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Connections from the lateral septum modulating reactivity in the rat
Physiology & Behavior, Vol. 21, pp. 761-767. Pergamon Press and Brain Research Publ., 1978. Printed in the U.S.A.
Connections from the Lateral Septum Modulating Reactivity in the Rat D. J. A L B E R T ,
K. N. BRAYLEY
A N D J. A . M I L N E R
Psychology Department, University of British Columbia ( R e c e i v e d 27 A p r i l 1978) A L B E R T , D. J., K. N. B R A Y L E Y A N D J. A. M I L N E R . Connections from the lateral septum modulating reactivity in the
rat. PHYSIOL. BEHAV. 21(5) 761-767, 1978.--Rats made hyperreactive by lesions of the medial hypothalamus had an electrode implanted unilaterally into the region ventral to the anterior septum or into the lateral septum. Some of the animals with electrodes in the lateral septum then received a bilateral lesion ventral to the anterior septum while some of the animals with electrodes ventral to the anterior septum received a bilateral lesion in the lateral septum. Following recovery, each animal's level of reactivity was scored 5 rain before, during, and 5 min following electrical stimulation at 20 /~A (60 Hz). The suppression of reactivity by stimulation ventral to the anterior septum was not altered by a lesion of the medial and lateral septum. However, the suppression of reactivity by stimulation of the lateral septum was significantly attenuated by a lesion ventral to the anterior septum. The effectiveness of the lesion ventral to the anterior septum in attenuating the suppression of reactivity by lateral septal stimulation suggests that the neural pathway from the lateral septum which normally modulates reactivity courses anteriorly and ventrally from the septum. Hyperreactivity
Lateral septum
Medial hypothalamus
T H E L A T E R A L septal nucleus and the region ventral to the anterior septum lying between the vertical arm of the diagonal band of Broca and the anterior horn of anterior commissure appear to be similarly involved in the modulation of reactivity and aggression. (See refs. [2, 6, 10] for more information on the boundaries of the critical area in the region ventral to the anterior septum.) Removal of either area by electrolytic lesions increases reactivity to an experimenter [2, 8, 11, 12, 13]. In more recent experiments, hyperreactivity, muricide, and intermale aggression have all been produced using intracranial infusions of a local anesthetic [3, 5, 6]. As further support of the inference that the lateral septum and the region ventral to its anterior portion both modulate reactivity and aggression it has been demonstrated that stimulation of either area will suppress the hyperreactivity induced by medial hypothalamic lesions [9,10]. The object of the present experiment was to use the demonstrated effectiveness of stimulation in either the lateral septum or the region ventral to the anterior septum in suppressing hyperreactivity to evaluate the possibility that a functional relationship exists between these areas. This was done by examining whether a lesion ventral to the anterior septum would block the suppression of hyperreactivity caused by stimulation of the lateral septum, and conversely, whether a lesion of the lateral septum would block the suppression of reactivity produced by stimulation ventral to the anterior septum. We were expecting that if the stimulation from one of the anterior forebrain areas suppressed the hypothalamic lesion-induced hyperreactivity by its action upon or by fibers passing through the other, a lesion in this
second area should prevent the suppression of the hyperreactivity by the stimulation. METHOD
Animals The data were obtained from 40 naive male hooded rats (Canadian Breeding Farms and Laboratories, Quebec, Canada) weighing 250--350 g at the start of the experiment. They were housed in individual cages following surgery.
Surgery While they were anesthetized with sodium pentobarbital, all animals sustained electrolytic lesions (stainless-steel anode) bilaterally in the medial hypothalamus. The animals were then divided into groups (Fig. 1). Eight animals had only an additional electrode implanted into the lateral septum; another group of 8 animals had only a single electrode implanted ventral to the anterior septum. Seven animals had a second lesion ventral to the anterior septum and an electrode in the lateral septum. Nine animals had the opposite, a second lesion in the lateral septum and a stimulating electrode ventral to the anterior septum. Following surgery, each animal was injected with penicillin (60,000 I.U.) intramuscularly and returned to an individual cage for a recovery period of 7 days.
Behavioral Testing The effect of stimulating the region ventral to the anterior
'Supported by a grant from the National R e s e r a c h Council o f Canada.
C o p y r i g h t © 1978 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/78/110761-07502.00/0
762
ALBERT, BRAYLEY AND MILNER
Lesion 1
Lesion 2
medial hypothalamus
Electrode
ventral to anterior
Site
Group
lateral
septum
LAV-SLS
lateral
septum
SLS
septum
lateral septum
v e n t r a l to a n t e r i o r septnm
LSL-SAV
ventr~l t o anterior septum
SAV
inoperative electrode '
USC
FIG. 1. The physiological manipulations produced in each group.
septum or the lateral septum on reactivity and aggressiveness was tested over a 3-day period, usually on Days 7, 8, and 9 postoperatively. Because of their hyperreactivity, each rat was given a very light dose of ether to permit attachment of the electrode lead. The animal was then placed in a gray box (60x60x60 cm) with an open top and wood shavings on the floor. Testing began 10 min later. An initial test of reactivity to a standard series of stimuli was used to obtain a behavioral baseline against which to compare the effects of the stimulation. To obtain the baseline rating the animal was given a rating of 0 to 3 (0 indicating little or no response, and 3 indicating a highly aggressive or reactive response) to each of a series o f stimuli (a tap on the back, a poke in the side, grasping by the tail, grasping by the abdomen, and others, for details see [1, 2, 3, 4, 5, 6, 9, 10]). If the baseline behavioral rating was 5 or greater out of a maximum of 24, the animal was retained in the experiment. Animals with lower scores (seven in this experiment) were discarded only because their lower scores limited the quantitative suppression that could be obtained with the stimulation. The rating scores for all animals were obtained by one experimenter but previous experiments have indicated that interrater reliability is very high [2]. Five min following the first behavioral test, stimulation at 60 Hz (sine wave), 20/xA(RMS), was begun and continued for about 30 sec while the animal's level of reactivity was reassessed. The stimulation was then turned off. Five min later the reactivity of the animal was assessed again, this time with the stimulation off. The 20 g A stimulation level was used because previous experiments have suggested that this is the lowest stimulation level which will produce a suppression in most animals on the first test [9,10]. The electrical activity at the stimulation site was recorded in all animals immediately before and following each stimulation (Grass, model 78D, 7P 511G amplifier).
Histology Following completion of testing, the brains of all animals were removed, placed in formol-saline, sectioned at 40/~ on a cryostat and stained with thionin.
Analysis of Results The group data from stimulating these animals was subjected to an overall analysis of variance for unequal cell frequencies [27]. Comparisons between conditions within groups were made with t-tests (all p values are two-tailed). RESULTS The overall analysis indicated that stimulation suppressed reactivity in some group s, F(8,70) = 13.2, p < 0.0001. Analysis of the results for the unstimulated control group (Table 1) showed that the repeated testing procedure itself did not give rise to any systematic variation in reactivity either within or between days.
Stimulation of the Lateral Septum with Lesions Ventral to the Anterior Septum In animals without lesions ventral to the anterior septum (N=8) stimulation of the lateral septum suppressed the hyperreactivity induced by medial hypothalamic lesions on all three test days (Table 1, Figs. 2 and 3). In addition, the strength of the stimulation effect increased over days, F(4,140)=6.7, p<0.0001; p<0.05 for the change in this group. In animals with a lesion ventral to the anterior septum (N=7) the suppression of hyperreactivity by the septal stimulation was greatly attenuated ('Fable 1, Figs. 2 and 13). F o r the first 2 test days, the reactivity ratings prior to, during, and following stimulation did not differ statistically. On the third test day the rating taken during stimulation was 7.7, which is reliably lower than the rating of 11.5 taken before stimulation 60<0.05) and the rating of 11.4 taken after stimulation (p<0.05). These results suggest that the suppression of reactivity by the lateral septal stimulation is attenuated by the lesion ventral to the anterior septum. However, examination of the individual scores reveals that of the 7 animals in the group, only 3 clearly showed a suppression of reactivity of the third test day, I animal showed a slight tendency toward suppression, and 3 animals showed no suppression (i.e., a change of 2 points or less between the stimulation rating and the
TABLE 1 MEAN REACTIVITYLEVELS FOR ANIMALSTESTED PRIOR TO (PRE), DURING(STIM), AND FOLLOWINGELECTRICALSTIMULATIONIN THE LATERALSEPTUM (SLS) OR IN THE REGION VENTRAL TO THE ANTERIOR SEPTUM (SAV).ONE GROUPOF STIMULATEDANIMALS HAD A LATERALSEPTAL LESION (LSL) AND ANOTHER GROUP HAD A LESION VENTRAL TO THE ANTERIORSEPTUM (LAV). USC IS AN UNSTIMULATED CONTROL GROUP Group
Day 1 Day 2 Day 3
pre.
SAV stim.
post.
pre.
10.9 11.6 11.8
7.3* 6.2* 5.0*
11.3 12.1 12.1
10.5 11.1 11.0
LSL-SAV stim. post. 6.1" 4.8* 3.7*
12.7 12.4 10.5
pre.
SLS stim.
post.
pre.
11.2 12.9 il.4
7.4* 5.4* 4.2*
12.8 14.3 13.2
10.7 11.3 11.5
Significantly different from pre- and post-stimulation reactivity level, p<0.01* p<0.05t
LAV-SLS stim. post. 10.2 9.9 7.7t
12.4 11.4 11.6
pre.
USC stim.
post.
10.9 10.5 10.1
11.3 11.3 10.2
12.1 11.6 10.4
LATERAL SEPTUM AND REACTIVITY
763 The mean reactivity rating obtained during stimulation was 10, and this was similar to the prestimulation rating of 11 and the poststimulation rating of 10. Thus, in 3 of the 7 animals the lesion ventral to the anterior septum completely blocked the suppression of reactivity produced by stimulation of the lateral septum. Examination of brain sections suggests that differences in the size of the lesions ventral to the anterior septum can account for most of the variation in the suppression of reactivity by the lateral septal stimulation. In 2 of the 3 animals in which lateral septal stimulation suppressed reactivity on the third test day, the lesions destroyed only part of the region between the rostral arm of the anterior commissure and the vertical arm of the diagonal band of Broca (Fig. 4). In contrast, the 3 animals showing no suppression of reactivity during the stimulation on all three days had lesions which destroyed a considerably large part of the region between the vertical arm of the anterior commissure and the vertical arm of the diagonal band of Broca (see Fig. 4).
15
(.9
z
-,
In,"
~
./..-j,- USC //
I0
Z <[ UJ '5
Stimulation Ventral to the Anterior Septum with Lesions of the Lateral Septum m
PRESTIMOLATION
STIMUI' A T I O N
P O S T ST'IMULATION
15
Z
~_.~=,~
I--
USC~,
o~.~-/-
I0 1-
~.~ ,,< uJ n," Z tzJ :E
In animals without lesions of the lateral septum, stimulation ventral to the anterior septum (N=8) on each test day suppressed the hyperreactivity caused by medial hypothalamic lesions (Table 1, Figs. 2 and 3). As with the stimulation of the lateral septum, the 20/xA stimulation ventral to the anterior septum became more effective in suppressing reactivity over the 3 test days (p <0.05). With lesions of the medial and lateral septum there was no change in the suppression of reactivity (N=9) caused by stimulation ventral to the anterior septum (Table 1, Figs. 2 and 3). The 20/zA stimulation again became more effective in suppressing reactivity over the three test days (.o<0.05). Examination of the septal lesions (Fig. 5) showed that in 5 of the 9 animals, the lesion included virtually the entire lateral septum and part of the medial septum. The remaining 4 animals had more than one-half of the lateral septum destroyed, the anterior portion being intact. In 3 animals the fornix was destroyed. The variation in lesion size was not related to the effectiveness of the stimulation in suppressing reactivity.
Electrical Recordings from Stimulation Site
PRESTIMULATION
STIMULATION
POSTST'IMULATION
FIG. 2. Mean reactivity ratings over 3 test days. Top. SAV: animals stimulated ventral to the anterior septum; LSL-SAV: animals with lesions in the lateral septum receiving stimulation ventral to the anterior septum, USC: unstimulated control animals. Botton. SLS: animals stimulated in lateral septum; LAV-SLS: animals with a lesion ventral to the anterior septum receiving stimulation in the lateral septum, USC: unstimulated control animals.
prestimulation and poststimulation ratings). To evaluate the possibility that the 20 p.A stimulating current was simply below threshold for suppressing reactivity in the 3 animals showing no suppression, the stimulating current was raised by 50% to 30/zA and the 3 animals tested on a fourth day.
Although none of the animals whose data have been cited showed electrographic indication of afterdischarge following electrical stimulation, data from 5 animals with lateral septal stimulating electrodes, 2 animals with lateral septal stimulating electrodes and a lesion ventral to the anterior septum, and 2 animals with stimulating electrodes ventral to the anterior septum were excluded for showing poststimulation afterdischarge. Examples of these electrophysiological abnormalities have been presented previously [9,10].
Medial Hypothalamic Lesions The lesions were similar across groups and entirely comparable to those produced in previous studies examining the effect of septal forebrain stimulation on medial hypothalamic lesion-induced hyperreactivity [9,10]. In most animals, the ventromedial nucleus was totally destroyed along with part or all of the arcuate and dorsomedial nuclei. The lesions did not extend lateral to the fornix or anterior to the descending columns of the fornix.
764
ALBERT, BRAYLEY AND MILNER
H
",~'k
/
sI
/
I I
l
I J
I~ )~
/ x,...f~
",L
'Tt..~pO-
-
//
j ----
_
AA*
Pill
i
//
7
FIG. 3. The location of the stimulating electrodes ventral to the anterior septum (top) and in the lateral septum (bottom). Filled circles are stimulation sites from animals with only an electrode in either the lateral septum or the region ventral to the anterior septum. Squares are stimulation sites in animals with a stimulating electrode in one of these areas and a lesion in the other. Brain sections are from Pellegrino and Cushman [9].
L A T E R A L SEPTUM AND REACTIVITY
•
765
, AI
,el
ts~
~
/
FIG. 4. Example of a small lesion (area enclosed with solid line) which blocked the stimulation-induced suppression of reactivity at the first 2 test sessions and attenuated it at the third. Also shown is a large lesion (stippled area) which abolished the suppression of hyperreactivity produced by lateral septal stimulation on all 3 test days at 20 pA and on a fourth day with 30/~A stimulation. DISCUSSION The neural pathway by which stimulation of the lateral septum suppresses the hyperreactivity induced by medial hypothalamic lesions appears to connect with, or pass through, the region ventral to the anterior septum. This is suggested by the observation that lesions ventral to the anterior septum attenuate the suppression of hyperreactivity caused by lateral septal stimulation (Fig. 1). Further, the effectiveness of the lesions in producing this effect appears to be related to the extent to which they destroy the region between the vertical arm of the diagonal band of Broca and the anterior horn of the anterior commissure. This region has previously been shown to be the most important part of the tissue ventral to the anterior septum controlling reactivity and aggression [2, 3, 5, 6, 10]. In contrast, septal lesions did not attenuate the effectiveness of stimulation ventral to the anterior septum in suppressing hyperreactivity. Apparently the lateral septum and the region ventral to the anterior septurn do not influence reactivity by pathways which course caudally into the hippocampus, the columns of the fornix, or the bed nucleus of the stria terminalis.
'
-_.-
FIG. 5. An example of a large lesion in the septum which did not alter the suppression of reactivity and aggression by stimulation ventral to the anterior septum.
In the groups which did not have a lesion in the septal forebrain area, the suppression of reactivity obtained with stimulation ventral to the anterior septum or in the lateral septum was similar to what we have observed previously [9,10]. Presumably, the suppression of reactivity caused by the stimulation was partial primarily because the stimulation was set at a level known to be close to the threshold for producing a suppression on the first trial (see ref. [3] for the reactivity level to be expected from unoperated control animals). As in the earlier experiments electrographic rec-
766
ALBERT, BRAYLEY AND MILNER
ordings immediately following the stimulation allowed us to exclude animals showing afterdischarge. Previous experiments have shown that the suppression of reactivity caused by stimulation in the septal forebrain area is not due to rewarding effects of the stimulation [9,10]. Further, it has been shown that the suppression of reactivity occurs only with stimulation in certain critical areas [1, 9, 10] and that the stimulation does not suppress other behaviors such as feeding and drinking [9,10]. The effectiveness Of the lesion ventral to the anterior septum in blocking the suppression of reactivity caused by lateral septal stimulation adds a measure of support to the suggestion that the outflow from the lateral septum modulating reactivity courses anteriorly and ventrally [3, 4, 5]. Fibers from the lateral septum are known to course ventrally and laterally from the anterior portion of the septum. Some of these fibers are in the vertical arm of the diagonal band of Broca while others lie in the medial accumbens nucleus [20,22]. From the region ventral to the anterior septum they are thought to course posteriorly in the medial forebrain bundle, giving off connections all along its course. Behavioral experiments have produced results which are generally congruent with these anatomical considerations. Tissue cuts ventrolateral to the septum produce hyperreactivity, as would be predicted if fibers from the lateral septum project anteroventrally and then laterally from the lateral septum [4]. Further, the subsequent passage of the fibers along the trajectory of the medial forebrain bundle leaves them conveniently positioned to interact with neurons in the lateral hypothalamus. Substantial evidence from experiments using electrical stimulation indicates that regions of the lateral hypothalamus are involved in initiating aggression and heightening reactivity [17, 26, 28].
Alternatively, it can be argued that the lesion ventral to the anterior septum produces an increase in reactivity which cannot be decreased by septal stimulation rather than blocking the suppression of medial hypothalamic lesion induced hyperreactivity. We have in fact found that stimulation of the lateral septum will not block the reactivity produced by lesions ventral to the anterior septum (unpublished results). However, the experiment is difficult to do because the level of reactivity produced by lesions ventral to the anterior septurn drops off very rapidly with the repeated testing required by the experimental design [2]. Because the reactivity caused by the lesion ventral to the anterior septum does drop off with repeated testing, it seems reasonable to argue as we have that the lesion ventral to the anterior septum in the present experiment is attenuating the effectiveness of lateral septal stimulation in blocking the hyperreactivity induced by the medial hypothalamic lesion. The observation that lesions of the septum do not block the suppression of reactivity by electrical stimulation ventral to the anterior septum is in agreement with other evidence suggesting that fibers coursing caudally through the lateral septum are not the fibers by which the lateral septum influences reactivity and aggression. The existence of such fiber tracts projecting into the hippocampus, columns of the fornix, the region of the stria terminalis and the stria medullaris are known [14, 21, 22]. However, in agreement with the present findings, lesions and/or cuts of the fornixhippocampal commissure area [16] the bed nucleus of the stria terminalis ([2,18], however, see also ref. [251) and the region posteroventral to the septum [6,18] have not been found to produce the strong hyperreactivity that would be expected if they were interrupting the fibers from the septum which influence the modulation of this behavior.
REFERENCES
1. Albert, D. J., K. N. Brayley and J. Milner. Medial hypothalamic electrical stimulation is ineffective in suppressing septal lesion induced hyperreactivity. Physiol. Behav. 21: 135-139, 1978. 2. 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. 3. Albert, D. J. and S. E. Richmond. Hyperreactivity and aggression following infusion of local anesthetic into the lateral septum or adjacent areas. Behav. Biol. 18: 211-226, 1976. 4. Albert, D. J. and S. E. Richmond. Neural pathways mediating septal hyperreactivity. Physiol. Behav. 17: 451-455, 1976. 5. Albert, D. J. and S. E. Richmond. Reactivity and aggression in the rat: Induction by a-adrenergic blocking agents injected ventral to the anterior septum but not into the lateral septum. J. comp. physiol. Psychol. 91: 886--896, 1977. 6. Albert, D. J. and R. C. K. Wong. Irritability, muricide, and intraspecific aggression in the rat produced by infusion of local anesthetic into the lateral septum and surrounding areas. J. comp. physiol. Psychol., 1978, in press. 7. Atrens, D. M., F. von Vietinghoff-Riesch, A. Der-Karabetian and E. Masliyah. Modulation of reward and aversion processes in the rat diencephalon by amphetamine. Am. J. Physiol. 226: 874-880, 1974. 8. Brady, J. V. and W. J. 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. 9. Brayley, K. N. and D. J. Albert. Suppression of VMH-lesion induced reactivity and aggressiveness by stimulation of the lateral septum but not medial septum or cingulate cortex in the rat. J. comp. physiol. Psychol. 91: 290-299, 1977.
10. Brayley, K. N. and D. J. Albert. Suppression of VMH lesion induced reactivity and aggressiveness by electrical stimulation ventral to the anterior septum in the rat. Physiol. Psychol. 18: 567-571, 1977. 11. Gottsick, J. E. and R. C. Marshall. Time course of the septal rage syndrome. Physiol. Behav. 9: 685-687, 1972. 12. King, F. A. Effects of septal and amygdaloid lesions on emotional behavior and CAR in the rat. J. nerv. ment. Dis. 126: 57-63, 1958. 13. Kreickhaus, E. E., H. J. Simmons, H. G. Thomas and K. Kenyon. Septal lesions enhance shock avoidance behavior in the rat. Expl. Neurol. 9: 107-113, 1964. 14. Miczek, K. A. and S. P. Grossman. Effects of septal lesions on inter- and intraspecies aggression in rats. J. comp. physiol. Psychol. 79: 37-45, 1972. 15. Nauta, W. J. H. An experimental study of the fornix system in the rat. J. Comp. Neurol. 104: 247-279, 1956. 16. Olton, D. S. and F. H. Gage. Role of the fornix in the septal syndrome. Physiol. Behav. 13: 269-279, 1974. 17. Panksepp, J. Aggression elicited by stimulation of the hypothalamus in albino rats. Physiol. Behav. 6: 321-329, 1971. 18. Paxinos, G. Interruption of septal connections: Effects on drinking, irritability, and copulation. Physiol. Behav. 17: 81-88, 1976. 19. Pellegrino, L. J. and A. J. Cushman. A Stereotaxic Atlas o f the Rat Brain. New York: Appleton-Century-Crofts, 1967. 20. Poweil, E. W. Septal efferents revealed by axonal degeneration in the rat. Expl. Neurol. 8: 406--422, 1963. 21. Powell, E. W. Septal efferents in the cat. Expl. Neurol. 14: 328-337, 1966. 22. Raisman, G. The connexions of the septum. Brain 89:317-348, 1966.
LATERAL SEPTUM AND REACTIVITY 23. Siegal, A. and J. P. Flynn. Differential effects of electrical stimulation and lesions of the hippocampus and adjacent regions upon attack behavior in cats. Brain Res. 7: 252-267, 1968. 24. Siegal, A., H. Edinger and S. Ohgami. The topographical organization of the hippocampus projections to the septal area: A comparative neuroanatomical analysis in the gerbil, rat, rabbit, and cat. J. comp. Neurol. 157: 359-378, 1974. 25. Turner, B. H. Neural structures involved in the rage syndrome of the rat. J. comp. physiol. Psychol. 71: 103-113, 1970.
767
26. Wasman, M. and J. P. Fiynn. Directed attack elicited from hypothalamus. Arch. Neurol., Chicago 6: 220-227, 1962. 27. Winer, B. J. Statistical Principles in Experimental Design. New York: McGraw-Hill, 1962. 28. Woodworth, C. H. Attack elicited in rats by electrical stimulation of the lateral hypothalamus. Physiol. Behav. 6: 345-353, 1971.
Connections from the Lateral Septum Modulating Reactivity in the Rat D. J. A L B E R T ,
K. N. BRAYLEY
A N D J. A . M I L N E R
Psychology Department, University of British Columbia ( R e c e i v e d 27 A p r i l 1978) A L B E R T , D. J., K. N. B R A Y L E Y A N D J. A. M I L N E R . Connections from the lateral septum modulating reactivity in the
rat. PHYSIOL. BEHAV. 21(5) 761-767, 1978.--Rats made hyperreactive by lesions of the medial hypothalamus had an electrode implanted unilaterally into the region ventral to the anterior septum or into the lateral septum. Some of the animals with electrodes in the lateral septum then received a bilateral lesion ventral to the anterior septum while some of the animals with electrodes ventral to the anterior septum received a bilateral lesion in the lateral septum. Following recovery, each animal's level of reactivity was scored 5 rain before, during, and 5 min following electrical stimulation at 20 /~A (60 Hz). The suppression of reactivity by stimulation ventral to the anterior septum was not altered by a lesion of the medial and lateral septum. However, the suppression of reactivity by stimulation of the lateral septum was significantly attenuated by a lesion ventral to the anterior septum. The effectiveness of the lesion ventral to the anterior septum in attenuating the suppression of reactivity by lateral septal stimulation suggests that the neural pathway from the lateral septum which normally modulates reactivity courses anteriorly and ventrally from the septum. Hyperreactivity
Lateral septum
Medial hypothalamus
T H E L A T E R A L septal nucleus and the region ventral to the anterior septum lying between the vertical arm of the diagonal band of Broca and the anterior horn of anterior commissure appear to be similarly involved in the modulation of reactivity and aggression. (See refs. [2, 6, 10] for more information on the boundaries of the critical area in the region ventral to the anterior septum.) Removal of either area by electrolytic lesions increases reactivity to an experimenter [2, 8, 11, 12, 13]. In more recent experiments, hyperreactivity, muricide, and intermale aggression have all been produced using intracranial infusions of a local anesthetic [3, 5, 6]. As further support of the inference that the lateral septum and the region ventral to its anterior portion both modulate reactivity and aggression it has been demonstrated that stimulation of either area will suppress the hyperreactivity induced by medial hypothalamic lesions [9,10]. The object of the present experiment was to use the demonstrated effectiveness of stimulation in either the lateral septum or the region ventral to the anterior septum in suppressing hyperreactivity to evaluate the possibility that a functional relationship exists between these areas. This was done by examining whether a lesion ventral to the anterior septum would block the suppression of hyperreactivity caused by stimulation of the lateral septum, and conversely, whether a lesion of the lateral septum would block the suppression of reactivity produced by stimulation ventral to the anterior septum. We were expecting that if the stimulation from one of the anterior forebrain areas suppressed the hypothalamic lesion-induced hyperreactivity by its action upon or by fibers passing through the other, a lesion in this
second area should prevent the suppression of the hyperreactivity by the stimulation. METHOD
Animals The data were obtained from 40 naive male hooded rats (Canadian Breeding Farms and Laboratories, Quebec, Canada) weighing 250--350 g at the start of the experiment. They were housed in individual cages following surgery.
Surgery While they were anesthetized with sodium pentobarbital, all animals sustained electrolytic lesions (stainless-steel anode) bilaterally in the medial hypothalamus. The animals were then divided into groups (Fig. 1). Eight animals had only an additional electrode implanted into the lateral septum; another group of 8 animals had only a single electrode implanted ventral to the anterior septum. Seven animals had a second lesion ventral to the anterior septum and an electrode in the lateral septum. Nine animals had the opposite, a second lesion in the lateral septum and a stimulating electrode ventral to the anterior septum. Following surgery, each animal was injected with penicillin (60,000 I.U.) intramuscularly and returned to an individual cage for a recovery period of 7 days.
Behavioral Testing The effect of stimulating the region ventral to the anterior
'Supported by a grant from the National R e s e r a c h Council o f Canada.
C o p y r i g h t © 1978 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/78/110761-07502.00/0
762
ALBERT, BRAYLEY AND MILNER
Lesion 1
Lesion 2
medial hypothalamus
Electrode
ventral to anterior
Site
Group
lateral
septum
LAV-SLS
lateral
septum
SLS
septum
lateral septum
v e n t r a l to a n t e r i o r septnm
LSL-SAV
ventr~l t o anterior septum
SAV
inoperative electrode '
USC
FIG. 1. The physiological manipulations produced in each group.
septum or the lateral septum on reactivity and aggressiveness was tested over a 3-day period, usually on Days 7, 8, and 9 postoperatively. Because of their hyperreactivity, each rat was given a very light dose of ether to permit attachment of the electrode lead. The animal was then placed in a gray box (60x60x60 cm) with an open top and wood shavings on the floor. Testing began 10 min later. An initial test of reactivity to a standard series of stimuli was used to obtain a behavioral baseline against which to compare the effects of the stimulation. To obtain the baseline rating the animal was given a rating of 0 to 3 (0 indicating little or no response, and 3 indicating a highly aggressive or reactive response) to each of a series o f stimuli (a tap on the back, a poke in the side, grasping by the tail, grasping by the abdomen, and others, for details see [1, 2, 3, 4, 5, 6, 9, 10]). If the baseline behavioral rating was 5 or greater out of a maximum of 24, the animal was retained in the experiment. Animals with lower scores (seven in this experiment) were discarded only because their lower scores limited the quantitative suppression that could be obtained with the stimulation. The rating scores for all animals were obtained by one experimenter but previous experiments have indicated that interrater reliability is very high [2]. Five min following the first behavioral test, stimulation at 60 Hz (sine wave), 20/xA(RMS), was begun and continued for about 30 sec while the animal's level of reactivity was reassessed. The stimulation was then turned off. Five min later the reactivity of the animal was assessed again, this time with the stimulation off. The 20 g A stimulation level was used because previous experiments have suggested that this is the lowest stimulation level which will produce a suppression in most animals on the first test [9,10]. The electrical activity at the stimulation site was recorded in all animals immediately before and following each stimulation (Grass, model 78D, 7P 511G amplifier).
Histology Following completion of testing, the brains of all animals were removed, placed in formol-saline, sectioned at 40/~ on a cryostat and stained with thionin.
Analysis of Results The group data from stimulating these animals was subjected to an overall analysis of variance for unequal cell frequencies [27]. Comparisons between conditions within groups were made with t-tests (all p values are two-tailed). RESULTS The overall analysis indicated that stimulation suppressed reactivity in some group s, F(8,70) = 13.2, p < 0.0001. Analysis of the results for the unstimulated control group (Table 1) showed that the repeated testing procedure itself did not give rise to any systematic variation in reactivity either within or between days.
Stimulation of the Lateral Septum with Lesions Ventral to the Anterior Septum In animals without lesions ventral to the anterior septum (N=8) stimulation of the lateral septum suppressed the hyperreactivity induced by medial hypothalamic lesions on all three test days (Table 1, Figs. 2 and 3). In addition, the strength of the stimulation effect increased over days, F(4,140)=6.7, p<0.0001; p<0.05 for the change in this group. In animals with a lesion ventral to the anterior septum (N=7) the suppression of hyperreactivity by the septal stimulation was greatly attenuated ('Fable 1, Figs. 2 and 13). F o r the first 2 test days, the reactivity ratings prior to, during, and following stimulation did not differ statistically. On the third test day the rating taken during stimulation was 7.7, which is reliably lower than the rating of 11.5 taken before stimulation 60<0.05) and the rating of 11.4 taken after stimulation (p<0.05). These results suggest that the suppression of reactivity by the lateral septal stimulation is attenuated by the lesion ventral to the anterior septum. However, examination of the individual scores reveals that of the 7 animals in the group, only 3 clearly showed a suppression of reactivity of the third test day, I animal showed a slight tendency toward suppression, and 3 animals showed no suppression (i.e., a change of 2 points or less between the stimulation rating and the
TABLE 1 MEAN REACTIVITYLEVELS FOR ANIMALSTESTED PRIOR TO (PRE), DURING(STIM), AND FOLLOWINGELECTRICALSTIMULATIONIN THE LATERALSEPTUM (SLS) OR IN THE REGION VENTRAL TO THE ANTERIOR SEPTUM (SAV).ONE GROUPOF STIMULATEDANIMALS HAD A LATERALSEPTAL LESION (LSL) AND ANOTHER GROUP HAD A LESION VENTRAL TO THE ANTERIORSEPTUM (LAV). USC IS AN UNSTIMULATED CONTROL GROUP Group
Day 1 Day 2 Day 3
pre.
SAV stim.
post.
pre.
10.9 11.6 11.8
7.3* 6.2* 5.0*
11.3 12.1 12.1
10.5 11.1 11.0
LSL-SAV stim. post. 6.1" 4.8* 3.7*
12.7 12.4 10.5
pre.
SLS stim.
post.
pre.
11.2 12.9 il.4
7.4* 5.4* 4.2*
12.8 14.3 13.2
10.7 11.3 11.5
Significantly different from pre- and post-stimulation reactivity level, p<0.01* p<0.05t
LAV-SLS stim. post. 10.2 9.9 7.7t
12.4 11.4 11.6
pre.
USC stim.
post.
10.9 10.5 10.1
11.3 11.3 10.2
12.1 11.6 10.4
LATERAL SEPTUM AND REACTIVITY
763 The mean reactivity rating obtained during stimulation was 10, and this was similar to the prestimulation rating of 11 and the poststimulation rating of 10. Thus, in 3 of the 7 animals the lesion ventral to the anterior septum completely blocked the suppression of reactivity produced by stimulation of the lateral septum. Examination of brain sections suggests that differences in the size of the lesions ventral to the anterior septum can account for most of the variation in the suppression of reactivity by the lateral septal stimulation. In 2 of the 3 animals in which lateral septal stimulation suppressed reactivity on the third test day, the lesions destroyed only part of the region between the rostral arm of the anterior commissure and the vertical arm of the diagonal band of Broca (Fig. 4). In contrast, the 3 animals showing no suppression of reactivity during the stimulation on all three days had lesions which destroyed a considerably large part of the region between the vertical arm of the anterior commissure and the vertical arm of the diagonal band of Broca (see Fig. 4).
15
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Stimulation Ventral to the Anterior Septum with Lesions of the Lateral Septum m
PRESTIMOLATION
STIMUI' A T I O N
P O S T ST'IMULATION
15
Z
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USC~,
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In animals without lesions of the lateral septum, stimulation ventral to the anterior septum (N=8) on each test day suppressed the hyperreactivity caused by medial hypothalamic lesions (Table 1, Figs. 2 and 3). As with the stimulation of the lateral septum, the 20/xA stimulation ventral to the anterior septum became more effective in suppressing reactivity over the 3 test days (p <0.05). With lesions of the medial and lateral septum there was no change in the suppression of reactivity (N=9) caused by stimulation ventral to the anterior septum (Table 1, Figs. 2 and 3). The 20/zA stimulation again became more effective in suppressing reactivity over the three test days (.o<0.05). Examination of the septal lesions (Fig. 5) showed that in 5 of the 9 animals, the lesion included virtually the entire lateral septum and part of the medial septum. The remaining 4 animals had more than one-half of the lateral septum destroyed, the anterior portion being intact. In 3 animals the fornix was destroyed. The variation in lesion size was not related to the effectiveness of the stimulation in suppressing reactivity.
Electrical Recordings from Stimulation Site
PRESTIMULATION
STIMULATION
POSTST'IMULATION
FIG. 2. Mean reactivity ratings over 3 test days. Top. SAV: animals stimulated ventral to the anterior septum; LSL-SAV: animals with lesions in the lateral septum receiving stimulation ventral to the anterior septum, USC: unstimulated control animals. Botton. SLS: animals stimulated in lateral septum; LAV-SLS: animals with a lesion ventral to the anterior septum receiving stimulation in the lateral septum, USC: unstimulated control animals.
prestimulation and poststimulation ratings). To evaluate the possibility that the 20 p.A stimulating current was simply below threshold for suppressing reactivity in the 3 animals showing no suppression, the stimulating current was raised by 50% to 30/zA and the 3 animals tested on a fourth day.
Although none of the animals whose data have been cited showed electrographic indication of afterdischarge following electrical stimulation, data from 5 animals with lateral septal stimulating electrodes, 2 animals with lateral septal stimulating electrodes and a lesion ventral to the anterior septum, and 2 animals with stimulating electrodes ventral to the anterior septum were excluded for showing poststimulation afterdischarge. Examples of these electrophysiological abnormalities have been presented previously [9,10].
Medial Hypothalamic Lesions The lesions were similar across groups and entirely comparable to those produced in previous studies examining the effect of septal forebrain stimulation on medial hypothalamic lesion-induced hyperreactivity [9,10]. In most animals, the ventromedial nucleus was totally destroyed along with part or all of the arcuate and dorsomedial nuclei. The lesions did not extend lateral to the fornix or anterior to the descending columns of the fornix.
764
ALBERT, BRAYLEY AND MILNER
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Pill
i
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7
FIG. 3. The location of the stimulating electrodes ventral to the anterior septum (top) and in the lateral septum (bottom). Filled circles are stimulation sites from animals with only an electrode in either the lateral septum or the region ventral to the anterior septum. Squares are stimulation sites in animals with a stimulating electrode in one of these areas and a lesion in the other. Brain sections are from Pellegrino and Cushman [9].
L A T E R A L SEPTUM AND REACTIVITY
•
765
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FIG. 4. Example of a small lesion (area enclosed with solid line) which blocked the stimulation-induced suppression of reactivity at the first 2 test sessions and attenuated it at the third. Also shown is a large lesion (stippled area) which abolished the suppression of hyperreactivity produced by lateral septal stimulation on all 3 test days at 20 pA and on a fourth day with 30/~A stimulation. DISCUSSION The neural pathway by which stimulation of the lateral septum suppresses the hyperreactivity induced by medial hypothalamic lesions appears to connect with, or pass through, the region ventral to the anterior septum. This is suggested by the observation that lesions ventral to the anterior septum attenuate the suppression of hyperreactivity caused by lateral septal stimulation (Fig. 1). Further, the effectiveness of the lesions in producing this effect appears to be related to the extent to which they destroy the region between the vertical arm of the diagonal band of Broca and the anterior horn of the anterior commissure. This region has previously been shown to be the most important part of the tissue ventral to the anterior septum controlling reactivity and aggression [2, 3, 5, 6, 10]. In contrast, septal lesions did not attenuate the effectiveness of stimulation ventral to the anterior septum in suppressing hyperreactivity. Apparently the lateral septum and the region ventral to the anterior septurn do not influence reactivity by pathways which course caudally into the hippocampus, the columns of the fornix, or the bed nucleus of the stria terminalis.
'
-_.-
FIG. 5. An example of a large lesion in the septum which did not alter the suppression of reactivity and aggression by stimulation ventral to the anterior septum.
In the groups which did not have a lesion in the septal forebrain area, the suppression of reactivity obtained with stimulation ventral to the anterior septum or in the lateral septum was similar to what we have observed previously [9,10]. Presumably, the suppression of reactivity caused by the stimulation was partial primarily because the stimulation was set at a level known to be close to the threshold for producing a suppression on the first trial (see ref. [3] for the reactivity level to be expected from unoperated control animals). As in the earlier experiments electrographic rec-
766
ALBERT, BRAYLEY AND MILNER
ordings immediately following the stimulation allowed us to exclude animals showing afterdischarge. Previous experiments have shown that the suppression of reactivity caused by stimulation in the septal forebrain area is not due to rewarding effects of the stimulation [9,10]. Further, it has been shown that the suppression of reactivity occurs only with stimulation in certain critical areas [1, 9, 10] and that the stimulation does not suppress other behaviors such as feeding and drinking [9,10]. The effectiveness Of the lesion ventral to the anterior septum in blocking the suppression of reactivity caused by lateral septal stimulation adds a measure of support to the suggestion that the outflow from the lateral septum modulating reactivity courses anteriorly and ventrally [3, 4, 5]. Fibers from the lateral septum are known to course ventrally and laterally from the anterior portion of the septum. Some of these fibers are in the vertical arm of the diagonal band of Broca while others lie in the medial accumbens nucleus [20,22]. From the region ventral to the anterior septum they are thought to course posteriorly in the medial forebrain bundle, giving off connections all along its course. Behavioral experiments have produced results which are generally congruent with these anatomical considerations. Tissue cuts ventrolateral to the septum produce hyperreactivity, as would be predicted if fibers from the lateral septum project anteroventrally and then laterally from the lateral septum [4]. Further, the subsequent passage of the fibers along the trajectory of the medial forebrain bundle leaves them conveniently positioned to interact with neurons in the lateral hypothalamus. Substantial evidence from experiments using electrical stimulation indicates that regions of the lateral hypothalamus are involved in initiating aggression and heightening reactivity [17, 26, 28].
Alternatively, it can be argued that the lesion ventral to the anterior septum produces an increase in reactivity which cannot be decreased by septal stimulation rather than blocking the suppression of medial hypothalamic lesion induced hyperreactivity. We have in fact found that stimulation of the lateral septum will not block the reactivity produced by lesions ventral to the anterior septum (unpublished results). However, the experiment is difficult to do because the level of reactivity produced by lesions ventral to the anterior septurn drops off very rapidly with the repeated testing required by the experimental design [2]. Because the reactivity caused by the lesion ventral to the anterior septum does drop off with repeated testing, it seems reasonable to argue as we have that the lesion ventral to the anterior septum in the present experiment is attenuating the effectiveness of lateral septal stimulation in blocking the hyperreactivity induced by the medial hypothalamic lesion. The observation that lesions of the septum do not block the suppression of reactivity by electrical stimulation ventral to the anterior septum is in agreement with other evidence suggesting that fibers coursing caudally through the lateral septum are not the fibers by which the lateral septum influences reactivity and aggression. The existence of such fiber tracts projecting into the hippocampus, columns of the fornix, the region of the stria terminalis and the stria medullaris are known [14, 21, 22]. However, in agreement with the present findings, lesions and/or cuts of the fornixhippocampal commissure area [16] the bed nucleus of the stria terminalis ([2,18], however, see also ref. [251) and the region posteroventral to the septum [6,18] have not been found to produce the strong hyperreactivity that would be expected if they were interrupting the fibers from the septum which influence the modulation of this behavior.
REFERENCES
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LATERAL SEPTUM AND REACTIVITY 23. Siegal, A. and J. P. Flynn. Differential effects of electrical stimulation and lesions of the hippocampus and adjacent regions upon attack behavior in cats. Brain Res. 7: 252-267, 1968. 24. Siegal, A., H. Edinger and S. Ohgami. The topographical organization of the hippocampus projections to the septal area: A comparative neuroanatomical analysis in the gerbil, rat, rabbit, and cat. J. comp. Neurol. 157: 359-378, 1974. 25. Turner, B. H. Neural structures involved in the rage syndrome of the rat. J. comp. physiol. Psychol. 71: 103-113, 1970.
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26. Wasman, M. and J. P. Fiynn. Directed attack elicited from hypothalamus. Arch. Neurol., Chicago 6: 220-227, 1962. 27. Winer, B. J. Statistical Principles in Experimental Design. New York: McGraw-Hill, 1962. 28. Woodworth, C. H. Attack elicited in rats by electrical stimulation of the lateral hypothalamus. Physiol. Behav. 6: 345-353, 1971.