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Medial hypothalamic electrical stimulation is ineffective in suppressing septal lesion induced hyperreactivity
Physiology & Behavior, Vol. 21, pp. 135--139. Pergamon Press and Brain Reesearch Pubi., 1978. Printed in the U.S.A.
BRIEF COMMUNICATION Medial Hypothalamic Electrical Stimulation is Ineffective in Suppressing Septal Lesion Induced Hyperreactivity 1 D. J. ALBERT, K. N. B R A Y L E Y A N D J. A. M I L N E R
Psychology Department, University of British Columbia, Vancouver, Canada (Received 17 October 1977) ALBERT, D. J., K. N. BRAYLEY AND J. A. MILNER. Medial hypothalamic electrical stimulation is ineffective in suppressing septal lesion induced hyperreactivity. PHYSIOL. BEHAV. 21(1) 135--139, 1978.--Animais made hyperreactive by lesions in the septal forebrain had bipolar electrodes implanted unilaterally in the medial hypothaiamus while animals made hyperreactive by lesions in the medial hypothalamus had electrodes implanted unilaterally ventral to the anterior septum. Postoperatively, the animals' reactivity level was scored 5 min before, during, and 5 rain following stimulation at 20/zA (60 Hz). Over 3 test days, unilateral electrical stimulation ventral to the anterior septum caused a mean decrease in reactivity of 42% while stimulation in the medial hypothalamus had no effect over a range of intensities (5-20 /xA). Electrical recordings revealed no poststimulus afterdischarges associated with the suppression of reactivity. Stimulation in the medial hypothalamus was slightly rewarding. It is suggested that the failure to suppress reactivity by electrical stimulation of the medial hypothalamus may be due to an organization of neural circuitry in that region which makes it difficult for a single behavior to be elicited by electrical stimulation.
Aggression
Hyperreactivity
Medialhypothalamus
THERE is a substantial correspondence in the behavioral effects of similar experimental manipulations of neural activity in the lateral septum, the region ventral to the anterior septum, and the medial hypothalamus. Electrolytic lesions in each area produce a strong increase in reactivity to the experimenter and a tendency to attack objects which the normal animal will ignore [I, 7, 10, 11, 14, 15, 16, 20]. One of the few differences that has been noted is that the recovery from the hyperreactivity is much slower in the animals with medial hypothalamic lesions [20]. There are some reports that electrolytic lesions in either the medial hypothalamus or the septum induce muricide as well as hyperreactivity [I0, 12, 13, 16, 17]. This has not been examined in animals with lesions ventral to the anterior septum. Temporary lesions produced by infusion of local anesthetic into the lateral septurn, the region ventral to the anterior septum, or the medial hypothalamus have been found to produce hyperreactivity and muricide, and, in addition, intermale aggression [2, 3, 4]. The converse manipulation, artificially increasing neural activity in the lateral septum or the region ventral to the anterior septum, suppresses the increase in reactivity caused by lesions of the medial hypothalamus [8,9]. Electrical activation of two other areas which have not been shown to be involved in the modulation of reactivity (i.e., the medial septum and the cingnlate cortex) do not cause a suppression of the medial hypothalamic lesion induced hyperreactivity. The
Septum
effect of medial hypothalamic stimulation on reactivity has not been examined. The substantial correspondence in the effects of comparable manipulations of the lateral septum, the region ventral to the anterior septum, and the medial hypothalamus points to a similar role of these areas in modulating reactivity and aggression. One important respect in which the pattern of evidence is incomplete is that the effectiveness of medial hypothalamic stimulation in suppressing reactivity has not been examined. This omission was addressed in the experiment reported here. Since in previous experiments, stimulation of the septal forebrain has been used to suppress the increase in reactivity caused by medial hypothalamic lesions, it was only reasonable to use stimulation of the medial hypothalamus to suppress septal lesion induced hyperreactivity. In other respects the stimulation and testing were done in the same way that proved effective with septal forebrain stimulation in earlier experiments [8,9]. METHOD
Animals The animals were 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. Food and
~Supported by a grant from the National Research Council of Canada (A 0192).
Copyright © 1978 Brain Research Publications Inc.--0031-9384/78/0701-0135502.00/0
136
ALBERT, B R A Y L E Y AND M I L N E R
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PRESTIMULATION
STIMULATION
POSTSTtMULATION
FIG. 1. Mean aggression-reactivity scores on the third test session for animals stimulated ventral to the anterior septum (AV), in the medial hypothalamus (MH), unstimulated controls with electrodes ventral to the anterior septum (AVC) or in the medial hypothalamus (MHC). Behavioral measures were taken 5 min bd~ore, during, and 5 min following stimulation.
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water were freely available and lighting was timed to a normal 12 hr light-dark cycle.
Surgery Electrolytic lesions (stainless steel anode) were made bilaterally in either the medial hypothalamus or the septum and the region ventral to its anterior portion (sodium pentobarbital anesthesia; septal lesions: anterior to bregma 2.4 mm, lateral: 1.0 mm, ventral to brain surface: 6.0 mm, [18]; 3.0 mA, 25 sec; medial hypothalamic lesions: anterior 0.0 mm, lateral; 0.6 mm, ventral: 9.3 mm [18], 2.0 mA 20 sec). At the same surgical session, a single bipolar nichrome electrode (Plastic Products, 0.6 mm dia) was implanted unilaterally. If the animal had received a lesion in the medial hypothalamus, the electrode was implanted ventral to the anterior septum; if the lesion was in the septal region the electrode was implanted in the medial hypothalamus (see Fig. 2 for location). Following surgery, each animal was injected with 0.2 cc penicillin (60,000 IU) intramuscularly and returned to an individual cage for a recovery period of at least 4 but more usually of 7 days.
Behavior Testing The effect of electrical stimulation on reactivity and aggression was examined over a three day period. Prior to testing, the rat was very lightly anesthetized with ether to permit attachment of the electrode lead. The animal was then placed in a gray box (60 x 60 x 60 cm) with an open top and wood shavings on the floor. Testing began about 10 min later. An initial test of aggressiveness and 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 score the animal was given a score of 0 to 3 (0 indicating little or no response, and 3 indicating a highly aggressive or reactive response) for its
FIG. 2. The placement of stimulating electrodes for all animals receiving stimulation and whose scores are included in Fig. 1. Top: stimulation sites ventral to the anterior septum. Bottom: stimulation siets in medial hypothalamus. Brain sections are from Pellegrino and Cushman [18].
response to each of 6 stimuli (presentation of a pencil, a tap on the back, presentation of a gloved hand, a poke in the side, grasping by the tail, and grasping by the abdomen) and on the amount of vocalization and amount of biting observed during testing (see [1,2] for details). 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 were discarded only because their lower scores limited the quantitative suppression that could be obtained with the stimulation. Twelve animals with lesions of the septal region and 9 animals with lesions of the medial hypothalamus were discarded for not meeting this criterion. Five minutes 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 and aggressiveness was reassessed. The stimulation was then turned off. Five minutes later the aggressiveness
SUPPRESSION OF REACTIVITY and reactivity of the animal was assessed again, this time with the stimulation off. The 20 t~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 [8,9].
Additional Observations Electrical activity at the stimulation site was recorded in all animals immediately before and following stimulation (Grass polygraph, Model 78D, Model 7P511G amplifier). To evaluate the extent to which the effects of stimulation ventral to the anterior septum or the medial hypothalamus on aggression and reactivity are confounded with rewarding or punishing effects of the stimulation, most animals were tested for self-stimulation in a shuttle box [5]. Because the threshold for the suppression of aggression goes down over test days the stimulating current in the shuttle box was set at 17/~A (RMS). The animals were given six 20 min sessions in the shuttle box. Current was available only on Days 1, 3 and 5. To ensure that each animal was exposed to the electrical stimulation on the days when current was available, the current was automatically turned on at 5 and 15 min, and off at 10 and 20 min. The timers were also turned on and offin this fashion on the days when the stimulating current was off. The shuttle box was used because it allows the animal to turn the stimulating current on if it is rewarding, and also to turn it off at the stimulus duration where the current becomes aversive.
137 is the reactivity rating attained during the stimulation reliably different from the pre- or poststimulation score (all p's>0.10). There is an increasing tendency over days for the hypothalamic stimulation to exacerbate the septal lesioninduced hyperreactivity. The location of the medial hypothalamic electrode tips are shown in Fig. 2. The placements fall within the area in which microinjection of local anesthetic causes an increase in reactivity and aggression
[3]. The pattern of scores for the unstimulated control groups with electrodes in the medial hypothalamus (N=8) did not show fluctuations comparable to those of the group receiving stimulation ventral to the anterior septum (see Fig. 1).
Medial Hypothalamic and Septal Area Lesions
Following completion of testing, the brains of all animals were removed and placed in formol-saline. Later they were sectioned at 30 microns on a cryostat and stained with thionin.
In the animals stimulated ventral to the anterior septum, the lesions of the medial hypothalamus included the area between the fornix and the midline and from the anterior tip of the ventromedial nucleus to the mammilothalamic tract in 6 of the 8 animals. In four of these animals the lesions also destroyed all or part of the dorsomedial nucleus. The remaining two animals had lesions approximately limited to the ventromedial nucleus. There was no relationship between the size of the medial hypothalamic lesion and the effectiveness of the stimulation in suppressing reactivity. In the animals given medial hypothalamic stimulation, the septal lesions were large and included the medial and lateral septum in all animals. In 11 of the animals, the region ventral to the anterior septum was destroyed at least down to the level of the anterior limb of the anterior commissure. The fornix was completely destroyed in two animals and partially destroyed in 3 others. The lesions in the unstimulated control animals were similar to those in the corresponding stimulation group.
Analysis of Results
Additional Observations
The group data from stimulating these animals was subjected to an overall analysis of variance for unequal cell frequencies [22]. Comparisons between conditions within groups were made with t tests (all p values are two-tailed).
Electrical recordings from stimulation site. None of the animals whose scores are included in Fig. 1 displayed after-discharge on any of the test trials. However, the scores of three animals with stimulation ventral to the anterior septurn but none in the medial hypothalamus were excluded for showing after-discharge on at least one test trial. Self-stimulation. For the group with medial hypothalarnic stimulation (N=8), the mean number of shuttle box crossings was 80 with the current available and 25 with the current off (t=2.9, p<0.05. The mean amount of time spent with the " o n " relay closed was 587 sec with the current available and 619 sec with the current off (t=0.3, p>0.20). The greater number of crossings on the days when crossings turn the current on and off argues for a rewarding effect of the stimulation in the medial hypothalamus. In the animals with electrodes ventral to the anterior septurn (N=6), the mean number of crossings was 13 on the days when stimulating current was available and 6 when it was not (t=2.1, p<0.10). The number of seconds spent with the on relay closed was 557 sec with the current available and 574 sec with current not available (t=0.1, p>0.20). There is little to indicate that the stimulation ventral to the anterior septum is rewarding at the current intensity used for suppressing reactivity and aggression. Mouse-killing. Inspection of the individual reactivity ratings of animals stimulated in the medial hypothalamus suggests that some animals may be made more, rather than less, reactive by the 20 /xA stimulation. In an attempt to
Histology
RESULTS Electrical stimulation of the region ventral to the anterior septum but not the medial hypothalamus suppressed reactivity and aggression, F(6,66) = 19.5, p <0.0001; Fig. 1). With stimulation ventral to the anterior septum (N=8), the prestimulation, stimulation, and poststimulation reactivity ratings were 10.9, 8.1, and 11.8 for the first day, 10.8, 6.1, and 11.5 for the second day, and 11.1, 4.6, and 12.3 for the third test day. In each case, the reactivity rating during stimulation is lower than either the pre- or poststimulation rating (all p's<0.01). The location of the electrode tips in these animals is shown in Fig. 2. The overall analysis also revealed a significant interaction of stimulation by days, F(12,132)=2.8, p<0.002, which means that the suppression of reactivity and aggression by the 20/xA electrical stimulation was becoming increasingly effective over days (t=2.4, p<0.05). Stimulation in the medial hypothalamus (N = 12) produced a slight increase in reactivity. The pre-stimulation, stimulation, and poststimulation ratings were 9.5, 9.9 and 9.8 for the first day, 10.0, 10.4 and 9.6 for the second day, and 9.0, 10.6 and 8.1 for the third test day. On none of the three test days
138
ALBERT, B R A Y L E Y AND M I L N E R
explore this more thoroughly, the last eight animals with medial hypothalamic electrodes were tested for muricide immediately following the third day of reactivity testing. None of these animals spontaneously killed an adult albino mouse which was placed beside it in the test box for 1 min. When the electrical stimulation was introduced, 4 rats killed the mouse within 1 rain and two attacked but did not kill. Only one rat continued to show no interest in the mouse. The pattern of the killing was that described by Woodworth [21] for stimulation of the lateral hypothalamus. It involves grasping the mouse with all four feet and leaping and biting ah the same time. The electrode placements for these animals were all medial to the fornix, with some in the ventromedial nucleus itself. Two animals with electrodes ventral to the anterior septum were tested for muricide in the same way. Neither of these animals was induced to kill a mouse by the electrical stimulation. Varying current intensity. The elicitation of muricide with medial hypothalamic stimulation raises the possibility that the 20 t*A stimulating current is spreading to the lateral hypothalamus. Accordingly, a separate group of 8 animals with lesions of the septal region and unilateral electrodes in the medial hypothalamus were tested on two successive days for reactivity and aggression to the experimenter in ascending 5 / , A current steps (0, 5, 10, 15, 20 and 0 / , A ) at 5 min intervals. There was no substantial suppression of reactivity or aggression at any of the current levels. The mean test scores for the stimulation sequence were 10.0, 9.3, 10.1, 9.4, 8.6, 9.4 on the first day of testing and 9.1, 8.6, 8.8, 8.4, 8.4, 9.0 on the second day of testing. Histological examination of the brains showed that the electrodes were in the medial hypothalamus and varied in location between the fornix and the middle of the ventromedial nucleus. Bilateral stimulation. In a final attempt to suppress septal lesion-induced reactivity, animals (N=4) with bilateral medial hypothalamic electrodes were tested at 0, 10, 20 and 0 p.A with a 5 min interval between each testing. There was no substantial effect of the bilateral stimulation on the septal lesion induced hyperreactivity. The mean reactivity scores during the stimulation sequence were 10.6, 10.0, 13.5 and 9.3. Histological examination of the brains showed all electrodes to be in the medial hypothalamus at positions similar to those plotted in Fig. 2. DISCUSSION
Some of the obvious reasons for the differential effectiveness of medial hypothalamic and anterior forebrain stimulation in suppressing reactivity and aggression seem to be excluded by the present findings. Thus, the differences do not appear to be due to variations in the induction of gross electrophysiological abnormalities such as seizuring and afterdischarging. The self-stimulation test revealed a slight difference between the two stimulation sites. However, the relatively greater tendency for animals with medial
hypothalamic electrodes to self-stimulate argues against the suggestion that rewarding effects are involved in the suppression of reactivity and aggression in the present instance. A critical aspect of the medial hypothalamic stimulation that bears close attention is the current intensity. In these experiments, we used 20/.tA after exploratory studies failed to yield any suppression with either stronger or weaker currents and in order that the medial hypothalamic stimulation would be comparable to that ventral to the anterior septum. However, the increase in reactivity and the induction of muricide that occurred in some animals suggests that the 20 izA current is sufficiently strong to spread to the area just lateral to the fornix which has been shown to produce these effects [21]. By stimulating at lower intensities (5, 10 and 15 /,A) we were able to avoid the exacerbation of the hyperreactivity caused by the 2 0 / , A current. Yet, the lower intensity currents were still not effective in suppressing the hyperreactivity induced by the septal forebrain lesion. Bilateral stimulation, which would intuitively seem to be a way of increasing the effectiveness of the stimulation by altering the activity of symmetrical parts of the brain stimultaneously, was also ineffective in producing a reduction of septal lesion induced hyperreactivity and aggression. There are at least two lines of reasoning that can be followed in explaining why medial hypothalamic stimulation is ineffective in suppressing hyperreactivity induced by anterior forebrain lesions. One of these is that the medial hypothalamic circuitry which modulates reactivity and aggression may have its effect on behavior through modulation of neural activity in the septal forebrain area. Accordingly, when the septal forebrain area is removed, the target area for the medial hypothalamic neurons is gone, and consequently, their impulses cannot alter behavior. While this line of reasoning cannot be ruled invalid with certainty, it would seem implausible. Lesions of the medial hypothalamus produce a long lasting increase in reactivity while lesions of the septal region produce a relatively transient increase in reactivity [20]. If the neurons in the medial hypothalamus had their effect through interaction with neurons in the septal region, the behavioral effects of septai lesions should be at least as enduring as those caused by medial hypothalamic lesions. A more plausible alternative is that the neural circuitry in the medial hypothalamus does not lend itself to obtaining simple behavioral effects with electrical stimulation. This would be in line with the tendency of the medial hypothalamic stimulation to induce muricide even though the main area for inducing mouse killing appears to be lateral to the fornix [21]. In a similar vein, stimulation at about 20 /xA sometimes induces a general increase in activity making it difficult to evaluate the animals' reactivity level. It may be that if slightly more intense currents could be used without generating conflicting behaviors, a suppression of reactivity to the experimenter could be demonstrated. This tendency for medial hypothalamic stimulation to produce multiple effects is very much in line with what others have observed in attempting to suppress feeding behavior in hungry rats [6,19]
REFERENCES !. Albert, J. D. and S. E. Richmond. Septal hyperreactivity: A comparison of lesions within and adjacent to the septum, Physiol. Behav. 15: 1975.
2. Albert, D. J. and S. E. Richmond. Hyperreactivity and aggressiveness following infusion of local anesthetic into the lateral septum and surrounding structures. Behav. Biol. 18: 211-226, 1976.
SUPPRESSION OF REACTIVITY 3. Albert, D. J. and R. C. K. Wong. Interanimal aggression and hyperreactivity following hypothalamic infusion of local anesthetic in the rat. Physiol. Behav. 20: 755-761, 1978. 4. 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., in press, 1978. 5. 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. 6. Ball, G. G. Self-stimulation in the ventromedial hypothalamus. Science 178: 72-73, 1972. 7. 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. 8. 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. 9. 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. Behav. 18: 567-571, 1977. 10. Eclancher, F. S. and P. Karli. Comportement d'aggression interspecific et comportement alimentaire du rat: effets de lesions des najaux ventromedians de l'hypothalamus. Brain Res. 26: 71-79, 1971. 11. Gotsick, J. E. and R. C. Marshall. Time course of the septal rage syndrome. Physiol. Behav. 9: 685-687, 1972.
139 12. Karli, P. Septum, hypothalamus posterieur et agressivite interspecifique rat-souds. J. Physiol. Paris 52: 135-136, 1960. 13. Karli, P., M. Vergnes and F. Didiergeorges. Rat-mouse interspecific aggressive behavior and its manipulation by brain ablation and by brain stimulation. In: Aggressive Behavior, edited by S. Garattini and E. B. Sigg. New York: John Wiley, 1969. 14. King, F. A. Effects of septal and amygdaloid lesions on emotional behavior and CAR in the rat. J. nerv. ment. Dis. 126: 5%63, 1958. 15. Kreickhaus, E. E., H. J. Simmons, G. J. Thomas and K. Kenyon. Septal lesions enhance shock avoidance behavior in the rat. Expl Neurol. 9: 10%113, 1964. 16. Malick, J. B. A behavioral comparison of three lesion-induced models of aggression in the rat. Physiol. Behav. 5: 679-681, 1970. 17. Miczek, K. A. and S. P. Grossman. Effects of septal lesions on inter- and intraspecies aggression in rats. J. comp. physiol. Psychol. 79: 39-45, 1972. 18. Pellegrino, L. J. and A. J. Cushman. A Stereotaxic Atlas of the Rat Brain. New York: Appleton-Century-Crofts, 1967. 19. Sclafani, A. and G. Maul. Does the ventromedial hypothalamus inhibit the lateral hypothalamus? Physiol. Behav. 12: 157-162, 1974. 20. Singh, D. Comparison of hyperemotionality caused by lesions in the septal and ventromedial hypothalamic areas in the rat. Psychon. Sci. 16: 3-4, 1969. 21. Woodworth, C. H. Attack elicited in rats by electrical stimulation of the lateral hypothalamus. Physiol. Behav. 6: 345-353, 1971. 22. Winer, B. J. Statistical Principles in Experimental Design. New York: McGraw-Hill, 1962.
BRIEF COMMUNICATION Medial Hypothalamic Electrical Stimulation is Ineffective in Suppressing Septal Lesion Induced Hyperreactivity 1 D. J. ALBERT, K. N. B R A Y L E Y A N D J. A. M I L N E R
Psychology Department, University of British Columbia, Vancouver, Canada (Received 17 October 1977) ALBERT, D. J., K. N. BRAYLEY AND J. A. MILNER. Medial hypothalamic electrical stimulation is ineffective in suppressing septal lesion induced hyperreactivity. PHYSIOL. BEHAV. 21(1) 135--139, 1978.--Animais made hyperreactive by lesions in the septal forebrain had bipolar electrodes implanted unilaterally in the medial hypothaiamus while animals made hyperreactive by lesions in the medial hypothalamus had electrodes implanted unilaterally ventral to the anterior septum. Postoperatively, the animals' reactivity level was scored 5 min before, during, and 5 rain following stimulation at 20/zA (60 Hz). Over 3 test days, unilateral electrical stimulation ventral to the anterior septum caused a mean decrease in reactivity of 42% while stimulation in the medial hypothalamus had no effect over a range of intensities (5-20 /xA). Electrical recordings revealed no poststimulus afterdischarges associated with the suppression of reactivity. Stimulation in the medial hypothalamus was slightly rewarding. It is suggested that the failure to suppress reactivity by electrical stimulation of the medial hypothalamus may be due to an organization of neural circuitry in that region which makes it difficult for a single behavior to be elicited by electrical stimulation.
Aggression
Hyperreactivity
Medialhypothalamus
THERE is a substantial correspondence in the behavioral effects of similar experimental manipulations of neural activity in the lateral septum, the region ventral to the anterior septum, and the medial hypothalamus. Electrolytic lesions in each area produce a strong increase in reactivity to the experimenter and a tendency to attack objects which the normal animal will ignore [I, 7, 10, 11, 14, 15, 16, 20]. One of the few differences that has been noted is that the recovery from the hyperreactivity is much slower in the animals with medial hypothalamic lesions [20]. There are some reports that electrolytic lesions in either the medial hypothalamus or the septum induce muricide as well as hyperreactivity [I0, 12, 13, 16, 17]. This has not been examined in animals with lesions ventral to the anterior septum. Temporary lesions produced by infusion of local anesthetic into the lateral septurn, the region ventral to the anterior septum, or the medial hypothalamus have been found to produce hyperreactivity and muricide, and, in addition, intermale aggression [2, 3, 4]. The converse manipulation, artificially increasing neural activity in the lateral septum or the region ventral to the anterior septum, suppresses the increase in reactivity caused by lesions of the medial hypothalamus [8,9]. Electrical activation of two other areas which have not been shown to be involved in the modulation of reactivity (i.e., the medial septum and the cingnlate cortex) do not cause a suppression of the medial hypothalamic lesion induced hyperreactivity. The
Septum
effect of medial hypothalamic stimulation on reactivity has not been examined. The substantial correspondence in the effects of comparable manipulations of the lateral septum, the region ventral to the anterior septum, and the medial hypothalamus points to a similar role of these areas in modulating reactivity and aggression. One important respect in which the pattern of evidence is incomplete is that the effectiveness of medial hypothalamic stimulation in suppressing reactivity has not been examined. This omission was addressed in the experiment reported here. Since in previous experiments, stimulation of the septal forebrain has been used to suppress the increase in reactivity caused by medial hypothalamic lesions, it was only reasonable to use stimulation of the medial hypothalamus to suppress septal lesion induced hyperreactivity. In other respects the stimulation and testing were done in the same way that proved effective with septal forebrain stimulation in earlier experiments [8,9]. METHOD
Animals The animals were 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. Food and
~Supported by a grant from the National Research Council of Canada (A 0192).
Copyright © 1978 Brain Research Publications Inc.--0031-9384/78/0701-0135502.00/0
136
ALBERT, B R A Y L E Y AND M I L N E R
LjIS-
//
f
co
~_lO-
o uJ -
,MHC
• t
i
I
PRESTIMULATION
STIMULATION
POSTSTtMULATION
FIG. 1. Mean aggression-reactivity scores on the third test session for animals stimulated ventral to the anterior septum (AV), in the medial hypothalamus (MH), unstimulated controls with electrodes ventral to the anterior septum (AVC) or in the medial hypothalamus (MHC). Behavioral measures were taken 5 min bd~ore, during, and 5 min following stimulation.
v,-.
F
water were freely available and lighting was timed to a normal 12 hr light-dark cycle.
Surgery Electrolytic lesions (stainless steel anode) were made bilaterally in either the medial hypothalamus or the septum and the region ventral to its anterior portion (sodium pentobarbital anesthesia; septal lesions: anterior to bregma 2.4 mm, lateral: 1.0 mm, ventral to brain surface: 6.0 mm, [18]; 3.0 mA, 25 sec; medial hypothalamic lesions: anterior 0.0 mm, lateral; 0.6 mm, ventral: 9.3 mm [18], 2.0 mA 20 sec). At the same surgical session, a single bipolar nichrome electrode (Plastic Products, 0.6 mm dia) was implanted unilaterally. If the animal had received a lesion in the medial hypothalamus, the electrode was implanted ventral to the anterior septum; if the lesion was in the septal region the electrode was implanted in the medial hypothalamus (see Fig. 2 for location). Following surgery, each animal was injected with 0.2 cc penicillin (60,000 IU) intramuscularly and returned to an individual cage for a recovery period of at least 4 but more usually of 7 days.
Behavior Testing The effect of electrical stimulation on reactivity and aggression was examined over a three day period. Prior to testing, the rat was very lightly anesthetized with ether to permit attachment of the electrode lead. The animal was then placed in a gray box (60 x 60 x 60 cm) with an open top and wood shavings on the floor. Testing began about 10 min later. An initial test of aggressiveness and 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 score the animal was given a score of 0 to 3 (0 indicating little or no response, and 3 indicating a highly aggressive or reactive response) for its
FIG. 2. The placement of stimulating electrodes for all animals receiving stimulation and whose scores are included in Fig. 1. Top: stimulation sites ventral to the anterior septum. Bottom: stimulation siets in medial hypothalamus. Brain sections are from Pellegrino and Cushman [18].
response to each of 6 stimuli (presentation of a pencil, a tap on the back, presentation of a gloved hand, a poke in the side, grasping by the tail, and grasping by the abdomen) and on the amount of vocalization and amount of biting observed during testing (see [1,2] for details). 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 were discarded only because their lower scores limited the quantitative suppression that could be obtained with the stimulation. Twelve animals with lesions of the septal region and 9 animals with lesions of the medial hypothalamus were discarded for not meeting this criterion. Five minutes 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 and aggressiveness was reassessed. The stimulation was then turned off. Five minutes later the aggressiveness
SUPPRESSION OF REACTIVITY and reactivity of the animal was assessed again, this time with the stimulation off. The 20 t~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 [8,9].
Additional Observations Electrical activity at the stimulation site was recorded in all animals immediately before and following stimulation (Grass polygraph, Model 78D, Model 7P511G amplifier). To evaluate the extent to which the effects of stimulation ventral to the anterior septum or the medial hypothalamus on aggression and reactivity are confounded with rewarding or punishing effects of the stimulation, most animals were tested for self-stimulation in a shuttle box [5]. Because the threshold for the suppression of aggression goes down over test days the stimulating current in the shuttle box was set at 17/~A (RMS). The animals were given six 20 min sessions in the shuttle box. Current was available only on Days 1, 3 and 5. To ensure that each animal was exposed to the electrical stimulation on the days when current was available, the current was automatically turned on at 5 and 15 min, and off at 10 and 20 min. The timers were also turned on and offin this fashion on the days when the stimulating current was off. The shuttle box was used because it allows the animal to turn the stimulating current on if it is rewarding, and also to turn it off at the stimulus duration where the current becomes aversive.
137 is the reactivity rating attained during the stimulation reliably different from the pre- or poststimulation score (all p's>0.10). There is an increasing tendency over days for the hypothalamic stimulation to exacerbate the septal lesioninduced hyperreactivity. The location of the medial hypothalamic electrode tips are shown in Fig. 2. The placements fall within the area in which microinjection of local anesthetic causes an increase in reactivity and aggression
[3]. The pattern of scores for the unstimulated control groups with electrodes in the medial hypothalamus (N=8) did not show fluctuations comparable to those of the group receiving stimulation ventral to the anterior septum (see Fig. 1).
Medial Hypothalamic and Septal Area Lesions
Following completion of testing, the brains of all animals were removed and placed in formol-saline. Later they were sectioned at 30 microns on a cryostat and stained with thionin.
In the animals stimulated ventral to the anterior septum, the lesions of the medial hypothalamus included the area between the fornix and the midline and from the anterior tip of the ventromedial nucleus to the mammilothalamic tract in 6 of the 8 animals. In four of these animals the lesions also destroyed all or part of the dorsomedial nucleus. The remaining two animals had lesions approximately limited to the ventromedial nucleus. There was no relationship between the size of the medial hypothalamic lesion and the effectiveness of the stimulation in suppressing reactivity. In the animals given medial hypothalamic stimulation, the septal lesions were large and included the medial and lateral septum in all animals. In 11 of the animals, the region ventral to the anterior septum was destroyed at least down to the level of the anterior limb of the anterior commissure. The fornix was completely destroyed in two animals and partially destroyed in 3 others. The lesions in the unstimulated control animals were similar to those in the corresponding stimulation group.
Analysis of Results
Additional Observations
The group data from stimulating these animals was subjected to an overall analysis of variance for unequal cell frequencies [22]. Comparisons between conditions within groups were made with t tests (all p values are two-tailed).
Electrical recordings from stimulation site. None of the animals whose scores are included in Fig. 1 displayed after-discharge on any of the test trials. However, the scores of three animals with stimulation ventral to the anterior septurn but none in the medial hypothalamus were excluded for showing after-discharge on at least one test trial. Self-stimulation. For the group with medial hypothalarnic stimulation (N=8), the mean number of shuttle box crossings was 80 with the current available and 25 with the current off (t=2.9, p<0.05. The mean amount of time spent with the " o n " relay closed was 587 sec with the current available and 619 sec with the current off (t=0.3, p>0.20). The greater number of crossings on the days when crossings turn the current on and off argues for a rewarding effect of the stimulation in the medial hypothalamus. In the animals with electrodes ventral to the anterior septurn (N=6), the mean number of crossings was 13 on the days when stimulating current was available and 6 when it was not (t=2.1, p<0.10). The number of seconds spent with the on relay closed was 557 sec with the current available and 574 sec with current not available (t=0.1, p>0.20). There is little to indicate that the stimulation ventral to the anterior septum is rewarding at the current intensity used for suppressing reactivity and aggression. Mouse-killing. Inspection of the individual reactivity ratings of animals stimulated in the medial hypothalamus suggests that some animals may be made more, rather than less, reactive by the 20 /xA stimulation. In an attempt to
Histology
RESULTS Electrical stimulation of the region ventral to the anterior septum but not the medial hypothalamus suppressed reactivity and aggression, F(6,66) = 19.5, p <0.0001; Fig. 1). With stimulation ventral to the anterior septum (N=8), the prestimulation, stimulation, and poststimulation reactivity ratings were 10.9, 8.1, and 11.8 for the first day, 10.8, 6.1, and 11.5 for the second day, and 11.1, 4.6, and 12.3 for the third test day. In each case, the reactivity rating during stimulation is lower than either the pre- or poststimulation rating (all p's<0.01). The location of the electrode tips in these animals is shown in Fig. 2. The overall analysis also revealed a significant interaction of stimulation by days, F(12,132)=2.8, p<0.002, which means that the suppression of reactivity and aggression by the 20/xA electrical stimulation was becoming increasingly effective over days (t=2.4, p<0.05). Stimulation in the medial hypothalamus (N = 12) produced a slight increase in reactivity. The pre-stimulation, stimulation, and poststimulation ratings were 9.5, 9.9 and 9.8 for the first day, 10.0, 10.4 and 9.6 for the second day, and 9.0, 10.6 and 8.1 for the third test day. On none of the three test days
138
ALBERT, B R A Y L E Y AND M I L N E R
explore this more thoroughly, the last eight animals with medial hypothalamic electrodes were tested for muricide immediately following the third day of reactivity testing. None of these animals spontaneously killed an adult albino mouse which was placed beside it in the test box for 1 min. When the electrical stimulation was introduced, 4 rats killed the mouse within 1 rain and two attacked but did not kill. Only one rat continued to show no interest in the mouse. The pattern of the killing was that described by Woodworth [21] for stimulation of the lateral hypothalamus. It involves grasping the mouse with all four feet and leaping and biting ah the same time. The electrode placements for these animals were all medial to the fornix, with some in the ventromedial nucleus itself. Two animals with electrodes ventral to the anterior septum were tested for muricide in the same way. Neither of these animals was induced to kill a mouse by the electrical stimulation. Varying current intensity. The elicitation of muricide with medial hypothalamic stimulation raises the possibility that the 20 t*A stimulating current is spreading to the lateral hypothalamus. Accordingly, a separate group of 8 animals with lesions of the septal region and unilateral electrodes in the medial hypothalamus were tested on two successive days for reactivity and aggression to the experimenter in ascending 5 / , A current steps (0, 5, 10, 15, 20 and 0 / , A ) at 5 min intervals. There was no substantial suppression of reactivity or aggression at any of the current levels. The mean test scores for the stimulation sequence were 10.0, 9.3, 10.1, 9.4, 8.6, 9.4 on the first day of testing and 9.1, 8.6, 8.8, 8.4, 8.4, 9.0 on the second day of testing. Histological examination of the brains showed that the electrodes were in the medial hypothalamus and varied in location between the fornix and the middle of the ventromedial nucleus. Bilateral stimulation. In a final attempt to suppress septal lesion-induced reactivity, animals (N=4) with bilateral medial hypothalamic electrodes were tested at 0, 10, 20 and 0 p.A with a 5 min interval between each testing. There was no substantial effect of the bilateral stimulation on the septal lesion induced hyperreactivity. The mean reactivity scores during the stimulation sequence were 10.6, 10.0, 13.5 and 9.3. Histological examination of the brains showed all electrodes to be in the medial hypothalamus at positions similar to those plotted in Fig. 2. DISCUSSION
Some of the obvious reasons for the differential effectiveness of medial hypothalamic and anterior forebrain stimulation in suppressing reactivity and aggression seem to be excluded by the present findings. Thus, the differences do not appear to be due to variations in the induction of gross electrophysiological abnormalities such as seizuring and afterdischarging. The self-stimulation test revealed a slight difference between the two stimulation sites. However, the relatively greater tendency for animals with medial
hypothalamic electrodes to self-stimulate argues against the suggestion that rewarding effects are involved in the suppression of reactivity and aggression in the present instance. A critical aspect of the medial hypothalamic stimulation that bears close attention is the current intensity. In these experiments, we used 20/.tA after exploratory studies failed to yield any suppression with either stronger or weaker currents and in order that the medial hypothalamic stimulation would be comparable to that ventral to the anterior septum. However, the increase in reactivity and the induction of muricide that occurred in some animals suggests that the 20 izA current is sufficiently strong to spread to the area just lateral to the fornix which has been shown to produce these effects [21]. By stimulating at lower intensities (5, 10 and 15 /,A) we were able to avoid the exacerbation of the hyperreactivity caused by the 2 0 / , A current. Yet, the lower intensity currents were still not effective in suppressing the hyperreactivity induced by the septal forebrain lesion. Bilateral stimulation, which would intuitively seem to be a way of increasing the effectiveness of the stimulation by altering the activity of symmetrical parts of the brain stimultaneously, was also ineffective in producing a reduction of septal lesion induced hyperreactivity and aggression. There are at least two lines of reasoning that can be followed in explaining why medial hypothalamic stimulation is ineffective in suppressing hyperreactivity induced by anterior forebrain lesions. One of these is that the medial hypothalamic circuitry which modulates reactivity and aggression may have its effect on behavior through modulation of neural activity in the septal forebrain area. Accordingly, when the septal forebrain area is removed, the target area for the medial hypothalamic neurons is gone, and consequently, their impulses cannot alter behavior. While this line of reasoning cannot be ruled invalid with certainty, it would seem implausible. Lesions of the medial hypothalamus produce a long lasting increase in reactivity while lesions of the septal region produce a relatively transient increase in reactivity [20]. If the neurons in the medial hypothalamus had their effect through interaction with neurons in the septal region, the behavioral effects of septai lesions should be at least as enduring as those caused by medial hypothalamic lesions. A more plausible alternative is that the neural circuitry in the medial hypothalamus does not lend itself to obtaining simple behavioral effects with electrical stimulation. This would be in line with the tendency of the medial hypothalamic stimulation to induce muricide even though the main area for inducing mouse killing appears to be lateral to the fornix [21]. In a similar vein, stimulation at about 20 /xA sometimes induces a general increase in activity making it difficult to evaluate the animals' reactivity level. It may be that if slightly more intense currents could be used without generating conflicting behaviors, a suppression of reactivity to the experimenter could be demonstrated. This tendency for medial hypothalamic stimulation to produce multiple effects is very much in line with what others have observed in attempting to suppress feeding behavior in hungry rats [6,19]
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