectrode array has been fabricatedand used to record neural events from the abdominal ganglion of themarine mollusk, Aplysia californica. The array consists of a pattern ofgold conductor lines on a glass substrate which is insulated with apolyimide. The 32 electrodes are 25 ,4m in diameter and are arrangedin a 4 x 8 matrix on 200 um centers. The array is durable and reusable,and can be safely autoclaved. The recording environment surroundingeach electrode is sufficiently uniform so as to permit spatial localizationof identified cells in the ganglion. The array can record large numbersof unique and often interrelated extracellular neural potentials in rel-atively simple experiments.

I. INTRODUCTIONR ESEARCHERS in the neurosciences have long rec-

ognized the potential value of simultaneously sensingthe individual electrical activities of large populations ofneurons. In addition to the increased volume of data ob-tained, important spatial information is gained that cannotbe obtained from a single microelectrode. Pairs of mi-croelectrodes have been used to gain much of the knowl-edge on the structure of neural networks, but as the num-ber of conventional electrodes needed to perform anexperiment increases, the experiment becomes extremelydifficult to perform consistently. Electrode arrays are idealfor such recordings because they provide reproducible ex-tracellular electrode geometries. These arrays can be fab-ricated using the technology already available in the mi-croelectronics industry. This newly emerging sensor arraytechnology should soon have a widespread impact in thebiomedical research community.

Several styles of electrode arrays have been demon-strated including probe type arrays [1], [2] for insertioninto neural tissue, regeneration electrodes [3], [4], andplanar arrays, the subject of this paper. Gross used a laserdeinsulation technique to define 10 Am diameter elec-trodes and reported on its use with an isolated molluscanganglion [5] and in tissue culture [6]. Using a tissue cul-ture dish substrate, Pine defined electrodes photolitho-graphically by etching a chemical vapor deposited layer ofsilicon dioxide over gold conductors [7]. To improve thesignal-to-noise ratio when recording from rat hippocam-pal slices, Jobling placed the slice directly over the gates

Manuscript received March 30, 1985; revised August 6, 1985. This workwas supported by the Whitaker Foundation.

The authors are with the Department of Electrical and Computer Engi-neering, University of Illinois, Urbana, IL 61801.

IEEE Log Number 8406346.

of an array of transistors [8]. A transsubstrate electrodearray, created by diffusing conducting channels through asilicon substrate, has also been reported [9]. The arrayreported here is similar to that of Pine [7] except that apolyimide insulating layer is used to simplify the fabrica-tion procedure. The physical and electrical characteristicsof this new array were investigated and are reported be-low.An investigation into the fidelity of the recording ability

of the planar electrode array in culture, by intracellularlystimulating individual cells and recording the action po-tentials, has been performed [7]. This report indicates that,for a relatively sparsely populated, two-dimensional prep-aration, it is reasonable to assume that the electrodes haveidentical, well behaved spatial sampling functions. Thesame need not be true for molluscan ganglia since theyare organized three-dimensional structures surrounded byfibrous sheaths, and since, in general, the electrical con-ductivity of neural tissues may be anisotropic or inhomo-geneous [10]. To investigate this possibility, we recordedfrom the abdominal ganglion of the Aplysia, which has arelatively thick sheath. Since many of its neurons havespiking somae and can be independently identified bycolor, firing pattern, or neural connections, this prepara-tion provides an independent means of locating the sourceof neural activity for investigating the array characteris-tics. Further recordings were made after enzymatic diges-tion of the sheath, as is done to facilitate intracellular re-cording, and which should reduce the sheath impedanceseparating the neural source from the electrodes and fromthe bath. The goal of this work was to show that the arraywas a reliable tool for surveying and locating cells in othersimilar preparations.

II. METHODS

The electrode array mask was created, for reasons ofeconomy, by superimposing a commercially producedelectrode mask (Towne Laboratories, Somerville, NJ) ona pattern of lead-ins [Fig. l(a)]. The electrode pads are25 ,um in diameter on 200 gm centers. The deinsulationmask consisted of 10 Itm diameter holes spaced similarly.

Glass plates (3 x 3 x 0.156 in) were used as the arraysubstrate. A 100 A layer of titanium was deposited byevaporation, followed by 3000 A of gold. Chromium wasrejected for use as the underlayer because it alloyed tooeasily with the gold during the insulation cure [11].

0018-9294/86/0200-0196$01.00 © 1986 IEEE

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NOVAK AND WHEELER: RECORDING FROM APLYSIA ABDOMINAL GANGLION

(a)

tU)

Fig. 1. (a) The conductor pattern for the interior of the el(Zm wide connectors lead from 25 Am diameter electrnAm2 bonding pads. The electrode pads are spaced on 20second pattern was superimposed on this to connect t}the edge contacts as seen in (b). (b) An insulated planaarray. The pattern of (a) occupies the center of the arrmof polyimide has been applied by hand over much of thethe center electrodes, four larger ground electrodes, awhich mate with an edge connector.

The metallized plates were photopatternecroposit 1450J positive photoresist (Shipleymask, and standard gold and titanium etcharpatterned plates were cleaned with acetone a

The insulation, Pyralin 2555 (DuPont), was applied fol-lowing the manufacturer's recommendations. This type of

/ Pyralin was used because of its faster curing rate, which7 reduced the titanium/gold alloying. First, an adhesion pro-

moter VM-651 (0.01 percent in 95 percent methanol/5percent DI water) was applied and spun at 3500 rpm for30 s. Next, 1 cc of Pyralin was spun for 60 s at 3500 rpm,

J producing a measured thickness of 3-5 ,tm. The plate wassoft-baked at 135°C for 10 min. After cooling, the platewas photosensitized with 1450J photoresist, soft-baked at135°C for 20 min, and exposed using the deinsulation

_ L mask. The photoresist was developed using MicropositDeveloper, which also acts as the etchant for the poly-imide, to create uniformly sized holes over the electrodes.

_ After removal of the photoresist, the glass plate was driedfor 30 min at 135°C.

\ ~< A thick layer of Pyralin 2555 was applied around theelectrode area to reduce shunt capacitance and improvedurability. The insulation was cured by baking at 90°Cand increasing the temperature 450 every 30 min untilreaching 270°C. If the temperature was increased morerapidly, unacceptable bubbling occurred. After curing at

I mm 270°C for 1 h, the oven was shut off and allowed to coolI I slowly to prevent cracking the glass. An insulated array

l u g is shown in Fig. 1(b).A colloidal deposit of platinum black was deposited on

each electrode tip by applying 1.0 ltA for 25 s using aplatinum anode and plating solution (3 percent platinum

l chloride and 0.025 percent lead acetate in 0.025 N hydro-chloric acid). Successful plating was indicated by a uni-form, dark black coating over the gold electrode tip.

.., : The array was mounted on an elevated mounting standand electrically connected, using modified PC board edgeconnectors, to a bank of 32 ac-coupled preamplifiers (gain= 100, 3 dB = 10 Hz), and eight variable gain, computer-controlled (LSI 11/02) intermediate amplifiers (gain = 50-10 000, BW = 100 Hz-3 kHz). Data were recorded (fourchannels at a time) on an FM tape recorder (HP 3960).Impedance measurements were performed by injecting

0.1-1.0 nA at 1 kHz. The resulting low electrode tip cur-rents prevent nonlinear effects [13]. Long duration imped-ance tests were done with the aid of the LSI 11/02 com-

puter. The system measured impedances to within 1percent. Impedance analysis assumed that the electrodescould be modeled as a parallel combination of a resistor(Re) and a capacitor (Ce), shunted by capacitance (CQ) be-

ectrode array. 15 tween the conductor leads and the saline bath, as well asade pads to 350 external wiring [13]. C, was measured using an electrode0 lim centers. Aie conductors to which had not been deinsulated. Ce and Re were computedr microelectrode from impedance measurements of the deinsulated elec-ay. A thick layer trodes and from the estimate of Cs. Lead resistances werearray, except forind the contacts neglected.

The abdominal ganglion of a marine mollusk (Aplysiacalifornica) was used for recording. A suitable saline so-

d using Mi- lution was prepared consisting of 420 mM NaCl, 25 mM'), the array MgCl2, 10 mM KCI, 10 mM CaCl2, and 5-10 mM MOPSits [12]. The buffer adjusted to pH 7.5. For some of the intracellularnd baked. recordings, the ganglion was treated in a 1 mg/ml pro-

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IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. BME-33, NO. 2, FEBRUARY 1986

tease in saline solution for 5 min to soften the sheath [14],permitting easier penetration with a glass microelectrode.-19 animals (50-100 g) were used and all experiments wereperformed at room temperature (15-200C).A chamber to contain the ganglion in a small pool of

saline was created from flexible magnetic stripping andwas attached to the array substrate with modeling clay.The ganglion was pinned down using insect pins whichhad been epoxied to small pieces of iron and placed on themagnetic strip. The reference Ag/AgCl electrode was in-serted through the clay. In order to minimize buoyancyand prevent dehydration, the saline level was adjusted tojust cover the ganglion.

The-preparation was viewed from beneath while record-ing with a dissecting stereomicroscope and a mirror lo-cated below the array. The final stage of an experimentconsisted of inverting the array with the ganglion pinnedout and most of the saline removed, permitting observa-tion at higher magnification using a Nikon Labophot trans-illuminating microscope. Photomicrographs taken usingboth methods showed that a well-pinned ganglion did notmove during the inversion. With this technique, photo-graphs were taken at magnifications large enough to ob-serve many of the cells in the ganglion.'

III. RESULTSA. Array PropertiesThe array is mechanically strong and can be used re-

peatedly. The insulating material remained stable, with nodegradation noticeable visually or electrically, for the du-ration of a 25 day test under saline. The polyimide insu-lation is durable and withstands swabbing and solvents thatsoften some other insulating materials, e.g., photoresist.Small holes created by a glass micropipette, as well asother incidental scratches, Ido not affect the integrity ofthe insulating film.The array may be autoclaved and rinsed with ethanol

without damage and is biologically inert. Another groupat the University of Illinois has successfully cultured rathippocampus explants and muscle cells on the surface [15].

It was helpful to electroplate the electrodes with plati-num black, as discussed below, to permit useful signal-to-noise ratios. 'However, the deposit does wear off after 5-7experiments, as indicated by a rise in impedance by asmuch as 500 percent, and by a corresponding 200-400percent increase in the recorded noise level. The electrodetips also change in color to a light grey-brown. Swabbingthe electrodes had a similar effect on both the impedanceand the color of the electrodes. Fortunately, replatinizingthe electrodes returned the impedance, the color, and thenoise levels to their original state.A problem inherent to the design of this type of array is

the' recessed electrode. The insulation is approximately 4Am thick, and the unplated electrode is actually located atthe bottom of a well over which the tissue rests. Gross [5]calculated that a layer of glial cells could possibly increasethe electrode impedance by 15-20 Mg, resulting in signalloss and increased noise. In this device, however, the plat-

inum black deposition nearly always grows out of the well,especially around the circumference, and in many cases,extends 3-5 ,um above the insulation surface. This out-growth moves the effective tip of the electrode out of thewell, maximizing the electrode surface presented to theneural tissue. We have found no instances in which gan-glion tissue covered a particular electrode and dramati-cally increased its impedance.The impedances of the electrodes on three different ar-

rays were tested for 24 h. One of these arrays was furthertested for 25 days. The average impedance of an unplatedgold electrode, at 1 kHz, stabilized after a 90 mm im-mersion in saline at 1.4 MQ with a phase of -75° andremained essentially constant for the duration of the 25-day test. The electrode resistance (Re), capacitance (Ce),and shunt capacitance (Cs) were measured as describedabove to be 5.1 Mg, 112 pF, and 5 pF, respectively. Theelectrode capacitance was 0.24 pF ptm2, which is in agree-ment with that given by Robinson [13] for bright platinum.Because the shunt impedance is relatively high (32 Mg at1 kHz), biological signals will not be attenuated appre-ciably. However, the large tip impedance resulted in anoise level between 20 and 50 ,uV. Since the signals to berecorded are within this range, typical noise levels of theunplated electrode tips are unacceptably high.The impedance of the platinum plated electrodes in-

creased by approximately 25 percent during the first 2 hof immersion. After this, the impedance stabilized andremained unchanged for the 25-day test. The steady-stateimpedance was between 12 and 14 kg at a phase of -30°to -35°, which is two orders of magnitude less than thatfor the unplated tips. Platinizing did not result in any long-term impedance instabilities. The equivalent model compo-nent values are Re = 15 kg and Ce = 6500 pF. Typicalnoise levels achieved with plated tips are 5-15 jAV, signif-icantly lower than those of unplated tips.

B. Recording CharacteristicsFig. 2 is a photomicrograph taken during an experiment

of the abdominal ganglion mounted on an array using thetechnique outlined above. The dorsal side of the ganglionwas positioned on the electrodes and the ganglion was il-luminated from the ventral side. The rostral end of theganglion was toward the top of the photograph. The elec-trodes were smaller than the majority of the cells and theirspacing and number were such that nearly the entire gan-glion surface could be covered by the matrix of elec-trodes. The spacing and geometry of the electrodes issuitable for recording from this ganglion.Superimposed on the photomicrograph is a map drawn

with the aid of the transmitted light microscope indicatingthe position of the entire ganglion (excluding the connec-tive tissue sheath) on the array and the general locationsof specific cells of interest [16], as well as the numberingof electrodes used in the following discussion. Not all ofthe identified cells are in the plane of focus.

Recordings were easily obtained when a ganglion waspositioned over the array of electrodes. Changes in saline

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Fig. 2. Photomicrograph of Aplysia abdominal ganglion mounted on an ar-

ray. The dorsal side is seen through the glass substrate supporting theelectrodes. The rostral end of the ganglion is toward the top of the figure.A map of identified Aplysia abdominal ganglion cells, as observed throughthe microelectrode array, has been superimposed over the photomicro-graph. The electrode numbers are as used in the text.

volume did not alter the recorded signals unless the gan-

glion was insufficiently pinned and began to float. Typi-cally, unique signals (amplitude 8-40 AiV) were presenton many of the recording channels. Large amplitude sig-nals (200 AiV) were occasionally present on several adja-cent electrodes. These, presumably, have a common ori-gin in a single neuron. However, the extracellularpotentials were usually more localized on the sheath andthe recorded activity shows only a modest amount of cross-talk between electrodes. The recording resolution of thearray can be as small as the interelectrode distance (200MIm).A center-of-mass calculation [5] can be used to deter-

mine the location of the neuron if the electrode imped-ances are the same and the electrical environment (e.g.,sheath thickness) is isotropic and is given by

Z (xi)(si)

zs.

)Z (Yi)(si)y =

E s

The variables x and y represent the computed rectangularcoordinates of the signal source; xi and yi are the distancesfrom the origin to the ith recording electrode and si is theamplitude of the ith recorded neural potential. By mea-

suring the amplitudes of the recorded bursts of action po-

tentials on electrodes 25, 29, and 30 (Fig. 3), the locationof the burst origin can be calculated to lie 120 jim to theright and 144 Am below electrode 26 in Fig. 2. Cell R15is known to have this bursting behavior [16] and since itssoma is isopotential [17], it was considered to be a source

centered at a point 137 jim to the right and 150 ,um belowelectrode 26 (Fig. 2), in close agreement with the pre-dicted location.The impedances of the electrodes were measured be-

fore, during, and after placement of an untreated ganglionon the array, in an attempt to quantify intimacy of the con-

tact. Cell membranes and glia exhibit a relatively highimpedance [18] and intimate contact between the ganglion

electrode 25

~ electrode 26

electrode 29

electrode 30

5 s

Fig. 3. Bursts of activity recorded on electrodes 25, 29, and 30. The burst-ing pattern and triangulated position of the source neuron correspond tothe observed location of cell R15 in Fig. 2.

and an electrode should result in an impedance increase[5]. The electrodes (Fig. 2) were divided into three groupsas follows: 1) those beneath sheath or nerve tissue and notcell bodies (electrodes 1, 2, 5, 6, 9-12), 2) those beneaththe main portion of the ganglion and cell bodies (elec-trodes 13-32), and 3) those with no tissue covering them(electrodes 3, 4, 7, 8). Upon placement of the ganglion onthe array, electrodes beneath the sheath exhibited a 4.6 +1.4 percent increase in impedance, electrodes directly be-neath cells had the greatest increase (9.2 + 4.2 percent),and electrodes with no contact showed only a 3.1 ± 0.4percent increase. After removal of the ganglion, theimpedances of all electrodes were found to have increasedby 2.3 ± 1.2 percent, suggesting that the above valueshave changed, in part, due to a process other than tissuecontact. Because of the small differences in impedancechanges, it appears that impedances measured at 1 kHzcannot be used as reliable indicators of the quality of tissuecontact with the array.The amplitude of recorded signals was adversely af-

fected in preparations in which the ganglia were treatedin the 1 percent protease solution to soften the sheath.This is significant because this treatment is routinely usedin some laboratories to facilitate glass micropipette pen-etration through the fibrous sheath. The intracellularlymeasured resting potentials of several neurons tested inboth untreated and treated ganglia were normal (-45 to-60 mV), as were the shapes and amplitudes of the actionpotentials. These measurements were made again at theend of each experiment to confirm ganglion viability. Un-treated ganglia possessed extracellular activity with am-

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plitudes from 10 to 100 AV for at least 24 h and for as longas 48 h. Those ganglia treated for 5 min in the proteasesolution initially exhibited extracellular activity similar tothe control. However, after 3-4 h, spike amplitudes haddecreased to between 0 and 20 ,uV. The reduced activitylevel was not improved by flushing the chamber to removeloosened tissue fragments. No activity was recorded ex-tracellularly in these treated preparations after 7 h. It ap-pears that the condition of the sheath is an important fac-tor in determining the signal detected with this type ofarray.

C. Recordings from Aplysia Abdominal GangliaActivity recorded was compared with the results of other

investigators regarding location, size, and firing patternsof specific cells or groups of cells in the ganglion. Thedorsal side of the ganglion was used for recording becausethe ventral sheath is thicker and three large nerves exitfrom this surface [16], [19], preventing both viewing ofthe cells and uniform contact with the flat surface.

Bursting cell R15 was located, as described above, inthe region near electrode 26 in Fig. 2. Cell R15 possessesburst patterns of 15-20 spikes occurring regularly every5-20 s [16]. A firing pattern of this type was recordedonly on electrodes 25, 26, 29, and 30 (Fig. 3).

Cell clusters RB and RC on the dorsal side of the gan-glion fire irregular spikes at rates that average 2 Hz. Thecells are also often light sensitive and can be inhibited byturning off the theater illumination [161. Recordings fromthe electrode array are consistent with these facts. Elec-trodes 25, 26, and 28 in Fig. 4 were located near the re-gion of the ganglion containing these clusters and re-corded neural signals that were light-sensitive, inhibitedupon light-offset, and possessed firing rates near 2 Hz dur-ing illumination.The rostral white cells of this ganglion possess regular

firing rates between 2 and 1 Hz [16]. These cells werelocated in the region of electrodes 14, 15, 18, and 19. Sig-nals from at least three unique, regularly firing neuronspossessing frequencies within the expected range were re-corded on these electrodes.

In addition to locating previously mapped cells by theirfiring characteristics, the interaction between cells in an-other experiment was recorded with the array (Fig. 5).The intracellular potential of cell L10 was observed whilesurveying the ongoing dorsal activity with the array. Dur-ing a burst recorded with the array, L1O underwent a largehyperpolarization which abolished its tonic activity. Theactivity resumed 1-2 s after the end of the burst. Neitherintracellular depolarization nor hyperpolarization of L1Oaltered the firing pattern observed extracellularly. Theelectrodes recording the bursts were located on the dorsalside of the ganglion near LIi. Center-of-mass calculationsand visual inspection confirmed this finding. It has beenobserved previously that Lll and L1O exhibit this syn-chronous activity [16], [20].

Interactions between activity on array electrodes canalso be observed. In another experiment, bursting activity

electrode 25

electrode 26

electrode 27

electrode 28

.8 ]IV

5 s

Fig. 4. Neural potentials recorded near cell clusters RB and RC on thedorsal surface of the ganglion. The signals recorded were stimulated bylight, inhibited upon light offset, and possessed irregular firing rates.

-I ~~~~~~~~~~~~~50 lV

o5 mv LIO

5 s

Fig. 5. Coupled activity observed between the signals on array electrodesand an intracellular electrode in cell LIO. The burst activity inhibits LIOand is unaffected by intracellular depolarization or hyperpolarization ofLl0.

recorded on one electrode was followed by another burston another electrode after a delay of about 3 s (Fig. 6).Tonic activity observed on the two lower extracellulartraces was abolished during the second burst. The tonicactivity also resumed before the end of either burst, incontrast to the data in Fig. 5. This same interaction wasobserved five times and occurred approximately every 3.5min. Unfortunately, the ganglion shifted during this ex-periment and the locations of these electrodes on the gan-glion are not known.

IV. DISCUSSIONThe microelectrode array described in this paper is suit-

able for long-term surveying of neuronal spike activitypresent in an isolated molluscan ganglion. The fabricationsequence uses standard equipment and is straightforward.The use of polyimide makes the process significantly eas-ier. (The use of newly introduced photosensitive poly-imides would further eliminate a photoresist application.)Once the high-resolution mask set is made (Fig. 1), pro-duction of the devices is moderately easy. The glass arraysubstrate permits visual identification of the position ofspecific cells located on the recording surface. The matrixof recording electrodes provides samples, taken at pointson the ganglion sheath, of the underlying electrical activ-ity of the neurons within the ganglion.

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20 pv

5- s

Fig. 6. Interrelated bursting activity recorded on four array electrodes. Theonset of bursting in two different cells can be observed. Activity on thelower two traces resumes before the end of either burst, in contrast tothe data of Fig. 5. The location of the electrodes relative to the ganglionis unknown.

The experiments in which recordings were made fromindependently identifiable neurons indicate that, exceptwhen enzymatic digestion is used, the electrodes' spatialsampling functions are sufficiently uniform so as to permitrelatively precise spatial location of individual cells. Noobservations were made where signals from the identifiedcells were recorded on any electrodes other than thosenearest the visually located cell body. In addition, trian-gulation of the signals from R15 and Lll generated esti-mates of the location of the neural signal source that laywithin the apparent cell body. The RB and RC cell clus-ters, as well as the rostral white cells, were located vi-sually and by electrical activity with good agreement. Theexistence of many unique spikes on adjacent electrodesindicates that it is possible to record from small cells andthat it would be profitable to reduce the interelectrode sep-aration to permit recordings from smaller cells lying be-tween our present electrodes.

In contrast, the experiments with the enzymatically di-gested sheath were particularly disappointing, since onemight expect that, in addition to making intracellular pen-etration easier, the procedure would effect the sheathisotropically, reducing the neuron to electrode impedanceand improving the signal quality. Instead, the neural cur-rents appear to have been shunted directly to the bath,thereby reducing the potentials across the sheath until theywere no longer detectable. Since the intracellularly re-corded action and resting potentials remained constant, itis not likely that neural injury was a factor. Pinsker useda much greater protease concentration (10 mg/ml) and a

longer soak time (10 min) in some experiments and didnot report any differences in intracellular potentials be-tween treated and untreated ganglia [14]. It appears thatthis procedure adversely affects the recording capabilityof the array.The capabilities of the planar electrode array comple-

ment the optical recording technique (see [21] for a re-view). In a typical recording session, the planar array re-corded 15-20 unique signals, or better than one signal perelectrode positioned beneath the ganglion. (We have alsofound this to be true in recordings from the pedal ganglion

of Pleurobranchia.) Optical techniques permit recordingsfrom a greater number of cells in different focal planes,and with greater spatial resolution. For instance, Londonet al. (cited in [21]) recorded from at least 48 neurons inthe Aplysia abdominal ganglion, and a resolution of 10 ttmhas been reported. Whereas the electrode array can beused for long-duration experiments and culturing, the in-tense illumination required for use with the voltage-sen-sitive fluorescent dyes limits recording sessions to 1-5 min[21]. The electrode array can be used for stimulation ofcells in culture [7], [22], while the optical technique ispassive. Our array has been used to stimulate culturedmuscle cells [10], and preliminary experiments in our lab-oratory indicate that it can record from, and stimulate,different fiber tracts in the rat hippocampal slice -prepa-ration.The capability of recording simultaneously from tens of

cells is clearly an improvement upon conventional tech-niques in which one or several cells are monitored. As arecording device, the array should serve as a survey tool,permitting experimenters to more rapidly focus upon areasof the ganglion in which to search for interactions betweenneurons with conventional techniques. The array permitsthe simultaneous observation of the activities of a numberof individual neurons only as long as these neurons showspiking activity and are located at or near the surface ofthe ganglion, in which case it substitutes for an equal num-ber of intracellular electrodes. Nevertheless, the planarelectrode array is unlikely to record from a large fractionof the neural population even in relatively simple struc-tures such as the invertebrate ganglion. While opticaltechniques may permit greater coverage, at present thelimitation on the duration of the recording session pro-hibits many interesting experiments. The combined use ofboth methods should offer significant advantages in theinvertebrate ganglion, and has already been reported fora cell culture preparation [22].The ability to routinely record from tens of neurons must

be followed by the development of techniques for analyz-ing the large number of interactions potentially present inthe data. Several researchers, notably Gerstein, have re-ported techniques for describing the interactions of pairsof units [23], [24] and for three units [25], [26]. Multi-variate statistical techniques have been applied to largerneural populations by Heetderks [27]. It may be difficultto compute and display these functions when, for example,a recording session with 20 neurons implies 400 pairwisecorrelations, each of which may have multiple parameters.Multiple unit extracellular electrode data include the sta-tistical probability that neural events are not detected,falsely detected, or misclassified. Although much has beenwritten about multiunit separation (see [28] for a review),only a few reports dealt with multiple channel data.The results presented here demonstrate that planar elec-

trode arrays are reliable reporters of simultaneous neuralactivity over large neural surfaces. These arrays could bemore widely used, provided a manufacturer offered a se-lection of interchangeable devices which mate with stan-

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IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. BME-33, NO. 2, FEBRUARY 1986

dard electrical connectors, amplifiers, and computer in-terfaces. Electrode size and spacing could be varied sothat arrays appropriate to a variety of preparations couldbe used efficiently. The fabrication method presented here,although by no means the only possible technique [29], isreliable and relatively inexpensive once the masks havebeen made. In particular, their durability should make theirpurchase attractive ta the neuroscience community, whilethe ease of construction should make them a commerciallyfeasible product.

V. CONCLUSION

The ability to correlate extracellular whole-ganglia ac-tivity with previously determined single neural patterns ofactivity using the array, demonstrates that it is suitablefor obtaining a survey of neural activity and connected-ness in an unknown ganglion. Preliminary experimentscan be performed relatively quickly and can reveal localfiring patterns as well as neural coupling.

ACKNOWLEDGMENTWe are very grateful to S. Smith and P. Cashman for

their work constructing the amplifier and testing system.The Pyralin polyimide was donated to us by DuPont.Thanks also to Dr. R. Gillette and D. Green for their dis-cussions concerning mollusks and for providing us with astorage tank.

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M ~~~~James L. Novak (S'80) was born in Berwyn, IL.,in 1961. He received the B.S. and M.S. degrees inelectrical engineering from the University of Illi-nois at Urbana-Champaign in 1983 and 1985, re-spectively.

Since 1983 he has been a Research Assistant inthe Department of Electrical and Computer En-gineering at the University of Illinois. Includedamong his research interests are the acquisition andanalysis of multiple-channel neurobiological sig-nals.

Mr. Novak is a member of Tau Beta Pi and Eta Kappa Nu.

Bruce C. Wheeler (S'75-M'80) was born inSchenectady, NY, in 1948. He received the S.B.degree from the Massachusetts Institute of Tech-nology, Cambridge, in 1971, and the M.S. andPh.D. degrees in electrical engineering from Cor-nell University, Ithaca, NY, in 1977 and 1981, re-spectively.

Since 1980 he has been with the University ofIllinois at Urbana-Champaign, where he is Assis-tant Professor of Electrical and Computer Engi-neering and of Bioengineering. His research in-

terests include the fabrication and use of microminiature sensors forneurobiological and other applications.

Dr. Wheeler is a member of Phi Beta Kappa.