Exp. Brain Res. 37, 49-63 (1979)
Experimental Brain Research 9
Springer-VerIag 1979
Septo-hippocampal Connections and the Hippocampal Theta Rhythm J. N. P. Rawlins, J. Feldon 1, and J.A. Gray Department of Experimental Psychology,South Parks Road, Oxford OX1 3UD, England
Summary. Recordings were made of spontaneous hippocampal theta activity in free-moving rats, before and after a variety of lesions. Three recording sites were used to monitor activity in the dorsal hippocampus, the ventral hippocampus, or close to the site of the hippocampal flexure. Electrolytic lesions were made in the medial septal area or the dorso-lateral septal area; surgical transections were made of the fimbria or dorso-medial area of the fornix. Following lesions restricted to the medial septal area, theta was abolished throughout the hippocampus; after lesions restricted to the dorso-lateral septal area theta was retained. Fimbria lesions abolished theta in the ventral, but not the dorsal hippocampus; dorso-medial fornix lesions abolished it in the dorsal, but not the ventral, hippocampus. In some subjects the hippocampal formation was subsequently stained for cholinesterase: cholinesterase staining loss was generally associated with theta loss, but this was not clear at the flexure recording site. It was confirmed that theta is dependent upon the integrity of the medial septal area. It was concluded that damage to hippocampal afferents from the septum does abolish theta, while damaging the feedback efferents does not.
Key words: Septum - Hippocampus - Theta - Cholinesterase Since the discovery of the hippocampal theta rhythm, this slow, large amplitude and almost sinusoidal activity has been the subject of a large number of behavioural and electrophysiological studies (Vanderwolf, 1971; Winson, 1974; Bland et al., 1975). The maintenance of the theta rhythm is thought to depend upon the rhythmic activity of pacemaker cells located in the medial septal nucleus and in the diagonal band nucleus (Petsche et al., 1962; Stumpf, 1965; ApoStol and Creutzfeldt, 1974). A large number of neuroanatomical studies have confirmed the existence of a projection from the medial septal area to the 1 Present address: Laboratory of Behavioural Neurochemistry, Department of Psychiatry and Behavioural Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA Offprint requests to: Dr. J. N. P. Rawlins (address see above)
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hippocampus via fibres travelling in the fimbria and the fornix (Raisman, 1966; Meibach and Siegal, 1977), and there is evidence to suggest that this projection m a y be cholinergic (Lewis and Shute, 1967; Dudar, 1975). It is uncertain how the rhythmic, bursting activity of the medial septal p a c e m a k e r cells is maintained, but it has been suggested that feedback via the fimbria from the hippocampus activates a frequency-gating mechanism in the lateral septal nucleus, which controls medial septal activity via interneurones (McLennan and Miller, 1974, 1976). This theory predicts that interruption of the hippocampal feedback loop to the septum at the level either of the fimbria or of the lateral septum will disrupt hippocampal theta activity as effectively as will lesions of the medial septal nucleus itself. The first aim of this study was to test these predictions. The second aim of the investigation was to compare the effects on hippocampal theta of fimbrial section to those of section of the dorso-medial region of the fornix. Myhrer (1975b) demonstrated that dorsal fornix section (leaving the fimbria intact) abolished hippocampal theta, although most of the fibres from the medial septum to the hippocampus have been reported to travel in the fimbria (Meibach and Siegal 1977). This suggests that the majority of septo-hippocampal fibres are not concerned with the control of theta. However, Myhrer's recording site was in the dorsal hippocampus, and there is evidence that the projection through the fornix is predominantly to that area, the fimbrial fibres projecting mostly to the ventral hippocampus (Meibach and Siegal, 1977). Histochemical studies of cholinesterase (ChE) staining point to the same conclusion (Lewis and Shute, 1967). Thus, if septal control of theta is cholinergic (Stumpf, 1965, Dudar, 1975), one would not expect Myhrer's (1975b) results using a ventral rather than a dorsal recording site. We, therefore, recorded hippocampal activity at b o t h sites after fimbrial and fornix section, as well as after lateral and medial septal lesions. In addition, we stained the brains of selected lesioned animals for C h E to see whether there was covariation between loss of theta and loss of ChE. The effects of fimbria and fornix lesions on hippocampal responses to septal stimulation were also investigated.
Methods Dorsal Recording Site Surgery. A total of 63 adult male rats were operated, weighing between 300 and 420 g, 29 of which
were Sprague-Dawley and 34 were Wistar strain. All surgical operations were carried out using chlor-nembutal anaesthesia, 3 ml/kg. Medial septal lesions were carried out in 29 subjects, lateral septal lesions in 20 subjects, fornix lesions in 9 subjects and bilateral fimbrial lesions in 5 subjects. Except in the fimbrial animals, recordings of hippocampal activity were made both before and after lesioning. In all subjects, recording was via bipolar twisted electrodes made of Diamel insulated stainless steel wire 150 Ix in diameter with a 2 mm vertical separation of the tips. One such electrode was implanted stereotaxically into each hemisphere using the following coordinates: 6 mm posterior to bregma, 2 mm lateral, and lower tip 4 mm deep from dura, with skull flat from bregma to lambda.
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The dorsal site actually straddles the cell body layer of the subiculum, but gives very clear recording of hippocampal theta activity (James et al., 1977). In addition to recording electrodes, the subjects intended for septal lesions were at the same time implanted with lesioning electrodes. For medial septal lesions these electrodes were either bipolar and made of twisted wire (as above except with the tips cut off square), or monopolar and made of 250 ~t diameter Diamel insulated wire. The stereotaxic coordinates were: 1 mm anterior to bregma, directly on the midline, and 5 mm deep from dura. For lateral septal lesions two 250 ~t monopolar electrodes were implanted bilaterally using the coordinates: 0.5 mm anterior, 0.7 mm lateral, and 4.5 mm deep from dura. Five of the fomix group, in addition to recording electrodes, were implanted with lateral septal electrodes for stimulation. These were operated identically to the lateral septal group, except that, when cementing the electrodes in place, access was left clear to a mark made on the skull, with coordinates 0.7 mm posterior to bregma and 0.5 mm lateral (Myhrer, 1975). The remainder were implanted for recording only and marked in the same way. All electrodes were led out to an Amphenol plug and secured to the skull with dental cement and stainless steel screws. A ground wire, wrapped around these screws, was also led out to the plug. Pre-lesion Recording. Pre-lesion recordings were taken from all but the fimbrial animals. After recovery from implantation, ink recordings were taken using a Grass 79D polygraph connected to the animal via a cable with a source-follower built into it. During the recording sessions, the subjects were flee to move about in an open-topped box (90 x 30 x 35 cm). Since movement is known reliably to correlate with theta in rats (Vanderwolf, 1971), subjects which had a tendency to freeze in the testing box were kept in there until they moved about freely. Often, the lights in the testing room were turned out, leaving only a low level of ambient light; under such conditions most subjects rapidly started to move around. In addition, rats were occasionally picked up and dropped from a height of some 15-20 cm, which reliably produced clear, high-amplitude theta prior to lesioning. Those subjects in the fornix and fimbria groups which had septal electrodes were stimulated 'using 0.5 ms pulses at an inter-pulse interval of 130 ms produced by an optically-isolated, constant-current, square-wave stimulator; this stimulation was delivered between the tips of the bipolar medial septal electrode, or the tips of the two lateral septal electrodes. The responses elicited were displayed on a Tektronix 502A oscilloscope, and photographed using a Grass Kymograph camera. Lesions. The lesions to the septal area were carried out under ether anaesthesia, by passing D C current from a constant-current source through the implanted electrodes. To make the medial septal lesions 1 m A was passed for 15 s using as an anode either the monopolar electrode or the bipolar electrodes connected together, the cathode being clips on the subject's ears. The lateral septal lesions were similarly made by passing 1 m A x 15 s through each of the two electrodes in turn. The fornix lesion was carried out under chlor-nembutal anaesthesia by placing the subject in a stereotaxic head holder, and drilling through the skull at the site of the mark made during implantation. A 27-gauge needle with a small adjustable collar was lowered vertically through the hole to a depth of 4 mm from skull surface, and was then moved in the frontal plane to an angle of 30 ~ from the vertical so that the tip of the needle moved across the midline. The needle was then returned to the vertical. This was repeated at a depth of 5 mm (Myhrer, 1975b). Subsequently, since the electrophysiological effects of the lesion were not marked, they were all re-operated as before, except that the needle angle was increased to 35 ~. In the fimbria group, bilateral openings were burred in the skull, the dura was cut and the overlying cortex aspirated until the fimbria was clearly visible. The fimbria was then bilaterally either aspirated, or cut with a very small knife and then aspirated. The wound was packed with Sterispon, the scalp stitched, and the subjects were allowed a 3-month recovery. A t the end of this period, they were implanted for recording and for stimulation of the septal area as described under Surgery: two rats were operated using lateral septal electrodes and three using bipolar medial septal electrodes. Post-lesion Recording. Electrophysiological testing, carried out as before, took place a week after lesioning in all but the fimbrial animals, which were tested 3 months after lesioning. Further testing took place up to 6 weeks later in the case of the fornix and lateral septal subjects and up to 6 months later in the case of the medial septal subjects. Assessment of Slow Waves. In the rat theta rhythm refers to hippocampal slow waves in the frequency range, 6-14 Hz (Vanderwolf et al., 1975); in our tests slow wave activity was generally in
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the 7-9 Hz range. Assessment of theta was accomplished by selecting a burst of l0 consecutive theta waves with as large an amplitude as possible in the EEG records, and measuring their deflection peak to peak, from which a mean amplitude in mV could be calculated. This figure was used as the basis for comparisons of pre-lesion and post-lesion activity. No attempt was made to quantify the proportion of time that theta was present in the record; this was often apparently changed following lesioning, the duration of bursts of theta activity being very much reduced. The estimates of post-lesion theta retention are thus optimistic, in that if any theta is present at all, even if the 10 consecutive waves are the only theta activity visible in the whole of a recording session, they are rated in exactly the same way as if there were theta activity present throughout. All the subjects for which pre- and post-lesion records were available were assigned to one of four classes. In the medial septal and fornix lesioned groups, the subjects were classified on the basis of the hemisphere with the greater amplitude after lesioning; the lateral septal lesioned subjects were assigned on the basis of the hemisphere with the smaller amplitude after lesioning. In view of our findings (see Results) this is the most conservative procedure. The classes were as follows: Class I: 0%-25 % retention Class II: 26%-50% retention Class III: 51%-75% retention Class IV: >76% retention.
Histology. At the conclusion of the experiments, the subjects were given an overdose of pentobarbitone and perfused through the heart with isotonic saline and 10% formalin. The brains were left in formol saline solution for a minimum of 2 weeks. They were embedded in celloidin, and sectioned at 30 p., every fifth section being stained. In all cases with lesions aimed at the septal nuclei, cresyl violet staining was used; four of the fornix lesion group were stained in the same way, but all the remaining subjects were stained with a solachrome cyanin fibre stain. The sections were then inspected microscopically, and any showing evidence of a lesion was drawn onto plates derived from K6nig and Klippel (1963).
Ventral Recording Sites A total of 40 adult male Sprague-Dawley rats, weighing 3 0 0 4 0 0 g, were operated. All methods were the same as in the dorsal recording experiment, with the exceptions noted below. Two additional recording sites were used, with coordinates 8 mm posterior to bregma, 4.5 mm lateral, and 6.5 mm deep from skull surface (the 'flexure' site: Fig. 5B); and 6 mm posterior, 4.2 mm lateral and 7.7 mm deep from skull surface (the 'ventral' site: Fig. 5A). The recording electrodes were identical to those used before, except that a 3-mm vertical tip separation was used at the flexure site. Ten animals were implanted bilaterally with ventral recording electrodes; five of these also had medial septal lesioning electrodes, and five had lateral septal electrodes. Twenty-five animals were implanted with both dorsal and ventral recording electrodes in the left hemisphere; ten of these were intended for fornix lesions and 15 for an ipsilateral flmbria lesion. In the latter group care was taken during implantation to leave the appropriate area of skull clear for the subsequent lesion. The lesion method itself remained unchanged. Recordings were taken from all subjects with ventral recording electrodes both before and after lesioning. Finally, five animals were implanted bilaterally with flexure recording electrodes. Three of these had recordings taken before and after a fornix lesion; the other two had sustained bilateral fimbrial lesions 3 months before implantation. In addition to the histological procedures described previously, the brains from two subjects in each lesion group were left overnight in cold formalin (4 ~ C) and were then cut so that the portion containing the lesioned area and the portion containing the bulk of the hippocampal formation were separated. The former portion was then treated in the same ways as were the brains of the other subjects; the latter portion was sectioned at 50 ~, using a frozen sectioning method. The sections were then stained for cholinasterase using the Koelle copper thiocholine method (Koelle, 1954).
Septo-hippocampal Connections and Theta
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Results
Dorsal Recording Site Our results demonstrate that lesions restricted to the medial septal area or to the dorso-medial portion of the fornix severely reduce dorsal hippocampal theta; lesions to the dorso-lateral septal nuclei or to the fimbria do not. In addition, the fornix section abolished or drastically altered the hippocampal response to septal stimulation in all subjects; while fimbria section did not prevent the elicitation of clear hippocampal responses. Medial septal lesions produced 20 subjects in Class I (i.e., almost total theta loss), 5 in Class II, 4 in Class III and 7 in Class IV. Lateral septal lesions produced 5 subjects in Class I, 4 in Class II, 6 in Class III and 5 in Class IV. Fornix lesions produced 7 subjects in Class I and 2 in Class II. The post-lesion recordings from animals with fimbrial lesions showed theta amplitudes within the range we saw in intact animals. Each of the two electrolytic septal lesions produced a characteristic outcome in terms of the shape and rostro-caudal extent of the damage. The variability was mostly in the vertical dimension. The fimbria lesions were very consistent with respect to damage to the target areas, although damage to nearby structures varied somewhat. The fornical damage varied in regard both to the volume of tissue destroyed and to its rostro-caudal and lateral position. The resemblance between subjects with any of the four lesions within each electrophysiological class was marked. It was therefore possible both to draw general conclusions; and to select subjects to represent the findings for each group as follows. To demonstrate (Figs. 1-3) the critical areas in the septum and fornix which must be damaged if theta is to be abolished, we chose Class I subjects with the most restricted septal or fornical d a m a g e found to be compatible with a total abolition of theta; in order more accurately to delineate these critical zones, a Class IV subject with medial septal damage and a Class II subject with fornical damage (in both cases as similar as possible to the lesions in the corresponding Class I subjects) are also illustrated. We further illustrate a lateral septal lesioned subject chosen as having the maximal damage to the critical zone in the medial septal area consonant with acceptable (Class III) theta retention; the other lateral septal subject is the one with the most restricted damage producing a total (Class I) abolition of theta. The fimbria lesioned animal illustrated demonstrates how little tissue needs to be left between the septum and the hippocampus, while still preserving a marked degree of theta activity. Figures 1 and 2 present reconstructions of the lesions2; Figure 3 presents relevant E E G records; and Fig. 4 presents photographs of the hippocampal responses to septal stimulation for the fornix and fimbria groups. Medial Septal Lesions. Two subjects are presented: JF165 (Class I) and JF144 (Class VI). It can be seen that the lesion in JF165 is deeper and more posterior than that in JF144, although both lesions involve the medial septal nucleus to 2 Photographs of sections through the area of maximal damage in the same subjects can be seen in Gray et al. (1978, p. 280)
J. N. P. Rawlins et al.
54
JF165
JF144
RC85
RC81
Fig. 1. Reconstructions of lesions drawn onto plates derived from K6nig and Klippel (1963). JF165 - Medial septal lesion (Class I). Plates 12-18. JF144 -Medial septal lesion (Class IV). Plates 11-16. RC85 - Fornix lesion (Class I). Plates 20-27. RC81 - Fornix lesion (Class II). Plates 23-27. The asterisks mark critical sections for the abolition of theta by a fornix lesion. The bilateral extent of damage here corresponds well with the bilateral extent of theta loss
s o m e extent. This result is typical of the results of the g r o u p as a whole: the d e e p e r the midline d a m a g e in the septal area, the m o r e t h o r o u g h the abolition of the theta activity. F u r t h e r m o r e , d a m a g e restricted to the anterior medial septal area p r o d u c e d less m a r k e d effects on theta activity than if the posterior part o f the medial septal nucleus was d a m a g e d . In o r d e r to be completely effective, the lesion had to e x t e n d at least as far ventrally as the tip o f the ventricles; effective lesions were often slightly d e e p e r than this, and sometimes e x t e n d e d as far ventrally as the Insula Calleja magna, although this was almost invariably u n d a m a g e d due to the restricted lateral extent of the lesions. W h e r e the lesion was not entirely symmetrical about the midline, there was never m o r e than a partial loss of theta, even o n the side ipsilateral to the heavier septal damage. In such lesions, d a m a g e in the midline was never as deep as in m o r e accurately located lesions. D a m a g e to the fornix was seldom visible, and was not a prerequisite for theta loss. In subjects tested 6 m o n t h s after lesioning, there was no sign of theta recovery. Fornix Lesion. Again, two subjects are presented, one Class I ( R C 8 5 ) and one Class II ( R C 8 ] ) . R C 8 5 ' s lesion was clearly bilateral and fairly symmetrical,
Septo-hippocampal Connections and Theta
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Fig. 2. Reconstructions of lesions. JF118 -Lateral septal lesion (Class III). Plates 11-517. JF123 Lateral septal lesion (Class I). Plates 12-18. RC58 - Fimbrial lesion: theta is still present. Alternate plates from 21-29, plus Plates 30 and 31
whereas R C 8 1 ' s was slightly m o r e posterior, being much m o r e extensive on the right of the midline than on the left. The a s y m m e t r y of this lesion was matched by an a s y m m e t r y in the reductions in theta amplitude: these were to 20 % in the right hemisphere and 5 0 % in the left. In both cases, there was damage to the dorsomedial fornix and to the triangular nucleus of the septum, but in neither was there damage extending as far anterior as the medial septal nucleus. In the group as a whole, there was a good correlation between the extent of theta loss on each side and the lateral extent of the lesion on that side, particularly in the sections m a r k e d with an asterisk in Fig. 1. To summarise the group results, it appears that to ensure total theta loss, there must be destruction of that area of the dorsomedial fornix whose ventral limit is the triangular septal nucleus on the asterisked sections. Whether d a m a g e to the triangular septal nucleus itself is essential is not clear, although good theta loss was observed in subjects with only minimal damage to this structure. Fimbria Lesion. The results from one subject (RC58) with a bilateral fimbria lesion are presented. In all subjects in this group, the fimbrial damage was very extensive and led to considerable shrinkage of the fornix-fimbria bundle. It was always possible to record hippocampal theta activity of a normal amplitude.
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Fig. 3. Hippocampal activity: dorsal recording site. Recordings from the left (above) and right (below) hemispheres: A before and a after a Class I medial septal lesion. B before and b after a Class III lateral septal lesion. C before and c after a Class I fornix lesion. D after a fimbrial lesion
Furthermore, the hippocampal activity on both sides may be seen to be in phase (Fig. 3D), in spite of the damage to the ventral hippocampal commissures. Lateral Septal Lesion. The results from two subjects are presented, one Class III (JF118) and one Class I (JF123). JF118 has marked dorsolateral septal damage, but little damage in the medial septal area. This reflects the results of the group as a whole, in that dorsolateral damage could be produced without marked medial septal damage; such subjects showed only small reductions in theta amplitude. We were unable to produce marked ventrolateral damage without considerably encroaching on the medial septal area; in such cases there was always a very marked theta loss. JF123 is typical of these subjects. Evoked Responses. The responses elicited by septal stimulation varied in appearance, ranging from relatively sharp evoked potentials, as illustrated by subject RC81, to almost sinusoidal activity, as illustrated by subject RC58 (Fig.
4).
Septo-hippocampal Connections and Theta
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RC58 demonstrated that responses to septal stimulation can be recorded in the dorsal hippocampus after bilateral section of the fimbria; similar responses were also observed in the other two fimbria lesioned subjects tested. The thresholds for a clear response ranged from 42 to 400 ~A, which is comparable to the range found in intact animals (James et al., 1977). Section of the dorso-medial fornix radically altered the response to septal stimulation of all five subjects tested. RC85 (Fig. 4) showed an evoked response after the lesion which was reversed in polarity and had a longer latency than the original response. One other subject showed an identical change. In two subjects the lesion altogether abolished the response to stimulation at the currents used in pre-lesion testing. RC81 was the only one of these five subjects to show partial retention of spontaneous theta (Class II), and it may be seen (Fig. 4) that scarcely any evoked responses remain.
Ventral and Flexure Recording Sites Of the 45 ventral electrode placements (10 bilateral and 25 unilateral implantations) usable records of theta activity were obtained in 31. This is a
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poorer success rate than with dorsal placements. Nonetheless, our most successful placements (Fig. 5A) produced recordings of theta as clear as in our dorsal site (Fig. 6). A t the flexure site (Fig. 5B) theta was recorded as easily as at the dorsal site. Our results demonstrate that ventrally recorded theta was abolished by medial septal lesions and spared by lateral septal lesions, as at the dorsal site. The effects of fornix and flmbria lesions, however, appeared to be the reverse of those seen at the dorsal site: fimbria section abolished theta; fornix section in most cases spared it. Abolition of ChE staining paralleled loss of theta. Usable ventral records were obtained from six placements in four animals which, upon histological examination, were found to have sustained some damage to the medial septal area. At these placements theta loss was total in three cases and partial (two Class II and one Class III) in the others. Three of the four animals had damage to the medial septal area similar to that illustrated in Fig. 1. The fourth animal (in which there was no theta after the lesion in one hemisphere but Class III retention in the other) had a markedly asymmetrical lesion with maximal damage ipsilateral to the side with total theta loss. In animals with lesions restricted to the dorso-lateral septal area usable records were obtained bilaterally from four animals. The lesion never abolished theta activity; the loss of theta could be classified as Class I V in all of them. In view of these results it seemed unnecessary to investigate the effects of septal lesions on theta recorded at the flexure. It proved possible to section the fimbria unilaterally in seven animals with good ipsilateral ventral and dorsal theta recordings. Ventral theta was totally abolished in five animals and reduced to 23 and 33%, respectively, in the remaining two. In no animal did the lesion affect dorsal theta as much as ventral theta (Fig. 6). Nonetheless, in five animals some toss of dorsal theta was observed (two Class IV, two Class I I I and one Class II). The extent of dorsal theta loss did not appear to correlate with the extent of ventral theta loss; indeed, in one animal, there was total loss of ventral theta with no reduction in dorsal theta.
Septo-hippocampal Connections and Theta
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Fig. 6. Hippocampal activity: a comparison of the dorsal and ventral recording sites. Recordings taken from the left hemisphere at the dorsal (above) and ventral (below)sites: A before and a after a fornix lesion. B before and b after a fimbrial lesion
Histologically verified section of the dorso-medial fornix was carried out in five animals with clear ventral and dorsal theta recordings. In all of them theta at the dorsal site was severely disrupted (four Class I, one Class II), while ventral theta was largely unaffected (four Class IV, one Class III). In three of these animals there was total loss of dorsal theta and no reduction in the amplitude of ventral theta (Fig. 6). The histological examination revealed damage similar to that shown in Fig. 1, although in all but one subject somewhat more extensive. Flexure Recording Site. The results after fimbrial and fornix section using the flexure recording site paralleled those obtained with dorsal, not ventral, recording electrodes. It was possible to obtain clear theta records in both hemispheres in two animals following bilateral section of the fimbria. Conversely, theta was abolished (Class I) bilaterally in three animals tested before and after fornix section. Cholinesterase Staining. Representative sections stained for ChE are shown in Fig. 7. Marked loss of ChE staining occurred throughout the hippocampus after medial septal lesions, and no loss after dorso-lateral septal lesions. After fornix section there was marked ChE depletion in the dorsal hippocampus only; there were no apparent changes in ventral hippocampal ChE. Unilateral fimbrial section produced a marked loss of ChE staining in the ipsilateral ventral hippocampus, but also reduced to a smaller degree the intensity of staining in the ipsilateral dorsal hippocampus. Both fornix and fimbria lesions appeared to reduce ChE staining to an equal extent at the site of the flexure recording electrode.
Discussion Our results confirm the work of Petsche and his collaborators (Stumpf, 1965) in showing that the hippocampal theta rhythm depends on the integrity of the
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Fig. 7. Cholinesterase staining. A Following a medial septal lesion. B Following a lateral septal lesion. C Following a fornix lesion (anterior section). D Following a fimbrial lesion (anterior section). E The same fornix lesioned subject (posterior section). F The same fimbrial lesioned subject (posterior section)
medial, but not the lateral, septal area. In addition, they show that this conclusion applies to ventral as well as dorsal theta activity and d e m o n s t r a t e it with s p o n t a n e o u s theta r e c o r d e d f r o m unanaesthetised, free-moving animals. T h e y also confirm M y h r e r ' s (1975b) r e p o r t that section of the d o r s o - m e d i a l p o r t i o n o f the fornix abolishes theta r e c o r d e d dorsally, but show that this result c a n n o t be generalised to ventral theta, which is controlled by fibres travelling in the fimbria, not in the medial fornix.
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It has recently been suggested by McLennan and Miller (1976) that the hippocampal theta rhythm is dependent on the hippocampal output to the lateral septum via the fimbria. This suggestion is ruled out by our data. First, fimbrial section failed to eliminate dorsal hippocampal theta in any animal studied. Second, dorso-lateral septal lesions, destroying the greater part of the site of termination of the hippocampo-septal output (Meibach and Siegal, 1977), failed to eliminate either dorsal or ventral hippocampal theta in any animal studied. Conversely, damage to the cells of origin of the septal projection to the hippocampus, located in the medial septal area, or to their axons which travel to the dorsal and ventral hippocampus via the dorso-medial fornix and fimbria, respectively (Meibach and Siegal, 1977), invariably caused severe and often total disruption of hippocampal theta in the deafferented area. McLennan and Miller's (1976) proposal was that feedback from the hippocampus passes through a frequency gating mechanism located in the lateral septum, and thus itself controls the bursting activity of medial septal neurones. While the present results demonstrate that this mechanism is not essential for the generation of theta, it may modulate theta frequency. When hippocampal theta is provoked by electrical stimulation of the septal area in free-moving rats, the function relating the threshold current to the stimulation frequency displays a minimum at a frequency of 7.7 Hz (James et al., 1977). Although this frequency-intensity function was not a primary object of study in the present investigation, we determined it in a group of rats with dorsal recording electrodes after bilateral fimbrial section. In no case was there a 7.7 Hz minimum. Thus, it is possible that McLennan and Miller's (1976) 'frequency gating mechanism' is concerned with the fine tuning of hippocampal theta frequency. Experiments in which hippocampal theta is elicited by septal stimulation (Gray and Ball, 1970) assume that electrically elicited hippocampal activity does not radically differ from spontaneous theta. Our results provide some support for this assumption, since, at our dorsal recording site, fornix lesions eliminating spontaneous theta also eliminated or drastically altered the response to septal stimulation. In the latter cases the reversal of polarity and change of latency suggest a changed location for the post-lesion hippocampal response, presumably mediated by other intact fibre systems. Conversely, our ability to elicit dorsal hippocampal responses in subjects with bilateral fimbria lesions demonstrates that such responses are not dependent upon antidromic activation of hippocampal efferent fibres running in the fimbria. Our observations of hippocampal staining for ChE after lesions to the septal area, the fornix or the fimbria are in accord with previous anatomical reports (Lewis and Shute, 1967; Mellgren and Srebro, 1973). They apparently conflict, however, with Dudar's (1975) observation that fimbria but not fornix section elimited hippocampal release of acetylcholine induced by septal stimulation. Our results, in contrast, clearly implicate the fornix as the cholinesterase-containing pathway to the dorsal hippocampus. There was a qualitative correspondence between loss of theta and loss of ChE staining, both between and within subjects (e.g., Figs. 6 and 7). However, at the flexure recording site, we could not distinguish between ChE depletion
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caused by fornix and fimbria section, although the former abolished theta while the latter did not. Thus, while our results are largely in agreement with the concept of septal cholinergic control of theta, they are not conclusive in this respect. We suggest that the fibres running in the dorso-medial fornix and those running in the fimbria should be considered as representing populations dispersed within a single, functional pathway. It is proposed that the fibres are densely packed as they emerge from the medial septal area, and start to fan out laterally as they run caudally. It might, therefore, be possible to sever the entire bundle with a single, midline cut located anterior to our dorso-medial fornix lesion, while the number of fibres damaged by such a cut would be smaller as the plane of section moves caudally. Thus, the ability to produce selective effects on dorsal and ventral theta activity would depend critically on the plane of sectioning. Selective lesions of the dorso-medial fornix and of the fimbria have already been investigated in behavioural experiments (Myhrer, 1975a,b~ Jarrard, 1976). Our data demonstrate that these results require more complex interpretation than has hitherto been realised.
Acknowledgements.
This research was supported by the United Kingdom Medical Research Council. J. Feldon was supported by a Sir Kenneth Lindsay Scholarship from the Anglo-Israel Association. We are grateful to Mr. I. Hughes for developing the cholinesterase stain, to Dr. J. J. B. Jack for the loan of the camera, and to Professor L.E. Jarrard for his help in the development of the fimbria lesion. A preliminary report using these data appeared in Feldon and Rawlins (1978).
References Apostol, G., Creutzfeldt, O.D.: Cross correlation between the activity of septal units and hippocampal EEG during arousal. Brain Res. 67, 65-75 (1974) Bland, B.H., Andersen, P., Ganes, T.: Two generators of hippocampal theta activity in rabbits. Brain Res. 94, 199-218 (1975) Dudar, J.D.: The effect of septal nuclei stimulation on the release of acetylcholine from the rabbit hippocampus. Brain Res. 83, 123-133 (1975) Feldon, J., Rawlins, J. N. P.: The anatomical basis of hippocampal theta rhythm. J. Physiol. (Lond.) 284, 81P (1978) Gray, J.A., Ball, G. G.: Frequency-specific relation between hippocampal theta rhythm, behaviour, and amobarbital action. Science 168, 1246-1248 (1970) Gray, J.A., Feldon, J., Rawlins, J.N.P., Owen, S., McNaughton, N.: The role of the septohippocampal system and its noradrenergic afferents in behavioural responses to non-reward. In: Whelan, J. (Ed.). Functions of the septo-hippocampal system. Ciba Foundation Symposium No. 58 (New Series), pp. 275-300. Amsterdam: Elsevier 1978 James, D.T.D., McNaughton, N., Rawlins, J.N.P., Feldon, J., Gray, J.A.: Septal driving of hippocampal theta rhythm as a function of frequency in the free-moving male rat. Neuroscience 2, 1007-1017 (1977) Jarrard, L.E.: Anatomical and behavioura! analysis of hippocampal cell fields in rats. J. Comp. Physiol. Psychol. 90, 1035-1050 (1976) Koelle, G.B.: The histochemical localization of cholinesterases in the central nervous system of the rat. J. Comp. Neurol. 100, 211-228 (1954) KOnig, J.F.R., Klippel, R.A.: The rat brain. Baltimore: Williams and Wilkins 1963
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Lewis, P.R., Shute, C. C. D.: The cholinergic limbic system: Projections to hippocampal formation, medial cortex, nuclei of the ascending cholinergie reticular formation, and the subfornical organ and supraoptic crest. Brain 901 521-540 (1967) Mellgren, S. I., Srebro, B.: Changes in acetylcholinesterase and distribution of degenerating fibres in the hippocampal region after septal lesions in the rat. Brain Res. 52, 19-36 (1973) MeLennan, H., Miller, J.J.: The hippocampal control of neuronal discharges in the septum of the rat. J. Physiol (Lond.) 237, 607--624 (1974) McLennan, H., Miller, J.J.: Frequency-related inhibitory mechanisms controlling rhythmical activity in the septal area. J. Physiol. (Lond.) 254, 827-841 (1976) Meibach, R.C., Siegal, A.: Efferent connections of the septal area in the rat: an analysis utilizing retrograde and anterograde transport methods. Brain Res. 119, 1-20 (1977) Myhrer, T.: Locomotor, avoidance, and maze behaviour in rats with selective disruption of hippocampal output. J. Comp. Physiol. Psychol. 89, 759-777 (1975a) Myhrer, T.: Normal jump avoidance performance in rats with the hippocampal theta rhythm selectively disrupted. Behav. Biol. 14, 489-498 (1975b) Petsche, H., Stumpf, Ch., Gogolak, G.: The significance of the rabbit's septum as a relay station between the midbrain and the hippocampus. I. The control of hippocampus arousal activity by the septum cells. Electroenceph. Clin. Neurophysiol. 14, 202-211 (1962) Raisman, G.: The connexions of the septum. Brain 89, 317-348 (1966) Stumpf, C.: Drug action on the electrical activity of the hippocampus. Int. Rev. Neurobiol. 8, 77-138 (1965) Vanderwolf, C.H.: Limbic diencephalic mechanisms of voluntary movement. Psychol. Rev. 78, 83-113 (1971) Vanderwolf, C.H., Kramis, R., Gillespie, L.A., Bland, B.H.: Hippocampal rhythmic slow activity and neocortical low-voltage fast activity: relations to behavior. In: Isaacson, R.L. and Pribram, K.H. (Eds.), The hippocampus. Vol. 2, pp. 101-128. New York: Plenum Press Winson, J.: Patterns of hippocampal theta rhythm in the freely moving rat. Electroenceph. Clin. Neurophysiol. 36, 291-301 (1974) Received October 17, 1978
Experimental Brain Research 9
Springer-VerIag 1979
Septo-hippocampal Connections and the Hippocampal Theta Rhythm J. N. P. Rawlins, J. Feldon 1, and J.A. Gray Department of Experimental Psychology,South Parks Road, Oxford OX1 3UD, England
Summary. Recordings were made of spontaneous hippocampal theta activity in free-moving rats, before and after a variety of lesions. Three recording sites were used to monitor activity in the dorsal hippocampus, the ventral hippocampus, or close to the site of the hippocampal flexure. Electrolytic lesions were made in the medial septal area or the dorso-lateral septal area; surgical transections were made of the fimbria or dorso-medial area of the fornix. Following lesions restricted to the medial septal area, theta was abolished throughout the hippocampus; after lesions restricted to the dorso-lateral septal area theta was retained. Fimbria lesions abolished theta in the ventral, but not the dorsal hippocampus; dorso-medial fornix lesions abolished it in the dorsal, but not the ventral, hippocampus. In some subjects the hippocampal formation was subsequently stained for cholinesterase: cholinesterase staining loss was generally associated with theta loss, but this was not clear at the flexure recording site. It was confirmed that theta is dependent upon the integrity of the medial septal area. It was concluded that damage to hippocampal afferents from the septum does abolish theta, while damaging the feedback efferents does not.
Key words: Septum - Hippocampus - Theta - Cholinesterase Since the discovery of the hippocampal theta rhythm, this slow, large amplitude and almost sinusoidal activity has been the subject of a large number of behavioural and electrophysiological studies (Vanderwolf, 1971; Winson, 1974; Bland et al., 1975). The maintenance of the theta rhythm is thought to depend upon the rhythmic activity of pacemaker cells located in the medial septal nucleus and in the diagonal band nucleus (Petsche et al., 1962; Stumpf, 1965; ApoStol and Creutzfeldt, 1974). A large number of neuroanatomical studies have confirmed the existence of a projection from the medial septal area to the 1 Present address: Laboratory of Behavioural Neurochemistry, Department of Psychiatry and Behavioural Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA Offprint requests to: Dr. J. N. P. Rawlins (address see above)
0 0 1 4 - 4 8 1 9 / 7 9 / 0 0 3 7 / 0 0 4 9 / $ 3.00
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hippocampus via fibres travelling in the fimbria and the fornix (Raisman, 1966; Meibach and Siegal, 1977), and there is evidence to suggest that this projection m a y be cholinergic (Lewis and Shute, 1967; Dudar, 1975). It is uncertain how the rhythmic, bursting activity of the medial septal p a c e m a k e r cells is maintained, but it has been suggested that feedback via the fimbria from the hippocampus activates a frequency-gating mechanism in the lateral septal nucleus, which controls medial septal activity via interneurones (McLennan and Miller, 1974, 1976). This theory predicts that interruption of the hippocampal feedback loop to the septum at the level either of the fimbria or of the lateral septum will disrupt hippocampal theta activity as effectively as will lesions of the medial septal nucleus itself. The first aim of this study was to test these predictions. The second aim of the investigation was to compare the effects on hippocampal theta of fimbrial section to those of section of the dorso-medial region of the fornix. Myhrer (1975b) demonstrated that dorsal fornix section (leaving the fimbria intact) abolished hippocampal theta, although most of the fibres from the medial septum to the hippocampus have been reported to travel in the fimbria (Meibach and Siegal 1977). This suggests that the majority of septo-hippocampal fibres are not concerned with the control of theta. However, Myhrer's recording site was in the dorsal hippocampus, and there is evidence that the projection through the fornix is predominantly to that area, the fimbrial fibres projecting mostly to the ventral hippocampus (Meibach and Siegal, 1977). Histochemical studies of cholinesterase (ChE) staining point to the same conclusion (Lewis and Shute, 1967). Thus, if septal control of theta is cholinergic (Stumpf, 1965, Dudar, 1975), one would not expect Myhrer's (1975b) results using a ventral rather than a dorsal recording site. We, therefore, recorded hippocampal activity at b o t h sites after fimbrial and fornix section, as well as after lateral and medial septal lesions. In addition, we stained the brains of selected lesioned animals for C h E to see whether there was covariation between loss of theta and loss of ChE. The effects of fimbria and fornix lesions on hippocampal responses to septal stimulation were also investigated.
Methods Dorsal Recording Site Surgery. A total of 63 adult male rats were operated, weighing between 300 and 420 g, 29 of which
were Sprague-Dawley and 34 were Wistar strain. All surgical operations were carried out using chlor-nembutal anaesthesia, 3 ml/kg. Medial septal lesions were carried out in 29 subjects, lateral septal lesions in 20 subjects, fornix lesions in 9 subjects and bilateral fimbrial lesions in 5 subjects. Except in the fimbrial animals, recordings of hippocampal activity were made both before and after lesioning. In all subjects, recording was via bipolar twisted electrodes made of Diamel insulated stainless steel wire 150 Ix in diameter with a 2 mm vertical separation of the tips. One such electrode was implanted stereotaxically into each hemisphere using the following coordinates: 6 mm posterior to bregma, 2 mm lateral, and lower tip 4 mm deep from dura, with skull flat from bregma to lambda.
Septo-hippocampal Connections and Theta
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The dorsal site actually straddles the cell body layer of the subiculum, but gives very clear recording of hippocampal theta activity (James et al., 1977). In addition to recording electrodes, the subjects intended for septal lesions were at the same time implanted with lesioning electrodes. For medial septal lesions these electrodes were either bipolar and made of twisted wire (as above except with the tips cut off square), or monopolar and made of 250 ~t diameter Diamel insulated wire. The stereotaxic coordinates were: 1 mm anterior to bregma, directly on the midline, and 5 mm deep from dura. For lateral septal lesions two 250 ~t monopolar electrodes were implanted bilaterally using the coordinates: 0.5 mm anterior, 0.7 mm lateral, and 4.5 mm deep from dura. Five of the fomix group, in addition to recording electrodes, were implanted with lateral septal electrodes for stimulation. These were operated identically to the lateral septal group, except that, when cementing the electrodes in place, access was left clear to a mark made on the skull, with coordinates 0.7 mm posterior to bregma and 0.5 mm lateral (Myhrer, 1975). The remainder were implanted for recording only and marked in the same way. All electrodes were led out to an Amphenol plug and secured to the skull with dental cement and stainless steel screws. A ground wire, wrapped around these screws, was also led out to the plug. Pre-lesion Recording. Pre-lesion recordings were taken from all but the fimbrial animals. After recovery from implantation, ink recordings were taken using a Grass 79D polygraph connected to the animal via a cable with a source-follower built into it. During the recording sessions, the subjects were flee to move about in an open-topped box (90 x 30 x 35 cm). Since movement is known reliably to correlate with theta in rats (Vanderwolf, 1971), subjects which had a tendency to freeze in the testing box were kept in there until they moved about freely. Often, the lights in the testing room were turned out, leaving only a low level of ambient light; under such conditions most subjects rapidly started to move around. In addition, rats were occasionally picked up and dropped from a height of some 15-20 cm, which reliably produced clear, high-amplitude theta prior to lesioning. Those subjects in the fornix and fimbria groups which had septal electrodes were stimulated 'using 0.5 ms pulses at an inter-pulse interval of 130 ms produced by an optically-isolated, constant-current, square-wave stimulator; this stimulation was delivered between the tips of the bipolar medial septal electrode, or the tips of the two lateral septal electrodes. The responses elicited were displayed on a Tektronix 502A oscilloscope, and photographed using a Grass Kymograph camera. Lesions. The lesions to the septal area were carried out under ether anaesthesia, by passing D C current from a constant-current source through the implanted electrodes. To make the medial septal lesions 1 m A was passed for 15 s using as an anode either the monopolar electrode or the bipolar electrodes connected together, the cathode being clips on the subject's ears. The lateral septal lesions were similarly made by passing 1 m A x 15 s through each of the two electrodes in turn. The fornix lesion was carried out under chlor-nembutal anaesthesia by placing the subject in a stereotaxic head holder, and drilling through the skull at the site of the mark made during implantation. A 27-gauge needle with a small adjustable collar was lowered vertically through the hole to a depth of 4 mm from skull surface, and was then moved in the frontal plane to an angle of 30 ~ from the vertical so that the tip of the needle moved across the midline. The needle was then returned to the vertical. This was repeated at a depth of 5 mm (Myhrer, 1975b). Subsequently, since the electrophysiological effects of the lesion were not marked, they were all re-operated as before, except that the needle angle was increased to 35 ~. In the fimbria group, bilateral openings were burred in the skull, the dura was cut and the overlying cortex aspirated until the fimbria was clearly visible. The fimbria was then bilaterally either aspirated, or cut with a very small knife and then aspirated. The wound was packed with Sterispon, the scalp stitched, and the subjects were allowed a 3-month recovery. A t the end of this period, they were implanted for recording and for stimulation of the septal area as described under Surgery: two rats were operated using lateral septal electrodes and three using bipolar medial septal electrodes. Post-lesion Recording. Electrophysiological testing, carried out as before, took place a week after lesioning in all but the fimbrial animals, which were tested 3 months after lesioning. Further testing took place up to 6 weeks later in the case of the fornix and lateral septal subjects and up to 6 months later in the case of the medial septal subjects. Assessment of Slow Waves. In the rat theta rhythm refers to hippocampal slow waves in the frequency range, 6-14 Hz (Vanderwolf et al., 1975); in our tests slow wave activity was generally in
52
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the 7-9 Hz range. Assessment of theta was accomplished by selecting a burst of l0 consecutive theta waves with as large an amplitude as possible in the EEG records, and measuring their deflection peak to peak, from which a mean amplitude in mV could be calculated. This figure was used as the basis for comparisons of pre-lesion and post-lesion activity. No attempt was made to quantify the proportion of time that theta was present in the record; this was often apparently changed following lesioning, the duration of bursts of theta activity being very much reduced. The estimates of post-lesion theta retention are thus optimistic, in that if any theta is present at all, even if the 10 consecutive waves are the only theta activity visible in the whole of a recording session, they are rated in exactly the same way as if there were theta activity present throughout. All the subjects for which pre- and post-lesion records were available were assigned to one of four classes. In the medial septal and fornix lesioned groups, the subjects were classified on the basis of the hemisphere with the greater amplitude after lesioning; the lateral septal lesioned subjects were assigned on the basis of the hemisphere with the smaller amplitude after lesioning. In view of our findings (see Results) this is the most conservative procedure. The classes were as follows: Class I: 0%-25 % retention Class II: 26%-50% retention Class III: 51%-75% retention Class IV: >76% retention.
Histology. At the conclusion of the experiments, the subjects were given an overdose of pentobarbitone and perfused through the heart with isotonic saline and 10% formalin. The brains were left in formol saline solution for a minimum of 2 weeks. They were embedded in celloidin, and sectioned at 30 p., every fifth section being stained. In all cases with lesions aimed at the septal nuclei, cresyl violet staining was used; four of the fornix lesion group were stained in the same way, but all the remaining subjects were stained with a solachrome cyanin fibre stain. The sections were then inspected microscopically, and any showing evidence of a lesion was drawn onto plates derived from K6nig and Klippel (1963).
Ventral Recording Sites A total of 40 adult male Sprague-Dawley rats, weighing 3 0 0 4 0 0 g, were operated. All methods were the same as in the dorsal recording experiment, with the exceptions noted below. Two additional recording sites were used, with coordinates 8 mm posterior to bregma, 4.5 mm lateral, and 6.5 mm deep from skull surface (the 'flexure' site: Fig. 5B); and 6 mm posterior, 4.2 mm lateral and 7.7 mm deep from skull surface (the 'ventral' site: Fig. 5A). The recording electrodes were identical to those used before, except that a 3-mm vertical tip separation was used at the flexure site. Ten animals were implanted bilaterally with ventral recording electrodes; five of these also had medial septal lesioning electrodes, and five had lateral septal electrodes. Twenty-five animals were implanted with both dorsal and ventral recording electrodes in the left hemisphere; ten of these were intended for fornix lesions and 15 for an ipsilateral flmbria lesion. In the latter group care was taken during implantation to leave the appropriate area of skull clear for the subsequent lesion. The lesion method itself remained unchanged. Recordings were taken from all subjects with ventral recording electrodes both before and after lesioning. Finally, five animals were implanted bilaterally with flexure recording electrodes. Three of these had recordings taken before and after a fornix lesion; the other two had sustained bilateral fimbrial lesions 3 months before implantation. In addition to the histological procedures described previously, the brains from two subjects in each lesion group were left overnight in cold formalin (4 ~ C) and were then cut so that the portion containing the lesioned area and the portion containing the bulk of the hippocampal formation were separated. The former portion was then treated in the same ways as were the brains of the other subjects; the latter portion was sectioned at 50 ~, using a frozen sectioning method. The sections were then stained for cholinasterase using the Koelle copper thiocholine method (Koelle, 1954).
Septo-hippocampal Connections and Theta
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Results
Dorsal Recording Site Our results demonstrate that lesions restricted to the medial septal area or to the dorso-medial portion of the fornix severely reduce dorsal hippocampal theta; lesions to the dorso-lateral septal nuclei or to the fimbria do not. In addition, the fornix section abolished or drastically altered the hippocampal response to septal stimulation in all subjects; while fimbria section did not prevent the elicitation of clear hippocampal responses. Medial septal lesions produced 20 subjects in Class I (i.e., almost total theta loss), 5 in Class II, 4 in Class III and 7 in Class IV. Lateral septal lesions produced 5 subjects in Class I, 4 in Class II, 6 in Class III and 5 in Class IV. Fornix lesions produced 7 subjects in Class I and 2 in Class II. The post-lesion recordings from animals with fimbrial lesions showed theta amplitudes within the range we saw in intact animals. Each of the two electrolytic septal lesions produced a characteristic outcome in terms of the shape and rostro-caudal extent of the damage. The variability was mostly in the vertical dimension. The fimbria lesions were very consistent with respect to damage to the target areas, although damage to nearby structures varied somewhat. The fornical damage varied in regard both to the volume of tissue destroyed and to its rostro-caudal and lateral position. The resemblance between subjects with any of the four lesions within each electrophysiological class was marked. It was therefore possible both to draw general conclusions; and to select subjects to represent the findings for each group as follows. To demonstrate (Figs. 1-3) the critical areas in the septum and fornix which must be damaged if theta is to be abolished, we chose Class I subjects with the most restricted septal or fornical d a m a g e found to be compatible with a total abolition of theta; in order more accurately to delineate these critical zones, a Class IV subject with medial septal damage and a Class II subject with fornical damage (in both cases as similar as possible to the lesions in the corresponding Class I subjects) are also illustrated. We further illustrate a lateral septal lesioned subject chosen as having the maximal damage to the critical zone in the medial septal area consonant with acceptable (Class III) theta retention; the other lateral septal subject is the one with the most restricted damage producing a total (Class I) abolition of theta. The fimbria lesioned animal illustrated demonstrates how little tissue needs to be left between the septum and the hippocampus, while still preserving a marked degree of theta activity. Figures 1 and 2 present reconstructions of the lesions2; Figure 3 presents relevant E E G records; and Fig. 4 presents photographs of the hippocampal responses to septal stimulation for the fornix and fimbria groups. Medial Septal Lesions. Two subjects are presented: JF165 (Class I) and JF144 (Class VI). It can be seen that the lesion in JF165 is deeper and more posterior than that in JF144, although both lesions involve the medial septal nucleus to 2 Photographs of sections through the area of maximal damage in the same subjects can be seen in Gray et al. (1978, p. 280)
J. N. P. Rawlins et al.
54
JF165
JF144
RC85
RC81
Fig. 1. Reconstructions of lesions drawn onto plates derived from K6nig and Klippel (1963). JF165 - Medial septal lesion (Class I). Plates 12-18. JF144 -Medial septal lesion (Class IV). Plates 11-16. RC85 - Fornix lesion (Class I). Plates 20-27. RC81 - Fornix lesion (Class II). Plates 23-27. The asterisks mark critical sections for the abolition of theta by a fornix lesion. The bilateral extent of damage here corresponds well with the bilateral extent of theta loss
s o m e extent. This result is typical of the results of the g r o u p as a whole: the d e e p e r the midline d a m a g e in the septal area, the m o r e t h o r o u g h the abolition of the theta activity. F u r t h e r m o r e , d a m a g e restricted to the anterior medial septal area p r o d u c e d less m a r k e d effects on theta activity than if the posterior part o f the medial septal nucleus was d a m a g e d . In o r d e r to be completely effective, the lesion had to e x t e n d at least as far ventrally as the tip o f the ventricles; effective lesions were often slightly d e e p e r than this, and sometimes e x t e n d e d as far ventrally as the Insula Calleja magna, although this was almost invariably u n d a m a g e d due to the restricted lateral extent of the lesions. W h e r e the lesion was not entirely symmetrical about the midline, there was never m o r e than a partial loss of theta, even o n the side ipsilateral to the heavier septal damage. In such lesions, d a m a g e in the midline was never as deep as in m o r e accurately located lesions. D a m a g e to the fornix was seldom visible, and was not a prerequisite for theta loss. In subjects tested 6 m o n t h s after lesioning, there was no sign of theta recovery. Fornix Lesion. Again, two subjects are presented, one Class I ( R C 8 5 ) and one Class II ( R C 8 ] ) . R C 8 5 ' s lesion was clearly bilateral and fairly symmetrical,
Septo-hippocampal Connections and Theta
JFl18
JF123
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55
RC58
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Fig. 2. Reconstructions of lesions. JF118 -Lateral septal lesion (Class III). Plates 11-517. JF123 Lateral septal lesion (Class I). Plates 12-18. RC58 - Fimbrial lesion: theta is still present. Alternate plates from 21-29, plus Plates 30 and 31
whereas R C 8 1 ' s was slightly m o r e posterior, being much m o r e extensive on the right of the midline than on the left. The a s y m m e t r y of this lesion was matched by an a s y m m e t r y in the reductions in theta amplitude: these were to 20 % in the right hemisphere and 5 0 % in the left. In both cases, there was damage to the dorsomedial fornix and to the triangular nucleus of the septum, but in neither was there damage extending as far anterior as the medial septal nucleus. In the group as a whole, there was a good correlation between the extent of theta loss on each side and the lateral extent of the lesion on that side, particularly in the sections m a r k e d with an asterisk in Fig. 1. To summarise the group results, it appears that to ensure total theta loss, there must be destruction of that area of the dorsomedial fornix whose ventral limit is the triangular septal nucleus on the asterisked sections. Whether d a m a g e to the triangular septal nucleus itself is essential is not clear, although good theta loss was observed in subjects with only minimal damage to this structure. Fimbria Lesion. The results from one subject (RC58) with a bilateral fimbria lesion are presented. In all subjects in this group, the fimbrial damage was very extensive and led to considerable shrinkage of the fornix-fimbria bundle. It was always possible to record hippocampal theta activity of a normal amplitude.
56
J.N.P. Rawlins et al.
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Fig. 3. Hippocampal activity: dorsal recording site. Recordings from the left (above) and right (below) hemispheres: A before and a after a Class I medial septal lesion. B before and b after a Class III lateral septal lesion. C before and c after a Class I fornix lesion. D after a fimbrial lesion
Furthermore, the hippocampal activity on both sides may be seen to be in phase (Fig. 3D), in spite of the damage to the ventral hippocampal commissures. Lateral Septal Lesion. The results from two subjects are presented, one Class III (JF118) and one Class I (JF123). JF118 has marked dorsolateral septal damage, but little damage in the medial septal area. This reflects the results of the group as a whole, in that dorsolateral damage could be produced without marked medial septal damage; such subjects showed only small reductions in theta amplitude. We were unable to produce marked ventrolateral damage without considerably encroaching on the medial septal area; in such cases there was always a very marked theta loss. JF123 is typical of these subjects. Evoked Responses. The responses elicited by septal stimulation varied in appearance, ranging from relatively sharp evoked potentials, as illustrated by subject RC81, to almost sinusoidal activity, as illustrated by subject RC58 (Fig.
4).
Septo-hippocampal Connections and Theta
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RC58 demonstrated that responses to septal stimulation can be recorded in the dorsal hippocampus after bilateral section of the fimbria; similar responses were also observed in the other two fimbria lesioned subjects tested. The thresholds for a clear response ranged from 42 to 400 ~A, which is comparable to the range found in intact animals (James et al., 1977). Section of the dorso-medial fornix radically altered the response to septal stimulation of all five subjects tested. RC85 (Fig. 4) showed an evoked response after the lesion which was reversed in polarity and had a longer latency than the original response. One other subject showed an identical change. In two subjects the lesion altogether abolished the response to stimulation at the currents used in pre-lesion testing. RC81 was the only one of these five subjects to show partial retention of spontaneous theta (Class II), and it may be seen (Fig. 4) that scarcely any evoked responses remain.
Ventral and Flexure Recording Sites Of the 45 ventral electrode placements (10 bilateral and 25 unilateral implantations) usable records of theta activity were obtained in 31. This is a
58
J. N. P. Rawlins et al.
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Fig. 5. Recording electrode placements. A The ventral recording site (tip separation 2 mm). B The flexure recording site (tip separation 3 mm). The locations of the lower tips of the electrodes are illustrated using plates 46 and 52 from K6nig and Klippel (1963)
poorer success rate than with dorsal placements. Nonetheless, our most successful placements (Fig. 5A) produced recordings of theta as clear as in our dorsal site (Fig. 6). A t the flexure site (Fig. 5B) theta was recorded as easily as at the dorsal site. Our results demonstrate that ventrally recorded theta was abolished by medial septal lesions and spared by lateral septal lesions, as at the dorsal site. The effects of fornix and flmbria lesions, however, appeared to be the reverse of those seen at the dorsal site: fimbria section abolished theta; fornix section in most cases spared it. Abolition of ChE staining paralleled loss of theta. Usable ventral records were obtained from six placements in four animals which, upon histological examination, were found to have sustained some damage to the medial septal area. At these placements theta loss was total in three cases and partial (two Class II and one Class III) in the others. Three of the four animals had damage to the medial septal area similar to that illustrated in Fig. 1. The fourth animal (in which there was no theta after the lesion in one hemisphere but Class III retention in the other) had a markedly asymmetrical lesion with maximal damage ipsilateral to the side with total theta loss. In animals with lesions restricted to the dorso-lateral septal area usable records were obtained bilaterally from four animals. The lesion never abolished theta activity; the loss of theta could be classified as Class I V in all of them. In view of these results it seemed unnecessary to investigate the effects of septal lesions on theta recorded at the flexure. It proved possible to section the fimbria unilaterally in seven animals with good ipsilateral ventral and dorsal theta recordings. Ventral theta was totally abolished in five animals and reduced to 23 and 33%, respectively, in the remaining two. In no animal did the lesion affect dorsal theta as much as ventral theta (Fig. 6). Nonetheless, in five animals some toss of dorsal theta was observed (two Class IV, two Class I I I and one Class II). The extent of dorsal theta loss did not appear to correlate with the extent of ventral theta loss; indeed, in one animal, there was total loss of ventral theta with no reduction in dorsal theta.
Septo-hippocampal Connections and Theta
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Fig. 6. Hippocampal activity: a comparison of the dorsal and ventral recording sites. Recordings taken from the left hemisphere at the dorsal (above) and ventral (below)sites: A before and a after a fornix lesion. B before and b after a fimbrial lesion
Histologically verified section of the dorso-medial fornix was carried out in five animals with clear ventral and dorsal theta recordings. In all of them theta at the dorsal site was severely disrupted (four Class I, one Class II), while ventral theta was largely unaffected (four Class IV, one Class III). In three of these animals there was total loss of dorsal theta and no reduction in the amplitude of ventral theta (Fig. 6). The histological examination revealed damage similar to that shown in Fig. 1, although in all but one subject somewhat more extensive. Flexure Recording Site. The results after fimbrial and fornix section using the flexure recording site paralleled those obtained with dorsal, not ventral, recording electrodes. It was possible to obtain clear theta records in both hemispheres in two animals following bilateral section of the fimbria. Conversely, theta was abolished (Class I) bilaterally in three animals tested before and after fornix section. Cholinesterase Staining. Representative sections stained for ChE are shown in Fig. 7. Marked loss of ChE staining occurred throughout the hippocampus after medial septal lesions, and no loss after dorso-lateral septal lesions. After fornix section there was marked ChE depletion in the dorsal hippocampus only; there were no apparent changes in ventral hippocampal ChE. Unilateral fimbrial section produced a marked loss of ChE staining in the ipsilateral ventral hippocampus, but also reduced to a smaller degree the intensity of staining in the ipsilateral dorsal hippocampus. Both fornix and fimbria lesions appeared to reduce ChE staining to an equal extent at the site of the flexure recording electrode.
Discussion Our results confirm the work of Petsche and his collaborators (Stumpf, 1965) in showing that the hippocampal theta rhythm depends on the integrity of the
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Fig. 7. Cholinesterase staining. A Following a medial septal lesion. B Following a lateral septal lesion. C Following a fornix lesion (anterior section). D Following a fimbrial lesion (anterior section). E The same fornix lesioned subject (posterior section). F The same fimbrial lesioned subject (posterior section)
medial, but not the lateral, septal area. In addition, they show that this conclusion applies to ventral as well as dorsal theta activity and d e m o n s t r a t e it with s p o n t a n e o u s theta r e c o r d e d f r o m unanaesthetised, free-moving animals. T h e y also confirm M y h r e r ' s (1975b) r e p o r t that section of the d o r s o - m e d i a l p o r t i o n o f the fornix abolishes theta r e c o r d e d dorsally, but show that this result c a n n o t be generalised to ventral theta, which is controlled by fibres travelling in the fimbria, not in the medial fornix.
Septo-hippocampal Connections and Theta
61
It has recently been suggested by McLennan and Miller (1976) that the hippocampal theta rhythm is dependent on the hippocampal output to the lateral septum via the fimbria. This suggestion is ruled out by our data. First, fimbrial section failed to eliminate dorsal hippocampal theta in any animal studied. Second, dorso-lateral septal lesions, destroying the greater part of the site of termination of the hippocampo-septal output (Meibach and Siegal, 1977), failed to eliminate either dorsal or ventral hippocampal theta in any animal studied. Conversely, damage to the cells of origin of the septal projection to the hippocampus, located in the medial septal area, or to their axons which travel to the dorsal and ventral hippocampus via the dorso-medial fornix and fimbria, respectively (Meibach and Siegal, 1977), invariably caused severe and often total disruption of hippocampal theta in the deafferented area. McLennan and Miller's (1976) proposal was that feedback from the hippocampus passes through a frequency gating mechanism located in the lateral septum, and thus itself controls the bursting activity of medial septal neurones. While the present results demonstrate that this mechanism is not essential for the generation of theta, it may modulate theta frequency. When hippocampal theta is provoked by electrical stimulation of the septal area in free-moving rats, the function relating the threshold current to the stimulation frequency displays a minimum at a frequency of 7.7 Hz (James et al., 1977). Although this frequency-intensity function was not a primary object of study in the present investigation, we determined it in a group of rats with dorsal recording electrodes after bilateral fimbrial section. In no case was there a 7.7 Hz minimum. Thus, it is possible that McLennan and Miller's (1976) 'frequency gating mechanism' is concerned with the fine tuning of hippocampal theta frequency. Experiments in which hippocampal theta is elicited by septal stimulation (Gray and Ball, 1970) assume that electrically elicited hippocampal activity does not radically differ from spontaneous theta. Our results provide some support for this assumption, since, at our dorsal recording site, fornix lesions eliminating spontaneous theta also eliminated or drastically altered the response to septal stimulation. In the latter cases the reversal of polarity and change of latency suggest a changed location for the post-lesion hippocampal response, presumably mediated by other intact fibre systems. Conversely, our ability to elicit dorsal hippocampal responses in subjects with bilateral fimbria lesions demonstrates that such responses are not dependent upon antidromic activation of hippocampal efferent fibres running in the fimbria. Our observations of hippocampal staining for ChE after lesions to the septal area, the fornix or the fimbria are in accord with previous anatomical reports (Lewis and Shute, 1967; Mellgren and Srebro, 1973). They apparently conflict, however, with Dudar's (1975) observation that fimbria but not fornix section elimited hippocampal release of acetylcholine induced by septal stimulation. Our results, in contrast, clearly implicate the fornix as the cholinesterase-containing pathway to the dorsal hippocampus. There was a qualitative correspondence between loss of theta and loss of ChE staining, both between and within subjects (e.g., Figs. 6 and 7). However, at the flexure recording site, we could not distinguish between ChE depletion
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caused by fornix and fimbria section, although the former abolished theta while the latter did not. Thus, while our results are largely in agreement with the concept of septal cholinergic control of theta, they are not conclusive in this respect. We suggest that the fibres running in the dorso-medial fornix and those running in the fimbria should be considered as representing populations dispersed within a single, functional pathway. It is proposed that the fibres are densely packed as they emerge from the medial septal area, and start to fan out laterally as they run caudally. It might, therefore, be possible to sever the entire bundle with a single, midline cut located anterior to our dorso-medial fornix lesion, while the number of fibres damaged by such a cut would be smaller as the plane of section moves caudally. Thus, the ability to produce selective effects on dorsal and ventral theta activity would depend critically on the plane of sectioning. Selective lesions of the dorso-medial fornix and of the fimbria have already been investigated in behavioural experiments (Myhrer, 1975a,b~ Jarrard, 1976). Our data demonstrate that these results require more complex interpretation than has hitherto been realised.
Acknowledgements.
This research was supported by the United Kingdom Medical Research Council. J. Feldon was supported by a Sir Kenneth Lindsay Scholarship from the Anglo-Israel Association. We are grateful to Mr. I. Hughes for developing the cholinesterase stain, to Dr. J. J. B. Jack for the loan of the camera, and to Professor L.E. Jarrard for his help in the development of the fimbria lesion. A preliminary report using these data appeared in Feldon and Rawlins (1978).
References Apostol, G., Creutzfeldt, O.D.: Cross correlation between the activity of septal units and hippocampal EEG during arousal. Brain Res. 67, 65-75 (1974) Bland, B.H., Andersen, P., Ganes, T.: Two generators of hippocampal theta activity in rabbits. Brain Res. 94, 199-218 (1975) Dudar, J.D.: The effect of septal nuclei stimulation on the release of acetylcholine from the rabbit hippocampus. Brain Res. 83, 123-133 (1975) Feldon, J., Rawlins, J. N. P.: The anatomical basis of hippocampal theta rhythm. J. Physiol. (Lond.) 284, 81P (1978) Gray, J.A., Ball, G. G.: Frequency-specific relation between hippocampal theta rhythm, behaviour, and amobarbital action. Science 168, 1246-1248 (1970) Gray, J.A., Feldon, J., Rawlins, J.N.P., Owen, S., McNaughton, N.: The role of the septohippocampal system and its noradrenergic afferents in behavioural responses to non-reward. In: Whelan, J. (Ed.). Functions of the septo-hippocampal system. Ciba Foundation Symposium No. 58 (New Series), pp. 275-300. Amsterdam: Elsevier 1978 James, D.T.D., McNaughton, N., Rawlins, J.N.P., Feldon, J., Gray, J.A.: Septal driving of hippocampal theta rhythm as a function of frequency in the free-moving male rat. Neuroscience 2, 1007-1017 (1977) Jarrard, L.E.: Anatomical and behavioura! analysis of hippocampal cell fields in rats. J. Comp. Physiol. Psychol. 90, 1035-1050 (1976) Koelle, G.B.: The histochemical localization of cholinesterases in the central nervous system of the rat. J. Comp. Neurol. 100, 211-228 (1954) KOnig, J.F.R., Klippel, R.A.: The rat brain. Baltimore: Williams and Wilkins 1963
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Lewis, P.R., Shute, C. C. D.: The cholinergic limbic system: Projections to hippocampal formation, medial cortex, nuclei of the ascending cholinergie reticular formation, and the subfornical organ and supraoptic crest. Brain 901 521-540 (1967) Mellgren, S. I., Srebro, B.: Changes in acetylcholinesterase and distribution of degenerating fibres in the hippocampal region after septal lesions in the rat. Brain Res. 52, 19-36 (1973) MeLennan, H., Miller, J.J.: The hippocampal control of neuronal discharges in the septum of the rat. J. Physiol (Lond.) 237, 607--624 (1974) McLennan, H., Miller, J.J.: Frequency-related inhibitory mechanisms controlling rhythmical activity in the septal area. J. Physiol. (Lond.) 254, 827-841 (1976) Meibach, R.C., Siegal, A.: Efferent connections of the septal area in the rat: an analysis utilizing retrograde and anterograde transport methods. Brain Res. 119, 1-20 (1977) Myhrer, T.: Locomotor, avoidance, and maze behaviour in rats with selective disruption of hippocampal output. J. Comp. Physiol. Psychol. 89, 759-777 (1975a) Myhrer, T.: Normal jump avoidance performance in rats with the hippocampal theta rhythm selectively disrupted. Behav. Biol. 14, 489-498 (1975b) Petsche, H., Stumpf, Ch., Gogolak, G.: The significance of the rabbit's septum as a relay station between the midbrain and the hippocampus. I. The control of hippocampus arousal activity by the septum cells. Electroenceph. Clin. Neurophysiol. 14, 202-211 (1962) Raisman, G.: The connexions of the septum. Brain 89, 317-348 (1966) Stumpf, C.: Drug action on the electrical activity of the hippocampus. Int. Rev. Neurobiol. 8, 77-138 (1965) Vanderwolf, C.H.: Limbic diencephalic mechanisms of voluntary movement. Psychol. Rev. 78, 83-113 (1971) Vanderwolf, C.H., Kramis, R., Gillespie, L.A., Bland, B.H.: Hippocampal rhythmic slow activity and neocortical low-voltage fast activity: relations to behavior. In: Isaacson, R.L. and Pribram, K.H. (Eds.), The hippocampus. Vol. 2, pp. 101-128. New York: Plenum Press Winson, J.: Patterns of hippocampal theta rhythm in the freely moving rat. Electroenceph. Clin. Neurophysiol. 36, 291-301 (1974) Received October 17, 1978
