Neuroethology deserves more study of evoked responses

0306.4522,81,!071203-13502CQjO Pergamon Press Ltd 0 1981 IBRO

COMMENTARY NEUROETHOLOGY DESERVES MORE EVOKED RESPONSES

STUDY

OF

T. H. BULLOCK Neurobiology

Unit, Scripps Institution of Oceanography and Department of Neurosciences, Medicine. University of California, San Diego, La Jolla, CA 92093, U.S.A.

School

of

CONTENTS Introduction Methods Variety in recording techniques Variety in stimulating techniques Variety in states of the organism Exemplary findings and advantages Variety in findings Variety in the advantages of average Summary

evoked

potentials

INTRODUCTION

UNCOVERINGthe neural basis of behavior is severely limited by applicable methods. We need both new methods and fuller exploitation of all those at hand. It is my first claim that the comparative neurological approach to what is now widely called neuroethology, has not made sufficient use of the methods of recording electrical responses time-locked to behaviorallyrelated events. These techniques are well established in psychophysical studies on humans and in the clinic but are little used beyond the standard laboratory animals. Event-related brain responses, especially stimulusevoked responses are recorded as single unit activity, multi-unit spikes or compound potentials that include slow fluctuations. There is a common prejudice in favor of single unit recording, which has obvious advantages and is the basis of a great part of our knowledge. It is, however only one window on the brain and not by itself adequate, even if we use available techniques for multichannel recording and extrapolate boldly. Adding the window that looks at cell population activity significantly widens our view although it is even so still very narrow, considering the complexity of the mechanism. The purpose of this commentary is to advocate further use of eventrelated recording as a tool in the study of animal behavior, especially to point out the choices to be made in using single unit and compound evoked potentials. It is written for those not already experienced and hence includes some elementary considerations important in research planning.

Ahhreuiation: AEP, average evoked potential. 1203

First, it may be useful to point out, for both single unit and compound potential recording, the range of their domains of applicability. I will mention four. These are in studying (i) responses to sensory stimuli, (ii) responses to direct electrical shock to nerves or to structures in the CNS, (iii) central preparations for motor action, and (iv) changes in brain-state such as may accompany attention, expectation or cognitive events. In each of these applications, the recording of activity time-locked to some known reference (start of stimulus or of movement) can provide several types of information. Responses to sensory-stimulated activity will be emphasized here; therefore, I mention examples mainly in this domain. (i) Information may be obtained more quickly than by any other method that a suspected but hitherto undemonstrated sensory modality is in fact present. (ii) Sensitivity, range of stimuli, discriminability of small differences in complex combinations and at least potentially, the biological importance of simulated natural stimuli can be estimated. (iii) The dynamic properties of such responses can be quantified. (iv) The anatomical sites and pathways most relevant to each response can be localized. In this paper, I will point out that recording from many units, using both spikes and slow events, provides information significantly different from single cell recording. These gross recordings are not predictable from single unit data and therefore are connected with the emergent properties of large systems. I will survey the recording methods, stimmating methods and data reduction methods. Limitations, advantages and representative findings will be discussed in order to assist in planning and making choices. I confine

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myself to comments especially relevant to comparative and ethological applications and to evaluation with respect to studies on different modalities, levels of the nervous system and major taxa of animals. A large and helpful literature is available since evoked potential methods are highly developed and extensively used in human and laboratory mammal studies (PERRY & CHILDERS, 1969; SHAC;ASS, 1972; REGAN, 1972; DESMEDT,1977; BEGLEITER,1979: OTTO. 1978; NAUNTON & FERNANDEZ, 1978; CALLAWAY. TUETING & KOSLOW, 1978; BARBER, 1980; an alerting bulletin classifying hundreds of current citations on evoked potentials was long issued periodically by the Brain Information Service, University of California. Los Angeles. Los Angeles, CA 90024).

finer. very flexible wires may be temporarily fastened together, for example, by caramelized sugar or low melting gelatin, inserted and dental cemented at the skull; when the sugar dissolves or the gelatin melts. each wire resumes its own flexibility and if there are brain movements, relative to the skull. the wires are less likely to damage the tissue than are stiff electrodes (O’KEEFE & BOUMA. 1969; CHOROVE:R & DEI.UCA, 1972; BABB vr trl.. 1973: BABB & CRANDALI.. 1976; PALMER. 1978). One may choose more elaborate forms of placement. Several skull-mounted devices have been described that permit a stiff microelectrode to be advanced with a fine screw. as in searching for units in the awake, virtually free animal (OOMURA, O~YAMA. NAKA & YAMAMOTO.1967; BAKER & YORK, 1972; VERTES, 1975; AINSWORTH & O’KEEFE. 1977; METHODS DEADWYLER, BIELA, ROSE, WEST & LYNCH. 1979; Variety in recording techniques MORROW, 1980). Other skull-mounted devices used in conjunction with head fixation and stereotaxic maniElectrodes of a wide array of sizes, materials, forms pulators, permit a new electrode penetration each and types of fabrication are used, and they differ day, anywhere in a considerable solid angle. Interwidely in properties and advantages. This is not the mediate forms have been used, for example a simple place to review the indications for use of various guide tulle fixed in the cranium, allowing an eleckinds of electrodes (GEDDES, 1972; THOMPSON& PATtrode to be inserted to any depth but without a fine TERSON,1973; PARKER, STRACHAN & WELKER, 1973; where mass-evoked potentials are BURGER, ESTABILLO, OSBORNE, STOLL & WALLACE, screw adjustment; sought instead of units this may suffice (BULLOCK & 1973: FERRIS, 1974; SKYDELL & CAPRANICZA,1975; BROWN & FLAMING, 1977; AINSWORTH,DOSTROVSKY. CORWIN, 1979). The several choices above imply, of course, MERRILL & MILLAR, 1977; C~~LEY & VANDERWOLF, recording. 1978 ; CORDON& GOODMAN. 1979; LAIRD, HERMANSEN another: single channel vs multichannel Obviously, the latter multiples the data obtainable 8c HUXTABLE, 1979; PICKARD, 1979; ROSE & from each subject but the complications of keeping WEISHAAR, 1979) but I will take the occasion to track of the subject. loci, electrodes. data reduction emphasize the empirical nature of these indications. and display often operate to keep the number of In spite of frequent and confident rationalization to channels down to a few, except in quite routine the effect that this or that measurable property such studies. Clearly, this is a major front for new developas impedance or diameter or material dictates the ment. especially in data reduction, handling and dissuitability of electrodes, their success is basically not understood or predictable. It is wise to try a variety. play. Electrodes need not be placed in the active brain especially different methods of fabrication rather than center to be useful for neuroethology. Several to choose solely on putative rules. methods look at a large volume of the brain. ExtenAnother set of alternatives that is important in the sively used on human subjects for diagnostic purposes present context is that between chronic and acute are electrodes placed on the skin that record activity, placement. The latter may be used when probing in with the help of computer averaging. from loci many many places for a ‘hot spot’ with only one or a few channels, in less valuable animals. However, chronic centimeters away. This has been especially useful in revealing the dynamics of activity at a number of placement and multichannel techniques are available brain stem auditory loci due to fortunate synchrony in several forms, with advantages wherever some and geometry of the neural elements (JEWETT& WILdegree of free behavior is desirable. One may choose, LISTON,1971; STARR & ACHOR, 1978, 1979; GALAMBOS as the simplest form, to place electrodes (under anes& HE~OX. 1977; GERKEN, 1978; HUANC~ & BUCHthesia), when possible with the benefit of previously WALD, 1978; MERZENITH. GARDI & VIVION, 1980) as determined stereotaxic coordinates, and then seal well as from the cortex. In some species, such as them in place. Under this technique, there are various manatee and dolphin we have bypassed some of the alternative forms of electrodes, besides the previously contact resistance problems, as well as some of the mentioned choices. I will mention only two that are distance, by inserting needles subcutaneously (BULmore likely to be overlooked. (i) A series of thin wires LOCK, DOMNINC & BEST 1980) or to the skull (RIDS;may be bundled together, with or without a stiffening BULLOCK, CARDER, SEELEY, WEEDS & WAY, member to keep the bundle straight, staggering the GALAMBOS,1980, 1981). In lower vertebrates, such as exposed tips so that each penetration into the brain sharks, fish and frogs, we have inserted wire elecoffers recordings at a series of depths (GEDDES, 1972; trodes through holes in the skull to record extracereBABB,CARR & CRANDALL, 1973). (ii) A series of much

Neuroethology deserves more study of evoked responses brally, in cerebrospinal fluid (CORWIN, 1980; CORWIN, BULLOCK & SCHWEITZER, 1981). Magnetic recording without touching the subject works (COHEN, 1972; BRENNER, LIPTON, KAUFMAN & WILLIAMSON, 1978; FARRELL,TRIPP, NORCREN & TAYLER, 1980) but is not yet in wide use. One may choose to have the animal severely restrained and the head immobilized, or lightly restrained with flexible leads, or untethered with telemetering of the brain potentials. An advantage of telemetering, even when it is not needed for freedom of movement is the decoupling of instrument ground and subject ground, which can aid in reducing power line ripple (BULLOCK & RIDGWAY, 1972). Dam processing. Choices must be made in respect to data processing, e.g. to use wide-band recording (usually meaning ca. 0.3 Hz-10 kHz) or to filter for the frequency band of spikes (ca. 300-3000 Hz), or for some low frequency band, to emphasize ‘slow potentials’ (e.g. l-100 Hz). Of course, if data is to be recorded, in analog or digital form, before reduction, it is wise to use wide-band filters, to preserve the options. Contrary to expectation, it is not necessarily better to use the wide-band for analog display, such as records of brain activity for visual inspection. Whatform ofactivity to look fir. A more important choice, which dictates the foregoing, is what form of activity to look for: i.e. whether to search (i) for single units, (ii) for a small number of units, thus permitting the use of a discriminator device that cuts out low voltage background, (iii) for ‘hash’ or ‘swish’ from the spikes of many units, too small to use an amplitude discriminator, or (iv) mainly for slow potentials such as the vertebrate electroencephalogram and evoked potentials in the usual usage of that term (between ca. 1 and 100 Hz). In the present context my emphasis is that no one of these should be neglected. They yield only partially overlapping not really redundant, information and each is a very inadequate window on the brain. Most of this paper is concerned with the methods of achieving item (iv) of this list. Choice of display. Data processing also depends upon choice of display. Single unit spikes or small numbers of units that can be converted into binary computer-recognized events permit various forms of analysis (WIEMER, KAACK & KEZDI, 1975; MUSCHAWECK & LOEVNER, 1978) especially the poststimulus time histograms (PSTH) that show the summed responses to some number of successive stimuli including earlier and later phases of response, on a suitable time base. Less used is the poststimulus latency histogram that sums only the first spike after each stimulus to display dispersion of the latency. ‘Hash’ and ‘slow potentials’ are generally computer-summed (the former after rectification) and spoken of as averaged. Evoked potentials were long studied by photographs of the cathode ray oscillograph or pen-writer electroencephalograph. With the availability of computers, the classic first look-single sweep voltage against time-has been to a large extent replaced

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where identical, clearly timed stimuli are used. A number of epochs, defined by stimuli, may be summed, to yield reproducible average records of stimulus-related as distinct from stimulus-unrelated potentials. Summing a reasonable number of epochs does not necessarily average out all the stimulus-unrelated fluctuations; this is most easily assessed by taking a second sum. If superimposed in an analog display. our eye is sensitive to the distinction between the congruent and the non-congruent deflections, so that in general it is better to divide the available number of epochs, if limited, between two or more averages than to accumulate them into one, smoother total. There are a number of choices in methods of data processing that go beyond simple averaging; they are aimed at bringing out different aspects of the morphology or properties of the temporally and spatially complex evoked potential. Details are beyond the present scope but some citations will be given as examples (MCDONALD, 1964; RUCHKIN, 1968; SPUNDA, WEISS-RADIL & RADILOVP; 1975; AUNON, 1978; AUNON & SENCAJ, 1978; SENCAJ. AUNON & MCGILLEM, 1979; AUNON & MCGILLEM, 1979; G~ijNEWALD-ZUBERBIER& GR~~NEWALD, 1978). These ‘sophisticated methods form another of the actively moving frontiers of new techniques, aiming at windows on hitherto hidden parameters of ensemble organization. Comparison among the forms of activity. By way of comparison among the forms of activity one can record, for the purposes given in the Introduction, the following statements can be made. (i) Single unit responses to sensory stimuli are the most nearly unequivocal, i.e. most nearly distinguishable from artifacts, but difficult to obtain. Furthermore, successively sampled units are often quite diversified in type. Since there is likely to be a spectrum of significantly different units, a good many must be accumulated, each held for sufficient time to characterize it, before a representative sample can be assumed. (ii) Multiunit responses are easier to obtain; they share these difficulties in a lesser degree, at the price of failing to distinguish clearly between kinds of units (e.g. submodalities). (iii) ‘Hash activity still requires that the electrodes lie among active cells or fibers, i.e. that one probes until a real ‘hot spot’ is found. (iv) Although any of these responses can be called evoked, the usual use of the term evoked potential is for the fourth form, characterized often but not exclusively by being slower than spikes and often, though not unambiguously, by being field potentials detectable beyond a few micrometers. Averaged evoked potentials (AEPs) are by far the easiest to obtain, do not require finding the actual hot spot, and do not present a serious sampling problem, once averaged, i.e. successive averages look much the same. Of course, the price is that nothing can be said about the units and often nothing about diversity of subpopulations. However, as we

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shall see shortly, there are situations where submodalities can make themselves evident. Whereas AEPs are the easiest of the four forms of response to obtain, they are the most liable to artifacts and require special care and controls. Details cannot be given here but only the strong recommendation that controls be multiple and aimed not only at suspected sources but also at seemingly quite unlikely sources of artifacts: mechanical, electrical, muscular and others according to the form of stimulus being used. For example, sometimes photoelectric potentials from the electrodes themselves contaminate ‘visual’ responses. Delayed mechanical or reflex artifacts can contaminate ‘auditory’ or even strobe flash responses. With proper respect for the possibilities, controls can be devised and a case built up along several lines that a candidate AEP is in fact due to brain activity. (i) Stimulus delivery can be manipulated, e.g. by blocking a strobe light with cardboard, to allow any click or electrical artifact to continue. (ii) Stimulus parameters can be manipulated according to the case, e.g. intensity, repetition rate, polarity, or duration. looking for non-linearities of response, since these arc unlikely with most non-biological artifacts. (iii) A small advance of the electrode can sometimes change the waveform markedly-which is unlikely for most forms of artifacts. (iv) A reversible physiological block or depression can delay and reduce an AEP without affecting non-biological artifacts. Cold, local anesthesia and too frequent stimulation are among the means of reversibly depressing. Since there can be no complete list of artifacts, and ruling out some does not rule out all, this is not an exhaustive list of controls In building up a case that observed potentials are uncontaminated evoked brain potentials, one can often strengthen the case by experiments using other ‘control’ forms of stimulus, e.g. standard flashes of light, to give positive evidence that the brain is active, based on recording AEPs of familiar form and latency in appropriate loci. Limirations ofaverage evoked potentiuls. Limitations of AEPs, even after adequate artifact control, should be understood. (i) A brain region with neurons active to a certain sensory input need not give an AEP-for example due to asynchrony or due to closed-field geometry. The AEP is a facultative form of response available at some of the relevant centers or pathways. (ii) The AEP is often difficult to associate unequivocally with a responsible locus of activity, that is localization of the active site can be quite uncertain. In other cases it is often clear and unambiguous, a small movement of the active electrode loses the AEP. Among the reasons for this diversity, we should note that the effective sources and sinks for the current through the tissue whose field we sample can be small or large; close together or far apart; monopole, dipole or complex multipole in geometry. The field therefore can be more or less open, oriented, stationary or moving, uniform or steeply changing in space. It is not

surprising therefore that the waveforms of some AEPs are altered substantially when the electrode is advanced a fraction of a millimeter, whereas others are unchanged over centimeters. A class of techniques for improving the localization and the resolution of detail in temporal shifts of sources and sinks uses multiple electrodes in a fixed array (or multiple successive depths of a single electrode advanced in steps normal to a well laminated structure that can maintain a stationary state throughout the depth series). Computation, either analog on-line or digital off-line. gives the average equivalent density and direction of current at a defined point in the array (NICHOLSON& FREEMAN, 1975; FREEMAN& NICHOLSON, 1975). This ‘current sources density’ (CSD) method acts as a spatial bandpass filter, to exclude uniform and large fields and sources and sinks at a distance of more than about one array diameter. as well as very small fields in which the source and the sink are both completely within the array. (iii) Spatial sampling limitations are inherent in any of the recording methods that use small electrodes within the brain. Stated ditferently, the resolution of the recording tip of an electrode small enough to avoid excessive damage traversing the brain can be a very small fraction of the volume of even a neuromere such as the mesencephalon, so that a correspondingly large number of loci would have to be occupied, within the survival time of the preparation, to realize the capacity of the method for mapping in three dimensions the full richness of structure of the fields over space and time. Authors who have probed, for example 200 electrode tracks per square millimeter, pausing to record at each of many depths in each track, have found abrupt changes in response or receptive field between one recording site and the next. Obviously, time limits the sample one can take. CSD use in particular, to realize its value, requires recording at many loci, long enough to average at each place, with a separation between array stations almost as small as the width of the array. Published works with CSD (FREEMAN& STONE, 1969; MITZLWRF& SINGER, 1979) have generally used 0.1 mm arrays-ideally requiring 1000 stations to plot the accessible information in 1 mm3! (iv) The AEP computed by summing trials referenced to a common trigger such as the stimulus start is entirely subject to the variance of the latency of response. Any fluctuation in latency will blur and broaden the AEP so that it is not the average response in the sense of the mean amplitude and typical form. In the usual case this limitation is ignored and latency fluctuation is unknown. Some special methods attempt to deal with it and are capable of preserving waves or shoulders otherwise lost (AUNON. 1978; AUNON & SENCAJ, 1978; SENCAJ et ul., 1979; AUNON & MCGILLEM, 1979). (v) The AEP may not manifest the activity of a large number of small cells and axons in the propor-

Neuroethology deserves more study of evoked responses

tion of their abundance. This is also true of unit, multiunit and hash recording, perhaps even more true. The composition of evoked field potentials is essentially unknown, in terms of the relative contribution of apparently relevant cells, parts of cells and types of cellular processes but it is quite possible that AEPs give at least some voice to small ceils and fibers, perhaps the best of any form of recording. (vi) The last limitation I shall mention is also inherent in other recording methods, namely, that the full appreciation of the discriminating power of the method for distinguishing responses to natural stimuli differing in subtle, ethologically important ways might require that the animal be unanesthetized, alert, in a certain mood or state of motivation, perhaps freely moving and in the presence of social partners! Variety in sfi~~l~ting rechniqMes Choice of stimulus regime can influence the kinds of information that will be obtained by recording evoked potentials, especially those that require considerable computer averaging to bring them up out of the background. (i) Direct shock to a sensory nerve or tract generally maximizes the amplitude of the response by exciting a high proportion of the fibers in synchrony. For many purposes it is not material that this is unnatural; the pathways, projection targets, dynamics in comparison to other systems, and similar topics are illuminated even by the artificial stimulation. (ii) Adequate stimuli, via the sense organs give the best AEPs if abrupt: thus clicks, flashes, rapid onset stretch or touch. This class of stimuli need not be entirely uninteresting to the subject or lightning boltlike. With ingenuity a wide variety of quasi-natural stimuli have been devised that share the feature of abruptness of onset (REGAN. 1977; DESMEDT,1977; O-rro, 1978). Especially with visual stimuli, one with less luminous flux but more structure---contrasts, profiles and angles, like a checkerboard-is more effective in respect to amplitude of evoked potentiai, compared to a high intensity flash or sustained light ON. (iii) Slow onset physiological stimuli nevertheless can sometimes work, especially if repeated at a suitable frequency. Thus sinusoidally modulated light intensity, low frequency tone bursts without abrupt onset, or vestibular stimulation by repeated small angular accelerations of the head are all effective for some recording loci and levels of the afferent system. This opens the possibility of sinusoidal analysis like that which has been successful, for example, in the small signal range for cephalopod electroretinograms (HARTLINE& LANGE, 1977). Where there is a strong time dependence, short trains of sinusoids could be delivered, separated by rest periods, so that the computer would add only those cycles of the same ordinai number. A difficulty with the most natural and powerful stimuli, such as ethologi~lly important signals is that responses to some of them may habituate. This is

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most likely in the forebrain and not usually serious in the midbrain or lower. A parameter of stimulation that is often overlooked but is capable of adding valuable characterization of dynamics is repetition rate. Most obvious and common is the use of stimulus pairs at different intervals, to plot the recovery rate after a first or ‘conditioning’ stimulus, as revealed by a second or ‘test’ stimulus. The result is typically not easily read when we have to deal with evoked potentials of several negative and positive deflections because there is no self-evidently appropriate measure; instead of merely being smaller, the AEP wave form may alter. Indeed, the stimulus intervals of most interest are likely to place a complex, drawn-out second AEP on top of the first, so that computer subtraction is necessary to compare the increment due to the second with the form of the first alone. instead of pairs I recommend short trains of 5-10 stimuli, the trains separated by a few seconds to allow nearly complete recovery, comparing various interstimulus intervals, according to the preliminary findings. This gives more information than pairs. It can sometimes distinguish one modality or center from another. Thus an initial depression or partial refractoriness may be followed by a later facilitation; or after several stimuli with poor responses the system may gradually develop a following response that persists up to some repetition rate unexpectedly high compared with the paired stimulus recovery curve. The sequence beginning with the first AEP after a rest period, through depressed stages, to a steady state ‘flicker’ response is rich in information when the AEP has several components of different dynamics. ‘White noise’ stimulation is useful in certain respects, Controlling light intensity or muscle stretch or head position or electric current with a band-limited white noise voltage, has been combined with single spike recording or intracellular slow potential and spike recording (MARMARELIS & MARMARELIS, 1978) and the stimulus cross correlated with the response. A newer technique, more tractable and greatfy improving the signal to noise ratio consists of stimulating with the sum of selected sine wave frequencies (VICTOR,1979). The value of the method is usually described as “system characterization’; it is adapted to assessing nonlinearities, especially that which can be accounted for in low order Wiener kernels (the second is a common limit). Leaving aside the intracellular use as beyond the present scope, the main application of extracellular single unit spike interval correlation with white noise stimulation has been to generate the average stimulus waveform preceding a spike. This can be a discrete wave that some consider equivalent to an ‘impulse response’, or the first kernel of a Wiener kernel series and useful because its Fourier transform approximates the filter curve of the system leading to spike initiation. The impulse response constructed in this way may however have little generality because it may depend heavily upon the mean stimulus inten-

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sity. For example, if spontaneous background firing of the unit tends to be rhythmic, low intensity noise causes little departure from the rhythmic background; this severe nonlinearity gets smaller with stronger stimuli (Bul;lo et al., 1980). and artifacts of evoked potential Limitations recording can be serious, depending on the form of stimulation and the recording situation. Unnoticed movement of tissue relative to electrode is a common cause. Electrical ‘stimulus escape’ may require special efforts to minimize it. Given awareness and care, these problems can generally be dealt with and controlled for. Some forms of adequate stimuli are not ordinarily studied with AEP methods, for example thermal, chemical and osmotic stimuli. The reasons for this are

several but may not be good reasons in all cases and I suspect it would pay to try them. Variety in states of the organism When considering possible uses of evoked potentials or other electrical signs in ethologically oriented studies it is important to think of the several possible states during which the animal could be examined. For some purposes it may be under anesthesia, as when the skull is opened and primary afferent responses are the objective. This will severely limit the kinds of responses of higher centers and even of lower levels that might normally be under descending control. Whether the preparation is quite free or is tethered or paralyzed may likewise govern some re-

Long.

Head+Tail + L

w

Trans. Right +

v

FIG. I. Evoked potentials indicate electroreception in lampreys (Lamprtra rridmta). Stimulating with a homogeneous electric field parallel to (Long.) or transverse to (Trans.) the longitudinal axis of the lamprey. Evoked potential responses recorded from a site in optic tectum. (A) Single unaveraged evoked potential responses to an electric field of 50 pV/cm and 20 ms duration (indicated by the line below the record) and for comparison, that to a 10 pts light flash. In each case the evoked potential is accompanied by a multiple unit response near the peak of the evoked wave. The results indicate convergence of visual and electrosensory modalities in the tectum. (B) Average evoked potential responses to an electric field of lOOpV/cm illustrate the selectivity of the response for field orientation and polarity. (C) Evoked potential response vs the electric field intensity. Repeatable responses were elicited by fields as weak as 0.1 pV/cm. Each record is the average of 64 responses. In (C) the vertical calibration bar = 20 /IV for the top three records and 10 PV for the remaining two records. Positive potentials in the tectum are upward deflections (BODZNITK & NORTHCUTT, 1981).

Neuroethology deserves more study of evoked responses sponses. Whether the subject is resting or active, or is primed by some prior stimulation, may also influence the AEP. One can make use of the influence of priming as von Hoist did in studying ‘Umstimmung’ (VON HOLST,1950). In his case the altered angle of postural tilt of a fish, to illumination from the side, depended on the presence or absence of food juice in the water, as though the chemical stimulus increased the valence of the visual. It might pay to compare the AEP elicited, for example, in a cat or chicken by a ‘miaow’ or ‘cheep’ with and without a stuffed hawk or a live kitten or chick in view, or before and after an injection of adrenaline, or before and after the amygdala is locally cooled or treated with novocaine, or applying one trial learning or rapid learning paradigms. The payoff will surely depend on the comparison of different recording sites and different means of manipulating the state of the organism.

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responses, for example in birds (BIEDERMAN-THORSON, 1967, 19691, dolphins (BULLOCK,GRINNELL,IKEZONO, KAMEDA, KAT~UKI, NOMOTO, SATO, SUGA & YANAGI-

SAWA,1968; BULLOCKand RIDGWAY,1972), sea lions (BULLOCK,RIDGWAY& SUGA, 1971), and bats (SUGA, 1969; GRINNELL,1970). For example, comparing five species of bats, GRINNELL(1970) found each species is most sensitive in approximately the same frequency range as its emitted pulses and those species are most sharply tuned that use predominantly constant frequency pulses. He discovered in the same species an ‘off’ response tuned to a few kHz below the ‘on’ response; the ‘off’ response seems to provide a better indication of the presence and time of arrival of ethos than does the ‘on’ response. Certain species are more sharply sensitive to changes in signal angle than others, as rapidly determined by collicular AEP. BROWN,GRINNELL& HARRISON(1978) found AEPs useful in following the ontogeny of hearing in young bats, including the development of frequency tuning, EXEMPLARY FINDINGS AND ADVANTAGES low temperature cut off, temporal resolution and Variety in jindings angular sensitivity. Most of these findings would have been impracticably slow by any other method. In this section, I cite some examples of evoked Another example is the finding of BATIJEV. potential studies whose results have some interest for & FATTER (1978) that tone zoology and comparative animal behavior as well as KULIKOV,KAMENSKAYA burst AEPs recorded in the posterior sigmoid somaneurophysiology and neuroanatomy. tosensory (Sl) cortex of the cat show reliable maxima ~~ectroreception in non-te~eQst fishes. Figure I at 0.8, 1.6 and 2-3 kHz which are energetically proshows that strong evidence can be obtained from AEPs for the possession of a sensory modality by a nounced frequencies in some biologically significant cat vocalizations. Many single units also show best species not known to have it; in this instance electroreception is demonstrated in Petromyzontzjmnes (lam- frequencies close to 0.8, 1.6 and 3.2 kHz. Apparently preys) (BODZNICK & NORTHCUTT,1981). Similar evi- this more associative frontal cortex is more specialized for cat sounds than the primary auditory cortex. dence has shown or confirmed the possession of this A special variety of AEP that has proved to be modality in elasmobranchs, holocephalans (chiuseful with human subjects is called the auditory maeras, ratfish), dipneustans (lungfish), chondrosteans brainstem response. Recording from the skin at the (sturgeons) and polypterifo~s (P~~ypterus, C&zmoichthys) (NORTHCUTT, B~DZNICK & BULLOCK, mastoid and vertex (top of the head) and averaging as 1980; BULLOCK, BODZNICK & NORTHCUTT, 1981). many as a few thousand trials, small but consistent click-evoked potentials appear that can be attributed Purely behavioral methods would have taken much to early (< 10 ms) parts of the auditory pathway such more time, as would recording from sensory nerves in as cochlear nerve, cochlear nucleus, medullary and several groups where the receptors are still quite unmidbrain levels, preceding the larger, later waves asknown. Acoustic reception in sharks and other groups. As sociated with the forebrain (GALAMBOS& HECOX examples, chosen from an evoked potential study, 1977; STARR& ACHOR,1978, 1979). Remarkably similar ABRs, apparently homologous wave for wave Fig. 2 shows that a significant portal for sound entry into a shark is the parietal fossa, and that the most from I to V and possibly VII, are found in other mammals: mouse, rat, guinea-pig, cat, monkey and effective masking tone frequency under our conditions dolphin (MERZENICHet al., 1980; RIDGWAY et u/., was 100 Hz (BULLOCK& CORWIN,1979). The method has permitted tracing the auditory 1980, 1981). Even the latencies of the peaks of the pathway to the telencephalon both in sharks and in waves are the same, within fractions of a millisecond, teleosts (ECHTELER& SAIDEL,1981). It has provided in brains as different in size as rat and dolphin. The the first physiological evidence of hearing in the dynamics of the ABR in the dolphin, compared to manatee (BULLOCK et al., 1980) and in the gar, that in the other species named shows its specializLepisosteus (Hofostei) (CORWIN, BIJLL~CK & ation for the kind of ultrasonic, high repetition rate, SCHWEITZER,1981). It permitted the demonstraabrupt onset, active echo-locating audition charactertion that the brain of snakes responds to sounds, istic of dolphins. as distinct from substrate-borne vibrations, although The method seems to be eminently suitable for behavioral proof of hearing was still lacking (HART- comparison of taxa with respect to some of the ethoLINE & CAMPBELL,1969; HARTLINE,19714b). It has logically important parameters of auditory adaptation been a useful component of many studies on auditory (see next section).

T. H. BULLOCK

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Over lateral

line,

pectoral

level

C Control:

click

alone

stimulus

potentials indicate properties of hearing in a shark. (A) Carchurhinus melanoprrrus. Localized stimulation of a blacktip reef shark with a weak acoustic click, delivered through a 2 x 16 cm tube with the open end held close to the body surface under water. Recording from the dorsal anterior midline medulla. The first deflection is an artifact of the stimulus. Note absence of response in lower trace. Average of 64 responses (BULLOCK & CORWIN,1979). (B) Negaprion rrcuridens. Airborne click stimulation of a lemon shark whose skin and connective tissue over the parietal fossa have been surgically removed to gain access to the fenestra ovalis. This is an opening in the otic capsule cartilage opposite the end of the posterior canal duct which contains the macula neglecta-one of the patches of sensory epithelium presumed to be sensitive to sound, in this case sound arriving via the fenestra ovalis and posterior canal duct. The AEP recorded from the VIIIth nerve is altered in form reversibly by occluding the fenestra ovalis, supporting the conclusions that it transmits sound and that the VIIIth nerve responses to sound originate at more than one detector in the ear. Averages of 64 responses; the

FIG. 2. Evoked

lowest trace is a microphone monitor of the click (CORWIN,1980). (C) C. melanoptrrus. AEP from the anterior dorsal medulla in response to clicks, suppressed by background tones of different frequencies. adjusted to give the same peak to peak hydrophone voltage output. Tones of 100 Hz were the most effective (BULLOCK & CORWIN, 1979).

We have begun to test the method on lower vertebrate taxa, retaining the feature of placing electrodes outside the brain instead of hunting for a small hot spot in the brain. The exact placement is relatively uncritical. We record intracranially, from the space between brain and skull, usually with one electrode over the medulla and one in front of the telencephaIon. Figure 3 shows complex AEPs having certain

similarities to the mammalian ABR, in that an early series of relatively sharp waves rides on a succession of slow waves; these have been recorded in elasmobranchs, teleosts, holosteans, amphibians, reptiles, birds and mammals (CORWIN. BULLOCK & SCHWEITZER, 1981). Other examples. Further AEP studies with an ethological context are those of BARRETT (1969), HARTLINE

Neuroethology deserves more study of evoked responses

1211

s Click stimulus

[

41)

FIG. 3. Auditory brainstem responses (ABRs) of various vertebrates. Acoustic clicks in the air (the trace at the lower left was the stimulus for the turtle immediately above); recording in the left column and in the dove between an electrode just intracranial in a midline hole above the posterior cerebellum and another in front of the telencephalon (telencephalon negative = upwards deflection), in the mammals between electrodes near the vertex and the mastoid region, extracranially (vertex negative up). All except the perch are close to their normal or acclimated temperature (19-22°C for ectotherms, on left; normal body temperature for bird and mammals, on right); the perch was at 11°C though acclimated to ca. 18°C. All records 40 ms long; voltage scale under the click record = ca. 2 /JVfor ray, perch, rat, guineapig, cat and dolphin, 5 pv for dove, ca. 0.5 pv for man. Man, dolphin, bird and turtle unanesthetized; cat, guinea-pig and rat under barbiturate; frog and fish under light MS 222. Amplifier filters: 1&3COOHz except dolphin, l-So00 Hz. The more conventional mammalian ABR technique would expand the first 10 ms and filter out the slow waves. This would make clearer the equivalence of the early teeth among the mammals, wave for wave. (Non-mammalian Species, by CORWIN, BULLOCK & SCHWEITZER,1981; mammals except dolphin from MERZENICHet al., 1980; dolphin from RIDGWAYet al., 1981).

(1974), GORIS & TERASHIMA (1973),

TERASHIMA &

GORIS (1975), on the infrared reception

of crotalid

boid snakes as seen in responses

brain

tectum

LOCK, CZ~H,

by physiological KORA~EVI~,

evoked in the midstimuli. PLAIT, BUL-

KONJEVI~ &

(1974) made some comparisons

and

GOJKOVIC

of the central process-

ing of photic input in rays vs sharks evoked potential dynamics.

on the basis of

Variety in the advantages of average evoked responses Although single or multiunit or even hash potentials can answer many of the same questions, AEPs are often the first convincing evidence or even the only practicable sign. I list here some of the kinds of

questions for which they are useful, to emphasize their diversity. (i) Determining whether a certain modality (e.g. hearing) or submodality (e.g. color vision) or higher processing (e.g. stereopsis) exists is satisfactorily accomplished if an AEP is found, with all due controls for artifacts. The converse is not true, i.e. failure to find an AEP does not mean the modality is absent. This should be of inexhaustible utility in neuroethology. (ii) Determination of sensitivity (SOKOL, 1978; BULLOCK, 1979), receptive range, dynamic intensity range, discriminable stimuli, including combinations simulating natural complex stimuli, as well as the effects of

1212

T.

H.

BULLOCK

conditioning events, masking, priming, hormones, seasonal or diurnal states is not only possible with AEPs but sometimes more easily achieved with them than by behavioral methods. Again, positive results are definitive; negative results unless well controlled mean little. By extension, AEPs offer a new and simple method, independent of overt behavior, to assess the readiness of the animal, defined operationally. The permutations of experimental possibilities within the parameters in this paragraph are many and potentially interesting to neuroethology. (iii) The physiology of the AEP as a form of response is rich in detail. The structure and fatencies of successive waves, their lability, dynamics and interactions with successive or simultaneous stimuli that incorporate spatial and temporal pattern are among the variables that have produced an extensive literature on humans and laboratory mammals, valuable not only because of correlations with behavioral, cognitive or clinical states but also as descriptive raw material for speculation, modeling and new experimental design on the cellular basis of organized activity. This is a frontier with unlimited horizons because responses to every stimulus or pattern or combination can be examined with electrodes in different positions, singly or in arrays. Arrays are important in opening a window on the fine structure of activity in the tissue in terms of size and spacing of equivalent dipoles or sources and sinks, their appearance, disappearance or movement. (iv) Anatomical pathways, loci of activity, evidence of parallel processing and differentiated subsystems comprise another domain of contribution, overlapping with the foregoing. AEPs can complement the modern experimental anatomical methods that generally show only direct connections, uninterrupted by interneurons, since suitable physiologica stimuli may elicit AEPs even through a series of synapses. (v) AEP waves that appear when the subject performs a cognitive act are well known in humans, though not yet accepted in other species (DONCHIN, 1979; GALAMBOS & HILLYARD, 1981). As objective signs that the subject has not only attended but recognized and categorized a stimulus (for example, as the

‘odd bail’ or unusual stimulus, whichever stimulus was actually presented more rarely), such waves may be a tool for studying the zoological distribution of certain higher brain functions. The AEP is not an end in itself but a glimpse, often a first look, at a response system to aid in designing further investigation, by single unit recording, high resolution 2-deoxyglucose uptake, cytochemistry, multi-barrel iontophoresis of drugs. microstimulation or other methods.

SUMMARY Evoked potentials in humans and laboratory mammals have proven to be valuable not only in clinical applications and in correlations with behavioral and cognitive states, but as a reservoir of higher order physiological phenomena to be explained at the cellular levei. Each of these domains suggests heuristic application to a range of zoologicai material, using species as a variable-. -animals with more primitive nervous systems, and those advanced species with special adaptations, or any species of ethological interest. Preliminary examples indicate the several kinds of questions for which evoked potentials have particular advantages. This commentary calls for exploitation of the wide array of electrophysiological recording methods for neuroethological studies. Modalities and submodalities of sensory reception and analysis, not readily demonstrated behaviorally, as well as quantification of sensitivity, of dynamic range, of discriminable stimuli, including natural or complex patterns, interactions among stimuli and states of the animal, the effects of conditioning events, masking, priming, hormonal, seasonal or circadian changes can be studied with evoked potentials. Physiological properties and bases of ethologically relevant potentials as well as anatomical structures and pathways can also be studied by these methods. An overview is provided of the methodological choices to be made, together with some practical considerations, from types of electrode placement to data processing.

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(Accepted 19 January 1981)