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Neural pathways mediating septal hyperreactivity
Physiology &Behavior, Voi; 17, pp. 451-455. Pergamon Press and Brain Research Publ., 1976. Printed in the U.S.A.
Neural Pathways Mediating Septal Hyperreactivity ' D. J. ALBERT 2 AND S. E. RICHMOND
Psychology Department, University of British Columbia, 2075 Wesbrook Place, Vancouver, Canada (Received 21 April 1975) ALBERT, D. J. AND S. E. RICHMOND. Neural pathways mediating septal hyperreactivity. PHYSIOL. BEHAV. 17(3) 451-455, 1976. - Cuts were made at various positions around the septum to interrupt septal connections with other structures. The effect of these cuts on reactivity was examined 2, 7, and 14 days postoperatively. Animals with anterior or lateral cuts scored significantly higher than control animals on each of the test days. It is concluded that the septal connections which mediate septal lesion-induced hyperreactivity are with the medial forebrain bundle and possibly with some area anterior and ventral to the septum. The lack of an effect with posterior cuts which severed the stria terminalis argues against the importance of this pathway. Septal lesions
Aggression Knife cuts
Stria terminalis
Hyperreactivity
While these results form a basis for speculation on the connections which mediate the septal lesion-induced increase in reactivity, their implications are sufficiently open to question that the conclusions must be reexamined with more definitive procedures. The experiments of Brady and Nauta [7] are not conclusive in demonstrating the irrelevance of septohabenular fibers. Clearly, the habenula may have a complex role in controlling behavior such that lesions there do not produce a simple increase in reactivity. The evidence supporting the importance of stria terminalis damage for the appearance of hyperreactivity is not conclusive because it is largely correlational and there are contrary findings [ 3 ]. The present experiment makes a fresh approach to the question of the septal connections involved in the hypero reactivity. Our method was to make cuts at different places around the septum, a method which lends itself to evaluating the importance of neural pathways. Since pathways are known to connect the septum with other areas by fibers coursing anteriorly, ventrolaterally, and posteriorly, cuts were made which severed fibers coursing in each of these directions. The importance of these pathways for hyperreactivity was evaluated by rating the animals' behavior at several times postoperatively.
LESIONS in the septal region are well known for increasing reactivity and aggressive behavior to stimuli which normally generate little or no response [6, 7, 12, 31]. As the most readily observable behavior change that occurs with septal lesions, this hyperreactivity and hyperaggressiveness has been examined quite extensively in terms of differences between species [8, 13, 30, 31], the factors affecting its severity [17], the action of other lesions and drugs in attenuating or accentuating it [10, 14, 16, 20, 21, 27, 28], and the relation of the hyperreactivity to other effects of septal lesions [ 1, 7, 11, 17, 21]. Yet, the pathways to, or from, the septum which mediate the hyperreactivity have received scant attention. This issue was first explored by Brady and Nauta [7] subsequent to their early experiment which demonstrated increased viciousness following septal lesions. On the basis of anatomical findings of strong septal connections with the habenula, they examined the effect of habenular lesions on reactivity. Failing to find any change, they concluded that these connections were not involved in the hyperreactivity produced by septal lesions. Connections between the septum and amygdala were the next to receive consideration. Two kinds of evidence bear directly on this suggestion. First, lesions of the amygdala attenuate the hyperreactivity produced by septal lesions showing that the amygdala is somehow involved in modulating the animal's responsivity [16,28]. Second, damage to the stria terminalis has been reported to be more highly correlated with increased reactivity than the amount of septal damage [33,34]. From this evidence one can argue that stria terminalis connections between the septum and amygdala may mediate the hyperreactivity which follows septal lesions.
METHOD
Animals The animals were male hooded rats weighing 3 0 0 - 4 0 0 g at the beginning of the experiment. They were obtained from the Canadian Breeding Farms and Laboratories (St.
1Supported by grants from the National Research Council of Canada (NRC 67-0192) and the University Research Committee. 2Send reprint requests to: D. J. Albert, Psychology Department, University of British Columbia, 2075 Wesbrook Place, Vancouver, B. C., Canada. 451
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Constant, Quebec). Housing was in group cages prior to surgery and in individual cages thereafter.
Surgical Procedure Our method of making cuts in brain tissue has been described in detail previously [2]. The knife consists of an outer guide cannula (21 ga stainless-steel hypodermic tubing) which is stereotaxically lowered into the brain of an anesthetized animal. With the guide cannula in place, a 26-ga insert with a cutting blade of stainless-steel wire (0.15 mm dia.) is lowered into the cannula. When the cutting blade reaches the lower slit, it extends and cuts through the neural tissue as it is lowered further. The cutting blade is then removed and the guide cannula raised out of the brain. Operated control animals were given sham cuts. These animals were treated in every way like the experimental animals, except that the knife was not lowered into the cannula to make the brain cut.
Behavioral Testing
<0.025). The highest level of reactivity was produced by cuts which were either lateral or anterior to the septum (Fig. 1). The median reactivity scores for the lateral cut group (n = 7) were 7, 6, and 1 for the 3 test days. These scores are each significantly different from the median scores of the control group (n = 6) which were 0.25, 0, and 0 for the corresponding test days (U -- 0, p < 0 . 0 0 2 ; U = 0, p<0.002; U = 4, p<0.02). 25w
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Statistics A one-way analysis of variance by ranks (Kruskal-Wallis, [29]) was performed for each test day to determine whether there were overall differences between groups. Individual pair comparisons were then made with a MannWhitney U-test [29]. All p values are two-tailed unless otherwise stated. RESULTS Cuts around the septal area caused an increase in reactivity as measured on each of the test days (Day 2, H = 14.2, p<0.01; Day 7, H = 13.0, p < 0 . 0 1 ; Day 14, H = 9.8,
"
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Animals were tested for reactivity 2, 7, and 14 days postoperatively. They were tested in a box measuring 60 x 60 x 60 cm. Its floor was covered with woodshavings and it was open on top. Lighting was provided by the room's florescent fixtures. On each of the test days the rat's home cage was first covered to prevent escape and then placed in the test box which rested on the floor of the colony room. The cage cover was then removed and the rat left for one minute before testing began. The animal's responses to six test stimuli were rated on a scale of 0 - 3 , 0 indicating little or no response and 3 indicating attack or a highly aggressive response. Each of the stimuli (except item 2, which was essentially a startle test) was repeated 3 times before moving on to the next. The average score for the 3 repetitions was recorded. In order of presentation the test items were: (1) presentation of a pencil just in front of the rat's snout; (2) a sharp tap on the back with a pencil; (3) presentation of a gloved hand before the snout; (4) gentle prods on the side of the rat's body with blunt one-inch alia. stick; (5) attempted capture by the tail; and (6) attempted grasping around the body. In addition, the rat was scored from 0 to 3 on both the amount of vocalization and biting observed during testing, 0 indicating none and 3 indicating vocalization and biting in response to all degrees of stimulation, from the visual presentation of a pencil to grasping during capture. The maximum reactivity score possible was 24. The rating scores presented in this experiment were all obtained by the same experimenter. Previous work has indicated that interrater reliability is extremely high.
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FIG. 1. The effect of cuts anterior (AS, n = 5), ventrolateral (VLS, n = 7), posterior (PS, n = 6) to the septum and sham cuts (Control, n = 6) on reactivity rated 2, 7, and 14 days postoperatively. The dotted lines indicate the level of reactivity obtained in animals of the same strain with septal lesions (SL) and septal lesions which also damage the region ventral to the anterior septum (SL-AV) [3]. The region severed by the cuts lateral to the septum is shown in Fig. 2. The cuts were all symmetrical about the midline and highly similar. They fell within a region bordered medially by the lateral edge of the septum and laterally about 0.5 mm to the side (see Fig. 2). The cuts began 4 mm anterior to bregma. In 5 of the animals, the cut ended at the posterior end of the septum. In the two remaining animals, the cut ended about 0.7 mm posterior to the septum. All of the cuts severed the entire region between the ventral edge of the lateral ventricle and the base of the brain. The median reactivity scores of the rats with cuts anterior to the septum (n = 5) were 8.5, 6.5, and 1.0 for test days 2, 7, and 14 respectively. These scores are each significantly different from those of the control group on the corresponding test days (U = 0, p<0.004; U = 4.5, p<0.04, one-tailed; and U = 3.5, p<0.04). These scores are almost identical to those found in the animals with cuts lateral to the septum. The tissue severed by these anterior cuts is shown in Fig. 3. The anterior-posterior pattern of the cuts was between 3.8 and 4.2 mm anterior to bregma, so that they cut through the anterior tip of the septum. Laterally, they invaded the medial edge of the caudate nucleus and ventrally they severed connections to the anterior portions of the diagonal band of Broca. The swath of the cuts in each case was very similar to that shown in Fig. 3. The smallest change in reactivity was that produced by
453
SEPTAL HYPERREACTIVITY
FIG. 3. The swath of the anterior cuts is shown by the arrows (top) and the stippling (bottom).
FIG. 2. The location of the lateral cuts is shown by the arrows (top) and stippling (bottom). All cuts extended at least from the level of the bottom of the ventricle to the base of the brain. The brain sections are from Pellegrino and Cushman [24]. cuts posterior to the septum. The median reactivity scores for the animals of this group (n = 6) were 0.25, 0.0, and 0.0 for the 3 test days (Fig. 1). None of these scores differed significantly from those of the control group. Each is different from the corresponding test score for the lateral cut (U = 3.5, p<0.01; U = 0, p<0.01; U = 7, p<0.05) and the anterior cut groups (U = 4, p < 0 . 0 5 ; U = 5.5, p<0.05, one-tailed; U = 5.5, p<0.05, one-tailed). The swath of the posterior cut is shown in Fig. 4. All of the cuts fell in the path of the stria terminalis and through the bed nucleus of the stria terminalis. Without exception they were located just anterior to the fornix. They did not interrupt any of the septal connections with the hippocampus, nor should they have disturbed septohabenular fibers since these are mixed with the fornix in the area of the cut [24,25]. Included in Fig. 1 for comparison purposes are the reactivity scores (from a previous experiment) of two groups of animals with different size septal lesions (see
Albert and Richmond [3] for a thorough presentation of this material). These scores, which were obtained at about the same time as the present results, show that both the anterior and lateral cuts cause a level of reactivity very similar to that of lesions restricted to the medial and lateral septum. However, the level of reactivity obtained with the cuts is substantially less than that found with a larger septal lesion which damages most of the medial and lateral septum and in addition parts of the accumbens nucleus and the diagonal band of Broca [ 3 ]. DISCUSSION The pathways involved in septal hyperreactivity course ventrolaterally and anteriorly with respect to the septum. This is shown by the increase in reactivity which follows cuts interrupting pathways with either of these orientations. The level of reactivity produced by each cut is substantially similar to what we have found previously with septal lesions [3 ]. Fiber tracts coursing through the region severed by the lateral cuts are well known. Efferent fibers leave the septum rostral to the anterior commissure and course ventrally and laterally [19, 25, 26]. They join the medial forebrain bundle and then course posteriorly through the lateral hypothalamus, giving off connections all along this route [26]. Another efferent pathway leads ventrally into the
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ALBERT AND RICHMOND
I FIG. 4. The swath of the posterior cuts is shown by the arrows (top) and stippling (bottom). medial arm of the diagonal band of Broca. From here some fibers appear to course laterally into the olfactory tubercle and then into the medial forebrain bundle [26]. Afferent pathways to the septum have been shown by degeneration studies to follow very similar routes. From their origin all along the medial forebrain bundle, they course around the rostral edge of the anterior commissure and terminate in both the lateral and medial septum [26]. Since lesions of the septum produce an increase in reactivity, it is reasonable to argue that the lesions are releasing the activity of areas which produce an increase in reactiveness when stimulated electrically or chemically. There is evidence that the effect of a septal lesion is mediated in this way. The septum is known to have strong efferent connections to the medial forebrain bundle. An it is well established that increased reactivity in the form of aggressive behavior can be elicited by electrical or chemical stimulation in the medial forebrain bundle region where these septal efferent fibers terminate [ 15, 22, 36]. Further, the forms of aggressiveness obtained include the explosive affective reactivity (attack) which occurs following septal lesions [15, 22, 36]. Alternatively, cuts which interfere with connections coursing laterally could also produce hyperreactivity by interrupting connections between the septum and the ventromedial nucleus of the hypothalamus. Activity from
the septum has been shown by electrophysiological methods to reach the ventromedial nucleus over a multiple synaptic pathway [33]. The pathway apparently courses laterally from the septum, follows the medial forebrain bundle caudally, and then enters the lateral edge of the ventromedial nucleus. Since lesions of the ventromedial hypothalamus are known to produce hyperreactivity [5,35], it may be that the septum normally has a role in modulating reactivity through its connections with the ventromedial hypothalamus. The removal of this modulation by destruction of the septum or by cuts lateral to it may result in increased reactiveness. There is support for this suggestion to the extent that cuts lateral to the medial hypothalamus are known to produce hyperreactivity [4,23]. From the circuitry controlling septal reactivity we have outlined thus far, one might argue that there is a single pathway which travels slightly anteriorly as well as laterally with respect to the septum and which is severed by the anterior as well as the lateral cuts. This would account for the observation that the change in reactivity is similar with both anterior and lateral cuts and that the effects of each of these in turn is similar to that which occurs with septal lesions. However, an inadequacy of this interpretation lies in the fact that while cuts lateral or anterior to the septum produce an increase in reactivity comparable to that of septal lesions, larger lesions which destroy the septum and in addition invade the region anteroventral to it produce a much greater and more long lasting effect (Fig. 1, also [3] ). Since the ventral region is within the region bounded by the anterior and lateral cuts, fiber connections to or from this ventral area are also being severed by these cuts. Hence, the change in reactivity which the cuts must account for may be that produced by the larger lesions which include the ventral area and not just that due to lesions restricted to the septum. Accordingly, it is reasonable to argue that while the lateral cuts are interrupting connections to the medial forebrain bundle, the fibers severed by the anterior cuts may constitute a separate pathway which terminates elsewhere. Cutting both the anterior pathway and the lateral connections to the medial forebrain bundle could cause the severe hyperreactivity shown by animals with lesions of the septum and the area ventral to its anterior region [ 3 ]. Anatomically, there is support for a separate pattern of connections with an anterior and ventral trajectory from the septum. One large efferent tract is known to lead into the diagonal band of Broca [26]. The suggestion that some of these fibers will make anterior connections with other areas involved in controlling reactivity receives some support from the finding of Cain [9], that lesions of the anterior olfactory area produce increased reactivity. Presumably there may be other fibers with similar anterior connections which originate in the region ventral to the anterior septum. Contrary to some previous suggestions [33,34], the present results offer no support for the possibility that fibers in the stria terminalis or other pathways which leave or enter the septum from a posterior-ventral direction are involved in controlling reactivity. In particular, the stria terminalis was consistently interrupted by the posterior cuts without producing an increase in reactivity. The status of fiber connections to the habenula is still uncertain since the posterior cuts did not disturb the fornix. The only evidence available shows that lesions in the region of the
SEPTAL HYPERREACTIVITY
455
fornix actually a t t e n u a t e septal lesion-induced hyperreactivity [211. The failure to p r o d u c e an increase in reactivity b y interrupting the stria terminalis is consistent w i t h our previous failure to produce such an effect with lesions of the stria terminalis and its bed nucleus [ 3 ] . A line o f reasoning by which one could object to our present result is that with our m e t h o d of severing the stria terminalis, there may be b o t h an increase in aggressiveness due to cutting the stria terminalis and an a t t e n u a t i o n of this effect due to cutting some o t h e r pathway. We did examine this in three rats by making septal lesions along w i t h the posterior cuts. We found the e x p e c t e d hyperreactivity in each o f the three animals.
The lack o f an increased reactivity w i t h stria terminalis cuts does not detract f r o m the existing evidence showing that the amygdala is i m p o r t a n t in the c o n t r o l of aggression. There is a b u n d a n t evidence to show this from h u m a n clinical data reporting increased aggressiveness w i t h abnormal discharging f r o m the amygdala [18] and animal studies showing an a t t e n u a t i o n of reactivity following amygdala lesions [ 1,1 1]. If the stria terminalis fibers are not the p a t h w a y through which these effects are mediated, the i m m e d i a t e alternative is that the influence of the amygdala on aggression and reactivity is via the ventral amygdalafugal p a t h w a y to the h y p o t h a l a m u s and brainstem [37].
REFERENCES
1. Ahmad, S. S. and J. A. Harvey. Long-term effects of septal lesions and social experience on shock elicited fighting in rats. J. comp. physiol. Psychol. 66: 596-602, 1968. 2. Albert, D. J. A simple method of making cuts in brain tissue. Physiol. Behav. 4: 863-864, 1969. 3. Albert, D. J. and S. E. Richmond. Septal hyperreactivity: A comparison of lesions within and adjacent to the septum. Physiol. Behav. 15: 339-347, 1975. 4. Albert, D. J. and L. H. Storlien. Hyperphagia in rats with cuts between the ventromedial and lateral hypothalamus. Science 165: 599-600, 1969. 5. Anand, B. K. and J. R. Brobeck. Hypothalamic control of food intake in rats and cats. Yale J. Biol. Med. 24: 123-140, 1951. 6. Brady, J. V. and W. H. Nauta. Subcortical mechanisms in emotional behavior: Affective changes following septal forebrain lesions in the albino rat. J. comp. physiol. Psychol. 46: 339-346, 1953. 7. Brady, J. V. and W. J. H. Nauta. Subcortical mechanisms in emotional behavior: Affective changes following septal and habenula lesions in the albino rat. J. comp. physiol. Psychol. 48: 412-420, 1955. 8. Buddington, R. W., F. A. King and L. Roberts. Emotionally and conditioned avoidance responding in the squirrel monkey following septal injury. Psychon. Sci. 8: 195-196, 1967. 9. Cain, D. P. Olfactory bulbectomy: Neural structures involved in irritability and aggression in the male rat. J. comp. physiol. Psychol. 86: 213-220, 1974. 10. Clark, S. M., P. M. Meyer, D. R. Meyer and D. A. Yutzey. Emotionality changes following septal and neocortical ablations in the albino rat. Psychon. Sci. 8: 125-126, 1967. 11. Corman, C. D., P. M. Meyer and D. R. Meyer. Open-field activity and exploration in rats with septal and amygdaloid lesions. Brain res. 5: 469-476, 1967. 12. Fried, P. A. The septum and hyper-reactivity: A review. Br. J. Psychol. 64: 267-275, 1973. 13. Green, J. D. and A. A. Arduini. Hippocampal electrical activity and arousal. J. Neurophysiol. 17: 533-557, 1954. 14. Harvey, J. A., A. Heller, R. Y. Moore, H. F. Hunt and L. J. Roth. Effect of central nervous system lesions on barbiturate sleeping time in the rat. J. Pharmac. exp. Ther. 144: 24-36, 1964. 15. King, M. B. and B. G. Hoebel. Killing elicited by brain stimulation in rats. Communs Behav. Biol. 2: 173-177, 1968. 16. Kleiner, F. B., P. M. Meyer and D. R. Meyer. Effects of simultaneous septal and amygdala lesions upon emotionality and retention of a black-white discrimination. Brain Res. 5: 459-468, 1967. 17. Krieckaus, E. E., H. J. Simons, G. J. Thomas and J. Kenyon. Septal lesions enhance shock avoidance behavior in the rat. ExplNeurol. 9: 107-113, 1964. 18. Mark, V. H., F. R. Ervin and W. H. Sweet. Deep temporal lobe stimulation in man. In: The Neurobiology of the Amygdala, Advances in Behavioural Biology, Vol. 2, edited by B. E. Eleftheriou. New York: Plenum Press, 1972, 485-510.
19. Nauta, W. J. H. An experimental study of the fornix system in the rat. J. comp. Neurol. 104: 247-271, 1956. 20. Novik, I. and R. Pihl. Effect of amphetamine on the septal rage syndrome in rats. J. cornp. Physiol. Psychol. 68: 220-225, 1969. 21. Olton, D. S. and F. H. Gage. Role of the fornix in the septal syndrome. Physiol. Behav. 13: 269-279, 1974. 22. Panksepp, J. Aggression elicited by stimulation of the hypothalamus in albino rats. Physiol. Behav. 6: 321-329, 1971. 23. Paxinos, G. and D. Bindra. Hypothalamic and midbrain pathways involved in eating, drinking irritability, aggression and copulation in rats. J. comp. physiol. Psychol. 82: 1-14, 1973. 24. Pellegrino, L. J. and A. J. Cushman. A StereotaxicAtlas of the Rat Brain. New York: Appleton-Century-Crofts, 1967. 25. Powell, E. W. Septal efferents revealed by axonal degeneration in the rat. ExplNeurol. 8: 406-422, 1963. 26. Raisman, G. The connexion of the septum. Brain 89: 317-348, 1966. 27. Schallek, W., A. Kuehn and N. Jew. Effects of chlordiazepoxide (librium) and other psychotropic agents on the limbic system of the brain. Ann. N. Y. Acad. Sci. 96: 303-314, 1962. 28. Schwartzbaum, J. S. and P. Gay. Interacting behavioral effects of septal and amygdaloid lesions in the rat. J. comp. physiol. Psychol. 61: 59-65, 1966. 29. Siegel, S. Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill: New York, 1956. 30. Sodetz, F. J., E. S. Matalka and B. N. Bunnell. Septal ablation and affective behavior in the golden hamster. Psychon. Sci. 7: 189-190, 1967. 31. Spiegel, E. A., H. R. Miller and M. J. Oppenheimer. Forebrain and rage reactions. £ Neurophysiol. 3: 538-548, 1940. 32. Sutin, J. An electrophysiological study of the hypothalamic ventromedial nucleus in the cat. Electroenceph. clin. Neurophysiol. 15: 786-795, 1963. 33. Thomas, J. B. and L. Van Atta. Hyperirritability, lever press avoidance, and septal lesions in the albino rat. Physiol. Behav. 8: 225-232, 1972. 34. Turner, B. H. Neural structures involved in the rage syndrome of the rat. J. comp. physiol. Psychol. 71: 103-113, 1970. 35. Wheatley, M. O. The hypothalamus and affective behavior in cats. ArchsNeurol. Psychiat. 52: 296-316, 1944. 36. Woodworth, C. H. Attack elicited in rats by electrical stimulation of the lateral hypothalamus. Physiol. Behav. 6: 345-353, 1971. 37. Zbrozyna, A. W. The organization of the defense reaction elicited from the amygdala and its connections. In: The Neurobiology of the Amygdala Advances in Behavioral Biology, Vol. 2, edited by B. E. Elftheriou. New York: Plenum Press, 1972, 597-606.
Neural Pathways Mediating Septal Hyperreactivity ' D. J. ALBERT 2 AND S. E. RICHMOND
Psychology Department, University of British Columbia, 2075 Wesbrook Place, Vancouver, Canada (Received 21 April 1975) ALBERT, D. J. AND S. E. RICHMOND. Neural pathways mediating septal hyperreactivity. PHYSIOL. BEHAV. 17(3) 451-455, 1976. - Cuts were made at various positions around the septum to interrupt septal connections with other structures. The effect of these cuts on reactivity was examined 2, 7, and 14 days postoperatively. Animals with anterior or lateral cuts scored significantly higher than control animals on each of the test days. It is concluded that the septal connections which mediate septal lesion-induced hyperreactivity are with the medial forebrain bundle and possibly with some area anterior and ventral to the septum. The lack of an effect with posterior cuts which severed the stria terminalis argues against the importance of this pathway. Septal lesions
Aggression Knife cuts
Stria terminalis
Hyperreactivity
While these results form a basis for speculation on the connections which mediate the septal lesion-induced increase in reactivity, their implications are sufficiently open to question that the conclusions must be reexamined with more definitive procedures. The experiments of Brady and Nauta [7] are not conclusive in demonstrating the irrelevance of septohabenular fibers. Clearly, the habenula may have a complex role in controlling behavior such that lesions there do not produce a simple increase in reactivity. The evidence supporting the importance of stria terminalis damage for the appearance of hyperreactivity is not conclusive because it is largely correlational and there are contrary findings [ 3 ]. The present experiment makes a fresh approach to the question of the septal connections involved in the hypero reactivity. Our method was to make cuts at different places around the septum, a method which lends itself to evaluating the importance of neural pathways. Since pathways are known to connect the septum with other areas by fibers coursing anteriorly, ventrolaterally, and posteriorly, cuts were made which severed fibers coursing in each of these directions. The importance of these pathways for hyperreactivity was evaluated by rating the animals' behavior at several times postoperatively.
LESIONS in the septal region are well known for increasing reactivity and aggressive behavior to stimuli which normally generate little or no response [6, 7, 12, 31]. As the most readily observable behavior change that occurs with septal lesions, this hyperreactivity and hyperaggressiveness has been examined quite extensively in terms of differences between species [8, 13, 30, 31], the factors affecting its severity [17], the action of other lesions and drugs in attenuating or accentuating it [10, 14, 16, 20, 21, 27, 28], and the relation of the hyperreactivity to other effects of septal lesions [ 1, 7, 11, 17, 21]. Yet, the pathways to, or from, the septum which mediate the hyperreactivity have received scant attention. This issue was first explored by Brady and Nauta [7] subsequent to their early experiment which demonstrated increased viciousness following septal lesions. On the basis of anatomical findings of strong septal connections with the habenula, they examined the effect of habenular lesions on reactivity. Failing to find any change, they concluded that these connections were not involved in the hyperreactivity produced by septal lesions. Connections between the septum and amygdala were the next to receive consideration. Two kinds of evidence bear directly on this suggestion. First, lesions of the amygdala attenuate the hyperreactivity produced by septal lesions showing that the amygdala is somehow involved in modulating the animal's responsivity [16,28]. Second, damage to the stria terminalis has been reported to be more highly correlated with increased reactivity than the amount of septal damage [33,34]. From this evidence one can argue that stria terminalis connections between the septum and amygdala may mediate the hyperreactivity which follows septal lesions.
METHOD
Animals The animals were male hooded rats weighing 3 0 0 - 4 0 0 g at the beginning of the experiment. They were obtained from the Canadian Breeding Farms and Laboratories (St.
1Supported by grants from the National Research Council of Canada (NRC 67-0192) and the University Research Committee. 2Send reprint requests to: D. J. Albert, Psychology Department, University of British Columbia, 2075 Wesbrook Place, Vancouver, B. C., Canada. 451
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Constant, Quebec). Housing was in group cages prior to surgery and in individual cages thereafter.
Surgical Procedure Our method of making cuts in brain tissue has been described in detail previously [2]. The knife consists of an outer guide cannula (21 ga stainless-steel hypodermic tubing) which is stereotaxically lowered into the brain of an anesthetized animal. With the guide cannula in place, a 26-ga insert with a cutting blade of stainless-steel wire (0.15 mm dia.) is lowered into the cannula. When the cutting blade reaches the lower slit, it extends and cuts through the neural tissue as it is lowered further. The cutting blade is then removed and the guide cannula raised out of the brain. Operated control animals were given sham cuts. These animals were treated in every way like the experimental animals, except that the knife was not lowered into the cannula to make the brain cut.
Behavioral Testing
<0.025). The highest level of reactivity was produced by cuts which were either lateral or anterior to the septum (Fig. 1). The median reactivity scores for the lateral cut group (n = 7) were 7, 6, and 1 for the 3 test days. These scores are each significantly different from the median scores of the control group (n = 6) which were 0.25, 0, and 0 for the corresponding test days (U -- 0, p < 0 . 0 0 2 ; U = 0, p<0.002; U = 4, p<0.02). 25w
8 20>-
Statistics A one-way analysis of variance by ranks (Kruskal-Wallis, [29]) was performed for each test day to determine whether there were overall differences between groups. Individual pair comparisons were then made with a MannWhitney U-test [29]. All p values are two-tailed unless otherwise stated. RESULTS Cuts around the septal area caused an increase in reactivity as measured on each of the test days (Day 2, H = 14.2, p<0.01; Day 7, H = 13.0, p < 0 . 0 1 ; Day 14, H = 9.8,
"
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Animals were tested for reactivity 2, 7, and 14 days postoperatively. They were tested in a box measuring 60 x 60 x 60 cm. Its floor was covered with woodshavings and it was open on top. Lighting was provided by the room's florescent fixtures. On each of the test days the rat's home cage was first covered to prevent escape and then placed in the test box which rested on the floor of the colony room. The cage cover was then removed and the rat left for one minute before testing began. The animal's responses to six test stimuli were rated on a scale of 0 - 3 , 0 indicating little or no response and 3 indicating attack or a highly aggressive response. Each of the stimuli (except item 2, which was essentially a startle test) was repeated 3 times before moving on to the next. The average score for the 3 repetitions was recorded. In order of presentation the test items were: (1) presentation of a pencil just in front of the rat's snout; (2) a sharp tap on the back with a pencil; (3) presentation of a gloved hand before the snout; (4) gentle prods on the side of the rat's body with blunt one-inch alia. stick; (5) attempted capture by the tail; and (6) attempted grasping around the body. In addition, the rat was scored from 0 to 3 on both the amount of vocalization and biting observed during testing, 0 indicating none and 3 indicating vocalization and biting in response to all degrees of stimulation, from the visual presentation of a pencil to grasping during capture. The maximum reactivity score possible was 24. The rating scores presented in this experiment were all obtained by the same experimenter. Previous work has indicated that interrater reliability is extremely high.
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DAY
FIG. 1. The effect of cuts anterior (AS, n = 5), ventrolateral (VLS, n = 7), posterior (PS, n = 6) to the septum and sham cuts (Control, n = 6) on reactivity rated 2, 7, and 14 days postoperatively. The dotted lines indicate the level of reactivity obtained in animals of the same strain with septal lesions (SL) and septal lesions which also damage the region ventral to the anterior septum (SL-AV) [3]. The region severed by the cuts lateral to the septum is shown in Fig. 2. The cuts were all symmetrical about the midline and highly similar. They fell within a region bordered medially by the lateral edge of the septum and laterally about 0.5 mm to the side (see Fig. 2). The cuts began 4 mm anterior to bregma. In 5 of the animals, the cut ended at the posterior end of the septum. In the two remaining animals, the cut ended about 0.7 mm posterior to the septum. All of the cuts severed the entire region between the ventral edge of the lateral ventricle and the base of the brain. The median reactivity scores of the rats with cuts anterior to the septum (n = 5) were 8.5, 6.5, and 1.0 for test days 2, 7, and 14 respectively. These scores are each significantly different from those of the control group on the corresponding test days (U = 0, p<0.004; U = 4.5, p<0.04, one-tailed; and U = 3.5, p<0.04). These scores are almost identical to those found in the animals with cuts lateral to the septum. The tissue severed by these anterior cuts is shown in Fig. 3. The anterior-posterior pattern of the cuts was between 3.8 and 4.2 mm anterior to bregma, so that they cut through the anterior tip of the septum. Laterally, they invaded the medial edge of the caudate nucleus and ventrally they severed connections to the anterior portions of the diagonal band of Broca. The swath of the cuts in each case was very similar to that shown in Fig. 3. The smallest change in reactivity was that produced by
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FIG. 3. The swath of the anterior cuts is shown by the arrows (top) and the stippling (bottom).
FIG. 2. The location of the lateral cuts is shown by the arrows (top) and stippling (bottom). All cuts extended at least from the level of the bottom of the ventricle to the base of the brain. The brain sections are from Pellegrino and Cushman [24]. cuts posterior to the septum. The median reactivity scores for the animals of this group (n = 6) were 0.25, 0.0, and 0.0 for the 3 test days (Fig. 1). None of these scores differed significantly from those of the control group. Each is different from the corresponding test score for the lateral cut (U = 3.5, p<0.01; U = 0, p<0.01; U = 7, p<0.05) and the anterior cut groups (U = 4, p < 0 . 0 5 ; U = 5.5, p<0.05, one-tailed; U = 5.5, p<0.05, one-tailed). The swath of the posterior cut is shown in Fig. 4. All of the cuts fell in the path of the stria terminalis and through the bed nucleus of the stria terminalis. Without exception they were located just anterior to the fornix. They did not interrupt any of the septal connections with the hippocampus, nor should they have disturbed septohabenular fibers since these are mixed with the fornix in the area of the cut [24,25]. Included in Fig. 1 for comparison purposes are the reactivity scores (from a previous experiment) of two groups of animals with different size septal lesions (see
Albert and Richmond [3] for a thorough presentation of this material). These scores, which were obtained at about the same time as the present results, show that both the anterior and lateral cuts cause a level of reactivity very similar to that of lesions restricted to the medial and lateral septum. However, the level of reactivity obtained with the cuts is substantially less than that found with a larger septal lesion which damages most of the medial and lateral septum and in addition parts of the accumbens nucleus and the diagonal band of Broca [ 3 ]. DISCUSSION The pathways involved in septal hyperreactivity course ventrolaterally and anteriorly with respect to the septum. This is shown by the increase in reactivity which follows cuts interrupting pathways with either of these orientations. The level of reactivity produced by each cut is substantially similar to what we have found previously with septal lesions [3 ]. Fiber tracts coursing through the region severed by the lateral cuts are well known. Efferent fibers leave the septum rostral to the anterior commissure and course ventrally and laterally [19, 25, 26]. They join the medial forebrain bundle and then course posteriorly through the lateral hypothalamus, giving off connections all along this route [26]. Another efferent pathway leads ventrally into the
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I FIG. 4. The swath of the posterior cuts is shown by the arrows (top) and stippling (bottom). medial arm of the diagonal band of Broca. From here some fibers appear to course laterally into the olfactory tubercle and then into the medial forebrain bundle [26]. Afferent pathways to the septum have been shown by degeneration studies to follow very similar routes. From their origin all along the medial forebrain bundle, they course around the rostral edge of the anterior commissure and terminate in both the lateral and medial septum [26]. Since lesions of the septum produce an increase in reactivity, it is reasonable to argue that the lesions are releasing the activity of areas which produce an increase in reactiveness when stimulated electrically or chemically. There is evidence that the effect of a septal lesion is mediated in this way. The septum is known to have strong efferent connections to the medial forebrain bundle. An it is well established that increased reactivity in the form of aggressive behavior can be elicited by electrical or chemical stimulation in the medial forebrain bundle region where these septal efferent fibers terminate [ 15, 22, 36]. Further, the forms of aggressiveness obtained include the explosive affective reactivity (attack) which occurs following septal lesions [15, 22, 36]. Alternatively, cuts which interfere with connections coursing laterally could also produce hyperreactivity by interrupting connections between the septum and the ventromedial nucleus of the hypothalamus. Activity from
the septum has been shown by electrophysiological methods to reach the ventromedial nucleus over a multiple synaptic pathway [33]. The pathway apparently courses laterally from the septum, follows the medial forebrain bundle caudally, and then enters the lateral edge of the ventromedial nucleus. Since lesions of the ventromedial hypothalamus are known to produce hyperreactivity [5,35], it may be that the septum normally has a role in modulating reactivity through its connections with the ventromedial hypothalamus. The removal of this modulation by destruction of the septum or by cuts lateral to it may result in increased reactiveness. There is support for this suggestion to the extent that cuts lateral to the medial hypothalamus are known to produce hyperreactivity [4,23]. From the circuitry controlling septal reactivity we have outlined thus far, one might argue that there is a single pathway which travels slightly anteriorly as well as laterally with respect to the septum and which is severed by the anterior as well as the lateral cuts. This would account for the observation that the change in reactivity is similar with both anterior and lateral cuts and that the effects of each of these in turn is similar to that which occurs with septal lesions. However, an inadequacy of this interpretation lies in the fact that while cuts lateral or anterior to the septum produce an increase in reactivity comparable to that of septal lesions, larger lesions which destroy the septum and in addition invade the region anteroventral to it produce a much greater and more long lasting effect (Fig. 1, also [3] ). Since the ventral region is within the region bounded by the anterior and lateral cuts, fiber connections to or from this ventral area are also being severed by these cuts. Hence, the change in reactivity which the cuts must account for may be that produced by the larger lesions which include the ventral area and not just that due to lesions restricted to the septum. Accordingly, it is reasonable to argue that while the lateral cuts are interrupting connections to the medial forebrain bundle, the fibers severed by the anterior cuts may constitute a separate pathway which terminates elsewhere. Cutting both the anterior pathway and the lateral connections to the medial forebrain bundle could cause the severe hyperreactivity shown by animals with lesions of the septum and the area ventral to its anterior region [ 3 ]. Anatomically, there is support for a separate pattern of connections with an anterior and ventral trajectory from the septum. One large efferent tract is known to lead into the diagonal band of Broca [26]. The suggestion that some of these fibers will make anterior connections with other areas involved in controlling reactivity receives some support from the finding of Cain [9], that lesions of the anterior olfactory area produce increased reactivity. Presumably there may be other fibers with similar anterior connections which originate in the region ventral to the anterior septum. Contrary to some previous suggestions [33,34], the present results offer no support for the possibility that fibers in the stria terminalis or other pathways which leave or enter the septum from a posterior-ventral direction are involved in controlling reactivity. In particular, the stria terminalis was consistently interrupted by the posterior cuts without producing an increase in reactivity. The status of fiber connections to the habenula is still uncertain since the posterior cuts did not disturb the fornix. The only evidence available shows that lesions in the region of the
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fornix actually a t t e n u a t e septal lesion-induced hyperreactivity [211. The failure to p r o d u c e an increase in reactivity b y interrupting the stria terminalis is consistent w i t h our previous failure to produce such an effect with lesions of the stria terminalis and its bed nucleus [ 3 ] . A line o f reasoning by which one could object to our present result is that with our m e t h o d of severing the stria terminalis, there may be b o t h an increase in aggressiveness due to cutting the stria terminalis and an a t t e n u a t i o n of this effect due to cutting some o t h e r pathway. We did examine this in three rats by making septal lesions along w i t h the posterior cuts. We found the e x p e c t e d hyperreactivity in each o f the three animals.
The lack o f an increased reactivity w i t h stria terminalis cuts does not detract f r o m the existing evidence showing that the amygdala is i m p o r t a n t in the c o n t r o l of aggression. There is a b u n d a n t evidence to show this from h u m a n clinical data reporting increased aggressiveness w i t h abnormal discharging f r o m the amygdala [18] and animal studies showing an a t t e n u a t i o n of reactivity following amygdala lesions [ 1,1 1]. If the stria terminalis fibers are not the p a t h w a y through which these effects are mediated, the i m m e d i a t e alternative is that the influence of the amygdala on aggression and reactivity is via the ventral amygdalafugal p a t h w a y to the h y p o t h a l a m u s and brainstem [37].
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