Spectral analysis of hippocampal EEG in the freely moving rat: Effects of centrally active drugs and relations to evoked potentials

Electroencephalographv and clinical Neurophvsiologv, 1985 6 0 : 6 5 - 7 7

65

Elsevier Scientific Publishers Ireland, Ltd.

SPECTRAL ANALYSIS OF HIPPOCAMPAL EEG IN THE FREELY MOVING RAT: EFFECTS OF CENTRALLY ACTIVE DRUGS AND RELATIONS TO EVOKED POTENTIALS t LAI-WO STAN L E U N G

Department of Psychology, UniversiO' of Western Ontario, London, Ont. N6A 5C2 (Canada) (Accepted for publication: June 7, 1984)

In previous studies, we have investigated the variation of the spontaneous hippocampal EEG and the hippocampal CA1 average evoked potentials (AEPs) following Schaffer collateral stimulation during different behaviors in the rat (Leung 1980; Leung and Vanderwolf 1980; Leung et al. 1982). We found that hippocampal AEPs and the EEG spectra show orderly changes across the following sequence of behavioral states (Leung 1982; Leung et al. 1982): slow wave sleep (SWS), awake-immobility, grooming and walking, rapideye-movement sleep (REMS). During SWS or awake-immobility, the hippocampal EEG spectrum with theta rhythm removed (the residue spectrum) showed large irregular slow activity of 0-30 c/sec (ISA) which declined smoothly with increasing frequency (Fig. 2 left column), and the Schaffer-collateral AEP displayed a large initial peak (N1) and a flat late component (N2) (Fig. 1D). During REMS or walking (Fig. 3 left colunto), the residue EEG spectrum had low ISA and high fast (30-70 c/sec) power, while the Schaffer collateral AEP showed a low initial N1 peak and a peak N2 component (Fig. 1D). The latter AEP appeared oscillatory at 20-50 c/sec, which was also the main frequency of the spontaneous fast EEG (Leung et al. 1982). A model of the hippocampal circuitry has been proposed to explain the covariation of the hippocampal AEP and EEG (Fig. 1A; Leung 1982). The model assumes that the hippocampus consists mainly of a recurrent inhibitory feedback circuit of ] Supported by grants from the Natural Sciences and Engineering Research Council of Canada U0052 and E6335, and in part by A0118.

pyramidal cells and interneurons linked by a saturation type non-linear gain (Fig. 1A). A tonic brain-stem input will shift the operating point vo and result in different dynamics in the circuitry. The EEG residue spectrum (Fig. 1B) and AEP (Fig. 1C) are the simulated outputs of the hippoOUT >

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0013-4649/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland, Ltd.

66 campal model (Leung 1982). EEG results from filtering of an input 'white noise' and AEP is a response to impulse stimulation. Since both AEP and EEG are responses of the same circuit, changing a single factor (the operating point Vo) can change both the EEG residue spectrum and the AEP. A continuum of EEG residue spectra (and AEP) can be generated that resembles the experimental data in different behavioral states. For example, the main characteristics of the power spectrum and AEP during SWS can be simulated by a low vo and those during REMS (or walking) by a high vo. In Fig. 1B and C, curve 'a' simulates conditions during SWS and curve 'c' conditions during REMS, while curve 'b' simulates behavioral states like grooming (presumably with intermediate vo, see sequence above). The present paper provides additional evidence that the model is largely successful in predicting the variation of hippocampal EEG and AEPs after administration of centrally active drugs. The effects of some of these drugs on the hippocampal EEG, especially on the theta rhythm, have been studied by polygraph recording. In acute, immobilized or in normal, immobile animals, cholinergic agonists or anticholinesterases have been shown to increase the theta rhythm while muscarinic cholinergic antagonists (scopolamine or atropine) block the rhythm (Stumpf et al. 1962; Stumpf 1965b; Kramis et al. 1975; Vanderwolf et al. 1978). However, freely moving rabbits or rats possess a theta rhythm which is resistant to atropine but sensitive to anesthetics such as urethane, ether and pentobarbital (Kramis et al. 1975; Leung 1984a). Reserpine depleted catecholamines but had relatively small effects on the theta rhythm (Vanderwolf et al. 1978; Leung 1984a). Both the atropineresistant and the atropine-sensitive theta inputs are presumably more active during walking than during immobility or SWS. Rats treated with phencyclidine or with entorhinal lesions appeared to possess only an atropine-sensitive theta during walking (Vanderwolf and Leung 1983). Behavior and many centrally active drugs modulate the AEPs in the hippocampal CA1 region following stimulation of the Schaffer collaterals (Leung 1980; Leung and Vanderwolf 1980). In correspondence to the suppression of the AEP

L.-W.S. LEUNG oscillations by atropine (Leung and Vanderwolf 1980), the above model predicts that fast hippocampal EEG should also be suppressed by atropine (Leung 1982). However, Stumpf (1965a) coneluded that scopolamine did not change the fast hippocampal EEG of curarized rabbits, as seen in polygraph records. It must be noted that polygraph records do not provide a quantitative measurement of the distribution of fast EEG across frequencies and that the effects of atropine (or other drugs) in behaving animals may be different from those in curarized ones. Therefore, this study uses power and coherence spectra to reveal and quantify drug effects on the hippocampal EEG of behaving rats. Power and coherence spectra have been shown to be sensitive indicators of hippocampal EEG changes (cf., Elazar and Adey 1967; Leung et al. 1982).

Method

Under pentobarbital anesthesia, electrodes were implanted in the dorsal hippocampus and the neocortex in 25 rats. Each electrode was a steel wire of 125 /~m diameter, insulated except at the cut end. Screws were inserted into the frontal bone to serve as ground and reference. Electrodes intended for recording hippocampal EEG were placed by electrophysiological criteria (Leung 1980) such that a dorsal electrode was in the deep neocortex (corpus callosum or layer 6) or alveus and a ventral electrode was at a depth from stratum radiatum to the granule cell layer of the dentate gyrus. Some animals were implanted with 2 pairs of electrodes, one in each hippocampus. An additional neocortical electrode was placed in the frontal neocortex in about layer 4. Electrode placements were verified by histology using cresyl violet stain. The procedure of recording EEG before and after drug injection (i.p.) has been described (Leung 1984a). Briefly, EEG was recorded during awakeimmobility (head up against gravity but with no discernible movement of the head, limbs or body) and 'walking' (includes walking, rearing, turning large postural shifts or induced struggling) before and after each drug. Cholinergic drugs (eserine

D R U G EFFECTS ON H I P P O C A M P A L EEG SPECTRA

sulfate, atropine methyl nitrate, atropine sulfate and scopolamine hydrochloride) and anesthetics (sodium pentobarbital, diethyl ether, urethane, and phencyclidine hydrochloride) and reserpine were used. Two or more drugs were given to each rat, with a minimal 4 day interval between drugs. The hippocampal EEG was analyzed off-line by a microcomputer. At least 2 channels of hippocampal EEG (dorsal and ventral) were filtered between 0.5 and 100 c/sec (24 dB/octave, 3 dB points) and sampled at 200 c/sec. Cosine-bell tapering, frequency smoothing, Fast Fourier Transform on the EEG segments (usually of 1024 points, i.e., 5.12 sec) have been described before (Leung et al. 1982). The logarithmic auto-power and the coherence z-transformed spectra were plotted. Significance of a drug effect was evaluated by a two-tailed sign test across the number of electrodes in rats treated in the same way. All spectra shown were derived for a representative, single preparation. Stimulations and recording electrodes were placed in stratum radiatum of the CA1 region of the dorsal hippocampus for the acquisition of the AEP following Schaffer collateral stimulation (Leung 1980). Averaging was done off-line by a microcomputer which calculated both the mean and the standard error of the mean. AEPs were recorded during immobility and walking in the same animals whose EEG was recorded.

Results

(1) Cholinergic drugs (a) Eserine. Twelve rats were given eserine (0.4-1 m g / k g i.p.). Eserine increased salvation and muscle twitching, increased periods of gross immobility (with spontaneous muscle twitching) and decreased spontaneous walking (horizontal) and rearing (vertical) movements. If immobility before and after drug was compared, theta rhythm was always found to increase (Fig. 2) following eserine at all doses above 0.4 m g / k g in all 12 rats (P < 0.01 sign test). A larger rhythm often accompanied a larger dose. Theta dorsoventral coherence increased in the same way as theta power. ISA was seen to decrease at 6 out

67

of 16 electrodes (7 rats) with a 0.4-0.5 m g / k g dose and at all 4 electrodes in 2 rats following a 1 m g / k g dose. No consistent changes in fast activity (30-60 c/sec) during immobility were observed following eserine of up to 1 mg/kg. Comparing normal walking with induced walking post-eserine, the theta amplitudes and dorsoventral coherence remained relatively unchanged (Fig. 3). There was a slight decrease (of 0.5-1 c/sec) in the average theta peak frequency after eserine (11 out of 12 rats, P < 0.01 sign test), especially obvious in the second theta harmonic (decrease of 1-2 c/sec). A small decrease in the power and coherence of the second theta harmonic was observed at about half of the electrodes (Fig. 3 left column). ISA remained low and unchanged after eserine, while a small increase in fast activity (30-50 c/sec) was found during walking for 1 m g / k g eserine (at all 12 electrodes in 5 rats, P < 0.01 sign test) but not for 0.4-0.5 m g / k g (at 8 out of 16 electrodes in 7 rats, P > 0.1). Atropine sulfate (25-50 m g / k g i.p.) increased motor activity, and decreased periods of immobility. During immobility or walking, 15 out of 18 electrodes in 8 rats showed an increase in ISA after atropine when compared to the same behavior before the drug (P < 0.01, Fig. 4A and C). While the absolute power at the peak theta frequency remained unchanged or slightly increased during walking after atropine, the rise of the theta peak above the ISA envelope was smaller. A small decrease in the dorsoventral theta coherence during walking was observed after drug in 6 out of 9 electrode pairs (P > 0.1; not shown in Fig. 4). If a theta power or coherence peak was observed during immobility before drug (as in the left column of Fig. 2, but not as clear in Fig. 4C and D) it would be obliterated by atropine, perhaps obscured by the large ISA during immobility after the drug. The increase in ISA power during immobility following atropine was similar at the neocortical and hippocampal electrodes (Fig. 4E) except that neocortical ISA was small at low (10 mg/kg) dose but increased up to 10-fold at 25-100 m g / k g dose, while hippocampal ISA typically increased only 2-4-fold. Hippocampal and neocortical ISA during immobility showed a practically maximal effect at 25 m g / k g dose of atropine

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Fig. 2. The effect of eserine on the hippocampal power and coherence spectra during immobility in one representative preparation. For each column, top and middle are logarithmic power spectra ventral and dorsal to CA1 pyramidal cells and the bottom is the dorsoventral coherence z-transform ('LOG COHR'). For immobile control (left), its EEG spectra (solid lines) are overlaid with the discontinuous lines which are spectra during immobility after eserine 1.0 mg/kg. For eserine 0.5 mg/kg (middle) spectra and eserine 1 mg/kg (right) spectra, the discontinuous, overlaid spectra are for control and eserine 0.5 mg/kg respectively, Note the decrease in ISA and increase in theta coherence and power with increasing doses of eserine.

sulfate. Fourteen out of 18 electrodes at various depths in the h i p p o c a m p u s showed a smaller fast activity ( 3 0 - 1 0 0 c / s e c ) during walking after atropine than before ( P < 0.05; Fig. 4A). Fast activity of 2 0 - 5 0 c / s e c during immobility was also decreased by atropine sulfate at 9 out of 18 sites tested (Fig. 4C). Atropine or scopolamine reversed the behavioral and electrographic effects of eserine. W h e n administered alone, atropine caused increases in ISA and decreases in fast activity which were opposite to the electrographic effects of eserine (Discussion). Scopolamine (2.5-5 m g / k g i.p.) gave similar results as atropine ( 2 5 - 5 0 m g / k g i.p.). Atropine methyl nitrate (50 m g / k g i.p.), which

does not cross the blood-brain barrier (Schweitzer et al. 1939), had no effect on hippocampal EEG.

(2) Anesthetics All anesthetics used suppressed m o t o r behavior. Movements of the hind limbs were affected first (occurrence of rearing decreased) and increasing dose affected trunk and neck movements. At high doses, all anesthetics caused complete paralysis and analgesia. (a) Sodium pentobarbital. L o w doses of pentobarbital (5-25 m g / k g i.p.) increased the E E G power at 1 5 - 4 0 c / s e c during immobility (12 out of 14 electrodes in 7 rats, P < 0.05), while power at other frequencies remained relatively unchanged

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Fig. 3. The effect of eserine on the hippocampal spectra during walking for same preparation as Fig. 2. Arrangement and lines as described in Fig. 2. Note a slight decrease in theta frequency and increase in 30-50 c/sec fast waves after eserine.

(Fig. 5). If a rat spontaneously walked or was induced to walk after pentobarbital, a theta rhythm of low frequency (3-7 c/sec) was always elicited ( P < 0.01, 9 rats; Figs. 5 and 6). Concomitant with the decrease in the theta frequency, the second theta harmonic was decreased or suppressed under pentobarbital (Figs. 5 and 6). Fast activity during 'walking' was slowed but increased in power after pentobarbital. This could be imagined as pushing the fast EEG power spectrum to the left (low frequency) side. The effect of pentobarbital on the fast EEG power spectrum was dose-dependent. Successive increments in dose 'pushed' the power spectrum further to the left and resulted in an enhancement of power below and a diminution above a 'cross-over' point of 20-60 c / s e c (Fig. 6). However, at doses below 25 m g / k g i.p., induced 'walking' still yielded larger

fast activity, lower ISA and a larger theta peak than immobility (Fig. 5). (b) Urethane. Urethane (1-1.5 g/kg) increased theta power at 5-6 c / s e c (at 9 out of 16 electrodes in 7 rats for 1 g / k g dose). The dorsoventral RSA coherence during spontaneous immobility was increased by urethane in all 7 rats ( P < 0.05) (Fig. 7). Theta power and coherence (at all 12 electrodes or 6 pairs, P < 0.05) during immobility were virtually abolished by 25-50 m g / k g i.p. atropine (right column, Fig. 7) while ISA was increased (at 11 out of 12 electrodes, P < 0.01). Theta could be elicited during induced struggling after 1 g / k g i.p. urethane (Fig. 8). However, the theta peak frequency was 1-3 c / s e c lower than in normal walking though similar in maximum power in all 8 rats ( P < 0.01). After urethane, fast activity during 'walking' was suppressed at half

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Fig. 4. A - D : power (at electrode in CA1 stratum radiatum) and dorsoventral coherence spectra during walking before (CON, discontinuous lines) and after (ATR, solid lines) atropine. Note increase in low frequency ISA and decrease in fast activity (30-70 c/sec) after atropine for the same behavior. E: logarithmic power increase of integrated ISA power (0.5-20 c/sec) during immobility for the right frontal neocortex (NC, layer V), and 3 hippocampal electrodes: 1, right CA1 alveus; 2, left granule cell layer; 3, right hippocampal fissure following doses of atropine sulfate. Absolute power ratios of integrated ISA at 0 dose are approximately 1:1.5:2.2:2.4 in the above electrode sequence. Only logarithmic power increase from the 0 dose condition is plotted. Note the general partial effect of 10 m g / k g and saturation at above 25 m g / k g for all electrode sites, but the largest power increase (1 log unit = 10-fold) was at the neocortical electrode.

the electrodes, especially at 50-100 c/sec (Fig. 8). Occasionally, power at 20-50 c/sec remained relatively similar to normal levels, and could even be increased. The administration of atropine (25-50 m g / k g i.p.) after urethane increased ISA and decreased fast activity (30-100 c/sec) during 'walking' for half of the electrode sites. This combination of

atropine and urethane had a greater effect on the residue spectrum than urethane or atropine alone (see Fig. 4). Theta during urethane struggling was strongly attenuated if not totally abolished by atropine in 13 out of 14 electrodes (P < 0.01). Dorsoventral coherence at the theta frequency was greatly reduced (to non-significant levels) following the combination of urethane and atropine (7 pairs of electrodes, P < 0.05) (Fig. 8 right column). (c) Ether. The actions of ether on the hippocampal EEG were similar to those of urethane. Theta was increased and ISA was decreased during immobility. During or slightly before the first R46

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Fig. 7. Effect of urethane and then atropine on the power at the electrode dorsal (top) and ventral (middle) to CA1 cell layer and dorsoventral z-transform coherence (' L O G C O H R ' ) (bottom) spectra during immobility. For immobile control (left), its EEG spectra (solid lines) are overlaid with the discontinuous lines which are spectra during immobility after urethane and atropine. The urethane 1 g / k g spectra (middle column, solid lines) are overlaid with control spectra (discontinuous). Subsequent addition of atropine sulfate (50 m g / k g i.p.) resulted in spectra on the right (solid lines), overlaid with spectra during urethane alone (discontinuous). Note an increase in the EEG power at the theta frequency but a general decrease at other frequencies after urethane. Atropine suppressed immobility theta, enhanced low frequency power (ISA) and suppressed fast waves ( > 50 c/sec).

spontaneous movements of a rat recovering from ether anesthesia, all 8 pairs of electrodes in 6 rats ( P <0.01) showed an increase in 20-50 c/sec power and dorsoventral coherence, while power above 50 c/sec was decreased• The combination of atropine and ether yielded spectra similar to those seen after the combination of atropine and urethane. After atropine, theta during immobility under ether was abolished, while clear theta was still observed during spontaneous, ataxic walking following withdrawal of ether• (d) Phencyclidine (PCP). The main effect of PCP on the theta first harmonic has been de-

scribed elsewhere (Vanderwolf and Leung 1983). After the rather variable anesthetic effect of a 10 mg/kg dose of PCP subsided, the first harmonic of the theta rhythm returned to a normal amplitude and frequency during stereotypic to-and-fro head movements and turning in circles (Fig. 9). However, concomitantly, the second harmonic of the theta rhythm was greatly suppressed in all 12 rats (P < 0.01) which showed this harmonic during undrugged walking (Fig. 9; arrow in left column). The fast EEG power in the hippocampus was also enhanced by PCP (20 out of 28 electrodes in 12 rats, P < 0.05), especially at the dorsal electrode•

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Fig. 8. Effect of urethane and then atropine on the spectra during 'walking.' Arrangement and lines as in Fig. 7. Note the decrease in theta frequency and fast EEG after urethane. Atropine practically abolished all theta power and coherence peak except the very small power peak at the ventral (middle row) electrode• Also note the very effective suppression by atropine of the high frequency power during 'walking' after urethane.

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Dorsoventral coherence in fast EEG, however, did not increase (Fig. 9) and in some cases, the dorsoventral phase clearly tended towards zero (not shown). The increase in fast EEG post-PCP began during the initial PCP anesthetic phase when the theta rhythm was suppressed, and did not change in power with behavior. Electrodes in the neocortex recorded even larger increases in fast EEG power than the hippocampal electrodes. The above results suggested that PCP probably acted to infrequency, power or coherence of the theta first harmonic, but great suppression of the theta second harmonic after PCP (arrows on left column). Subsequent injection of scopolamine (5 m g / k g i.p.) (right column) abolished the theta power and coherence peak (arrow) and suppressed the enhanced fast EEG above 50 c/sec.

DRUG EFFECTS ON HIPPOCAMPAL EEG SPECTRA crease fast EEG in the neocortex, though direct effects on hippocampal fast E E G could not be excluded. Atropine (25-50 m g / k g i.p.) or scopolamine (5 m g / k g i.p.) has been shown to block or severely suppress the theta rhythm after 10 m g / k g PCP (Vanderwolf and Leung 1983; Fig. 9). The dorsoventral theta coherence was severely attenuated after the combination of PCP and atropine (13 pairs of electrodes in 11 rats, P < 0.01). Fast EEG after PCP was suppressed by atropine at half of the recorded sites.

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(3) Reserpine In all 5 animals given reserpine 10 m g / k g i.p., the animals were cataleptic 16-18 h after the drug. The power spectra after reserpine showed the following: (1) Theta at 5 - 6 c / s e c could occur during immobility. (2) There was a 1 1.5 c / s e c decrease in the average peak theta frequency during induced 'struggling.' (3) Spectra during immobility and struggling following reserpine still showed large differences in ISA, theta and fast EEG, as in undrugged animals.

(4) Relations of EEG to AEPs following stimulation of the Schaffer collaterals As reported elsewhere (Leung and Vanderwolf 1980), the Schaffer collateral AEP at the distal apical dendritic layers of CA1 region in the freely moving rat consisted of an early negative peak (N1), a positive trough (P1) and a late negative (N2) wave. During active movements (e.g., walking) as compared to immobility, N1 and P1 decreased in amplitude and the N2 peak became more pronounced and appeared at an earlier latency (Fig. 10). Eserine (0.4 m g / k g i.p.) reduced the N1 peak and slightly enhanced the late N2 peaks during immobility or walking (Fig. 10A). Atropine (25 m g / k g i.p.) increased N1 and blocked late oscillations, i.e., the N2 wave became flat (Fig. 10B). Pentobarbital (20 m g / k g i.p.) increased the late N2 wave but also delayed its peak, with an inconsistent effect on the N1 peak (Leung 1981; Fig. 10C). Urethane increased the N2 peak latency, especially during walking, with a small decline in the N1 (Fig. 10D). After reserpine, the N1 peak was decreased but the N2 peak remained large or

Fig. 10. The effect of different drugs on the AEP following stimulation of the Schaffer collaterals. Left column, AEPs during immobility, right column, AEPs during walking. Solid lines are after drug and dotted ones are control tracings before drugs. Each AEP is displayed with its mean (lower curve) and its mean+S.E.M. (standard error of the mean). Effects are shown for different rats following intraperitoneal dose of (A) 0.4 mg/kg eserine, (B) 25 mg/kg atropine sulfate, (C) 10 mg/kg pentobarbital, (D) 1 g/kg urethane and (E) 10 mg/kg reserpine. The largest AEP magnitude in each rat (N = 16-20 stimuli) was of 1-1.5 mV, the complete trace was 128 msec with shock artifact labeled by dot.

even slightly larger (Fig. 10E). AEPs still remained correlated with behavior after reserpine. Discussion

Effects of drugs on theta In undrugged rats, theta peaks during immobility were small or absent (Leung et al. 1982). Eserine, ether or urethane induced a regular theta rhythm during immobility in a dose-dependent manner. The rhythm under eserine appeared the most regular, while under urethane or ether, irregular waves could alternate or be mixed with theta during spontaneous immobility. Atropine or scopolamine severely attenuated or abolished the immobility theta, in accordance with the previous literature (Stumpf 1956b; Kramis et al. 1975; Vanderwolf et al. 1978). Atropine (up to 50 m g / k g i.p.) or scopolamine (up to 5 m g / k g i.p.) alone was unable to abolish

74 theta during walking (Kramis et al. 1975; Vanderwolf et al. 1978). However, the following spectral characteristics of EEG were affected by atropine: (1) The rise of the theta above the 'residue' spectrum was reduced after atropine. (2) ISA was increased by atropine compared to the same behavior (immobility or walking) before the drug. (3) The theta dorsoventral coherence peak was usually smaller after atropine. The increase in theta frequency after scopolamine reported by Buzshki et al. (1980) was usually but inconsistently found and could reflect a general increase in average motor activity after atropine. The action of atropine on hippocampal theta during walking is further supported by other evidence: the theta phase in CA1 stratum radiatum was affected by atropine (Leung 1984a). Theta units in the hippocampus sometimes stopped firing over several theta cycles, which was rarely found in undrugged rats (Buzsb&i et al. 1983). The above results suggest that atropine-sensitive as well as atropine-resistant inputs are present during walking, as previously proposed (Leung and Vanderwolf 1980; Vanderwolf and Leung 1983; Leung 1984a, b). The second theta harmonic was commonly found in spectra during walking when the peak theta frequency (first harmonic) was high. The appearance of a second (or higher) harmonic makes the theta rhythm appear more like a sawtooth than a sinusoidal wave. Theta of low frequency accompanying grooming or immobility in undrugged animals (Leung et al. 1982) or following ether, urethane or eserine infrequently possessed a second harmonic. PCP, and to a smaller degree eserine, had an interesting effect in suppressing the theta second harmonic during walking when the fundamental theta frequency and amplitude remained unchanged. This signifies that even at high theta frequency, the first harmonic is not necessarily associated with the second harmonic. Since PCP, ether or urethane suppresses the atropine-resistant theta (and eserine enhances the atropine-sensitive theta), it may be suggested that the second theta harmonic more frequently accompanied the atropine-resistant than the atropinesensitive rhythm (however, see also Fig. 8). After administration of a high dose of an anesthetic or reserpine, rats had to be induced to

L.-W.S. LEUNG struggle. In normal undrugged rats, vigorous movements such as struggling increase the theta frequency, the theta second harmonic and less consistently the fast EEG power (Leung 1984b). Therefore the effects of anesthetics in decreasing the theta frequency, the theta second harmonic and the >50 c/sec fast EEG (below) should be greater if struggling before and after an anesthetic is compared (instead of walking before and struggling after drug).

Activation hierarchy of hippocampal cortex A sharp distinction between ISA (defined to be <30 c/sec) and fast activity (>30 c/sec) is difficult, especially after drugs. The model predicts a continuum of residue spectra (Fig. 1; Leung 1982) corresponding to a range of tonic brain-stem input. A low operating point (Vo) results in a residue spectrum of large ISA and small fast activity and an evoked response of a large initial peak and a flat late component (curve 'a' of Fig. 1). Increasing the brain-stem tonic input increases vo, reduces the magnitude of the decline of spectral power with frequency, increases fast EEG activity, decreases the initial AEP peak and increases late AEP oscillation (with emergence of an N2 peak) (curves 'b' and 'c' in Fig. 1). Experimental data can be qualitatively organized in a sequence corresponding to increasing 'activation' (presumed equivalent to a vo increase). The lowest hippocampal activation (presumably low brain-stem tonic input) occurs after the combination of ether/urethane/PCP plus atropine/ scopolamine and is manifested by a residue spectrum of extremely large slow EEG and very small fast EEG. A slightly higher activation gives residue spectra of smaller ISA but little fast activity as seen during immobility after atropine or during SWS (Leung et al. 1982). Further activation gives lower ISA as seen during normal, undrugged immobility and during immobility after eserine, ether or urethane (Figs. 2 and 7). Still higher activation (large Vo) is accompanied by increased fast activity and decreased ISA: walking after ether or urethane gives a small increase in fast activity, normal walking a larger increase, and walking after eserine the largest increase of fast activity. The effect of drugs on the hippocampal AEP is

D R U G EFFECTS ON HIPPOCAMPAL EEG SPECTRA

generally similar to the sequence proposed above. AEPs after atropine had the largest N1 peak, followed by AEPs during undrugged immobility, grooming and immobility after eserine/ether/ urethane (see also Leung and Vanderwolf 1980), An N2 peak in the AEP was seen with increasing prominence and earlier latencies, corresponding to the increasing high activation.

Action of drugs Hippocampal activation can be partly ascribed to a muscarinic cholinergic input through the septum (Lewis and Shute 1967; Dudar 1977; StormMathisen 1977). Eserine, a cholinesterase inhibitor gave responses indicative of 'high activation' and atropine gave responses indicative of 'low activation.' Our results differ from those of Stumpf (1965a) who found no change in the rabbit's fast hippocampal EEG following scopolamine, based on inspection of polygraph records. However, not all spectral (or AEP) changes can be ascribed to a muscarinic cholinergic input. For example, after a large dose of atropine (up to 50 mg/kg i.p.), presumably sufficient to block all muscarinic cholinergic receptors, ISA and the N1 peak of the Schaffer-collateral AEP still varied with behavior though fast EEG and late AEP oscillations were suppressed (Figs. 4 and 10). Reserpine, at doses which deplete forebrain norepinephrine, dopamine and serotonin levels by 80-90% (Vanderwolf and Pappas 1980), did not greatly affect the relation of the hippocampal EEG or AEP to behavior. Perhaps only minute quantities of monoamines are necessary for behavioral modulation of hippocampal EEG and AEP or perhaps they are not critically important (cf., Vanderwolf et al. 1978). The hippocampal 'acitivation' may depend on the sum total of multiple inputs, each of a different neurotransmitter (Leung 1982). Effects of neurotransmitters other than acetylcholine still remain to be studied in detail. Using EEG spectra alone, it is difficult to distinguish whether a drug acts directly on the hippocampus or on its modulating inputs originating downstream. Similar difficulties may be encountered with the use of evoked responses (extra- or intracellular) when the tonic modulation or the intrinsic feedback is strong (cf., Leung 1982). While

75

cholinergic drugs (e.g., eserine or scopolamine) may affect septohippocampal and intrinsic hippocampal synapses, their effects on the theta rhythm are probably exerted primarily on the pacemaker cells of the medial septum (Stumpf et al. 1962). Similarly, effects of cholinergic drugs on the hippocampal residue spectrum could also be interpreted as a modification of tonic inputs from the brain-stem. Barbiturates (Nicoll et al. 1975; Leung 1981), urethane (Ben-Ari et al. 1981) and ether (Leung 1981) have been shown to act on hippocampal synapses, especially inhibitory ones, besides acting on the rhythmic generation of theta in the septum (Stumpf et al. 1962). This may account for the general direction of change in the residue spectrum (by reduction of tonic inputs and Vo) after pentobarbital, urethane and ether, but not for the large increase in fast activity (or the large N2 component of the Schaffer-collateral AEP) seen particularly after pentoarbital. Direct drug action on the hippocampal circuit itself could not be fully accounted for by means of changing a single model parameter Vo. The relative importance of direct and indirect action of each drug would be elucidated by simultaneously studying activities in the hippocampus and those structures afferent to it (e.g., medial septum as in Stumpf et al. (1962)), preferably in the behaving rat.

Summary Hippocampal EEG signals derived from chronically implanted electrodes in the freely moving rat were recorded before and after administration of centrally acting drugs, and analyzed by power and coherence spectra. Eserine, ether or urethane induced a low frequency (3-6 c/sec) theta power and coherence peak in the immobile rat, which was sensitive to atropine or scopolamine. After phencyclidine, theta that occurred during walking (7-8 c/sec) was virtually abolished by atropine while in the normal rat, absolute theta power was not affected by atropine. The residue spectrum, defined as the EEG spectrum with the theta harmonics removed, was sensitive to centrally acting drugs. Ether, urethane and pentobarbital suppressed fast waves of 50-100 c/sec, and under

76

some conditions, enhanced 15-50 c/sec waves. Eserine enhanced (30-60 c/sec) fast waves during walking while atropine suppressed fast waves and increased irregular slow activity (<30 c/sec). The main effects of drugs and behavior on the residue spectra and on the average evoked potentials following stimulation of the Schaffer collaterals could be explained by a previously proposed model (Leung 1982) which suggests a continuum of hippocampal 'activation' (tonic input) under the various conditions.

Resum~ Analyse spectrale de I'EEG hippocampique chez le rat libre: effets de drogues ~ action centrale et relations avec les potentiels bvoqubs

L'EEG hippocampique a 6t6 enregistr6 h partir d'61ectrodes implantres chroniquement chez le rat libre, avant et aprrs administration de drogues action centrale; puis analys6 h l'aide des spectres de puissance et de cohrrence. L'rsrrine, l'rther et l'urrthane ont provoqu6 un pic de cohrrence et de puissance thrta h basse frrquence (3 h 6 c/sec), qui a 6t6 sensible b, l'atropine ou h la scopolamine. Aprrs phencyclidine, le thfta qui apparait pendant la locomotion (7 ~t 8 c/sec) a 6t6 pratiquement supprim6 par l'atropine alors que chez le rat normal l'atropine n'a pas affect6 la puissance absolue du thdta. Le spectre rrsiduel, drfini comme 6tant le spectre EEG aprrs 61imination des harmoniques du thrta, a 6t6 sensible au drogues h action centrale. L'rther, l'urrthane et le pentobarbital ont supprim6 les ondes rapides de 50 h 100 c/sec, et dans certaines conditions ont augment6 les ondes de 15 ~t 50 c/sec. L'rsrrine a augment6 les ondes rapides (30 ~ 60 c/sec) au cours de la locomotion alors que l'atropine les a supprim6 et a augment6 l'activit6 lente et irrrgulirre (<30 c/sec). Les principaux effets des drogues et du comportement sur le spectre rrsiduel et sur les potentiels 6voqurs, moyennrs aprrs stimulation des collatrrales de Schaffer, pourrait &re expliqurs par un modrle propos6 prrcrdemment (Leung 1982) qui suggrre un continuum d'activation (entrre tonique) de l'hippocampe sous diverses conditions.

L.-W.S. LEUNG I thank Dr. Case Vanderwolf for comments and discussions, Sue Rumble, Lynne Mitchell and Barb Mills for typing the manuscript, and Derrick MacFabe and Debbie Stewart for histology.

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