HTPPOCAMPAL THETA ACTIVITY IN THE ACUTE PRETRIGEMINAI, CAT

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1 ACTA NEUROBIOL. EXP. 1976, 36: HTPPOCAMPAL THETA ACTIVITY IN THE ACUTE PRETRIGEMINAI, CAT T. RADIL-WEISS, B. ZERNICKI and A. MICHALSKI Department of Neurophysiology, Nencki Institute of Experimental Biology Warsaw, Poland Abstract. In five cats with a pretrigeminal brainstem transection ocular behavior and cortical and hippocampal EEG activity were recorded. The ECoG desynchronization was usually accompanied by clear hippocampal theta activity. The episodes of ECoG desynchronization and hippocampal theta activity lasted in some cases for several hours. Visual stimuli evoked two types of hippocampal EEG response: (I) increase of regularity and frequency of theta activity (11) EEG desynchronization. The intensity of these responses was positively correlated with the intensity of ocular and ECoG components of the orienting reflex (the most conspicuous phenomenon being their habituation with stimulus repetition). Electrical stimulation of the midbrain reticular formation evoked theta activity of a frequency proportional to stimulus voltage; for maintaining it constant stimulus strength had to be elevated gradually. Depriving the brain of influences from the lower brainstem does not influence qualitafi~el~ the function of reticulo-septo-hippocampal system. INTRODUCTION Several lines of evidence indicate that the majority of acute pretrigeminal cats are continously awake (2, 11, 16). In such preparations visual stimuli evoke clear-cut ocular orienting reflex consisting of following movement, pupillary dilatation and accommodation. This reflex habituates rapidly with repetition of the stimulus. It has been also shown that both classical and instrumental ocular conditioned reflexes

2 can be easily elaborated in the pretrigeminal cat (1, 10, 19). The neocortical EEG activity of the pretrigeminal cat was intensively investigated (11, 16). In the acute stage two types of ECoG activity were distinguished: type I almost continuous low-voltage activity, and type I1 - high-voltage activity mixed in different proportions with low-voltage activity. It is assumed that in the awake preparation these two types of ECoG activity correspond to the states of alert wakefulness and drowsiness, respectively. Spontaneous transitions from one to another type of ECoG activity are infrequent: a given type may last for several hours. During type I1 ECoG activity, visual stimuli produce clear-cut ECoG arousal. This paper deals with hippocampal EEG activity in the acute pretrigeminal cat. Both spontaneous EEG activity and EEG responses to visual stimuli as well as to stimulation of the midbrain reticular formation were analysed. The aim of the experiments was to find out whether disconnection of the brain from the lower brainstem influences the activity of the reticula-septo-hippocampal system responsible for the generation of the regular slow hippocampal "theta" activity (6) and whether a correlation exists between its function on the one side and cortical EEG and behavioral manifestations present in the acute pretrigeminal cat on the other side. METHODS Five cats were used. The pretrigerninal brainstem transection was performed with a blunt spatula under ether anesthesia (16). Two pairs of silver electrodes were placed on the dura mater in the right occipital and frontal areas. The interelectrode distance in each pair was 10 mm. "Bipolar" electrodes were placed in the left dorsal hippocampus and in the left midbrain reticular formation. The electrodes were made from 0.3 mm diameter Nichrome wire with sharpened, uninsulated tips 1 mm apart. Position of all electrodes was determined histologically (Fig. 1). Lowering the hippocampal electrode in place was often heralded by seizure activity (which might spread to the cortex) followed by flattening of the hippocampal EEG. In two cases the pathological activity in the hippocampus resembling spreading depression (14) lasted several minutes (Fig. 2). The upper and the third eyelid were removed. A thread was attached to the lower margin of the cornea of the right eye and the eye movements were recorded with a tensometric method (3). Three visual stimuli were usually used: a feather duster (20" X 35"), a black stick (lo X 1" X 4"), and a black card (10" X 15"). The stimuli

HIPPOCAMPAL THETA ACTIVITY 519 moved vertically against white screen located 57 cm from

The stick and card were usually presented automatically: they appeared in the window (10"

The brush was moved by hand with a similar speed.

(frequency 300/s, duration 1 ms), of a voltage either adjusted by the experimenter or

3 HIPPOCAMPAL THETA ACTIVITY 519 moved vertically against white screen located 57 cm from the cat's eyes. The stick and card were usually presented automatically: they appeared in the window (10" X 40") of the screen (see 18) and moved up or down with a speed of 10 /s. The brush was moved by hand with a similar speed. The mesencephalic reticular formation was stimulated by a series of electrical pulses (frequency 300/s, duration 1 ms), of a voltage either adjusted by the experimenter or controlled automatically Fig. 1. Representative example of electrode placement used for recording dorsal hippocampal EEG' activity. Arrow indicates location of the tip of the electrode. Kliiver-Barrera stain. Cat 1 (AP+5, L5, H-t-9).

4 520 T. RADIL-WEISS, B. ZERNICKI AND A. MICHALSKI - 4 * F Front. ',;:"&~*~'1,)~'~ +, 'y4;v'u*%~+v., k c t-!rode owed C5mm L Fig. 2. Seizure activity in the hippocampus and cerebral cortex evoked by the penetration of the electrode into the hippocampus. In the hippocampal trace the seizure action is followed by EEG flattening lasting several minutes. After 8 min theta activity appeared in the hippocampal record. Cat 1. by means of an external feedback device (12), increasing or decreasing the voltage in an indirectly proportional way according to the instantaneous hippocampal theta frequency (13). The effective stimulation voltage was constantly monitored during the experiment. All EEG recordings have been evaluated visually and the frequency of theta acti-

5 ~ type HIPPOCAMPAL THETA ACTIVITY <521 vity counted and statistically processed from randomly selected samples. Typical recordings in three cats have been recorded on FM modulated tape and processed off-line by means of a DEC LAB 8 computer for autocorrelation and spectral density functions. RESULTS Main features of the preparations are summarized in Table I. Cats 1-3 were awake as shown by the presence of adequate ocular following reflex. Cat 4 was semicomatous and cat 5 comatous: the ocular Ocular responsiveness and spontaneous EEG activity of preparations Dominating Hippocampal theta activity Cata 1 Following rerex ( ECoG typeb AmountC / Frequency: mean & SD (n) 3 After amphetamine 4 After amphetamine 5 After amphetamine!, adequate adequate adequate adequate I ) abortive I adequate I absent, absent I I1 j ++ 1 type I ++ i type I1 I type I ++ ' I type I ++ I type I ++ type I1 type I, type 11 / + + a In three cats amphetamine was given a few hours after the pretrigeminal transection. b Type 1: almost continous low-voltage activity; type 11: high voltage activity mixed in different proportions with low voltage activity. c Symbols: +. theta activity mixed with low-voltage activity; + +. domination of a pure or almost pure theta activity. following reflex was respectively abortive and absent in these cats. In confirmation of our previous results (ll), the wakefulness of the preparations was not correlated with the type of ECoG activity. Type I dominated in cats 2 and 4, and type I1 in cats 1, 3 and 5. In all preparations theta activity was observed in the hippocampus (Table I). The presence of clear theta activity was correlated with cortical EEG desynchronization, but in cat 1 well developed theta activity was accompanied by type I1 ECoG activity. The episodes of ECoG desynchronization and clear hippocampal theta activity were usually long-lasting and occasionally they were observed for several hours. In

6 Fig. 3. The effect of amphetamine on the cortical and hippocampal EEG activity. Native and band path filtered hippocampal EEG is recorded. Arrow indicates the moment of intravenous administration of 0.5 mg of amphetamine. After about 20 s, ECoG activity became continuously desynchronized (type I), and hippocampal theta activity became much more regular. Cat 5.

HIPPOCAMPAL THETA ACTIVITY 523 cats 1, 2 and 4 well developed theta activity was present virtually during the whole experiment. The frequency of theta activity was slow about 3.5-4 cyclels (Table I).

7 HIPPOCAMPAL THETA ACTIVITY 523 cats 1, 2 and 4 well developed theta activity was present virtually during the whole experiment. The frequency of theta activity was slow about cyclels (Table I). In cats 3-5, 0.5 mg of amphetamine was administered intravenously a few hours after the transection. Then cat 4 became fully awake (the adequate following reflex appeared), but cat 5 remained comatous. Moreover, in cats?, and 5, type I ECoG activity became dominant and hippocampal theta activity became clear (Table I, Fig. 3). Ocular following reflex was accompanied by pupillary dilatation, ECoG arousal (clearly seen against the background of type I1 ECoG activity) and hippocampal EEG response (Fig. 4-7). The intensity of all these components of the orienting reflex was positively correlated. Two types of hippocampal EEG response to visual stimuli were observe'd. The first type will be called "theta response": the theta activity became more regular, its frequency often increased (maximally to 6 cycle/s), and its amplitude occassionally increased as well (Fig. 4-7). The second type consisted simply of EEG desynchronization (Fig. 6). In cat 4 there was usually present the theta response, whereas in cat 1, the EEG desynchronization. In cats 2 and 3, both types of hippocampal EEG response were observed with even frequency. With repetition of the visual stimulus the orienting reflex habituated rapidly (Fig. 4, 6). All components of the reflex habituated parallely. In one or two cases during the course of habituation the hippocampal theta response was replaced by the desynchronization response (Fig. 5). Electrical stimulation of the midbrain reticular formation evoked clear-cut response consisting of pupillary dilatation, ECoG desynchronization, and hippocampal EEG response (Fig. 8). In some cases a vertical tonic eye-movement also appeared. The frequency of hippocampal activity increased (within a certain range) proportionally to the stimulus voltage. Very strong stimuli induced in some cases hippocampal desynchronization. During stimulation with unchanged voltage the theta frequency decreased gradually. Occasionally, the response consisted of two stages: theta response was followed by EEG desynchronization (Fig. 8). Maximal increase of theta frequency was up to 7 cyclels. In pilot experiments with the feedback device we succeeded in stabilizing the hippocampal theta frequency for several minutes. To maintain it, a constant stimulus voltage had to be increased gradually (Fig. 9). At the end of the experiment mg of Nembutal was administrated intravenously to three preparations. In all cats cortical EEG b'ecame synchronized and hippocampal theta activity reduced for several 4 - Acta Neurobiologiae Experimentalis

8 T. RADIL-WEISS, B. ZERNICKI AND A. MICHALSKI

9 ,, ",..," -....,,,,,.,,,,,, ^.. I,,,,",,.,,,...", "-,.,...,,...I After 3 min Fig. 4. Habituation of the hippocampal "theta" response and ocular following reflex to a feather brush moved downward by hand. Black dots indicate presentations of the brush which was seen by the cat for about 1 s each time. Intertrial intervals were 30 s. Note that duration of theta response corresponded roughly with that of the ocular response. In trial 1, both responses lasted several seconds. The eyes made a saccadic movement upward and followed the object smoothly downward. After disappcarence of the object the cat still looked downward for a few seconds and then made a few additional movements. In trial 19, theta and ocular responses were already poor but recovered spontaneously after 3 min break (trial 20). Cat 2.

10 T. RADIL-WEISS, B. ZERNICKI AND A. MICHALSKI R. Eye Fig. 5. Replacement of a hippocampal theta response by a desynchronization response with repetition of the brush stimulus. Intertrial intervals were 30 s. Note the habituation of movements occurring after the stimulation has ceased. Explanations as in Fig. 4. Cat 1.

11 R Eye - Fig. 6. Habituation of the hippocampal EEG desynchronization and ocular following reflex to a black stick moved automatically dovrnward. The moments when the stick enters or leaves the screen window are indicated by arrows. The stick (lo X 4') was oriented horizontally and moved with a spead of 1O0/s. Intertrial intervals were 20 s. Cat 1.

12 h ' 1 ' l ", ' ', ' f ' l " ', I, " " ' f f l,, ' ' 1, '',';,'I ' 4 I ' l fl " J, I"' I :$..!,I...~,\:l.~~...,./i,i,,.:,,.,,, l.,oa ~,JA/!~/., , 1,r;~v 1,,,,/1 l,'d,"4't " I ~I~.:P~ri~..l Fig. 7. The hippocampal and ocular effects of long-lasting action of the brush-stimulus moved up and down by hand. A - Gradual habituation of the response. The stimulus was presented for 90 s but only the first 60 s are seen on the Figure. B - Partial recovery after 20 min of rest. Cat 4.

13 Fig. 8. The effect of electrical stimulation of midbrain reticular formation on hippocampal EEG activity. Small-voltage stimulation increased frequency and amplitude of theta activity, middle-voltage increased frequency but decreased amplitude of theta, and high-voltage evoked EEG desynchronization which was followed by frequent- low-amplitude theta activity. Cat 2. CD

14 Filh L. Hipp. Fig. 9. The effect of electrical stimulation of midbrain reticular formation on hippocampal EEG activity performed by means of a feedback device increasing automatically stimulus voltage when theta frequency decreased and vice versa. Native and band path filtered EEG is recorded in the first and second row. The amplitude of the third one represents the actual value of instantaneous hippocampal frequency expressed by means of an analog electrical circuit from the filtered recording. The amplitude of the fourth row indicates the actual stimulation voltage (controlled automatically according to the value of instantaneous frequency) as well as the instant of switching on and off the stimulation. Stabilization of hippocampal frequency by means of the feedback device requires a gradual increase of stimulation voltage. Cat 2.

15 HIPPOCAMPAL THETA ACTIVITY 531 minutes (Fig. 10). Simultaneously visual stimuli stopped to evoke ocular and EEG orienting response. The results of correlation and spectral analysis of hippocampal EEG a 1 2"' Fig. 10. The effect of Nembutal on EEG activity and ocular following reflex. Arrows indicate the moments of intravenous administrations of 10 mg of Nembutal. Dots indicate the presentations of the brush-stimulus. After the first dose of Nembutal, the cortical EEG activity became partly synchronized, but the hippocampal theta activity seemed to be unaffected and presentation of the brush still evoked the ocular following reflex. The second dose of Nembutal synchronized the cortical EEG, reduced regularity of hippocampal theta activity, and abolished the following reflex. After 17 min, partial recovery was observed. Cat. 4.

confirmed the above described results. Theta frequency in acute pretrigeminal cats is relatively slow but regular. The degree of regularity increases further when the feedback device is used.

16 confirmed the above described results. Theta frequency in acute pretrigeminal cats is relatively slow but regular. The degree of regularity increases further when the feedback device is used. After Nembutal administration the activity becomes slower and more dispersed in time. DISCUSSION Our results showed that in the acute pretrigeminal cat the cortical EEG desynchronization is usually correlated with well developed hippocampal theta activity. In this respect the pretrigeminal cat is similar to the intact cat (4, 6, 7) in spite of the fact that the brain is deprived of influences from structures below the pretrigeminal section. In the preparation studied neither the generation of theta activity by means of the reticula-septo-hippocampal system, nor its modulation (of excitatory and inhibitory nature) correlated with other components of the orienting reflex to visual stimuli, is influenced in a dramatic way by the transection. However, two differences between the acute pretrigeminal cat and the intact cat should be noted: (I) The amount of EEG desynchronization and hippocampal theta activity is much larger in the pretrigeminal cat. Previous data (11) showed that in the acute pretrigeminal cat episodes of ECoG desynchronization may last for several hours, and the present results revealed that the same is true for the hippocampal theta activity. As activation of the reticulo-septo-hippocampal systems is a sign of arousal its presence proves that the brain of the pretrigeminal cat is awake. The reactivity of theta activity to visual and midbrain stimulation is a strong argument against the assumption that the state of preparation might correspond to paradoxical sleep. (11) The frequency of theta activity is lower in the pretrigerninal than in the intact cat (4, 6, 7). It is hard to decide, without performing further experiments whether it is a consequence of a decrease of the general activation level due to disconnection of the brain from a part of the reticular formation, or is caused by some type of active inhibitory phenomenon related to immobilization. Experimental results obtained in rats (9) seem to support the second assumption. It is generally believed that hippocampal theta response and hippocampal EEG desynchronization have different physiological meaning (4, 5, 8, 151. Surprisingly, in our cats both types of responses were observed in the same experimental situation, and both seemed to be simply a component of the orienting reflex to visual stimuli. It is possible, however, that the motivational state (andlor the "attitude"

17 HIPPOCAMPAL THETA ACTIVITY 533 toward the stimulus) might change in the pretrigeminal cat which is capable of learning (1, 10, 16, 17, 19). Special experiments would be required, however, for testing this possibility. We thank Dr. M. S16sarska, Mr. J. Folga and Mrs. J. Rokicka for assistance in conducting the experiments. This work was performed within the Intermozg collaboration and was supported by Project of the Polish Academy of Sciences. REFERENCES AFFANNI, J., MARCHIAFAVA, P. L., and ZER.NICK1, B Conditioning in the midpontine pretrigeminal cat. Arch. Ital. Biol. 100: BATINI, C., MORUZZI, G., PALBTINI, M., ROSSI, G. F., and ZANCHETTI, A Effects of complete pontine transection on the sleep-wakefulness rhythm: the midpontine pretrigeminal preparation. Arch. Ital. Biol. 97: FOLGA, J., MICHALSKI, A., TURLEJSKI, K., and ZERNICKI, B Eye movement recording with a tensometric method ill the pretrigeminal cat. Acta Neurobiol. Exp. 33: GRASTYAN, E., LISSAK, K., MADARASZ, I., and DONHOFFER, H Hippocampal electrical activity during the development of conditioned reflexes. Electroenceph. Clin. Neurophysiol. 11: GRASTYAN, E., KARMOS, G., VERECZKEY, L., and KELLENYI, E. E The hippocampal electrical correlates of the homeostatic regulation of motivation. Electroenceph. Clin. Neurophysiol. 21: GREEN, J. D., and ARDUINI, A. A Hippocampal electrical activity in arousal. J. Neurophysiol. 17: KEMP, I. R., and KAADA, B. R The relation of hippocampal theta activity to arousal, attentive behaviour and somatomator movements in unrestrained cats. Brain Res. 95: LOPES da SILVA, F. H., and KAMP, A Hippocampal theta frequency shifts and operant behaviour. Electroenceph. Clin. ~europhysiol. 26: RADIL-WEISS, T., and HLAVICKA, P To the function of the reticulosepto-hippocampal subsystem of the brain. Acta Sci. Hung. (in press). SHLAER, F., and MYERS, M. E Operant conditioning of the pretrigeminal cat. Brain Res. 38: SLOSARSKA, M., and ZERNICKI, B Wakefulness and sleep in the isolated cerebrum of the pretrigeminal cat. Arch. Ital. Biol. 111: SPUNDA, J., and RADIL-WEISS, T Technique for feedback control of brain stimulation according to the dominant EEG frequency. Physioi. Bohemosl. 21: 441. SPUNDA, J., and RADIL-WEISS, T A simple device for measuring the instantaneous frequency of the dominant EEG activity. Electroenceph. Clin. Neurophysiol. 32: WEISS, T., and FIFKOVA, E The use of spreading depression to analyse the mutual relationship between the neocortex and the hippocampus. Electroenceph. Clin. Neurophysiol. 12:

18 15. YOKOTA, T., and FUJIMORI, B Effects of brain stem stimulation upon hippocampal electrical activity somatomotor reflexes and autonomic functions. Electroenceph. Clin. Neurophysiol. 16: ZERNICKI, B Isolated cerebrum of the pretrigeminal cat. Arch. Ital. - Biol. 112: ZERNICKI, B., and DREHEH, B Studies on the visual fixation reflex. I. General properties of the orientation fixation reflex in pretrigeminal and intact cats. Acta Biol. Exp. 25: ZERNICKI, B., and MICHALSKI, A Single-unit responses to natural objects in visual areas 17 and 18 of cats reared under different visual experiences. Acta Neurobiol. Exp. 34: ZERNICKI, B., and OSETOWSKA, E Conditioning and differentiation in the chronic midpontine pretrigeminal cat. Acta Biol. Exp. 23: Accepted 25 May 1976 TomaS RADIL-WEISS, Institute of Physiology, Czechoslovak Academy of Sciences, Budejovicka 1083, Prague 4, Czechoslovakia. Boguslaw ZERNICKI, and Andrzej MICHALSKI, Nencki Institute of Experimental Biology, Pasteura 3, Warsaw, Poland.

A BLOOD FLOW INCREASE TO A VISUAL STIMULUS IN THE OCCIPITAL LOBE OF THE CAT WITH BRAINSTEM TRANSECTION AT THE PRETRIGEMINAL LEVEL

ACTA NEUROBIOL. EXP. 1977, 37: 5-14 A BLOOD FLOW INCREASE TO A VISUAL STIMULUS IN THE OCCIPITAL LOBE OF THE CAT WITH BRAINSTEM TRANSECTION AT THE PRETRIGEMINAL LEVEL K. SKOLASINSKA, L. KR~LICKI and B.