Physiology & Behavior, Vol. 16, pp. 349-354. Pergamon Press and Brain Research Publ., 1976. Printed in the U.S.A.
Effects of Subtotal Hippocampal Lesions upon Hippocampal Electrical Activity E. HELMES AND C. H. VANDERWOLF 1
Department o f Psychology, University o f Western Ontario London, Canada (Received 10 March 1975) HELMES, E. AND C. H. VANDERWOLF. Effects of subtotal hippocampal lesions upon hippocampal electrical activity. PHYSIOL. BEHAV. 16(3) 349-354, 1976. - Hippocampal electrical activity was recorded during spontaneous behavior in 20 rats before and after the production of electrolytic lesions of the hippocampus. Electrographic seizures resulted from one-third of the lesions but hippocampal activity returned to normal within 24 tar and showed no further abnormalities in 1-5 months of observation. Hippocampal electrographic seizure activity did not result in any observed behavioral seizures in any rat. Electrolytic lesions of the neocortex overlying hippocampus caused neither behavioral seizures nor electrographic seizures in the hippocampus during the period of recording. Hippocampus Seizures Brain lesions Cortical spindles Cortical EEG
Hippocampal RSA
IT has frequently been suggested that subtotal electrolytic lesions of the hippocampus may produce foci of abnormal electrical activity in the remaining hippocampal tissue [2, 7, 9, 17]. This implies that the results of experiments using the electrolytic lesion technique are ambiguous because it is not known whether postsurgical effects are due directly to the removal of tissue or to seizure discharges. Recently this suggestion has been extended as an explanation for the differential behavioral effects of hippocampus damage in man and in other mammals [9]. There are reports [10,15] that hippocampal lesions and epileptogenic foci in the hippocampus produce different behavioral effects. It is also possible that these may interact in such a way to produce the deficit in long-term memory formation th t is reported in man but not found in animals [3, 11, 16]. However, there is little direct evidence on the degree to which electrolytic lesions of the hippocampus induce long term abnormal electrical activity there. Partial hippocampal damage has been reported to result in seizures in cats [8]. However, small electrolytic lesions have also been reported not to result in seizure activity [13]. While electrolytic lesions may result in irritative foci induced by ion deposition [14,20], the existence of such foci has not been demonstrated after lesions which led to altered behavior. The lesions in the cats of Porter et al. [ 13] did not produce altered behavior, while those of Green et al. [8] did. This study was designed to assess the effects of relatively large hippocampal lesions upon hippocampal electrical activity in rats. It is possible that the effects of electrolytic lesions of the hippocampus are different in cats and rats. In addition, the effects in rats are of especial importance since
EEG effects of hippocampal lesions
most behavior-hippocampus lesion studies have used this species. However the primary interest was in determining the extent to which such lesions would produce abnormal electrical activity. Lesions were also made in the neocortex overlying the hippocampus to control for inadvertent damage there. METHOD
Animals Twenty-eight male Long-Evans hooded rats weighing 4 2 0 - 5 2 0 g were used. In 20 of these rats, slow wave recordings were taken during waking behavior. Recordings were taken at varying intervals from daily to bimonthly, over periods ranging from 1 to 6 months. All these animals received at least 1 hippocampal lesion. Five of them received lesions to the neocortex overlying the hippocampus. Eight other rats were used in acute experiments.
Apparatus Recording electrodes consisted of pairs of 0.25 mm nichrome wire, insulated with Teflon (Leico Industries), and soldered to miniature Winchester connectors. One electrode of the pair was 0 . 5 - 1 . 0 mm longer than the other. Electrodes to be used in making lesions were chronically implanted and consisted of No. 00 stainless steel insect pins, soldered to Winchester connectors. The pins were insulated with epoxy except for 1 mm at the tips. Records of hippocampal activity were made while the rats were i n a 4 0 x 40 × 31 cm high plywood box with a
1This research was supported by National Research Council of Canada grant A0118 to C. H. Vanderwolf. We would like to thank Dr. B. Kolb for his comments on an earlier version of this manuscript. 349
350
HELMES A N D V A N D E R W O L F
foam m o u n t e d plexiglas floor and an open top. O u t p u t from an electromagnetic m o v e m e n t sensor (Electrocraft) m o u n t e d beneath the plastic floor, was fed into one channel of a Grass Model 7 polygraph. Procedure Electrodes were implanted during pentobarbital anaesthesia (55 mg]kg IP) using a stereotaxic instrument. The skull was bared through a midline incision and 1.0 m m burr holes drilled through the skull for the electrodes. F o u r stainless steel jewellers screws and a ground connection were attached to the skull at this time. Electrodes were implanted bilaterally at the following coordinates adapted from the atlas of deGroot [1] : A - P : 2.2 mm posterior to bregma, L: _+2.0, V: - 2 . 5 from dura and at A - P : 4.0 m m posterior to bregma, L: +_5.0 and V: - 6 . 0 f r o m dura. Each rat received either 2 insect pins and 1 bipolar electrode or 2 insect pins and 2 bipolar electrodes. Both anterior (dorsal) and posterior (ventral) sites in the h i p p o c a m p u s were used to implant either insect pins or bipolar electrodes, such that all permutations of recording electrode and lesion site locations a m o n g the 4 implant sites were obtained. Neocortical electrodes were implanted at the same coordinates as for the hippocampus, except that V = - 1 . 0 from dura in all cases. Daily 1 0 - 3 0 rain samples of hippocampal activity were obtained over a period of 2 30 days in order to obtain baseline data. Then the rats were briefly etherized and were given anodal electrolytic lesions (2 mA for 20 sec) via one or both of the insect pins. They were then immediately reconnected to the polygraph and recordings were taken for a period lasting 3 0 - 9 0 min. Recordings were made every day for 5 days postlesion and at irregular intervals thereafter for up to 5 months. Twelve animals received 2 lesions to different areas of the hippocampus, separated by intervals of 3 to 90 days (median = 40 days). Since each animal was etherized at the time of the lesion, it is possible that the procedure lowered the probability of seizure activity. In order to test this possibility, 8 rats were tested acutely under different levels of ether anaesthesia. Electrodes were placed as described above with the rat in the stereotaxic device. Incision and pressure points were infiltrated with Xylocaine hydrochloride (1%, Astra). The h i p p o c a m p u s was either damaged by electrolysis (n = 4) or stimulated until seizure activity was induced (n = 4) by 60 Hz square wave bipolar stimulation from a Grass $6C stimulator through a Grass stimulus isolation unit. Recordings were made from the hippocampus contralateral to that being stimulated or receiving a lesion. Each site in the hippocampus was lesioned once or stimulated several times. Lesions were produced as before at light and deep levels of anaesthesia, and stimulations at light, m e d i u m and deep levels. Light anaesthesia was defined as a state in which a m o d e r a t e tail pinch would evoke a vigorous tail twitch with some b o d y m o v e m e n t , medium anaesthesia as a state in which only a slight tail m o v e m e n t resulted from a pinch and deep anaesthesia as a state in which no response at all could be evoked by a strong tail pinch. These recording sessions lasted about 2 hr and all seizure activity was recorded while the animals were anaesthetized. RESULTS Most animals in b o t h chronic and acute groups had some
damage to overlying cortex as well as varying a m o u n t s of hippocampal damage resulting from the lesions. These ranged in size from small ones restricted to one layer of h i p p o c a m p u s to large ones destroying most of h i p p o c a m p u s plus dentate area. Representative large and small hippocampal lesions are shown in Fig. 1. Seventeen lesions produced some damage to thalamus, either to the dorsal part of the lateral nucleus or to the lateral dorsal area of lateral geniculate nucleus. Five rats received a total o f 6 lesions in cortex overlying the h i p p o c a m p u s (Fig. 1). These lesions occasionally damaged the corpus callosum, but not the h i p p o c a m p u s itself or cingulate cortex. Lesions averaged about 4 mm in dia. Recording electrodes were in various areas: anterior electrodes were primarily in regio superior near the dentate gyrus, posterior electrodes were in the vicinity o f the dentate gyrus or slightly ventrolateral to this. Recording sites are shown in the right side of Fig. 1. Electrographic seizures immediately following the creation of an electrolytic lesion occurred in 12 of 34 lesions in the chronic animals and in 1 of 7 lesions in the 4 acute animals. A seizure might be recorded either ipsilateral or contralateral to the lesion or might be recorded b o t h ipsiand contralaterally. Table I gives the frequency of occurrence of recorded unilateral or bilateral seizure activity with reference to the locus of the lesion. The value of chi-square for such a contingency table is less than 1, indicating no significant difference in the occurrence of seizures among the various recording sites, in b o t h dorsalventral and ipsilateral-contralateral dimensions. In addition, there were no apparent differences in the location of sites which recorded seizure activity and those sites which did not as indicated in Fig. 1. TABLE1 DISTRIBUTION OF ELECTRODE SITES DISPLAYING S E I Z U R E ACTIVITY Dorsal Ventral Hippocampus Hippocampus Sites lpsilateral to Lesion Sites Contralateral to Lesion Total
2 3 5
7 4 I1
Total 9 7 16
Notes: There is a discrepancy in numbers of sites displaying seizure activity between Fig. I and Table 1. This discrepancy is a result of seizure activity being recorded both ipsilaterally and contralaterally simultaneously in 2 rats following a unilateral lesion and the same occurring following a bilateral single stage lesion in one rat. Dorsal hippocampus refers to electrodes implanted at the anterior set of coordinates and ventral hippocampus refers to electrodes implanted at the posterior set of coordinates. Seizure activity following a lesion lasted no longer than 30 min after the lesion, except in one animal which also had a seizure on the day after the lesion. Typical bilateral seizure activity is shown in Fig. 2. Seizures began in b o t h chronic and acute animals with single m o n o p h a s i c spikes which increased in f r e q u e n c y and amplitude over a period of several seconds to a m a x i m u m of about 3/sec and an amplitude 2 - 3 times that of the original spike. They then declined in f r e q u e n c y and amplitude to either a relatively normal pattern or to a flat period of depressed hippocampal activity which was followed by either normal activity or a
EEG E F F E C T S O F H I P P O C A M P A L L E S I O N S
351
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FIG. 1. Anatomical results. The left side of each brain section shows a coronal view of 1 representative large (lines) and 1 small lesion (dots) at both the anterior and posterior sites. Note that lesions from 4 different animals are shown. A representative neocortical lesion is shown on the right side. Recording electrode sites are shown on the right side of each section. Those sites which showed electrographic seizure activity after either a contralateral or ipsilateral lesion are shown as open squares. Those sites which never showed seizure activity are shown as solid circles. To save space, sites are distorted in the anterior-posterior plane up to 0.5 mm. Drawings adapted from K6nig and Klippel [12].
repetition of the spike pattern. On occasion there would be as m a n y as 4 such bursts of spike activity in succession before activity returned to normal. The animals were usually motionless during this t y p e of electrical activity and in no case were behavioral effects o f the abnormal h i p p o c a m p a l activity seen e x c e p t for a r h y t h m i c twitching of the vibrissae. There was no indication o f unusual posture or rigidity o t h e r than this. There were no apparent long t e r m effects of hippocampal lesions u p o n h i p p o c a m p a l electrical activity. Figure 2 shows h i p p o c a m p a l electrical activity during walking and sitting still after a unilateral h i p p o c a m p a l lesion. The frequency o f the r h y t h m i c waves seen w h e n the rats walked was 7 . 0 - 8 . 5 Hz and did not alter during the experiment. A m p l i t u d e ranged f r o m 5 0 - 5 0 0 uV in different animals and remained unchanged as well. Nine of 10 electrodes
within the posterior/ventral h i p p o c a m p u s (excluding dentate) showed clear r h y t h m i c a l slow activity w h e n the animals walked about. Electrical activity was irregular w h e n the animals were still, as shown in the lower traces of Fig. 2. This situation is identical to that seen in the anterior part of the h i p p o c a m p u s [ 18]. Electrographic seizures in the h i p p o c a m p u s did not result f r o m control lesions placed in overlying neocortex. F o u r t e e n recording electrodes in n e o c o r t e x and hippocampus in 10 animals showed repeated bouts of 5 - 1 0 Hz r h y t h m i c a l waves, 2 - 3 times the resting amplitude, which varied in duration f r o m a single 1 sec burst to 2 - 3 rain c o n t i n u o u s trains. These were seen as a rule only when the rats were absolutely still, except for a r h y t h m i c twitching of the vibrissae. The rats were awake, as indicated by their having their heads up and eyes open, and generally stood
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FIG. 2. Representative recordings taken from rat 16, taken 2 months before the lesion (top 3 traces), 30 sec following an electrolytic lesion to the left posterior hippocampus (middle 3 traces) showing the development of bilateral seizure activity, and 50 days following the lesion (bottom 3 traces). This lesion damaged parts of neocortex and lateral geniculate nucleus in addition to most of the dentate gyrus and adjacent hippocampus. The left recording electrode is anterior and ipsilateral to the lesion; the right recording electrode is contralateral to the lesion and at approximately the same level as the lesion. Note the depressed flat trace immediately preceding the seizure activity in the left channel. This is the second seizure recorded from that electrode, and the amplitude of the spontaneous slow waves had not returned to normal when the second seizure began. Calibration: 1 sec and 300 ~V. with t h e i r feet close t o g e t h e r a n d b a c k s arched. These spindle waveforms resembled those described by V a n d e r w o l f [ 19] as o r i g i n a t i n g in t h e n e o c o r t e x of n o r m a l rats and described as o c c u r r i n g o n l y in t h e a b s e n c e o f m o v e m e n t s such as head m o v e m e n t s a n d walking. This was c o n f i r m e d b y o b s e r v a t i o n s m a d e here. H o w e v e r , following h i p p o c a m p a l damage, at 4 r e c o r d i n g sites in 3 rats, such spindles o c c u r r e d while t h e a n i m a l was walking or m o v i n g its head. This a b n o r m a l i t y persisted for a p e r i o d of u p to 6
days f o l l o w i n g a lesion to t h e h i p p o c a m p u s . In t h e m o s t e x t r e m e case, this spindle activity, at a f r e q u e n c y o f 4 - 6 Hz, was a l m o s t c o n t i n u o u s a n d o c c u r r e d regardless of t h e rat's behavior. This c o n d i t i o n persisted for 3 days following the lesion. This was f o l l o w e d b y 2 days in w h i c h b r i e f (1 sec) b u r s t s of 5 Hz spindles o c c u r r e d while the a n i m a l m o v e d a n d longer d u r a t i o n , 6 - 7 Hz spindles o c c u r r e d w h e n the rat was still. F o l l o w i n g this, 4 - 1 0 Hz spindles o c c u r r e d only while t h e animal was c o m p l e t e l y still. Spindling
EEG EF F EC TS OF HIPPOCAMPAL LESIONS
353
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FIG. 3. Cortical spindles. A. Cortical spindle in a normal unlesioned rat. B. Long duration spindle in a rat standing motionless the day following an electrolytic lesion of the hippocampal formation. Lower trace is movement sensor. C. Spindles during movement in the same rat as (B), shortly following the lesion. Lower trace is movement sensor. Calibrations: 100 ~aV and 1 sec. during movement in the other two rats was seen only briefly and only on the 1 or 2 days immediately following a hippocampal lesion. Examples of spindles in normal and lesioned rats are shown in Fig. 3. These cortical spindles are different from sleep spindles in that they are observed in awake animals, and have a lower frequency than sleep spindles. In the acute animals, there was no difference in either the frequency of o c c u r r e n c e of seizures or of the duration of stimulation required to elicit the seizure among the 3 levels of ether anesthesia. All but 1 site in the 4 rats showed at least 1 (and usually more) seizure in response to repeated stimulations. Seizures occurred following 23 of 37 stimulations with a mean onset latency of 16.9 sec during deep anaesthesia, 26 of 55 (latency of 15.9 sec) during medium anaesthesia and 30 of 55 stimulations (latency of 16.4 sec) during light anaesthesia. These proportions are not signif-
icantly different as tested with a rat × anaesthesia level analysis of variance. Seizures induced by stimulation differed in form from those seen following electrolytic lesions in chronic animals. These seizures consisted of a brief ( 0 . 1 - 0 . 2 5 sec) burst of biphasic spikes at a frequency of about 40 Hz, followed by a slow wave of about 1 sec duration. This was repeated approximately 6 - 1 0 times, then followed either by a resumption of normal activity or a period of depressed activity lasting several seconds, which was followed by a return to normal activity. DISCUSSION These data contradict a c o m m o n impression that one of the chronic effects of electrolytic lesions of the hippocampus is the creation of epileptogenic foci [2, 7, 9, 17]. Seizures are present at most for one day following
354
HELMES A N D V A N D E R W O L F
electrolytic damage to the h i p p o c a m p u s in b o t h rat (as shown here) and cat [ 1 3 ] , and are not an invariable result of damage to the hippocampus. The fact that seizures were recorded f r o m several areas of h i p p o c a m p u s would tend to indicate that there is no area of the h i p p o c a m p u s which was recorded f r o m that was differentially sensitive to seizure activity induced by electrolytic damage. Failure to observe more extensive seizure activity was probably not a result of anaesthesia at the t i m e the lesions were made, as there was no increased difficulty in producing a seizure by electrical stimulation at increasing levels of anaesthesia in the acute animals. A l t h o u g h the hippocampus is highly susceptible to seizure activity [ 4 ] , this does not necessarily imply that seizure activity is inevitable following any manipulation of the hippocampus. It is perhaps not surprising that stimulation and lesions of the h i p p o c a m p u s lead to seizure activity that is different in b o t h f o r m and time course, as the manner in which neural firing b e c o m e s synchronized is presumably quite different in the 2 cases, one being a forced entrainment and the other irritative. There was a difference b e t w e e n the
effects of neocortical and hippocampal lesions: no seizures were observed following neocortical lesions; hippocampal lesions were followed by hippocampal seizures. This difference can be a c c o u n t e d for in terms of the differential sensitivity of these brain areas to seizure activity [4], the h i p p o c a m p u s being more sensitive than n e o c o r t e x . It is also rather difficult to elicit behavioral seizures by electrical stimulation of the h i p p o c a m p u s relative to other limbic structures and n e o c o r t e x [ 5,6 ]. The significance of cortical spindle activity has been discussed elsewhere [ 19]. N o r m a l l y 6 - 1 0 Hz spindles occur only during behavioral i m m o b i l i t y and are never observed during walking or extensive head movements. However, following hippocampal damage spindling activity does occur in the cortex during such behavior, indicating some form of generalized cortical electrical abnormality lasting up to 6 days following a lesion. Spindle activity occurring during walking has also been observed after lateral h y p o t h a l a m i c lesions (I.Q. Whishaw, unpublished observations).
REFERENCES 1. deGroot, J. The rat forebrain in stereotaxic coordinates. Verh K. ned Akad. Wet. Natuurkunde. 52: 1-40, 1959. 2. Deutsch, J. A. and D. Deutsch. Physiological Psychology, Rev. ed'n. Homewood, Ill.: Dorsey, 1973. p. 378. 3. Douglas, R. J. The hippocampus and behavior. Psychol. Bull. 67: 416-442, 1967. 4. Gastaut, H. and M. Fischer-Williams. The physiopathology of epileptic seizures. In: Handbook o f Physiology sec. 1 Neurophysiology, vol. 1 edited by J. Field. Washington, D. C.: American Physiological Society. 1959, 329-363. 5. Goddard, G. V. Development of epileptic seizures through brain stimulation at low intensity. Nature 214: 1020-1021, 1967. 6. Goddard, G. V., D. C. Mclntyre and C. K. Leach. A permanent change in brain function resulting from daily electrical stimulation. Expl Neurol. 25: 295-330, 1969. 7. Green, J. D. The hippocampus. Physiol. Rev. 44: 561-608, 1964. 8. Green, J. D., C. D. Clemente and J. deGroot. Experimentally induced epilepsy in the cat with injury of cornu Ammonis. A.M.A. Archs Neurol. Psychiat. 78: 259-263, 1957. 9. Isaacson, R. L. Hippocampal destruction in man and other animals. Neuropsychologia 10: 47-64, 1972. 10. Isaacson, R. L., R. J. Douglas and R. Y. Moore. The effects of radical hippocampal ablation on acquisition of avoidance responses, ar. comp. physiol. Psychol. 54: 625-628, 1961. 11. Kimble, D. P. Hippocampus and internal inhibition. Psychol. Bull. 70: 285-295, 1968.
12. K6nig, J. F. R. and R. A. Klippel. The Rat Brain: A Stereotaxic Atlas o f the Forebrain and Lower Parts of the Brainstem. Baltimore: Williams and Wilkins, 1963. 13. Porter, R., W. R. Adey and T. S. Brown. Effects of small hippocampal lesions on locally recorded potentials and on behavior performance in the cat. Expl NeuroL 10: 216-235, 1964. 14. Reynolds, R. W. An irritative hypothesis concerning the hypothalamic regulation of food intake. Psychol. Rev. 72: 105-116, 1965. 15. Schmaltz, L. W. Deficit in active avoidance learning in rats following penicillin injection into hippocampus. PhysioL Behav. 6: 667-674, 1971. 16. Scoville, W. B. and B. Milner. Loss of recent memory after bilateral hippocampal lesions. J. NeuroL Neurosurg. Psychiat. 20: 11-21, 1957. 17. Thompson, R. F. Foundations of Physiological Psychology. New York: Harper and Row, 1967, pp. 565-566. 18. Vanderwolf, C. H. Hippocampal electrical activity and voluntary movement in the rat. Electroeneeph. clin. Neurophysiol. 26:407 418, 1969. 19. Vanderwolf, C. H. Neocortical and hippocampal activation in relation to behavior: Effects of atropine, eserine, phenothiazines and amphetamine. J. comp. physiol. Psychol. 88: 300-323, 1975. 20. Whishaw, I. Q. and T. E. Robinson. Comparison of anodal and cathodal lesions and metal deposition in eliciting postoperative locomotion in the rat. Physiol. Behav. 13: 539-551,1974.
Effects of Subtotal Hippocampal Lesions upon Hippocampal Electrical Activity E. HELMES AND C. H. VANDERWOLF 1
Department o f Psychology, University o f Western Ontario London, Canada (Received 10 March 1975) HELMES, E. AND C. H. VANDERWOLF. Effects of subtotal hippocampal lesions upon hippocampal electrical activity. PHYSIOL. BEHAV. 16(3) 349-354, 1976. - Hippocampal electrical activity was recorded during spontaneous behavior in 20 rats before and after the production of electrolytic lesions of the hippocampus. Electrographic seizures resulted from one-third of the lesions but hippocampal activity returned to normal within 24 tar and showed no further abnormalities in 1-5 months of observation. Hippocampal electrographic seizure activity did not result in any observed behavioral seizures in any rat. Electrolytic lesions of the neocortex overlying hippocampus caused neither behavioral seizures nor electrographic seizures in the hippocampus during the period of recording. Hippocampus Seizures Brain lesions Cortical spindles Cortical EEG
Hippocampal RSA
IT has frequently been suggested that subtotal electrolytic lesions of the hippocampus may produce foci of abnormal electrical activity in the remaining hippocampal tissue [2, 7, 9, 17]. This implies that the results of experiments using the electrolytic lesion technique are ambiguous because it is not known whether postsurgical effects are due directly to the removal of tissue or to seizure discharges. Recently this suggestion has been extended as an explanation for the differential behavioral effects of hippocampus damage in man and in other mammals [9]. There are reports [10,15] that hippocampal lesions and epileptogenic foci in the hippocampus produce different behavioral effects. It is also possible that these may interact in such a way to produce the deficit in long-term memory formation th t is reported in man but not found in animals [3, 11, 16]. However, there is little direct evidence on the degree to which electrolytic lesions of the hippocampus induce long term abnormal electrical activity there. Partial hippocampal damage has been reported to result in seizures in cats [8]. However, small electrolytic lesions have also been reported not to result in seizure activity [13]. While electrolytic lesions may result in irritative foci induced by ion deposition [14,20], the existence of such foci has not been demonstrated after lesions which led to altered behavior. The lesions in the cats of Porter et al. [ 13] did not produce altered behavior, while those of Green et al. [8] did. This study was designed to assess the effects of relatively large hippocampal lesions upon hippocampal electrical activity in rats. It is possible that the effects of electrolytic lesions of the hippocampus are different in cats and rats. In addition, the effects in rats are of especial importance since
EEG effects of hippocampal lesions
most behavior-hippocampus lesion studies have used this species. However the primary interest was in determining the extent to which such lesions would produce abnormal electrical activity. Lesions were also made in the neocortex overlying the hippocampus to control for inadvertent damage there. METHOD
Animals Twenty-eight male Long-Evans hooded rats weighing 4 2 0 - 5 2 0 g were used. In 20 of these rats, slow wave recordings were taken during waking behavior. Recordings were taken at varying intervals from daily to bimonthly, over periods ranging from 1 to 6 months. All these animals received at least 1 hippocampal lesion. Five of them received lesions to the neocortex overlying the hippocampus. Eight other rats were used in acute experiments.
Apparatus Recording electrodes consisted of pairs of 0.25 mm nichrome wire, insulated with Teflon (Leico Industries), and soldered to miniature Winchester connectors. One electrode of the pair was 0 . 5 - 1 . 0 mm longer than the other. Electrodes to be used in making lesions were chronically implanted and consisted of No. 00 stainless steel insect pins, soldered to Winchester connectors. The pins were insulated with epoxy except for 1 mm at the tips. Records of hippocampal activity were made while the rats were i n a 4 0 x 40 × 31 cm high plywood box with a
1This research was supported by National Research Council of Canada grant A0118 to C. H. Vanderwolf. We would like to thank Dr. B. Kolb for his comments on an earlier version of this manuscript. 349
350
HELMES A N D V A N D E R W O L F
foam m o u n t e d plexiglas floor and an open top. O u t p u t from an electromagnetic m o v e m e n t sensor (Electrocraft) m o u n t e d beneath the plastic floor, was fed into one channel of a Grass Model 7 polygraph. Procedure Electrodes were implanted during pentobarbital anaesthesia (55 mg]kg IP) using a stereotaxic instrument. The skull was bared through a midline incision and 1.0 m m burr holes drilled through the skull for the electrodes. F o u r stainless steel jewellers screws and a ground connection were attached to the skull at this time. Electrodes were implanted bilaterally at the following coordinates adapted from the atlas of deGroot [1] : A - P : 2.2 mm posterior to bregma, L: _+2.0, V: - 2 . 5 from dura and at A - P : 4.0 m m posterior to bregma, L: +_5.0 and V: - 6 . 0 f r o m dura. Each rat received either 2 insect pins and 1 bipolar electrode or 2 insect pins and 2 bipolar electrodes. Both anterior (dorsal) and posterior (ventral) sites in the h i p p o c a m p u s were used to implant either insect pins or bipolar electrodes, such that all permutations of recording electrode and lesion site locations a m o n g the 4 implant sites were obtained. Neocortical electrodes were implanted at the same coordinates as for the hippocampus, except that V = - 1 . 0 from dura in all cases. Daily 1 0 - 3 0 rain samples of hippocampal activity were obtained over a period of 2 30 days in order to obtain baseline data. Then the rats were briefly etherized and were given anodal electrolytic lesions (2 mA for 20 sec) via one or both of the insect pins. They were then immediately reconnected to the polygraph and recordings were taken for a period lasting 3 0 - 9 0 min. Recordings were made every day for 5 days postlesion and at irregular intervals thereafter for up to 5 months. Twelve animals received 2 lesions to different areas of the hippocampus, separated by intervals of 3 to 90 days (median = 40 days). Since each animal was etherized at the time of the lesion, it is possible that the procedure lowered the probability of seizure activity. In order to test this possibility, 8 rats were tested acutely under different levels of ether anaesthesia. Electrodes were placed as described above with the rat in the stereotaxic device. Incision and pressure points were infiltrated with Xylocaine hydrochloride (1%, Astra). The h i p p o c a m p u s was either damaged by electrolysis (n = 4) or stimulated until seizure activity was induced (n = 4) by 60 Hz square wave bipolar stimulation from a Grass $6C stimulator through a Grass stimulus isolation unit. Recordings were made from the hippocampus contralateral to that being stimulated or receiving a lesion. Each site in the hippocampus was lesioned once or stimulated several times. Lesions were produced as before at light and deep levels of anaesthesia, and stimulations at light, m e d i u m and deep levels. Light anaesthesia was defined as a state in which a m o d e r a t e tail pinch would evoke a vigorous tail twitch with some b o d y m o v e m e n t , medium anaesthesia as a state in which only a slight tail m o v e m e n t resulted from a pinch and deep anaesthesia as a state in which no response at all could be evoked by a strong tail pinch. These recording sessions lasted about 2 hr and all seizure activity was recorded while the animals were anaesthetized. RESULTS Most animals in b o t h chronic and acute groups had some
damage to overlying cortex as well as varying a m o u n t s of hippocampal damage resulting from the lesions. These ranged in size from small ones restricted to one layer of h i p p o c a m p u s to large ones destroying most of h i p p o c a m p u s plus dentate area. Representative large and small hippocampal lesions are shown in Fig. 1. Seventeen lesions produced some damage to thalamus, either to the dorsal part of the lateral nucleus or to the lateral dorsal area of lateral geniculate nucleus. Five rats received a total o f 6 lesions in cortex overlying the h i p p o c a m p u s (Fig. 1). These lesions occasionally damaged the corpus callosum, but not the h i p p o c a m p u s itself or cingulate cortex. Lesions averaged about 4 mm in dia. Recording electrodes were in various areas: anterior electrodes were primarily in regio superior near the dentate gyrus, posterior electrodes were in the vicinity o f the dentate gyrus or slightly ventrolateral to this. Recording sites are shown in the right side of Fig. 1. Electrographic seizures immediately following the creation of an electrolytic lesion occurred in 12 of 34 lesions in the chronic animals and in 1 of 7 lesions in the 4 acute animals. A seizure might be recorded either ipsilateral or contralateral to the lesion or might be recorded b o t h ipsiand contralaterally. Table I gives the frequency of occurrence of recorded unilateral or bilateral seizure activity with reference to the locus of the lesion. The value of chi-square for such a contingency table is less than 1, indicating no significant difference in the occurrence of seizures among the various recording sites, in b o t h dorsalventral and ipsilateral-contralateral dimensions. In addition, there were no apparent differences in the location of sites which recorded seizure activity and those sites which did not as indicated in Fig. 1. TABLE1 DISTRIBUTION OF ELECTRODE SITES DISPLAYING S E I Z U R E ACTIVITY Dorsal Ventral Hippocampus Hippocampus Sites lpsilateral to Lesion Sites Contralateral to Lesion Total
2 3 5
7 4 I1
Total 9 7 16
Notes: There is a discrepancy in numbers of sites displaying seizure activity between Fig. I and Table 1. This discrepancy is a result of seizure activity being recorded both ipsilaterally and contralaterally simultaneously in 2 rats following a unilateral lesion and the same occurring following a bilateral single stage lesion in one rat. Dorsal hippocampus refers to electrodes implanted at the anterior set of coordinates and ventral hippocampus refers to electrodes implanted at the posterior set of coordinates. Seizure activity following a lesion lasted no longer than 30 min after the lesion, except in one animal which also had a seizure on the day after the lesion. Typical bilateral seizure activity is shown in Fig. 2. Seizures began in b o t h chronic and acute animals with single m o n o p h a s i c spikes which increased in f r e q u e n c y and amplitude over a period of several seconds to a m a x i m u m of about 3/sec and an amplitude 2 - 3 times that of the original spike. They then declined in f r e q u e n c y and amplitude to either a relatively normal pattern or to a flat period of depressed hippocampal activity which was followed by either normal activity or a
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FIG. 1. Anatomical results. The left side of each brain section shows a coronal view of 1 representative large (lines) and 1 small lesion (dots) at both the anterior and posterior sites. Note that lesions from 4 different animals are shown. A representative neocortical lesion is shown on the right side. Recording electrode sites are shown on the right side of each section. Those sites which showed electrographic seizure activity after either a contralateral or ipsilateral lesion are shown as open squares. Those sites which never showed seizure activity are shown as solid circles. To save space, sites are distorted in the anterior-posterior plane up to 0.5 mm. Drawings adapted from K6nig and Klippel [12].
repetition of the spike pattern. On occasion there would be as m a n y as 4 such bursts of spike activity in succession before activity returned to normal. The animals were usually motionless during this t y p e of electrical activity and in no case were behavioral effects o f the abnormal h i p p o c a m p a l activity seen e x c e p t for a r h y t h m i c twitching of the vibrissae. There was no indication o f unusual posture or rigidity o t h e r than this. There were no apparent long t e r m effects of hippocampal lesions u p o n h i p p o c a m p a l electrical activity. Figure 2 shows h i p p o c a m p a l electrical activity during walking and sitting still after a unilateral h i p p o c a m p a l lesion. The frequency o f the r h y t h m i c waves seen w h e n the rats walked was 7 . 0 - 8 . 5 Hz and did not alter during the experiment. A m p l i t u d e ranged f r o m 5 0 - 5 0 0 uV in different animals and remained unchanged as well. Nine of 10 electrodes
within the posterior/ventral h i p p o c a m p u s (excluding dentate) showed clear r h y t h m i c a l slow activity w h e n the animals walked about. Electrical activity was irregular w h e n the animals were still, as shown in the lower traces of Fig. 2. This situation is identical to that seen in the anterior part of the h i p p o c a m p u s [ 18]. Electrographic seizures in the h i p p o c a m p u s did not result f r o m control lesions placed in overlying neocortex. F o u r t e e n recording electrodes in n e o c o r t e x and hippocampus in 10 animals showed repeated bouts of 5 - 1 0 Hz r h y t h m i c a l waves, 2 - 3 times the resting amplitude, which varied in duration f r o m a single 1 sec burst to 2 - 3 rain c o n t i n u o u s trains. These were seen as a rule only when the rats were absolutely still, except for a r h y t h m i c twitching of the vibrissae. The rats were awake, as indicated by their having their heads up and eyes open, and generally stood
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FIG. 2. Representative recordings taken from rat 16, taken 2 months before the lesion (top 3 traces), 30 sec following an electrolytic lesion to the left posterior hippocampus (middle 3 traces) showing the development of bilateral seizure activity, and 50 days following the lesion (bottom 3 traces). This lesion damaged parts of neocortex and lateral geniculate nucleus in addition to most of the dentate gyrus and adjacent hippocampus. The left recording electrode is anterior and ipsilateral to the lesion; the right recording electrode is contralateral to the lesion and at approximately the same level as the lesion. Note the depressed flat trace immediately preceding the seizure activity in the left channel. This is the second seizure recorded from that electrode, and the amplitude of the spontaneous slow waves had not returned to normal when the second seizure began. Calibration: 1 sec and 300 ~V. with t h e i r feet close t o g e t h e r a n d b a c k s arched. These spindle waveforms resembled those described by V a n d e r w o l f [ 19] as o r i g i n a t i n g in t h e n e o c o r t e x of n o r m a l rats and described as o c c u r r i n g o n l y in t h e a b s e n c e o f m o v e m e n t s such as head m o v e m e n t s a n d walking. This was c o n f i r m e d b y o b s e r v a t i o n s m a d e here. H o w e v e r , following h i p p o c a m p a l damage, at 4 r e c o r d i n g sites in 3 rats, such spindles o c c u r r e d while t h e a n i m a l was walking or m o v i n g its head. This a b n o r m a l i t y persisted for a p e r i o d of u p to 6
days f o l l o w i n g a lesion to t h e h i p p o c a m p u s . In t h e m o s t e x t r e m e case, this spindle activity, at a f r e q u e n c y o f 4 - 6 Hz, was a l m o s t c o n t i n u o u s a n d o c c u r r e d regardless of t h e rat's behavior. This c o n d i t i o n persisted for 3 days following the lesion. This was f o l l o w e d b y 2 days in w h i c h b r i e f (1 sec) b u r s t s of 5 Hz spindles o c c u r r e d while the a n i m a l m o v e d a n d longer d u r a t i o n , 6 - 7 Hz spindles o c c u r r e d w h e n the rat was still. F o l l o w i n g this, 4 - 1 0 Hz spindles o c c u r r e d only while t h e animal was c o m p l e t e l y still. Spindling
EEG EF F EC TS OF HIPPOCAMPAL LESIONS
353
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FIG. 3. Cortical spindles. A. Cortical spindle in a normal unlesioned rat. B. Long duration spindle in a rat standing motionless the day following an electrolytic lesion of the hippocampal formation. Lower trace is movement sensor. C. Spindles during movement in the same rat as (B), shortly following the lesion. Lower trace is movement sensor. Calibrations: 100 ~aV and 1 sec. during movement in the other two rats was seen only briefly and only on the 1 or 2 days immediately following a hippocampal lesion. Examples of spindles in normal and lesioned rats are shown in Fig. 3. These cortical spindles are different from sleep spindles in that they are observed in awake animals, and have a lower frequency than sleep spindles. In the acute animals, there was no difference in either the frequency of o c c u r r e n c e of seizures or of the duration of stimulation required to elicit the seizure among the 3 levels of ether anesthesia. All but 1 site in the 4 rats showed at least 1 (and usually more) seizure in response to repeated stimulations. Seizures occurred following 23 of 37 stimulations with a mean onset latency of 16.9 sec during deep anaesthesia, 26 of 55 (latency of 15.9 sec) during medium anaesthesia and 30 of 55 stimulations (latency of 16.4 sec) during light anaesthesia. These proportions are not signif-
icantly different as tested with a rat × anaesthesia level analysis of variance. Seizures induced by stimulation differed in form from those seen following electrolytic lesions in chronic animals. These seizures consisted of a brief ( 0 . 1 - 0 . 2 5 sec) burst of biphasic spikes at a frequency of about 40 Hz, followed by a slow wave of about 1 sec duration. This was repeated approximately 6 - 1 0 times, then followed either by a resumption of normal activity or a period of depressed activity lasting several seconds, which was followed by a return to normal activity. DISCUSSION These data contradict a c o m m o n impression that one of the chronic effects of electrolytic lesions of the hippocampus is the creation of epileptogenic foci [2, 7, 9, 17]. Seizures are present at most for one day following
354
HELMES A N D V A N D E R W O L F
electrolytic damage to the h i p p o c a m p u s in b o t h rat (as shown here) and cat [ 1 3 ] , and are not an invariable result of damage to the hippocampus. The fact that seizures were recorded f r o m several areas of h i p p o c a m p u s would tend to indicate that there is no area of the h i p p o c a m p u s which was recorded f r o m that was differentially sensitive to seizure activity induced by electrolytic damage. Failure to observe more extensive seizure activity was probably not a result of anaesthesia at the t i m e the lesions were made, as there was no increased difficulty in producing a seizure by electrical stimulation at increasing levels of anaesthesia in the acute animals. A l t h o u g h the hippocampus is highly susceptible to seizure activity [ 4 ] , this does not necessarily imply that seizure activity is inevitable following any manipulation of the hippocampus. It is perhaps not surprising that stimulation and lesions of the h i p p o c a m p u s lead to seizure activity that is different in b o t h f o r m and time course, as the manner in which neural firing b e c o m e s synchronized is presumably quite different in the 2 cases, one being a forced entrainment and the other irritative. There was a difference b e t w e e n the
effects of neocortical and hippocampal lesions: no seizures were observed following neocortical lesions; hippocampal lesions were followed by hippocampal seizures. This difference can be a c c o u n t e d for in terms of the differential sensitivity of these brain areas to seizure activity [4], the h i p p o c a m p u s being more sensitive than n e o c o r t e x . It is also rather difficult to elicit behavioral seizures by electrical stimulation of the h i p p o c a m p u s relative to other limbic structures and n e o c o r t e x [ 5,6 ]. The significance of cortical spindle activity has been discussed elsewhere [ 19]. N o r m a l l y 6 - 1 0 Hz spindles occur only during behavioral i m m o b i l i t y and are never observed during walking or extensive head movements. However, following hippocampal damage spindling activity does occur in the cortex during such behavior, indicating some form of generalized cortical electrical abnormality lasting up to 6 days following a lesion. Spindle activity occurring during walking has also been observed after lateral h y p o t h a l a m i c lesions (I.Q. Whishaw, unpublished observations).
REFERENCES 1. deGroot, J. The rat forebrain in stereotaxic coordinates. Verh K. ned Akad. Wet. Natuurkunde. 52: 1-40, 1959. 2. Deutsch, J. A. and D. Deutsch. Physiological Psychology, Rev. ed'n. Homewood, Ill.: Dorsey, 1973. p. 378. 3. Douglas, R. J. The hippocampus and behavior. Psychol. Bull. 67: 416-442, 1967. 4. Gastaut, H. and M. Fischer-Williams. The physiopathology of epileptic seizures. In: Handbook o f Physiology sec. 1 Neurophysiology, vol. 1 edited by J. Field. Washington, D. C.: American Physiological Society. 1959, 329-363. 5. Goddard, G. V. Development of epileptic seizures through brain stimulation at low intensity. Nature 214: 1020-1021, 1967. 6. Goddard, G. V., D. C. Mclntyre and C. K. Leach. A permanent change in brain function resulting from daily electrical stimulation. Expl Neurol. 25: 295-330, 1969. 7. Green, J. D. The hippocampus. Physiol. Rev. 44: 561-608, 1964. 8. Green, J. D., C. D. Clemente and J. deGroot. Experimentally induced epilepsy in the cat with injury of cornu Ammonis. A.M.A. Archs Neurol. Psychiat. 78: 259-263, 1957. 9. Isaacson, R. L. Hippocampal destruction in man and other animals. Neuropsychologia 10: 47-64, 1972. 10. Isaacson, R. L., R. J. Douglas and R. Y. Moore. The effects of radical hippocampal ablation on acquisition of avoidance responses, ar. comp. physiol. Psychol. 54: 625-628, 1961. 11. Kimble, D. P. Hippocampus and internal inhibition. Psychol. Bull. 70: 285-295, 1968.
12. K6nig, J. F. R. and R. A. Klippel. The Rat Brain: A Stereotaxic Atlas o f the Forebrain and Lower Parts of the Brainstem. Baltimore: Williams and Wilkins, 1963. 13. Porter, R., W. R. Adey and T. S. Brown. Effects of small hippocampal lesions on locally recorded potentials and on behavior performance in the cat. Expl NeuroL 10: 216-235, 1964. 14. Reynolds, R. W. An irritative hypothesis concerning the hypothalamic regulation of food intake. Psychol. Rev. 72: 105-116, 1965. 15. Schmaltz, L. W. Deficit in active avoidance learning in rats following penicillin injection into hippocampus. PhysioL Behav. 6: 667-674, 1971. 16. Scoville, W. B. and B. Milner. Loss of recent memory after bilateral hippocampal lesions. J. NeuroL Neurosurg. Psychiat. 20: 11-21, 1957. 17. Thompson, R. F. Foundations of Physiological Psychology. New York: Harper and Row, 1967, pp. 565-566. 18. Vanderwolf, C. H. Hippocampal electrical activity and voluntary movement in the rat. Electroeneeph. clin. Neurophysiol. 26:407 418, 1969. 19. Vanderwolf, C. H. Neocortical and hippocampal activation in relation to behavior: Effects of atropine, eserine, phenothiazines and amphetamine. J. comp. physiol. Psychol. 88: 300-323, 1975. 20. Whishaw, I. Q. and T. E. Robinson. Comparison of anodal and cathodal lesions and metal deposition in eliciting postoperative locomotion in the rat. Physiol. Behav. 13: 539-551,1974.
