Effects of median raphe nucleus lesions on hippocampal EEG in the freely moving rat

Brain Research, 163 (1979) 223-234 © Elsevier/North-Holland Biomedical Press

223

EFFECTS OF M E D I A N R A P H E N U C L E U S LESIONS ON H I P P O C A M P A L E E G IN T H E F R E E L Y M O V I N G RAT

EIICHI MARU*, LOREY K. TAKAHASHI* * and SHINKURO IWAHARA*** Department of Psychology, Tokyo University of Education, 3-29-1 Otsuka Bunkyoku, Tokyo and the University of Tsukuba, Ibaraki Pref 300-31 (Japan)

(Accepted June 29th, 1978)

SUMMARY The effects of median raphe lesions on the hippocampal EEG were examined in freely moving rats. First, median raphe lesions, including those restricted to the median raphe nucleus, unequivocally produced hippocampal low-frequency theta activity (5.8 Hz, SD = 0.47 Hz) during relaxed immobility which was not observed under normal conditions. This lesion-induced theta activity during immobility continued for at least 20 days, and was markedly suppressed by atropine sulfate (10 mg/kg, i.p.). On the other hand, reticular formation lesions had little effect on either hippocampal EEG patterns during immobility, movement or PS. Second, the mean frequency of theta activity was significantly reduced during movement and PS on the day following the median raphe lesion. These findings suggest a raphe-hippocampal pathway in which the median raphe nucleus plays a major role in hippocampal desynchronization (irregular pattern) by exerting an inhibitory influence on the hippocompal theta generating or facilitating mechanism. Thus the theta activity will be induced by the disinhibition following median raphe lesions.

INTRODUCTION Recent anatomical studies, using autoradiographic, silver staining and horse* To whom reprint requests should be addressed at: Department of Psychology,University of Tsukuba, Ibaraki Pref. 300-31, Japan. ** Japanese National Research Exchange Fellow. Present address: Department of Psychology, University of Hawaii, Honolulu, Hawaii, U.S.A. *** The untimely death of the third author, Dr. Shinkuro Iwahara, occurred while this work was in progress.

224 radish peroxidase techniques, have shown that the hippocampus receives a monosynaptic projection from the median raphe nucleusZ,4,5,14,is. The majority of the ascending projections from the raphe nuclei has been demonstrated by Conrad et al. 4 to run into the medial forebrain bundle and joining the diagonal band of Broca, pass through the septal nuclei or the cingulum bundle to finally reach the subiculum and the hippocampus. Electrophysiological and neurochemical data 1~, moreover, have indicated that electrical stimulation of the raphe nuclei and application of serotonin (5-HT) iontophoretically, exert a strong inhibitory influence upon the firing of hippocampal pyramidal cells. Since the raphe nuclei contain predominant numbers of 5-HT cell bodies, these findings, in addition to others 1°,17, support the view that serotonin might be an inhibitory neurotransmitter for a raphe-hippocampal neural pathway. Furthermore, Lindsley and co-workers 1,3,11,12 have found that high-frequency electrical stimulation of the raphe nucleus (nucleus raphe centralis superior) or the nucleus reticularis pontis caudalis produces a hippocampal desynchronization (lowvoltage fast activity). This desynchronizing system continues rostrally into the lateral hypothalamic area or medial forebrain bundle. Thus, this hippocampal desynchronizing system may correspond to the serotonin ascending pathway 2,4,5,14,18. In contrast, hippocampal synchronization (rhythmic theta activity) was induced by stimulating diffusely distributed regions of the midbrain and pontine tegmentum, including the nucleus reticularis pontis oralis, the nucleus locus coeruleus, the nuclei of giant cells in the pontine tegmental field and the periaqueductal grey substance of the midbrain 12. This synchronizing theta system ascends through the medial hypothalamic area (the dorsal fasciculus of Schutz). Although there is now little information concerning the interaction of the hippocampal synchronizing and desynchronizing systems, it was suggested that the amplitude of hippocampal theta activity can be enhanced by the destruction of the median raphe nucleus, a part of the desynchronizing system (Ueki, Kyushu University, personal communication, 1975). Furthermore, in our previous study tz, it was shown that the hippocampal theta activity, usually not observed during immobility in normal rats 23, appeared continuously during immobility following large brain stem lesions which included the median raphe nucleus. The following experiments were therefore undertaken to determine whether discrete lesions of the median raphe nucleus would induce the hippocampal theta activity during immobility and to examine the effects of the median raphe lesions on the frequency of hippocampal theta activity during voluntary movement and paradoxical sleep (PS). Furthermore, it has been suggested by Vanderwolf and his coworkers 9,24 that there are two types of hippocampal theta activity in rats and rabbits. Atropinesensitive theta activity occurs in rats during freezing and during the immobility that is produced by midbrain tegmentum stimulation or by certain drugs, i.e. ethyl ether, ethyl urethane, and physostigmine 9,2~,z4,26. In contrast, atropine-resistant theta activity occurs in association with 'voluntary movement 'z4,26 or during PS 9,2~,z4,26.

225 Thus the effect of atropine sulfate on hippocampal theta activity following median raphe lesions will be examined. MATERIALSAND METHODS

Animals Fifty-one male Wistar albino rats, weighing 250--390 g at the start of the experiment, were used. Animals were individually housed, and were given ad libitum access to both food and water.

General procedure Animals were given a 7-day recovery period following surgery and on the 8th day, all animals were tested for hippocampal EEGs. Only those animals which showed clear hippocampal theta activity during movement were used for the experiments. EEG recordings during various behaviors including PS were taken both before and after the brain stem lesion. All rats received either a median raphe or a unilateral midbrain reticular formation lesion. The animals which showed motor disturbances or abnormal postures with continuous muscle twitchings following these brain stem lesions were discarded. Immediately after the lesion, cortical and hippocampal EEGs were recorded during various behaviors except for PS and again after 24 h, EEG recordings during various behaviors including PS were taken in the same manner as in the pre-lesion recording session. In addition, 3 raphe-lesioned animals were observed at 3, 6, 11, and 20 days after the lesion.

Surgery, lesion and histology Animals were anesthetized with sodium pentobarbital (35 mg/kg, i.p.) and placed in a stereotaxic instrument. Bipolar electrodes were implanted in the dorsal hippocampus. A monopolar electrode was placed in either the median raphe nucleus or the reticular formation. Both hippocampal and brain stem electrodes consisted of glass-coated stainless steel wire 150/zm in diameter, exposed 0.5 mm at the tip. Implantation coordinates were followed according to the atlas of Pellegrino and Cushman 15. Coordinates used were: dorsal hippocampus: AP 3.3, ML 2.5, DV +2.0; median raphe nucleus: AP 0.0, ML 0.0, DV 4.5; midbrain reticular formation (MRF): AP 0.0, ML 2.0, DV 4.5 or--3.0. Cortical and reference electrodes consisted of miniature stainless steel screws 1 mm in diameter. The cortical electrode was placed over the sensory motor area. The reference electrode was attached to the nasal bones. Electromyographic (EMG) recordings were taken from stainless steel wires, 150 #m in diameter, chronically implanted in the neck muscles bilaterally. All lead wires were soldered to miniature connector pins and were fixed to the skull with screw anchors and dental cement.

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Fig. 2. Hippocampal electrical activities before and after the median raphe lesion. This rat (RPH-39) had a discrete (minimal) lesion restricted to the median raphe nucleus (see Fig. 1). Before median raphe lesions, the hippocampal irregular pattern appeared predominantly during immobility. Following the lesion, however, the low-frequency hippocampal theta activity appeared continuously during immobility. Although the hippocampal EEG rhythmic pattern during movement and PS continued after median raphe lesions, the mean frequency of theta activity decreased significantly(see text and Fig. 5). and placed in a 10 ~ formalin solution for at least one week. Frozen sections were cut at 50/~m and lesion cavitation and glial scarring were determined microscopically and plotted on the rat brain atlas 15.

EEG recordings and behavioral observations Recording was done on a polygraph (Sanei model 1A11) with a multiple flexible cable. Rats were placed in a small observation chamber (20 × 35 × 35 cm) and observed through the frontal glass portion of the chamber. A 2-3 cm layer of sawdust was placed on the chamber floor. Discrete behavioral episodes including head movement, body movement, walking, rearing, struggling (when held from the back), face washing, grooming, scratching and immobility were recorded concurrently with the EEG. Behavioral immobility was recorded when the animal assumed a relaxed immobile-lying or sitting posture accompanied with a low voltage EMG. Slow wave sleep and freezing (tonic immobility with no vibrissae movements) were excluded from this immobile behavioral category. Paradoxical sleep was determined by combined behavioral, EEG and E M G activity. All other behaviors are self-explanatory.

Pharmacological procedure In order to examine the effects of atropine on the hippocampal theta activity

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following median raphe lesions, 5 operated animals were first observed in the manner described above without drug during the awake state. The animals were subsequently given a dose of atropine sulfate (10 mg/kg, i.p.). Fifteen minutes following atropine administration, behavioral and EEG recordings were again taken. At the end of this recording session, a lesion was produced in the raphe nucleus. After 24 h, behavioral and EEG recordings were again taken under non-drug and atropine states in the same manner as during the pre-lesion recording session.

Data analysis Recordings of hippocampal theta activity during various behaviors were analyzed by inspection and mean frequencies of hippocampal theta activity were measured by a transparent ruler (ram units) at l-sec intervals. In order to assess the mean theta frequency, 10 samples (during movement) and 60 samples (during PS) of a 1-sec hippocampal EEG were taken. Similarly, 60 l-sec hippocampal EEG samples were taken during immobility. RESULTS

In the course of the experiment, 8 animals with poorly developed hippocampal theta activity during movement and 6 animals with motor disturbances (abnormal postures with continuous muscle twitchings) resulting from the reticular formation lesion were discarded. Thus data were obtained from the remaining 37 animals.

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Histological results Animals could he divided into 3 groups according to histological examination: the median raphe lesion group (21 animals), the reticular formation lesion group (14 animals) and the posterim pons and medulla oblongata large lesion group (2 animals). Fig. 1 shows the largest and smallest extent of damage for raphe and reticular formation lesioned groups. Four animals received discrete lesions restricted to the median raphe nucleus while 17 rats suffered raphe lesions which included partial damage to the brachium conjunctivum, the tegmental nucleus of pons and the ventral tegmental nucleus. In some animals, additional destruction extended into parts of the medial longitudinal fasciculi, the locus coeruleus, the facial nerve, the medial vestibular nucleus, the interpeduncular nucleus and the central tegmental nucleus. All 21 animals had at least 6 0 ~ of their median raphe nucleus destroyed. The second group of 14 animals received variable damage to the reticular formation. In some cases, damage extended to parts of the lateral lemniscus, the mesencephalic nucleus of the trigeminal nerve, the medial longitudinal fasciculi, the trapezoid body, the locus coeruleus, or the most caudal portion of the raphe nucleus. The last group of 2 animals received extremely large destruction in the posterior pontine and the central portion of the medulla oblongata. However, due to the small number of subjects, the data were considered only as supplementary.

Changes of hippocampal EEG patterns following median raphe lesions In pre-lesion recordings, relatively high mean frequency hippocampal theta activity (7.5 Hz, S.D. = 0.43 Hz) was always observed during movement (body

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Fig. 5. The mean frequencies of hippocampal theta activity following either median raphe or reticular formation lesions. Mean theta frequency per rat was indicated either by solid circles (movement), open circles (PS) or solid triangles (immobility following raphe lesions). See text for details.

movement, walking, rearing, etc.) or during PS (7.3 Hz, S.D. = 0.36 Hz) in all animals. In contrast, a large amplitude irregular pattern or a mixed pattern consisting of both large amplitude slow waves and low frequency indistinct hippocampal theta activity (hippocampal desynchronization) appeared during immobility. This irregular hippocampal EEG pattern, however, was replaced by hippocampal theta rhythmic activity during immobility following median raphe lesions (Fig. 2) but not after reticular formation lesions (Fig. 3). In all raphe-lesioned animals, this effect on the hippocampal EEG pattern during immobility appeared immediately and could be observed unmistakably after 24 h. The mean frequency during immobility following median raphe lesions taken from the mean values of 21 animals was 5.8 Hz (S.D. = 0.47 Hz). On days 3, 6, 11, and 20 following the median raphe lesions, we conducted further behavioral observations and EEG recordings using 3 animals. Although the behaviors of these animals were not studied in a quantifiable fashion, these animals showed a characteristic hyperactivity behavior pattern on the 3rd day following the lesion. They would dart from corner to corner in the small observation box and would occasionally rear or dig while remaining in the corner. After about 30 min they would become inactive and would sometimes suddenly fall into a catalepsy-like immobility assuming whatever posture they held previously. Animals which fell into this

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Fig. 6. The effects of atropine sulfate on hippocampal activity. The median raphe lesion-induced hippocampal rhythmic pattern during immobility was suppressed by atropine sulfate (10 mg/kg, i.p.), whereas the hippocampal theta activity during movement was left unaffected. catalepsy-like immobility as well as those which remained quietly immobile showed the characteristic slow hippocampal rhythmic pattern. Although the large amplitude irregular hippocampal activity was sometimes seen on day 3 after the median raphe lesion, the hippocampal rhythmic pattern dominated the E E G recording and was still observable even on day 20 following the median raphe lesion (see Fig. 4). Similarly, animals with large lesions in the posterior pons and the central portions of the medulla oblongata also produced a characteristic low frequency theta pattern during immobility. However, since these lesions were too large to identify the essential loci responsible for these effects and only two rats were used, the data were not further analyzed, as previously noted.

Changes in hippocampal theta frequency following median raphe lesions Mean theta frequency during movement was reduced from 7.4 Hz (SD = 0.49 Hz) to 6.8 Hz (SD = 0.65 Hz) by raphe lesions, while it remained relatively unchanged following reticular formation lesions; 7.6 Hz (SD = 0.29 Hz) to 7.5 Hz (SD = 0.26 Hz). The interaction between the two (lesion site × frequency changes) was significant (F (1/33) ---- 19.18) (see Fig. 5). Similarly, the mean theta frequency during PS was reduced from 7.2 Hz (SD ----0.38 Hz) to 6.6 Hz (SD = 0.59 Hz) following raphe

232 lesions, while remaining relatively the same following reticular formation lesions (7.5 Hz, SD = 0.21 Hz and 7.3 Hz, SD = 0.25 Hz respectively), and again the interaction was significant (F (1/33) -- 6.82).

Effects of atropine on the median raphe lesion-induced hippocampal theta activity Five rats with raphe lesions were used to examine the effects of atropine sulfate on hippocampal electrical activity. Fig. 6 shows the typical effects of atropine sulfate (10 mg/kg, i.p.) on hippocampal electrical activity during the awake state. Before median raphe lesions, atropine sulfate produced no apparent change on hippocampal EEG patterns during immobility and movement. Following median raphe lesions, however, the hippocampal theta pattern which appeared during immobility was suppressed by atropine sulfate, thus reverting back to the irregular pattern, whereas the hippocampal rhythmic pattern during movement remained unchanged. DISCUSSION The main finding of the present study is that median raphe lesions, including those restricted to the median raphe nucleus, consistently induced hippocampal lowfrequency theta activity during immobility in the rat, which was not observed under normal conditions. Reticular formation lesions placed lateral to the median raphe nucleus did not show such an effect. This hippocampal rhythmic pattern during immobility continued for at least 20 days following the lesion. Large destruction of the posterior pons and the central portions of the medulla oblongata, also produced a similar hippocampal rhythmic pattern during immobility. However, since the data from these animals (two rats) were insufficient to identify the essential loci responsible for the effects on hippocampal electrical activity and behaviors, further experiments are necessary to determine the posterior extent responsible for production of theta activity during immobility. While it has been reported that a similar hippocampal rhythmic pattern can be induced during immobility following electrical stimulation of the midbrain reticular formationTM, the possibility that the present results could be due to the irritation of the reticular formation caused by median raphe lesions must be ruled out by the following findings: (1) unilateral destruction of the reticular formation immediately lateral to the median raphe nucleus failed to bring about such a theta activity accompaying immobility; (2) this raphe-lesion theta activity was observed for a long period of time, even 20 days after the lesions, whereas reticular formation irritation effects, if any, would not last that long. Another possibility is that the observed hippocampal theta activity was the same as that which occurs during freezing in an inescapable situationTM. However, our animals showed no freezing behaviors and instead remained in relaxed immobile sitting or lying positions. Although Stumpf20 proposed earlier a theta-depressing mechanism, as one of the three basic mechanisms modulating hippocampal electrical activity, he did not ascertain the locus responsible for this depression. Our data suggest that the median

233 raphe nucleus is responsible for hippocampa~ oesynchronization by exerting a strong inhibitory influence on the hippocampal theta generating mechanism. Thus, the theta activity will be induced by the disinhibition caused by the destruction of the raphe nucleus. In contrast, Lindsley and his coworker 1, have assumed two separate and independent systems mediating hippocampal theta and desynchronizing patterns. However, a close examination of their data (Fig. 5 and Fig. 9A of Anchel and Lindsley 1) indicated that the stimulation of the desynchronizing system following bilateral lateral hypothalamic lesions or cryogenic blockades in the ventrolateral hypothalamus no longer elicited a desynchronizing pattern but instead produced a theta rhythmic pattern. Thus, their data also appear to support our hypothesis that the raphe nucleus exerts a tonic inhibitory influence on the theta-generating or facilitating mechanism and is therefore responsible for hippocampal desynchronization. Recent pharmacological and electrophysiological evidence also appears to support the present hypotheses. Sega116 found in the rat that electrical stimulation of the median or dorsal raphe nucleus strongly inhibited the firing of hippocampal pyramidal cells and that this inhibitory effect was absent when the rats were pretreated with p-chlorophenylalanine (PCPA), a serotonin synthesis blocker. Thus, the monosynaptic pathway from raphe nuclei to hippocampus in addition to the oligosynaptic pathway, for example, via medial septal nucleus 19, seems to play an important role in hippocampal electrical activities 16. The present study has also shown that theta activity, assumed to be disinhibited by median raphe lesions, could be depressed by atropine sulfate. Thus the theta activity occurring during relaxed immobility in raphe-lesioned rats is atropinesensitive and is not unlike that which occurs during freezing as well as the immobility produced by stimulation of the midbrain tegmentum and some drugsT,24,2~. Although our data are insufficient to evaluate Vanderwolf's dualistic theory of hippocampal theta activityg,24, e6, further examination of the interaction of the serotonergic and cholinergic systems on the pyramidal cells in the hippocampus will elucidate the behavioral functions and neural mechanisms concerning these two types of hippocampal theta activity. Our data also shows a significant decrease in hippocampal theta activity during movement and PS following median raphe lesions. Since Vanderwolf 24 indicated that ether treatment which induced low-frequency theta activity during immobility also slightly reduced the frequency of theta activity during movement, and that this frequency reduction may be due to underlying autonomic functioning (i.e. decrease in body temperature), the reduction oftheta frequency during movement or PS may also be due to the changes in body conditions following median raphe lesions. Finally, recent behavioral studies6-S, 25 have indicated that the raphe lesion- or PCPA-induced hyperactivity appears to be mediated by the septum or the hippocampus. Our final goal, therefore, is to elucidate the behavioral role of the raphe-hippocampal system on the basis of the present finding that damage to the median raphe nucleus produces marked changes in hippocampal electrical activity.

234 REFERENCES 1 Anchel, H. and Lindsley, D. B., Differentiation of two reticulo-nypothalamic systems regulating hippocampal activity, Electroenceph. clin. Neurophysiol., 32 (1972) 209-226. 2 Bobillier, P., Seguin, S., Petijean, F., Salvert, D., Touret, M. and Jouvet, M., The raphe nuclei of the cat brain stem: A topographical atlas of their efferent projections as revealed by autoradiogiaphy, Brain Research, 113 (1976) 449-486. 3 Coleman, J. R. and Lindsley, D. B., Hippocampal electrical correlates of free behavior and behavior induced by stimulation of two hypothalamic-hippocampal systems in the cat, Exp. NeuroL, 49 (1975) 506-528. 4 Conrad, C.A.,Leonard, C. M. andPfaff, D.W.,Connectionsofthemediananddorsalraphenuclei in the rat: an autoradiographic and degeneration study, or. comp. Neurol., 156 (1974) 179-206. 5 Hedreen, J. C.,A direct projection fromtegmentum to cortex and hippocampus demonstrated with the Nauta and Fink-Heimer methods, Anat. Rec., 175 (1973) 340. 6 Jacobs, B. L. and Cohen, A., Differential behavioral effects of the median or dorsal raphe nuclei in rats: open field and pain-elicited aggression, J. comp. physiol. Psychol., 90 (1976) 102-108. 7 Jacobs, B. L., Trimbach, C., Eubanks, E. E. and Trulson, M., Hippocampal mediation of raphe lesion- and PCPA-induced hyperactivity in the rat, Brain Research, 94 (1975) 253-261. 8 Jacobs, B. L., Wise, W. D. and Taylor, K. M., Differential behavioral and neurochemical effects following lesions of the dorsal or median raphe nuclei in rats, Brain Research, 79 (1974) 353-361. 9 Kramis, R., Vanderwolf, C. H. and Bland, B. H., Two types ofhippocampal rhythmical slow activity in both the rabbit and the rat: Relations to behavior and effects of atropine, diethyl ether, urethane, and pentobarbital, Exp. Neurol., 49 (1975) 58-85. 10 Kuhar, M. S., Aghajanian, G. K. and Roth, R. H., Tryptophan hydroxylase activity and synaptosomal uptake of serotonin in discrete brain regions after midbrain raphe lesion. Correlations with serotonin levels and histochemical fluorescence, Brain Research, 44 (1972) 165 176. 11 Lindsley, D.B. andWilson, C. L.,Brainstem-hypothalamicsystemsinftuencinghippocampalactivity and behavior. In The Hippocampus: A Comprehensive Treatise, R. L. Isaacson and K. H. Pribrain (Eds.), Plenum, New York, 1975, pp. 247-278. 12 Macadar, A. W., Chalupa, L. M. and Lindsley, D. B., Differentiation of brain stem loci which affect hippocampal and neocortical electrical activity, Exp. Neurol., 43 (1974) 449-514. 13 Maru, E. and Iwahara, S., Effects of brainstem and hypothalamic lesions on the hippocampat electrical activity, Jap. J. Neurosci. Res. Ass., 3 (1977) 42-43 (in Japanese). 14 Moore, R. Y. and Halaris, A. E., Hippocampal innervation by serotonin neurons of the midbrain raphe in the rat, J. eomp. Neurol., 164 (1975) 171-184. 15 Pellegrino, L. J. and Cushman, A. J., A Stereotaxic Atlas of the Rat Brain, Appleton-CenturyCrofts, New York, 1967. 16 Segal, M., Physiological and pharmacological evidence for a serotonergic projection to the hippocampus, Brain Research, 94 (1975) 115-131. 17 Segal, M., 5-HT antagonists in rat hippocampus, Brain Research, 103 (1976) 161-166. 18 Segal, M. and Landis, S. C., Afferents to the hippocampus of the rat studied with the method of retrograde transport of horseradish peroxidase, Brain Research, 78 (1974) 1-15. 19 Segal, M. and Landis, S. C., Afferents to the septal area of the rat studied with the method of retrograde axonal transport of horseradish peroxidase, Brain Research, 82 (1974) 263-268. 20 Stumpf, C. H., The fast component in the electrical activity of rabbit's hippocampus, Electroenceph. clin. Neurophysiol., 18 (1965) 477-486. 21 Teitelbaum, H., Lee, J. F. and Johannessen, J. N., Behaviorally evoked hippocampal theta waves: a cholinergic response, Science, 188 (1975) 1114-1116. 22 Usui, S. and Iwahara, S., Effects of atropine upon the hippocampal electrical activity in rats with special reference to paradoxical sleep, Electroenceph. clin. Neurophysiol., 42 (1977) 510-517. 23 Vanderwolf, C. H., Hippocampal electrical activity and voluntary movement in the rat, Electroenceph, clin. Neurophysiol., 26 (1969) 407-418. 24 Vanderwolf, C. H., Neocortical and hippocampal activation in relation to behavior: effects of atropine, eserine, phenothiazines and amphetamine, J. comp. physiol. Psychol., 88 (1975) 300-323. 25 Vergnes, M. et Penot, C., Effets comportementaux des lesions du raphe chez des Rats prives du septum, Brain Research, 115 (1976) 154-159. 26 Whishaw, I. Q., The effects of alcohol and atropine on EEG and behavior in the rabbit, Psychopharmacology, 48 (1976) 83-90.