Decreased temporal variability in hippocampal theta rhythms of cats administered methylphenidate

BEHAVIORAL BIOLOGY 13, 497-503 (1975), Abstract No. 4116

BRIEF REPORT Decreased Temporal Variability in Hippocampal Theta Rhythms of Cats Administered Methylphenidate 1

NEAL HALFON, DANIEL GOLDOWITZ, STEVEN GLOTZBACH and RALPH BERGER

Division of Natural Sciences, University of California, Santa Cruz, California 95064 Hippocampal theta activity, after an intraperitoneal injection of methylphenidate, was monitored in cats with chronically implanted hippocampal recording electrodes. Over a ninety minute control recording session, the hippocampal theta rhythm exhibited random fluctuations in activity levels. Following methylphenidate injections the temporal variability of the theta rhythm was significantly reduced. Hypotheses are presented on the possible role of the hippocampus in relation to the hyperkinetic syndrome observed in children.

Whole and partial ablations of cat, rat and monkey hippocampi produce behavioral effects characterized by reduced attentiveness, heightened activity, and consequent learning deficits (see Altman, Brunner and Bayer, 1973) resembling the symptoms characteristic of hyperkinesis (also called hyperactivity and minimal brain dysfunction) in children (Capute, Nidermeyer and Richardson, 1968). This resemblance suggests that the hippocampus might possibly be involved in the hyperkinetic syndrome. A further approach to a correlation between the hippocampus and the hyperkinetic syndrome is based upon a recent proposal by Altman et al. (1973). According to this proposal, the hippocampus acting as a braking mechanism to the arousal response, is functionally immature in the young animal. Development of this system in the older animal allows for response inhibition and the phenomenon of socialization. This proposal of Altman et al. (1973), has two interesting parallels with the human hyperkinetic syndrome. 1)Hyperkinesis is a juvenile syndrome that usually disappears around the time of puberty. 2)Multiple signs of central nervous system immaturity 1This study was made possible by a President of the University of California Undergraduate Research Fellowhip. We thank Marshall Sylvan for statistical advice and John W. Haycock for his thoughtful criticisms of the manuscript. Request for reprints should be sent to Dan Goldowitz, University of California, Irvine, CA 92664. 497 Copyright Q 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

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have been found in hyperactive children (Satterfield et al., 1973). The abnormal EEG activity of the hyperkinetic child includes excessive slow wave activity and long latency and low amplitude evoked cortical responses. It may be, that this apparent immaturity of central nervous system activity reflects a deficit in the arousal inhibiting network of the hippocampus. Drugs have been commonly administered in the treatment of the hyperkinetic child. Amphetamines have had varied success, but the drug of choice for the treatment of hyperkinesis is methylphenidate hydrochloride, or Ritalin (Knights and Hinton, 1969; Denhoff and Millichap, 1971). Considerable success .has been claimed for methylphenidate treatment of children whose learning is apparently hampered by their inattention and hyperactivity. The purpose of the present study was to investigate the effects of methylphenidate on the hippocampal electroencephalogram of the cat. The dominant pattern of electrical activity in the hippocampus is the low frequency (approximately 4-7 Hz), high amplitude, rhythmic activity, or theta rhythm. Since Green and Arduini's original work (1954) on the rhythmic activity of the hippocampus, investigators have tried to elucidate the electrophysiological and behavioral correlates of the theta rhythm. The general consensus has been that theta activity is correlated with attention and arousal. Subsequent investigations have proceeded to define further the nature of the hippocampal theta rhythm in relationship to orienting responses and attention (Grastyan et al., 1959; Bennett and Gottfried, 1970), to learning and consolidation of memory (Elazar and Adey, 1967; Landfield et al., 1972), and to negative inhibition (Parmeggiani, 1967; Kimble, 1968). The therapeutic effects of methylphenidate in the treatment of hyperkinesis indicate that this drug may influence processes underlying attention and arousal. In the present study we investigated the effects of methylphenidate on hippocampal EEG of the cat in order to clarify the mode of action of methylphenidate on the central nervous system and, hopefully, also offer insights into behavioral activity mediated by the hippocampal theta

rhythm. Six female cats weighing an average of 3 kg had bipolar concentric electrodes (with a 1 mm tip separation stereotaxically implanted into the dorsal hippocampus (from Jasper and Ajmone-Marsan, 1954-Fr=4.2,L=5.3, H=7.8). Surgery was aseptically performed under sodium pentobarbital (35 mg/kg) anesthesia. The bipolar electrodes were affixed to the skull with dental cement in a conventional manner. Stainless steel screws were imbedded in the skull to anchor the implant. One of the screws served as a gross cortical electrode. Electrode connections were made through a miniature radio plug and shielded microdot recording cable. Animals were allowed at least 7 days to recover from surgery prior to experimentation. The recording chamber was a 2 X 3 X 3 ft box with a

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Plexiglas front enclosed in a Faraday cage. The cats were placed in this chamber for 7 hr prior to experimentation in order to adapt them to the surroundings. During recording, food and water were freely available. Recordings were taken on a Grass Model 7 polygraph. The six cats were divided into two groups of three. One group first received an intraperitoneal injection of methylphenidate (1.5 mg/kg) and a week later an equal volume saline (0.9%) injection. (This methylphenidate dose falls between those dose levels used to obtain human psychic changes and animal behavioral activation (Plummer, 1971).) The other group was treated in the reverse order, that is, saline followed a week later by methylphenidate administration. The recording period was divided into two intervals: 1) a baseline recording of 30 min prior to injection, and 2)the experimental period of approximately 90 rain post-injection. EEG and behavior were continuously monitored during each session. The records were then coded and scored blind as to treatment by three independent judges. Three criteria were necessary in order to score hippocampal electrical activity as theta activity: (1)predominance of low frequency (4-7 Hz), high amplitude waves, (2)the absence of superimposed fast frequency activity, and (3)sustained theta activity for a minimum interval of 1 sec (see Fig. 1). Hippocampal activity was included as theta rhythm if scored as theta by two of the three judges. The baseline and experimental periods were divided into 5-min segments and theta activity was expressed in terms of the number of seconds that the hippocampus exhibited theta rhythm. The first 5-min interval after injection was eliminated from analysis and the following 18 5-min segments were analyzed. There were no significant differences in the absolute amount of postinjection theta between the control and experimental_periods (methylphenidate, 1~= 17.6 -+ 5.6 sec of theta/300 sec; saline, X -- 20.5 + 7 sec of theta/300 sec), (Table 1). Similarly when the pre-injection baseline period was compared to the post-injection period, there were no significant differences. Owing to large standard deviations caused by individual differences the group means yielded little information about differences in the amount of theta from one animal to another. L.DHI

E.DH2

l~ec

Fig. 1. Two sample recordings from the left dorsal hippocampus in two different animals. The underlined segments denote those periods which were scored as theta in accordance with the criteria listed in the text.

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HALFON E T AL. TABLE 1

Preinjection and Postinjection Grand Means -+ SEM's of Theta for Methylphenidate and Saline-Control Conditions (Quantities are expressed as the number of seconds of theta activity out of 300 seconds) Cat

Saline Pre (~')

1. 2. 3. 4. 5. 6.

Cheshire Sylvester Amy Leslie Vanessa George

4.2 25.5 65.8 2.8 23.8 54.5 29.43 -+ 10.58

Methylphenidate Post (~-) 1.7 23.9 48.3 4.4 17.2 27.7 20.53 -+ 6.99

Pre (.~)

Post (~')

8.3 22.3 46.8 2.6 9.8 24.8

12.9 41.4 23.4 4.7 5.1 18.2

19.1 -+ 6.56

17.6 -+ 5.62

However, examination of the variability of theta from 1-min period to the next revealed a significantly lower variance following the methylphenidate injection when compared to saline-control time periods (see Fig. 2). An analysis of variance was computed for each animal comparing the saline and methylphenidate conditions, and computations were made across all the animals. The data were transformed to measure the change in theta from one 5-min period to the next. This transformed variance was computed by subtraction of one point from the next, thereby obtaining the mean movement of theta levels. The null hypothesis was that there would be no difference in the transformed variance between the control and experimental groups. A two-way analysis of variance for unrelated measures (equal n's) allowed for variance comparisons on both individual and population levels. For individual animals a variance of ratio score was also computed. F values in each analysis were obtained. The variance of ratio scores indicated a significant difference between methylphenidate and saline produced variability (three animals with P < 0.001; one a n i m a l - P < 0 . 0 1 ; and two animals whose data did not show significance but revealed a trend in that direction). Across the six animals the two-way analysis of variance showed significant individual differences between animals ( P < 0 . 0 0 1 ) with theta levels during saline periods showing a significantly greater variance as compared to the methylphenidate periods ( P < 0.001). There appeared to be no significant difference in frequency of theta between the two conditions. Frequency levels were maintained at 4-5.5 Hz. The behavioral effects of each condition were observed. The animals administered with methylphenidate characteristically adopted a semi-rigid posture and rarely moved throughout the 90-min recording session.

METHYLPHENIDATE AND HIPPOCAMPAL VARIABILITY 70

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Fig. 2. Each graph denotes the amount of raw theta for a given animal following both the injection of methylphenidate and saline. Figure 2A illustrates a case (George) where overall saline-theta is greater than methylphenidate-theta. Figure 2B illustrates a case (Sylvester) where overall saline-theta is less than methylphenidate-theta. For both cases, however, there was less of a variability of theta from one 5-rain period to the next in the methylphenidate condition as compared to the saline-condition.

The principle findings o f this study is that the h i p p o c a m p a l theta r h y t h m b e c o m e s less variable w i t h time f o l l o w i n g the administration of m e t h y l p h e n i d a t e w h e n compared to the saline levels o f theta. In lieu o f the i n v o l v e m e n t of theta r h y t h m activity in processes of arousal and attention,

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stabilization of theta activity produced by methylphenidate has behavioral implications. When considering the beneficial effects from methylphenidate treatment of hyperkinetic children, our findings suggest that a regularized theta may be correlated with the maintenance of selective arousal and attention. The reduction of temporal variability of theta activity might reflect an important background role of gross hippocampal activity in the processing of nervous system information. If this is the case, then a variable theta activity level would interfere with the effective registration and processing of selected environmental cues. A regularized theta level would increase the probability of an organism's successful registration and processing of selected environmental cues while operating to screen out non-essential signals. An inability of the central nervous system to entrain theta levels might be one of the neural bases of the human hyperkinetic syndrome. We have previously mentioned the important developmental aspects of hyperkinesis and how they may relate to hippocampal function. Work by Campbell et al. (1969) suggests a developmental relationship between arousal levels and brain stem adrenergic and forebrain cholinergic systems. Apart from possible specific neural pathways that are affected, methylphenidate is known to increase brain norepinephrine levels (Cart, 1970). Increased levels of norepinephrine may represent a restoration of a chemically imbalanced and immature nervous system (as might be the case in the hyperkinetic syndrome). Studies by Carlton (1963) and Janowitz et al. (1972) suggest that a cholinergic/adregenic balance is important for proper nervous system activity and behavioral functioning. This study is a preliminary attempt to look at a neuronal population's activity as related to an organism's behavior. Further studies would undoubtedly benefit from 1) detailed histological analysis of electrode placements to determine if such placements are responsible for individual differences in theta levels, 2)more quantitative analysis of EEG activity and 3)dose-dependent investigation of methylphenidate's effects upon behavioral and EEG responses. Future work must determine how generalized the methylphenidate effect is upon the electrical activity of the hippocampus as related to behavior.

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Carlton, P. L. (1963). Cholinergic mechanisms in the control of behavior by the brain. Psych. Rev. 70, 19-39. Carr, L. (1970). Release of norepinepherine and normetanehrine from cat brain by central nervous stimulants. Biochem. PharmacoL 19, 2671-2675. Cohen, J. J., Douglas, V. I., and Morgenstern, G. (1971). The effects of methylphenidate on attentive behavior and autonomic activity in hyperactive children. Psychopharmacol. 22,282-294. Denhoff, E., and Millichap, J. G. (1971). "The Hyperactive Child." New Jersey: Medcon. Elazar, Z., and Adey, W. R. (1967). Spectral analysis of low frequency components in the electrical activity of the hippocampus during learning. Electroencephalogr. Clin. Neurophysiol. 23, 225-240. Grastyan, E., Lissak, K., Madarasz, I., and Donhoffer, H. (1959). Hippocampal electrical activity dur.'mg the development of conditioned reflexes. Electroencephalogr. Clin. Neurophysiol. 11,409-430. Green, J. D., and Mduini, A. (1954). Hippocampal electrical activity in arousal. J. Neurophysiol. 17,533-557. Janowsky, D. S., E1-Yousef, M. K., Davis. J. M., and Sekerke, H. J. (1972). Cholinergic antagonism of methylphenidate4nduced stereotyped behavior. PsychopharmacoL 27, 295-303. Jasper, H. H., and AjCmone-Marsan, C. (1954). "A Stereotaxic Atlas of the Diencephalon of the Cat." Ottawa, Canada: Nat. Res. Council. Kimble, D. P. (1968). Hippocampus and internal inhibition. Psychol. Bull. 70, 285-295. Landfield, P. W., McGaugh, J. L., and Tusa, R. J. (1972). Theta rhythm: A temporal correlate of memory storage processes in the rat. Science 175, 87-89. Parmeggiani, P. L. (1967). On the functional significance of the hippocampal theta rhythm. Prog. Brain. Res. 27, 413-441. Plummet, A. J. (1971). Pharmacological difference between Ritalin and amphetamine. CIBA pamphlet, Exhibit B. Satterfield, J. H., Cantwell, D. P., Lesser, L. I., and Podosin, R. L. (1972). Physiological studies of the hyperkinetic child: I. Amer. J. Psychiat. 128, 1418-1424. Satterfield, J. H., Cantwell, D. P., Saul, R. E., and Lesser, L. I. (1973). Response to stimulant drug treatment in hyperactive children: Prediction from EEG and neurological findings. J. Aut. Child. Schiz. 3, 36-42.