Computer measurements of hippocampal fast activity in cats with chronically implanted electrodes

ELECTROENCEPHALOGRAPHY AND CLINICAL NEUROPHYSIOLOGY

165

COMPUTER MEASUREMENTS OF HIPPOCAMPAL FAST ACTIVITY IN CATS W I T H C H R O N I C A L L Y I M P L A N T E D E L E C T R O D E S JAMES C. BOUDREAU1

U. S. Army Medical Research Laboratory, Fort Knox, Ky. (U.S.A.) (Accepted for publication: August 9, 1965)

INTRODUCTION

The electrical output from the hippocampus is extremely complex. For the cat, the primary component is a 4-7 c/see "theta" wave activity, which was perhaps first described by Saul and Davis (1933). Much is known about the origin of this activity and the neural circuits controlling it (Green 1964). Its measurement in cats with chronically implanted electrodes has provided an index of hippocampal function during controlled behavioral conditions. In contrast, relatively little is known of the higher frequencies present in hippocampal activity. Brooks (1962) has demonstrated that a maximum potential gradient of fast activity is found in the hippocampal pyramidal cell layer. The various estimates that have been made of the spectral composition of this fast activity have rarely been in agreement. Dominant spectral components of 15-25 c/see and 35-40 c/see have been reported in lightly anesthetized cats (Brooks 1962), 30-40 c/see in enc~phale isol~ cat preparations (Bradley and Nicholson 1962), 15-30 c/see in cats with chronically implanted electrodes (Grasty/m et al. 1959; Passouant and Cadilhac 1961), and 40-60 c/see in unanesthetized rabbits (Petsche and Stumpf 1960; Stumpf 1965). Tokizane et al. (1959), using multiple narrow band pass filtering, have reported that the spectral energy of fast activity recorded from cats with chronically implanted electrodes is concentrated in the 10-14 c/see and 20-25 c/see bands with a 40 c/see component appearing occasionally. Adey and Walter (1963), who applied comx Present address: Veterans Administration Hospital, Leech Farm Road, Pittsburgh, Pa. (U.S.A.)

puter techniques similar to those used in the present investigation, found no concentration of spectral energy other than theta in the power spectra. It is the purpose of this paper to demonstrate that there exists a dominant fast component which can be precisely measured under controlled behavioral conditions. METHODS

The preparation

Bipolar electrodes (constructed from Formvar insulated stainless steel wire, No. 32 gauge) were chronically implanted in the dorsal hippocampus of 13 adult cats. The 2 electrode leads were cemented together with a vertical tip separation of 1.5 ram. Only the tips of the wires were bared. A silver wire soldered to a screw set in the bone overlying the frontal sinus served as a referential lead. The wires were soldered to a 9 pin socket which was fixed to the skull with stainless steel screws and dental cement. The technique of placing the electrodes to record potentials of maximal amplitude from specific areas of the hippocampus is presented in detail in the section on Results. A month or more after implantation of the electrodes, the cats were trained to perform a simple food response consisting of orientation to a food dish with the sounding of a 2500 c/see tone followed by lapping of milk which appeared in the dish from 4 to 10 sec after onset of the tone. Training of the animals was continued until they consistently oriented. This behavior took place in a sound-insulated, ventilated box equipped with a viewing window. Samples of hippocampal activity were recorded during the period immediately following onset of the tone Electroenceph. clin. Neurophysiol., 1966, 20: 165-174

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and while the animal was lapping milk without tone. These two behavioral periods are designated "waiting" and "lapping", respectively. A 24 h deprivation schedule for both food and milk was regularly employed. Five of the animals have been sacrificed and the electrode tip locations have been determined from serial sections by the Prussian blue marking technique. In other cases the animals are still being used experimentally.

exception: "coherence" here refers to the square of the "coherence" he describes. RESULTS

Electrode implantation Many evoked potentials in the hippocampus can be characterized as the products of a dipole field in which one pole is in the surface layers with a maximal amplitude near the pyramidal somata and the pole of opposing polarity is located deeper with a maximal amplitude in the

Recording and data analysis The recorded signals were amplified and passed through Krohn-Hite band pass filters, Model 330 MR with cut-off frequencies of 10 and 100 c/sec, a nominal attenuation slope of 24 db per octave and a peaking factor to reduce attenuation at the cut-off frequencies. The filtered signals were recorded at 50 in./sec on a Minneapolis Honeywell Visicorder, Model 906B. A timing signal was recorded simultaneously with the hippocampal signal on a separate Visicorder channel. The duration of a single recording period varied from about 3 to 8 sec. The amplitude of the recorded signals was measured every 6.25 msec (provided by the recorded timing pulse) with a Gerber GOAT Model B, punched as digital data onto IBM cards, and analyzed on an IBM 7090 computer. Two types of data decks were analyzed: those derived from a single record and those obtained by combining records from the same pair of electrodes from the same cat during the same behavioral period but on different days. These latter records are termed "composites". Records were made of the spontaneous activity of 13 cats (in the figures to be presented, each cat is identified by a letter and a number). The total number of digital measurements for each analyzed record is given as "N". The method of spectrum estimation used in this study has been described by others (Blackman and Tukey 1959; Walter 1963). The computer program (G2 BC COQD, available from U. C. Berkeley Computer Center) provided auto-correlation and cross-correlation functions, auto-spectra and cross-spectra, and phase and coherence functions for comparing two simultaneously recorded signals. The terminology of Walter (1963) is applicable to this report with one

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100 msec 0.1 mV Fig. 1 Examples of different types of electrical activity recorded monopolarly from a bipolar electrode straddling the pyramidal cell layer of the hippocampus. The upper tracing in each frame is from the deep lead, the lower tracing is from the surface lead. A shows the commissural evoked potential used for implantation. B and C are examples of theta and fast wave activity recorded from the bipolar electrode after the animal had recovered from the operation. The fast activity was filtered from the signals shown in the center frame and the theta wave was filtered from the signals in C.

Electroenceph. clin. NeurophysioL, 1966, 20:165-174

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HIPPOCAMPAL FAST WAVES

pyramidal apical dendritic layer (Green and Petsche 1961 ; Green 1962; Andersen et al. 1963; Gloor et al. 1963). Thus it is possible to utilize an evoked potential as guidance for implanting a pair of electrodes in such a manner that they straddle the pyramidal cell layer of the hippocampus. (This technique has been used for electrode implantation into the pre-pyriform cortex by Freeman - 1959 - and into the olfactory bulb by Boudreau and Tsuchitani - 1963.) The actual steps of implantation (carried out routinely) were as follows: a pair of recording electrodes was first lowered stereotaxically to within 3 or 4 mm of the hippocampus. A pair of stimulating electrodes was then inserted slowly into either the contralateral or ipsilateral hippocampus with the advanced lead negative until an evoked potential was recorded from each of the two recording leads. The recording electrodes

were then lowered farther until potentials of opposite polarity were recorded from the two leads, indicating that the hippocampal generator had been straddled and the leads were in opposite poles of a dipole field (Fig. 1). Even if this procedure were faithfully followed, successful implantation did not always occur due to a variety of reasons (e.g. unreliability of stereotaxic coordinates; variability in the electrically evoked potentials themselves; difficulties of hitting the curved hippocampal surface at an angle parallel to the dipole axis of the field; volumeconducted potentials from other brain regions or from separate portions of the hippocampus; local hippocampal depression induced by mechanical trauma, etc.). The major cause of failure, however, appeared to be a lesion at the recording site. Despite these failures, 1 or 2 successful implants were usually achieved when 4 pairs of

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o.1s e c Fig. 2 Oscillographic tracings of fast activity recorded from various pairs of electrodes (A & B, C & D, F & H, J & K) implanted into the dorsal hippocampus of 4 cats. The electrodes of cats AI3, A18 and A28 were located at unilateral loci; those of A40 were bilaterally located at approximately homotopic loci. See Table I for information on the distance between any 2 pairs of electrodes. Letters such as " J & K " refer to a single electrode pair.

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electrodes were implanted with the method described. Success in implantation could only be judged after the animal had recovered from the operation, since the out-of-phase evoked potential was not the final criterion. The theta activity had to be approximately 180 ° out of phase to establish the fact that the recording leads of an electrode pair were located in opposite poles of an active dipole generator. Such a situation is pictured in Fig. 1. Monopolar recordings from each lead of an electrode pair yield out-of-phase theta potentials of large amplitude. The advanced lead always recorded potentials of larger amplitude than the less advanced or surface lead (a natural consequence of the geometric configuration of the hippocampus, according to Gloor et al. 1963). Note in Fig. l that the fast activity is also out of phase and that the amplitude distribution is similar to that of the theta (arguing for contiguous if not identical generators). The phase relationship of the theta and fast activity was similar regardless of whether they were in or out of phase. This finding, which suggests that both activities are products of dipole generators located in or near the pyramidal cell layer, is in agreement with the observations of Stumpf (1965). Histological confirmation disclosed that an electrode pair straddled the dorsal hippocampal pyramidal cell zones hi or h2 (Green 1960) with the most advanced lead located in the stratum radiatum and the least advanced lead in the alveus or stratum oriens. It is believed, from the stereotaxic coordinates of implantation and the types of wave forms seen, that the electrodes of the animals still under investigation are also located in these hippocampal areas. The greatest success was achieved in the Horsley-Clarke AP plane from A 1.0 to A 5.0. Monopolar recordings from the two leads of a more anteriorly placed electrode pair usually revealed potentials of mixed phase relationships (probably because of the great degree of convolution of the anterior dorsal hippocampus). Attempts to implant into the ventral hippocampus were unsuccessful. With electrodes implanted in this manner it is possible, using a difference amplifier, to record locally generated spontaneous activity of extreme-

ly large amplitude (Fig. 2). In the sections to follow, some finer aspects of hippocampal fast activity are described. The criterion for selection of recording electrodes was near perfect superimposition of the beta activity recorded at the two leads of an electrode pair when one signal was shifted 180 ° in polarity and adjusted for disparity in amplitude. Auto-correlograms

It is apparent in Fig. 2 that even with the theta removed, it is difficult to determine even the approximate spectral composition of the fast activity. Averaging techniques are required to bring some degree of simplification of wave form. From the auto-correlograms of records of hippo-

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Fig. 3 Auto-correlograms of fast activity recorded from 10 cats while they performed the learned food response. A "composite" record contains all the "single" records for that cat taken under the specified behavioral condition on different days. N: total number of digital amplitude measurements taken every 6.25 msec for each record. "Waiting" and "lapping" refer to the behavioral condition. Eleetroenceph. clin. Neurophysiol., 1966, 20:165-174

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Fig. 4 Cross-correlograms of fast activity recorded simultaneously from 2 unilateral electrode placements. The time between recording sessions is expressed in terms of days from the first recording session.

campal fast activity in Fig. 3, it can be clearly seen that many of the signals exhibit a dominant periodicity. In some of these auto-correlograms, the dominant fast periodicity is partially obscured by other spectral components, whereas in others, a single narrow band frequency component is present.

Cross-correlograms With cross-correlation techniques it is possible to obtain measures of shared activity from two separate recording loci. In the case of crosscorrelograms a strong dominant fast rhythm was also seen, often more clearly, since unshared activity had been eliminated from the averaged record.

In Fig. 4 are presented all the cross-correlograms computed from records collected under a given behavioral condition from the 3 cats most extensively studied. Each of these 3 cats had 2 pairs of electrodes implanted unilaterally in the dorsal hippocampus (one always more anteriorly placed than the other - - see Table I for distances between electrodes). It is apparent from Fig. 4 that a com.mon dominant fast rhythm is present at each of the two recording loci. For these 3 cats a single short record during a stable behavioral condition provides a fairly accurate sample of the fast frequency components shared by the hippocampal signals. In 2 cats with bilateral, approximately homotopic implantation of electrodes, crossElectroenceph. clin. NeurophysioL, 1966, 20:165-174

170

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correlograms indicated a dominant narrow band shared fast component (Fig. 5). Cross-spectrograms To render more graphic the spectral compot27 (SIM;LE) A~8, A ~ B, d EL K. V ~ T ~ N • 1168

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sition of shared hippocampal activity, crossspectrograms were calculated. Some examples of the cross-spectrograms computed from the crosscorrelograms of Fig. 4 and 5 are presented in Fig. 6, where the concentration of spectral energy within the 10-30 c/sec range is evident. Little shared activity is seen above 40 c/sec, especially in the¢ross,spectrograms of bilateral recordings. Finer measures were made of the peak spectral energy concentrations of the cross-spectrograms by adding to the estimate with the greatest amount of spectral energy (the "dominant frequency band") the energy contained in two adjacent estimates on either side and then calculating a median value in c/sec. The median values (termed "peak frequencies") were calculated from every single "waiting" record from each of 5 cats and are plotted in Fig. 7. The peak frequencies from cats A13, A18, and A38 vary within a relatively small range. The peak frequencies of A28 exemplify that which was observed in the single record cross-spectrograms of this animal: the presence of two high energy periodicities, one at 14 and another at 20 c/sec. These two spectral components were alternatively dominant. Some of the single record crossspectrograms of A28 clearly exhibited a bimodal distribution of spectral energy, although the 20 c/sec component predominated in the composite record cross-spectrogram (Table I). No explanation can be given for the single deviant peak frequency of A40, which lowered the dominant frequency band of the composite record cross-spectrogram for this animal (Table I).

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171

HIPPOCAMPAL FAST WAVES TABLE ! Cross-spectral measurements of phase and coherence calculated from the "waiting" records of 6 cats

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*** Rate of anterior to posterior movement of the wave at the dominant frequency band of the cross-spectrograms (Electrodes were implanted bilaterally in cats A38 and A40 at approximately homotopic loci). Phase and coherence measures When the phase angle between two signals and the distance between the recording electrodes are known, it is possible to estimate the rate of spread of the signal at each estimated frequency band. The signal recorded from an anterior electrode pair consistently exhibited a phase lead over the posterior electrode pair. Such a lead suggests a moving field, sweeping across the hippocampus in an antero-medial to postero-lateral direction. The coherence, a value which ranges from 0.0 to 1.0, is a measure of the proportion of shared variability at each frequency band and is analogous to a reliability coefficient or the square of the correlation coefficient between two spectral variables (Adey and Walter 1963; Walter 1963). Table I presents measurements from the crossspectrograms of composite records from AI3, A18, and A28 and of a single record from A25. Measurements from the composite records of the 2 cats with bilateral implantation, A38 and A40, are provided for comparison. Estimates of the rate of spread of a wave at the dominant frequency band range from 0.34 to 1.44 m/sec. Three of these estimates correspond fairly closely with the 0.34 m/sec given by Petsche and Stumpf (1960; 1962) for the conduction velocity of the tbeta wave. Estimates regarding the absolute phase angles of signals recorded with bipolar technique are subject to error from several sources. The differentially recorded signal is a vector sum of the potential variations present at each lead.

Even with special selection of electrodes as in this study, the monopolarly recorded signals were not precisely 180 ° out of phase and thus phase ambiguity was introduced. The potential recorded from the lead located deep in the hippocampus is most subject to phase distortion since the hippocampus is curved and potentials generated from other hippocampal areas contribute to some extent to the final product (Gloor et al. 1963). It must also be realized that a phase lag greater than 180 ° would be calculated as a phase lead. That this excessive lag did not exist is evident from Fig. 2 in which no extreme disparity is seen between the two signals. Also, since the electrodes were placed fairly randomly about the dorsal hippocampus, a phase lag should be as common as a phase lead if an excessive lag existed. Behavior and hippocampal fast activity The cats were trained to perform a response to food primarily because the statistical spectral techniques required relatively invariant behavioral conditions. Nevertheless, changes in hippocampal spectral composition such as the appearance of new components, shifts in frequency of the dominant frequency band, and amplitude changes occurred when the animal shifted from "waiting" to "lapping". It often proved impossible to quantify these changes which varied from cat to cat and from record to record, especially when "lapping" auto-spectrograms were compared. Because of this variability, most of the Electroeneeph. clin. NeurophysioL, 1966, 20: 165-174

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J. c. BOUDREAU

figures in this report were chosen from "waiting" records. Notwithstanding this variability, consistent changes were detected in the records of the animals that exhibited fairly stable peak frequencies in their cross-spectrograms (Fig. 7). In all the 9 "lapping" cross-spectrograms of A13 and A18, the peak frequencies shifted downward with respect to the "waiting" cross-spectrograms. The decrease in frequency ranged from 0.8 to 3.8 c/sec and from 0.4 to 4.5 c/sec respectively. A single "lapping" record was taken for each of the 2 cats with bilateral implantation (Fig. 7). The peak frequency of the "lapping" crossspectrogram of A38 was 2.4 c/sec less than that measured from the cross-spectrogram of the record from the immediately preceding "waiting" period. Correspondingly, a decrease of 4.1 c/sec was found in the peak frequency of the "lapping" cross-spectrogram of A40. The amplitude of the dominant frequency band sometimes increased during "lapping" and sometimes decreased with respect to that of the "waiting" cross-spectrograms. The "waiting" and "lapping" crossspectrograms of A28 were complex and inconsistent (Fig. 7) and could not be compared. Despite the limited scope of the present study with regard to behavioral correlates of hippocampal fast activity, it is apparent from the consistent spectral changes found for the 4 cats that even short records (some less than 4 sec in duration) provide a fairly reliable index of the dominant fast component present in the hippocampal signal during a defined behavioral state Notes on artifacts

A computer printout is only as reliable as the data fed into the computer. If the signal to be analyzed becomes distorted or contaminated in any way prior to being digitized, the spectral estimates will reveal more about the recording system than about the biological preparation. Some signal distortions are not easily extracted from the computer analyzed records. For example, if the two separate channels (i.e., pre-amplifiers, filters, amplifiers, and galvanometers) for the recording of two simultaneous signals are not equated in all respects, the computer comparisons are apt to be misleading. This possibility was tested by feeding a signal

from the same pair of electrodes into both recording channels with the usual filter settings for eliminating theta. The outcome was that the autospectrograms were for all practical purposes identical and the R 2 values were greater than 0.90 from 6 to 58 c/sec and greater than 0.98 from 8 to 34 c/sec, the region in which most of the spectral energy is confined. In the range from 0 to 80 c/sec, the only phase shift greater than 8 ° was at 62 c/sec. The theta wave was removed from the record to provide greater excursions of fast activity for digital analysis and to reduce the statistical artifacts introduced by the presence of a narrow band, high energy component (Blackman and Tukey 1959). However, high pass filtering itself can induce artifacts into the record if the theta wave departs widely from sinusoidal form. Inspection ofauto-correlograms calculated from unfiltered records and the averaged signals reported by others (Adey et al. 1960; Adey and Walter 1963) revealed that the theta wave does in fact strongly resemble a sinusoid and therefore filtering is a safe procedure. Nevertheless, the fear remained that the theta wave departed from a sinusoid to a degree sufficient to induce artifacts into the filtered record. To check on this possibility, a 5 c/sec triangular wave and a 5 c/sec sawtooth wave, larger in amplitude than any theta normally encountered, were passed through the filter at the usual cutoff settings, recorded, and analyzed with the computer. The calculated correlograms and spectrograms did not resemble those computed from filtered hippocampal activity in many respects. The conclusion was drawn that departure of theta from a sine wave contributed little in the way of artifact to the spectral estimates. DISCUSSION

It has been established in this report that a narrow band fast component can be found in the hippocampal spontaneous activity of cats with chronically implanted electrodes. This component usually could not be detected if the electrodes did not meet certain standards and were not oriented precisely with respect to the local generator, in which case they would record signals from diffuse areas or of such small amplitude that a dominant periodicity could not be detected. Electroenceph. clin. NeurophysioL, 1966, 20:165-174

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HIPPOCAMPAL FAST WAVES

This dominant component and the well known theta wave do not complete the description of the spectral composition of hippocampal spontaneous activity. It is evident even from the autoand cross-correlograms and the cross-spectrograms that there exists other small amplitude wave forms of different frequency. There is also a strong possibility that any radical change from the present experimental conditions would result in a completely different spectral composition of the hippocampal signal. In this work a change in the animal's behavior was correlated with a measurable frequency shift in the dominant fast activity. It is likely that different behavioral conditions, the utilization of a different species of animal or the administration of various drugs would induce even greater changes in fast activity. Nevertheless, this report demonstrates for the first time a dominant fast rhythm, generated bilaterally, and susceptible to precise measurements. It would be interesting to speculate on the neural mechanisms producing and influencing these dominant fast waves. Eidelberg et al. (1959), Torii (1961), Yokota and Fujimori (1964) and Stumpf (1965) have provided ample evidence that the fast and theta activity of the hippocampus can be dissociated, the former involving neural circuitry that bypasses the septum. The unanalyzed fast activity is so complex that these investigators would often lump together all frequencies outside the theta range and treat it as one entity. The "regular fast activity" reported by Stumpf (1965) had a frequency between 40 and 50 c/sec. The dominant 15-20 c/sec component reported here could be detected in the presence of theta and "irregular" activity and was of a frequency greatly attenuated by the high pass filter settings used by Stumpf. So the neural circuitry responsible for the dominant fast waves described in this report probably remain undetermined. SUMMARY An evoked potential technique was used for implanting bipolar electrodes to straddle the pyramidal cell layer of the dorsal hippocampus. The fast wave spontaneous activity recorded from awake cats was analyzed with computers. The salient features of this study were as follows:

1. Further evidence was provided that hippocampal fast activity is a product of a dipole generator located in the pyramidal cell layer. 2. Computer analysis of records taken during the performance of a simple behavioral food response revealed a dominant rhythm of about 15 to 20 c/sec. This dominant rhythm varied within a fairly narrow frequency range for any one cat, with the modal frequency highly dependent upon the individual cat. 3. The fast waves could be detected in both hemispheres and appeared to propagate in an antero-medial to postero-lateral direction at a rate of less than 1 m/sec. 4. Consistent frequency changes occurred in the dominant fast waves when recordings were taken under different behavioral conditions. J. W. Rohwer assisted in the tedious process of data reduction and animal training. R. Wallace helped in the preparation of the figures. I am especially indebted to F. P. Testa of Computer Sciences Center, Purdue University, for programming assistance. REFERENCES ADEY,W. R., DUNLOP,C. W. and HENDRIX,C. E. Hippocampal slow waves. Arch. Neurol. (Chic.), 1960, 3:

74-90. ADEY, W. R. and WALTER,n . O. Application of phase

detection and averaging techniques in computer analysis of EEG records in the cat. Exp. Neurol., 1963, 7: 186-209. ANDERSEN,P., ECCLES,J. C. and LOYN1NG,Y. Recurrent inhibition in the hippocampus with identification of the inhibitory cell and its synapses. Nature (Lond.), 1963, 198: 540-542. BLACKMAN,R. B. and TUKEY,J. W. The measurement of power spectra. Dover, New York, 1959, 190 p. BOUDREAU,.1. C. and TSUCHITANI,C. Chronic implantation into the olfactory bulb of cat. Nature (Lond.), 1963, 198: 614. BRADLEY,P. B. and NICHOLSON,A. N. The effects of drugs on the electrical activity of the hippocampus. In J. CADILHAC(Ed.), Physiologie de l'hippocampe. Centre National de la Recherche Scientifique, Paris, 1962: 445-462. BROOKS, n. C. Rhinencephalic spikes and beta waves in the cat under pentobarbital. Amer. J. Physiol., 1962, 202: 1221-1229. E1OELBERO,E., WHITE,3". C. and BRAZIER,M. A. B. The hippocampal arousal pattern in rabbits. Exp. NeuroL, 1959, 1: 483-490. FREEMAN, W. J. Distribution in time and space of prepyriform electrical activity. J. Neurophysiol., 1959, 22: 644--665. Electroenceph. clin. NeurophysioL, 1966, 20:165-174

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