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Cholinergic synapses in the lateral hypothalamus for the control of predatory aggression in the rat
BRAIN RESEARCH
409
CHOLINERGIC SYNAPSES IN THE LATERAL HYPOTHALAMUS FOR THE CONTROL OF PREDATORY AGGRESSION IN THE RAT
R I C H A R D J. BANDLER, JR.
Yale University School of Medicine, Department of Psychiatry, Connecticut Mental Health Center, New Haven, Conn. 06519 (U.S.A.) (Accepted December 15th, 1969)
INTRODUCTION
Predatory aggression is a most distinct kind of aggression. The aggressive response whether naturally-occurring or electrically-elicited is both stereotyped and stimulus specific. Karli 7 in the rat and Wasman and Flynn ~7 in the cat have identified the aggressive response by both the lack of affective display and the characteristic biting directed at the neck region of the prey. In the cat, the stimulus specific character of the aggressive response is indicated by Wasman and Flynn's 17 report that the electrically-elicited aggressive response, while fatal to the rat, will not result in an attack on the experimenter. Levinson and Flynn 1~ have further shown that the elicited aggressive response rapidly deteriorates as the prey object changes from anesthetized rat, to stuffed toy dog, to foam rubber block. A similar stimulus specificity is present in the rat. Bandler and Moyer2 have reported that while 95 ~ of all tested male hooded rats kill frogs or turtles, less than 6 0 ~ kill baby chicks, and only 15~ kill white mice. Nearly all of these rats are quite tame and easily handled by the experimenter. The limited number of stimuli capable of eliciting the aggressive response and the stereotyped nature of the response, then, make predatory aggression unconfusable with other kinds of aggression 14. The participation of the lateral hypothalamus in the regulation of predatory aggression is now well established. Wasman and Flynn 17, Roberts and Kiess 15, and Hutchinson and Renfrew 6 have reported that electrical stimulation of the lateral hypothalamus of the cat elicits predatory aggression. Electrical stimulation of the lateral hypothalamus of the rat has both facilitated9 and elicited 10 predatory aggression. In addition, Karli and Vergnes s have reported that extensive bilateral lesions of the lateral hypothalamus block predatory mouse-killing in natural killer rats. Little is known, however, about the neurochemical factors involved in the facilitation and inhibition of predatory aggression. Earlier research by Bandler 1 found that direct cholinergic (carbachoi) stimulation of the lateral hypothalamus facilitated predatory aggression in natural killer rats. Controls for osmotic effects, Brain Research, 20 (1970) 409-424
410
R.J. BANI)LER
non-specific neural effects, and changes in general activity level failed to duplicate the carbachol effect, suggesting that the carbachol-induced facilitation of predator) aggression might be due to the cholincrgic action of the drug in the hypothalamus. This suggested, that the regulation of predator)' aggression at the lateral hypothalamus was cholinergic: acetylcholine functioned to transmit nervous impulses across the relevant synapses and was then promptly destroyed by cholincsterase. To test this hypothesis a number of predictions were made concerning the effect of central stimulation with acetylcholine, and cholinesterase inhibitors, and central and peripheral stimulation with cholinergic blocking agents: (I) direct. central stimulation of positive lateral hypothalamic sites, those sites at which carbachol facilitates predatory aggression, with acetylcholine should facilitate predatory aggression; (2) direct, central stimulation of positive lateral hypothalamic sites with cholinesterase inhibitors should potentiate a facilitation brought about by stimulation with acetylcholine; (3) given that endogenous acetylcholine is always being released at cholinergic synapses, central stimulation of positive lateral hypothalamic sites with cholinesterase inhibitors might allow enough of an accumulation of acetylcholine to facilitate predatory aggression; (4) peritoneal injections of atropine sulfate (a cholinergic blocking agent which readily crosses the blood brain barrier) 60-75 min before testing should inhibit 'natural" predatory aggression or block a cholinergically-induced facilitation of predatory aggression; (5) peritoneal injections of atropine methyl-nitrate (a cholinergic blocking agent which does not readily cross the blood brain barrier) should result in less interference with 'natural" predator S' aggression and cholinergically-facilitated predatory aggression than atropine sulfate. That is, the blocking effects of atropine sulfate would reflect both its action in the brain and on peripheral structures. The blocking action of atropine methyl-nitrate would reflect only its action on peripheral structures; (6) direct, central stimulation of positive lateral hypothalamic sites with either atropine sulfate or atropine meth)lnitrate should suppress 'natural" predatory aggression. By testing these hypotheses then, this study asked questions concerning, (I) the mechanism responsible for the carbachol-induced facilitation of aggression; (2) the identity of the neurotransmitter which regulates predatory aggression at the lateral hypothalamus. METHODS
Twenty-eight rats selected from a population of Long Evans derived male hooded rats weighing between 275 and 400 g at the start of testing were used in this experiment. All of the selected rats were natural frog-killers. Six of the rats were, as well, natural mouse-killers. The 28 rats were stereotaxically implanted with chemical stimulation cannulas while under pentobarbital anesthesia. Each cannula, after Grossman 4, consisted of a 23 gauge outer guide cannula which was permanently implanted and a 30 gauge inner cannula which could be raised and lowered within the guide cannula, allowing for repeated stimulation of the same brain site. Each of the rats received 2 cannulas Brain Research, 20 (1970)409--424
411
CONTROL OF PREDATORY AGGRESSION
bilaterally aimed at the lateral hypothalamus. The coordinates were anterior-posterior 6.3-6.7, lateral 1.5-1.7, dorsal-ventral 7.3-7.7, from dura, with the incisor bar set 2.4 mm below the ear bar. Each rat was to be used in a later experiment and had 1-3 additional cannulas implanted at this time. Following a 3-5 day recovery period the subjects were retested for frog-killing and mouse-killing. A training period designed to stabilize attack and kill latencies was then initiated. All of the testing was conducted in each subject's home cage, a 10 in. x 7 in. x 7 in. metal cage with a plexiglass back which allowed for unobstructed observation of the subject. All observations were made from behind a oneway mirror. All subjects were maintained on an ad libitum food and water schedule throughout the experiment. The training procedure was as follows. On successive days each of the subjects was presented with 5 successive prey objects, frogs for the nonmouse-killers, mice for the mouse-killers. For each trial a metal barrier was inserted into each subject's home cage. This barrier isolated the subject in the back of the cage. The prey was then placed on the opposite side of the barrier. The removal of the barrier signaled the start of the trial. For each trial the latencies of attack, i.c., a bite on the body, and kill were measured with stopwatches and recorded. Either a kill or the passage of 6 min terminated the trial. The dead prey was removed immediately after the kill, the subject never being allowed to eat the prey. Each trial was separated by a 1 min inter-trial interval. For each nonmousekiller a final 6 min trial with a mouse was included. After 3-9 days of such training all subjects reached a stable level of performance. That is, the latencies of kill were not statistically different from one another for the last 3 days. Chemical stimulation treatments were then begun.
Cholinergic, anticholinergic and noradrenergic treatments Three different chemical stimulation treatments were administered twice each to each of the 28 subjects: (a) carbachol stimulation, (b) norepinephrine stimulation, (c) atropine application. For carbachol and norepinephrine stimulation, the inner cannula was removed from the outer guide cannula, packed with a 3 tap dose of the drug, and reinserted 7 min before the start of testing. For atropine application, the inner cannula packed with a 3 tap dose was inserted 12 min before the start of testing, removed 5 min before the start of testing, repacked with a 3 tap dose and reinserted 2 min before the start of testing. Microscopic analysis of cannulas had ascertained that 5-7 min was enough time for the drug to diffuse from the cannula. Thus the drug dosage, and hence the magnitude of the atropine effect could be gradually increased. This double stimulation method has been successfully used by Margules and Stein 1~. Atropine methylnitrate was used as preliminary work had indicated that it had more potent blocking effects than atropine sulfate. The drug dose was crudely quantified by the number of taps. The amount of drug injected per number of taps was determined in the following way. Ten empty cannulas were weighed twice each on a Mettler M5 microbalance. Each cannula was
Brain Research, 20 (1970) 409--424
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R.J. BANDLER
then packed, tapped once into a glass mortar in which carbachol had been finely ground, and reweighed. This procedure was repeated for 2 and 3 tap dosages. The dosages for one tap varied from I to 5 fzg, mean of 3.2 /~g; for 2 taps. 3 to 7 !~g, mean of 5.7 ug; for 3 taps. 3 to 10 ug, mean of 7.2 t~g. The rats, since they could be stimulated via two cannulas received one stimulation via one cannula first, the next chemical stimulation via the other cannula, and so on in an alternating fashion. The order of chemical stimulations was randomized with the condition that the same site should not be stimulated with the same drug on two successive chemical stimulation days. Control days intervened between successive chemical stimulation days. On control days the subjects were stimulated with sodium chloride or no chemical (sham). Thus. 48 h intervened between successive chemical stimulation treatments unless a drug effect persisted. If a drug effect persisted, the latencies of kill for the control days preceding and following the chemical day were statistically different, the rat received only control stimulation until performance stabilized. That is, there had to be no statistical difference between latencies of kill for two additional successive control days before chemical stimulation was resumed. The amount of food and water consumed was recorded for the 1 h period following each stimulation. The subjects were closely observed throughout the 1 h test period and the data from any day on which a subject displayed abnormal behavior (e.g. convulsions) were discarded.
Stimulation with acetylcholine and cholinesterase inhibitors Six subjects with positive hypothalamic placements were selected for this experiment. For those subjects with positive bilateral placements all of the testing was done via the 'best" implant, the one with the most significant carbachol-induced decrease in latency of kill. Four different chemical stimulation treatments were administered twice each to each subject, (a) stimulation with acetylcholine, (b) stimulation with acetylcholine mixed with physostigmine sulfate. (c) stimulation with physostigmine sulfate, (d) stimulation with neostigmine bromide. Again, in order to increase the magnitude of the drug effect, two doses were administered before the presentation of the first prey object. For the acetylcholine and acetylcholine mixed with physostigmine treatments, 3 tap doses were administered 12 rain and 2 min before the start of testing. For the physostigmine and neostigmine treatments the 2 doses were administered 30 min and 7 min before the start of testing. All of the chemical stimulation treatments were administered in a randomized order. Control stimulation days intervened between chemical stimulation days. Food and water consumptiol, monitoring of each subject's test behavior and control for the persistence of the drug effects were the same as above.
Cholinergic stimulation in combination with systemically administered blocking agents Six subjects with positive hypothalamic placements were selected for this
Brain Research, 20 (1970) 409-424
CONTROL OF PREDATORY AGGRESSION
413
experiment. A total of 8 stimulation treatments were administered twice each to each of the subjects: (al, a2) peritoneal injections of atropine sulfate, I0 and 25 mg/kg, (bb b2) peritoneal injections of atropine methyl-nitrate, 10 and 25 mg/kg; administered 60-75 min before, (c) carbachol stimulation and (d) control stimulation. As preliminary testing confirmed Grossman's 5 report that high doses of atropine sulfate (50 mg/kg) produce extremely irritable and hyper-reactive subjects, 10 and 25 mg/kg doses were employed. The I0 mg/kg dose produced neither irritability nor hyper-reactivity, while the 25 mg/kg dose produced some irritability. Atropine sulfate and atropine methyl-nitrate were dissolved in distilled water and injected in solutions of both 10 and 25 mg/ml. That is, th.e amount of solution injected was constant for each subject regardless of the dose. Carbachol and control stimulation procedures were the same as before. All treatments were administered in a randomized order with the condition that the same treatment not be administered on 2 successive experimental days. Two control days (72 h) intervened between successive experimental treatments. On half of the control days, the subjects received peritoneal injections of appropriate amounts of distilled water to determine if any of the behavioral effects were due solely to the injection procedure. At the end of testing each subject was stimulated with carbachol on a subsequent chemical stimulation day to assure that predatory aggression was still facilitated.
Histology At the end of the experimental testing the subjects were sacrificed and perfused. The brains were removed, sectioned on a freezing microtome at 50/,m and stained with cresyl violet. Microscopic inspection determined the location of each implant. RESULTS
Cholinergic and noradrenergic stimulation Effects on predatory aggression. Complete test results were obtained from 16 of the 28 subjects. The attrition represents a loss of 5 rats following surgery, 2 rats with inner-ear disease, 3 rats that became 'sporadic' frog-killers during training, and 2 rats that became convulsive and then weak during testing and were sacrificed. Evaluation of the data was based on correlated t tests, with the data of each trial of each chemical day compared with the same trial of the preceding and following control day. It is seen in Table I, that of the 16 subjects from which complete test results were obtained, carbachol stimulation of 13 hypothalamic sites (Nos. 20-46 in Table 1) produced a consistent and reliable facilitation of predatory aggression. Data from the 19 sites at which carbachol stimulation failed to facilitate predatory aggression are not presented. Table I also includes complete data from 10 hypothalamic sites (Nos. I-18 in Table 1) at which it was previously reported 1 that carbachol stimulation facilitated predatory aggression. Data from 8 additional sites at which carbachol stimulation failed to facilitate predatory aggression are not presented. Brain Research, 20 (1970) 409-~124
414
R.J.
BANDLER
TABLE 1 MEAN LATENCIES OF ATTACK AND KILL (IN SECONDS) FOR CARBACHOL-INDUCED FACILITATION OF PREDATORY AGGRESSION AT HYPOTHALAMUS
All t tests on 4 df, * P < 0.05, t = 2.78; ** P < 0.01, t ~ 4.60; *** P < 0.001, t = 8.61. Sites at which carbachol induced significant increases in water consumption are printed in italics. R and L refer to stimulation at the right or left implant. Abbreviations: Ca, carbachol stimulation; Ct, control stimulation. S
Attack latency Ca
Killlatency Ct
t
37.0 6.3 71.5 84.8 25.8
103.3 139.7 224.8 181.4 263.9
2.20 7.22** 4.15" 3.52* 5.53**
4.21 * 3.14" 6.52** 1.88 I 1.10"**
5.5 9.5 67.0 131.0 149.0
92.3 106.0 201.4 216.8 253.2
3.73* 2.40 7.75** 2.82* 5.15"*
193.5 262.5 253.0 114.0 111.0
2.52 6.51 ** 10.92":'* 3.10" 2.51
40.0 45.0 83.0 56.0 43.0
266.0 300.0 274.0 155.5 134.5
4.02* 6.64** 5.25** 2.73 2.71
37R 37L
164.0 11.0 13.0 46.0 35.0
360.0 41.0 31.0 281.0 244.0
2.85* 5.08** 2.78* 7.38** 5.15 * *
! 78.0 22.0 28.0 55.0 40.0
360.0 47.5 51.5 305.0 262.0
2.88* 3.22* 2.58 7.10"* 6.74" *
41L 46R 46L
24.0 29.5 34.0
71.0 72.3 52.0
2.81" 3.81 * 2.66
32.0 84.7 56.0
171.0 137.5 87.5
2.83* 3.84* 2.77
1R
2R 7R 9R 12R 15R
15L 17R
18R 18L
20L 26R 26L
27R 27L 30R
35R 35L
Ct
t
4.7 3.3 62.0 74.0 9.0
29.2 82.2 167.8 154.3 146.8
2.61 3.98* 8.13"* 3.38' 8.47**
1.0 5.0 48.5 108.0 67.7
76.8 74.5 168.0 137.3 197.2
7.0 34.0 31.0 17.0 32.0
Ca
F i v e o f the s u b j e c t s w i t h p o s i t i v e h y p o t h a l a m i c p l a c e m e n t s ( N o s .
I, 2, 20,
30 a n d 35) w e r e m o u s e - k i l l e r s . T h e r e m a i n i n g 11 s u b j e c t s w i t h p o s i t i v e h y p o t h a l a m i c p l a c e m e n t s were n o n m o u s e - k i l l e r s . S e v e n o f these 16 subjects, I m o u s e - k i l l e r and 6 nonmouse-killers, had positive bilateral hypothalamic placements. That
the c a r b a c h o l - i n d u c e d
f a c i l i t a t i o n o f a g g r e s s i o n was a f a c i l i t a t i o n o f
p r e d a t o r y a g g r e s s i o n was s u g g e s t e d by the s t e r e o t y p e d killing r e s p o n s e (the d i r e c t e d biting in the neck r e g i o n o f the prey), a n d the n o n - i r r i t a b i l i t y o f the subjects. A f t e r c h o l i n e r g i c s t i m u l a t i o n , as b e f o r e , the subjects w e r e easily h a n d l e d : o n e c o u l d p l a c e a finger in m o s t s u b j e c t s ' m o u t h s w i t h o u t f e a r o f b e i n g bitten. T h e c a r b a c h o l - i n d u c e d f a c i l i t a t i o n o f p r e d a t o r y a g g r e s s i o n was r e p l i c a t e d on, at least, 2 successive c h o l i n e r g i c s t i m u l a t i o n days at e a c h p o s i t i v e site. T h e r e v e r s i b i l i t y o f the d r u g effect was e v i d e n c e d by the less t h a n I0°//o o f the t i m e the d r u g effect persisted. Brain Research, 20 (1970) 409-424
CONTROL OF PREDATORY AGGRESSION
415
The same unilateral cholinergic stimulation which facilitated frog-killing, however, failed to elicit mouse-killing in 10 of the I I nonmouse-killers. The one exception, No. 17, attacked and killed mice on each of 3 carbachol stimulation days and failed to kill on all control days. Bilateral cholinergic stimulation of the 6 nonmousekillers with two positive hypothalamic implants failed as well to elicit mouse-killing. The fact that mouse-killing could be elicited in only one nonmouse-killer, when the facilitation of 'naturally occurring' predatory aggression was easily obtained, deserves special consideration. Whereas King and Hoebe110 report that the electrical stimulation of the lateral hypothalamus elicits mouse-killing in nonmousekillers, Karli et alp report that while electrical stimulation of the lateral hypothalamus elicits an immediate killing response in the natural mouse-killer, it 'never elicits any attack against a nearby mouse' in the nonmouse-killer. The failure to elicit mousekilling in the nonmouse-killer while easily facilitating the killing of prey previously killed is not peculiar, then, to direct chemical stimulation. The elicitation of the killing of prey not previously killed may be dependent on variables such as the strain of rat selected, the parameters of stimulation, the kind of aggression elicited, or the criterion used to classify the rat as a natural non-killer of certain prey. (See Addendum.) Histological examination, as seen in Fig. 1, showed the cannula tip locations for the sites at which predatory aggression was facilitated to be between the mammillothalamic tract and the optic tract, lateral to the fornix at the level of the ventromedial hypothalamus. In addition, the histology indicated that the 'best' predatory aggression points were consistently ventral to the fornix. Compare (in Table I) the carbachol-induced facilitation following stimulation of points ventral to the fornix, for example 2R, 18L, 26R, 26L, 35R, with points dorsal to the fornix, for example, IR, 18R, 27L, 30R and 41L. Anatomically, the positive hypothalamic sites of this study correspond well to those sites at which King and Hoebel l° elicited mouse-killing by electrical stimulation. Fig. 2 is a representative photomicrograph of a hypothalamic section showing the sites of the cannula tips for a subject with two positive hypothalamic placements. As previously reported 1, norepinephrine stimulation at only 2 of 10 positive (carbachol-sensitive) sites, I R and 17R, produced a statistically significant suppression of predatory aggression, increase in attack and/or kill latencies. Similarly, norepinephrine stimulation at only 2 of the 13 new positive placements produced a significant increase in latencies of attack (46R, P < 0.05, two-tailed) and kill (35R, P < 0.05, two-tailed; 46R, P < 0.05, two-tailed). Bilateral norepinephrine stimulation for 3 subjects (Nos. 26, 27 and 37) failed to suppress predatory aggression. Norepinephrine stimulation at each of the 27 negative (carbachol-insensitive) sites failed to suppress predatory aggression. Effects on consummatory behavior. It is seen in Table i that carbachol stimulation at I I of the 23 placements at which carbachol facilitated predatory aggression produced significant increases in water consumption, 3.5-7.0 ml over control levels. While not seen in Table I, carbachoi stimulation at 9 of the 27 placements at which carbachol failed to facilitate predatory aggression yielded carbachol-induced drinking. Brain Research, 20 (1970) 409-424
4t6
R . J . BANDLER
j,
Fig. 1. Sections A - H are from K6nig and KlippeP 1 (Figures 29b, 31-36b, 38b). Cannula tip locations for cholinergic facilitation of predatory aggression (dot), drinking (asterisk), predatory aggression and drinking (dot with asterisk), and no effect (triangle). The number beside each symbol refers to the subject.
is
O 7~
m
0
,-.] 0 ,<
0
0
0 -I
418
R.J. BANI)t.I~R
•
,,
.~
"
•
,
,
~"." .,"
,
,o
,
. .
¶
, •
Fig. 2. Representative photomicrograph of hypothalamic section showing sites of cannula tips f~r subject No. 46.
Carbachol stimulation at some hypoth.alamic sites, then, can both elicit drinking and facilitate predatory aggression. However, this is not necessarily the case, as 12 of the 23 hypothalamic aggression sites were specific to only aggression, and 9 of the 20 hypothalamic drinking sites yielded only to drinking• In accordance with these behavioral data, the histology (as seen in Fig. It indicated some anatomical separation of the hypothalamic neural elements which regulate drinking and predatory aggression. It is seen that those points which proved positive for only drinking are posterior to most of th.e predatory aggression points• Further, while most of the drinking points are dorsal to the fornix, it will be recalled that the 'best" aggression points were consistently ventral to the fornix. The behavioral and histological data suggest, then, that the lateral hypothalamic neural elements which regulate predatory aggression are distinct from those which regulate drinking. No other effects on consummatory behavior were elicited by any stimulation treatment. Stimulation with acet.vlcholine and cholinesterase inhibitors As is seen in the first row of Table II acetylcholine stimulation at the 6 positive hypothalamic sites (Nos. 20L, 26R, 27R, 35R. 37R and 46R) failed to elicit a reliable Brain Research, 20 (1970) 409~,24
CONTROL OF PREDATORYAGGRESSION
419
TABLE II MEAN LA'FENCIES OF ATTACK AND KILL (IN SECONDS) FOLLOWING EACH STIMULATION TREATMENT AT HYPOTHALAMUS
All t tests on 5 df; * P < 0.05; ** P < 0.01. Abbreviations: ACh, acetylcholine stimulation ; Phy, physostigmine stimulation; Neo, neostigmine stimulation; Ct, control stimulation; Ex, experimental condition. Condition (N = 6)
Attack latency
Kill latency
Ex
Ct
t
Ex
Ct
t
ACh ACh/Phy Phy Neo
101.7 72.3 80.7 83.7
130.0 126.1 129.5 125.3
0.69 2.84* 1.93 2.86*
147.0 104.5 123.2 117.8
182.5 185.7 185.8 171.5
1.96 5.60"* 2.10 2.97*
facilitation of predatory aggression. The elicited behavior could be described as sporadic, with a decrease in attack and kill latencies for only 2 or 3 of the presented prey. It is seen in row 2, however, that stimulation with acetylcholine when mixed with physostigmine did effect a reliable decrease of both latencies of attack (P < 0.05, two-tailed) and kill (P < 0.01, two-tailed). This decrease was neither as pronounced nor as dramatic as the decrease induced by carbachol stimulation. Rows 3 and 4 of Table I! indicate that direct, central stimulation with the cholinesterase inhibitors had mixed results. Physostigmine sulfate stimulation failed to elicit a significant facilitation of predatory aggression, but stimulation with neostigmine bromide significantly decreased both latencies of attack (P < 0.05, two-tailed) and kill (P < 0.05, two-tailed). A recent study by Margules and Stein la has reported effects following stimulation with neostigmine to be significantly greater than those following physostigmine stimulation. These results concur with theirs. That acetylcholine mixed with physostigmine facilitated predatory aggression, suggests that the failure of acetylcholine alone was due to the rapid rate of destruction of acetylcholine by cholinesterase. Both this finding and the facilitation of predatory aggression subsequent to direct stimulation with neostigmine bromide is strong evidence for the participation of acetylcholine in the regulation of predatory aggression at the lateral hypothalamus. Since 5 of the 6 placements tested were negative for carbachol-induced drinking, it was to be expected that none of the 4 stimulation treatments produced significant increases in drinking. There were, as well, no changes in food consumption. Cholinergic stimulation in combination with systemically administered blocking agents
It is seen in the first and third columns of Table III that peritoneal injections of either atropine sulfate (10 and 25 mg/kg) or atropine methyl-nitrate (10 and 25 Brain Research, 20 (1970) 409-424
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R.J. BANDLER
TABLE llI MEAN LATENCIESO1- ATTACK AND KILL (IN SECONDS) FOLLOWINGEACH COMBINED CENTRAL AND PERII'HERALSTIMULATIONTREATMEhT All t tests on 5 dr; * P < 0.05; * * P < 0.01 ; ** * P < 0.001. Significant differences between the placebo condition and the 4 experimental conditions are noted in the table. Abbreviations: Ca, carbachol stimulation; Ct, control stimulation; AS-10, atropine sulfate 10 mg/kg; AS-25, atropine sulfate 25 mg/kg; AMN-10, atropine methyl-nitrate 10 mg/kg; AMN-25, atropine methyl-nitrate 25 mg/kg. Co;Iditioa ( N -- 6)
Placebo AS-10 AMN-10 AS-25 AMN-25
Attack lateney
(A) (B) (C) (D)
Kill latency
Ct
Ca
Ct
Ca
48.8 224.2*** 123.6" 187.6"* 158.5"*
-104.1" 21.8 146.3"* 75.6
87.8 305.8*** 178.5' 263.9*** 192.3*
-196.5"* 58.5 261.5'* 88.7
3.01 * 2.51 *
4.09** 2.62*
!
A vs. B C vs. D
3.24* 0.78
6.93*** 4.33**
mg/kg) resulted in a t t a c k and kill latencies significantly greater than those in the placebo condition. It is also seen in the first a n d third c o l u m n s t h a t at the l0 m g ] k g dose, a t r o p i n e methyl-nitrate interfered less with the latencies o f a t t a c k ( P < 0.05, two-tailed) and kill ( P < 0.01, two-tailed) than the same dose o f a t r o p i n e sulfate; while at the 25 mg/kg dose, a t r o p i n e m e t h y l - n i t r a t e p r o v e d a less effective suppressant than atropine sulfate for latencies o f kill ( P < 0.05, two-tailed) but not a t t a c k (t = 0.78, N.S.). Since the injected a t r o p i n e m e t h y l - n i t r a t e w o u l d n o t be expected to pass the b l o o d brain barrier in any a p p r e c i a b l e a m o u n t s , the differential suppression o f ' n a t u r a l ' p r e d a t o r y aggression is a t t r i b u t a b l e to the central action o f the injected a t r o p i n e sulfate and is viewed as evidence f a v o r i n g the hypothesis o f the cholinergic regulation o f p r e d a t o r y aggression. That a t r o p i n e sulfate was a m o r e effective s u p p r e s s a n t o f p r e d a t o r y aggression at the 10 mg/kg dose than at the 25 mg/kg dose was a surprising result. A f t e r the 25 m g / k g i n j e c t i o n the subjects seemed s o m e w h a t i r r i t a b l e and were difficult to handle. S u c h an increased irritability m a y have f a c i l i t a t e d their aggressiveness t o w a r d the p r e y a n d thus lessened the suppressive effects o f the a t r o p i n e sulfate on a t t a c k and kill latencies. W i t h respect to the c a r b a c h o l - i n d u c e d facilitation o f p r e d a t o r y aggression, c o l u m n s 2 a n d 4 reveal t h a t the peritoneal injections o f a t r o p i n e sulfate (10 a n d 25 m g / k g ) not only blocked the c a r b a c h o l - i n d u c e d facilitation, b u t signific a n t l y increased the a t t a c k and kill latencies. The p e r i t o n e a l injections o f a t r o p i n e methyl-nitrate, while they blocked the c a r b a c h o l - i n d u c e d facilitation, did not signifiBrain Research, 20 (1970) 409-424
CONTROL OF PREDATORY A G G R E ~ I O N
421
cantly increase either attack or kill latencies. The relevant comparisons, as seen in columns 2 and 4, show that the 10 mg/kg dose of atropine sulfate more effectively interfered with the carbachol-induced facilitation of attack (P < 0.05, two-tailed) and kill latencies (P < 0.001, two-tailed) than the 10 mg/kg dose of atropine methyl-nitrate. Similarly, at the 25 mg/kg dose, the carbachol-induced decrease in latencies of attack (P < 0.05, two-tailed) and kill (P < 0.01, two-tailed) are more interfered with by atropine sulfate injection than by atropine methyl-nitrate injection. Since the differential effects are thought to reflect the unique action of atropine sulfate on the brain, the differential blockage of the hypothalamic, carbacholinduced facilitation of predatory aggression suggests that the carbachol-induced facilitation is due to the cholinergic action of the drug in the brain. Injections of both atropine sulfate (10 and 25 mg/kg) and atropine methylnitrate (10 and 25 mg/kg) blocked all carbachol-induced drinking. Atropine treatment
Direct, central atropine application produced a consistent and reliable suppression of attack ($35R, P < 0.01, two-tailed; $46R, P < 0.05, two-tailed) and kill latencies ($35R, P < 0.05, two-tailed ; $46R, P < 0.001, two-tailed) at only 2 of the 13 positive placements tested and none of the 19 negative placements tested. Bilateral atropine application for 3 subjects (Nos. 26, 27 and 37) also failed to suppress predatory aggression. It is probably not coincidental that both atropine and norepinephrine suppressed predatory aggression at the same two sites (35R and 46R). It is clear, however, from the lack of inhibition at all but these two sites that both the atropine and norepinephrine-induced inhibitions reflect more than a direct inhibitory influence on lateral hypothalamic synapses. Further, it is unlikely that the inhibitions are due only to spread of the chemicals to distal sites, as both atropine and norepinephrine application failed to suppress predatory aggression at all negative sites tested. The fact that neither atropine nor norepinephrine administration at the positive contralateral hypothalamic placement for each of the subjects (35L and 46L) suppressed predatory aggression suggests that the inhibitions do not reflect individual differences. One remaining possibility is that the inhibitions reflect a simultaneous drug effect at the lateral hypothalamus and by unique diffusion to other distal sites. No conjectures are offered concerning other possible sites. Atropine application had no effect on consummatory behavior. DISCUSSION
It is clear from the results of the study that the lateral hypothalamic regulation of predatory aggression is sensitive to cholinergic stimulation. The principle question which arises is what the data can tell us about the neurotransmitter which regulates predatory aggression at lateral hypothalamic synapses. The data of this study, in particular the differential inhibition of the carbacholBrain Research, 20 (1970)409-424
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R.J. RANI)I+t R
induced facilitation of predatory aggression by the two systemically administercd cholinolytic agents, atropine sulfate and atropine methyl-nitrate, leave little doubt that the carbachol effect is due to the cholinergic action of the drug ill the brain. Further, the data favor the view that the action of the drug is a local one and does not reflect spread of the drug to other brain sites as has been suggested for dircct cholinergic and anticholinergic stimulation effects on water consumption in the rat 1G and direct cholinergic stimulation effects on affective aggression in the cat 3. That the facilitation is not the result of spread of the drug to the third ventricle and then by diffusion to other sites is suggested by the fact that direct cholinergic stimulation of brain sites medial to the fornix, but in the same anterior-posterior and dorsalventral planes as the positive aggression sites, has proved negative for predatory aggression. That the facilitation is not due to spread of the drug upward along the cannula shaft to dorsal sites is suggested by the fact that cholinergic and electrical stimulation t° of sites dorsal to the positivc sites have proved negative for aggression. In addition, it will be recalled that the 'better" aggression sites were ventral to the fornix, not dorsal to it. That the effects are not the result of extensive spread even within the hypothalamus is suggested by the fact that sites negative for predatory aggression or specific to drinking are often in close proximity to positive aggression sites. Finally, the fact that predatory mouse-killing has been elicited by electrical stimulation 10 of the +same' lateral hypothalamic area, which has proved positive for the cholinergic facilitation of predatory aggression, suggests that there is no need to postulate extensive drug diffusion in order to explain the facilitation of predatory aggression following cholinergic stimulation. The data presented, then, are certainly compatible with the notion that acetylcholine is involved in the regulation of predatory aggression at lateral hypothalamic synapses. In particular, the finding that predatory aggression is facilitated by direct central stimulation of positive hypothalamic sites with either neostigmine bromide or acetylcholine mixed with physostigmine sulfate rules in favor of acetylcholine involvement. The finding that the central application of atropine to positive hypothalamic sites failed to suppress predatory aggression is, however, inconsistent with this conclusion. Two explanations, the blocking action of the injected atropine being too temporary, or the diffusion of the atropine within the anterior-lateral h.ypothalamus being insufficient to block enough relevant neural elements, are unlikely explanations considering the amount of drug injected by the double-stimulation technique, and the failure of bilateral as well as unilateral atropine application. The data are compatible, however, with at least one alternative explanation: there might be such a redundancy of predation related neural circuitry in the brain that a functional lesion (cholinergic blockade) of the anterior-lateral hypothalamus is not disruptive enough to suppress predatory aggression. Karli and Vergnes's 8 report that only extensive, rostral to caudal, bilateral hypothalamic lesions block predatory mouse-killing is supportive of this conjecture. That is, it suggests that more extensive hypothalamic interference than a blockade of only anterior-lateral hypothalamic synapses is necessary to inhibit predatory aggression. This suggests the Brain Research,
20 (1970) ~09-424
CONTROL OF PREDATORY AGGRESSION
423
involvement of an additional lateral hypothalamic area in predatory aggression, possibly posterior to the level of the ventromedial hypothalamus (an area not extensively explored in this study). The involvement of just such an area in the regulation of predatory aggression is supported by the finding that electrical stimulation of posterior-lateral hypothalamus facilitates mouse-killing in natural mouse-killers (DeSisto, personal communication). It should be noted, however, that the failure of atropine to suppress predatory aggression at those hypothalamic sites at which carbachol facilitated predatory aggression suggests that the unique blocking effects of the systemically administered atropine sulfate must reflect anticholinergic action at sites other than, or in addition to, the anterior-lateral hypothalamus. This suggests, of course, the hypothesis of the cholinergic regulation of predatory aggression in other parts of the central nervous system. The results of this study, then, are interpreted to mean that acetyicholine is involved in the regulation of predatory aggression at, no less than, the anteriorlateral hypothalamus. ADDENDUM
Since the acceptance of this paper for publication, Smith, King and Hoebel (Science, 167 (1970) 900-901) reported that cholinergic stimulation (carbachol,
neostigmine) of the lateral hypothalamus elicited mouse-killing in nonmouse-killers. However, in contrast to the 3-10/zg doses used in this study, Smith et al. used doses of 50/zg 4- 25/~g. This suggests that their results are due to stimulation effects at both the lateral hypothalamus, and by spread of the drug, because of the high doses, to other brain structures. That such spread is in fact critical to their results is indicated by the long latencies for the reported drug effects: the mean elicitation of kill latency for carbachol stimulation was 45 min, for neostigmine stimulation 2.75 h. These latencies contrast with the 7 rain latency for the carbachol-induced facilitation of aggression and the 30 min latency for the neostigmine-induced facilitation of aggression reported in this paper. SUMMARY
Direct central stimulation, via chronically implanted cannulas, of sites in the lateral hypothalamus with either carbachol, acetylcholine mixed with physostigmine, or neostigmine, facilitated frog-killing and mouse-killing in natural killer rats. The cholinergic stimulation was believed to have affected predatory aggression because of (a) the stereotyped nature of the killing response, (b) the lack of increased irritability in nearly all subjects, (c) the neuroanatomical correspondence to the hypothalamic areas in rat and cat which, when stimulated electrically, elicit predatory aggression. These results, as well as the effects of systemically administered cholinergic blocking agents, were found to be compatible with the hypothesis that acetylcholine is involved in the control of predatory aggression at the lateral hypothalamus. Brain Research, 20 (1970)409--424
424
R. J. BANDLER The study also indicated that the lateral h y p o t h a l a m i c neural elements which
regulate predatory aggression are distinct from those which regulate drinking, although both are sensitive to cholinergic stimulation. ACKNOWLEDGEMENTS I am indebted to Dr. K. E. M o y e r for his guidance, s u p p o r t and e n c o u r a g e m e n t of this research. Dr. L. E. Jarrard c o n t r i b u t e d helpful advice and criticism. Dr. C. C. Chi and Dr. J. P. F l y n n reviewed the manuscript. 1 wish to t h a n k Fay G o m e s and Mildred Groves for p r e p a r a t i o n of the microscope slides. This research was supported by N a t i o n a l Science F o u n d a t i o n G r a n t GB 6652 awarded to Dr. K. E. Moyer, and was completed while R.J.B. was a predoctoral fellow in Psychology of C a r n e g i e - M e l l o n University, Pittsburgh, Pa.. U.S.A.
REFERENCES 1 BANDLER, R. J.. Facilitation of aggressive behavior in rat by direct cholinergic stimulation of the hypothalamus, Nature (Lond.), 224 0969) I035-1036. 2 BANDLER, R. J., AND MOYER, K. E., The objects naturally attacked by the rat, in preparation. 3 BAXTER, B. L., Comparison of behavioral effects of electrical or chemical stimulation applied at the same brain loci, Exp. Neurol., 19 (1967) 412-430. 4 GROSSMAN,S. P., Direct adrenergic and cholinergic stimulation of hypothalamic mechanisms, Amer. J. Physiol., 202 (1962) 872-882. 5 GROSSMAN, S. P., Effects of adrenergic and cholinergic blocking agents on hypothalamic mechanisms, Amer. J. Physiol., 202 0962) 1230-1236. 6 HUTCHINSON,R. R., AND RENFREW,J. W., Stalking attack and eating behavior elicited from the same sites in the hypothalamus, J. comp. physiol. Psycho/., 61 (1966) 360-367. 7 KARL1, P., The Norway rat's killing response to the white mouse, Behaviour, I0 (1956) 81-103. 8 KARLI, P., ET VERGNES, M., Dissociation exp~rimentale du comportement d'agression intersp6cifique rat-souris et du comportement alimentaire, C. R. Soc. Biol. (Paris), 158 (1964) 650-653. 9 KARLI, P., VERGNES, M., AND DIDIERGEORGES,F., Rat-mouse interspecific aggressive behavior and its manipulation by brain ablation and brain stimulation. In E. B. SIc~ AND S. GARATrIM (Eds.), Biology of Aggressive Behavior, Excerpta Medica Foundation, Amsterdam, 1969, pp. 47-55. 10 KING, M. B., AND HOEaEL, B. G., Killing elicited by brain stimulation in rat, Commun. Behav. Biol., 2 (1968) 173-177. 11 K/3NIG,J. F. R., AND KLIPPEL, R. A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, Md., 1963. 12 LEVINSON,P. K., AND FLVr~N,J. P., The objects attacked by cats during stimulation of the hypothalamus, Anita. Behav., 13 (1965) 217-220. 13 MARGULES,D. L., AND STEIN, L., Cholinergic synapses in the ventromedial hypothalamus for the suppression of operant behavior by punishment and satiety, J. eomp. physiol. Psychol., 67 (1969) 327-335. 14 MoveR, K. E., Kinds of aggression and their physiological basis, Commun. Behav. Biol., 2 (1968) 65-87. 15 ROaER'rS,W. W., Ar,~DKIESS, H. O., Motivational properties of hypothalamic aggression in cats, J. comp. physiol. Psychol., 57 (1964) 187-193. 16 RotrrrESBERG, A., Drinking induced by carbachol: thirst or ventricular modification, Science, 157 (1967) 838-839. 17 WASMAN, M., A~D FLVNN, J. P., Directed attack elicited from hypothalamus, Arch. Neurol. (Chic.), 6 (1962) 220-227. Brain Research, 20 (1970) 409-424
409
CHOLINERGIC SYNAPSES IN THE LATERAL HYPOTHALAMUS FOR THE CONTROL OF PREDATORY AGGRESSION IN THE RAT
R I C H A R D J. BANDLER, JR.
Yale University School of Medicine, Department of Psychiatry, Connecticut Mental Health Center, New Haven, Conn. 06519 (U.S.A.) (Accepted December 15th, 1969)
INTRODUCTION
Predatory aggression is a most distinct kind of aggression. The aggressive response whether naturally-occurring or electrically-elicited is both stereotyped and stimulus specific. Karli 7 in the rat and Wasman and Flynn ~7 in the cat have identified the aggressive response by both the lack of affective display and the characteristic biting directed at the neck region of the prey. In the cat, the stimulus specific character of the aggressive response is indicated by Wasman and Flynn's 17 report that the electrically-elicited aggressive response, while fatal to the rat, will not result in an attack on the experimenter. Levinson and Flynn 1~ have further shown that the elicited aggressive response rapidly deteriorates as the prey object changes from anesthetized rat, to stuffed toy dog, to foam rubber block. A similar stimulus specificity is present in the rat. Bandler and Moyer2 have reported that while 95 ~ of all tested male hooded rats kill frogs or turtles, less than 6 0 ~ kill baby chicks, and only 15~ kill white mice. Nearly all of these rats are quite tame and easily handled by the experimenter. The limited number of stimuli capable of eliciting the aggressive response and the stereotyped nature of the response, then, make predatory aggression unconfusable with other kinds of aggression 14. The participation of the lateral hypothalamus in the regulation of predatory aggression is now well established. Wasman and Flynn 17, Roberts and Kiess 15, and Hutchinson and Renfrew 6 have reported that electrical stimulation of the lateral hypothalamus of the cat elicits predatory aggression. Electrical stimulation of the lateral hypothalamus of the rat has both facilitated9 and elicited 10 predatory aggression. In addition, Karli and Vergnes s have reported that extensive bilateral lesions of the lateral hypothalamus block predatory mouse-killing in natural killer rats. Little is known, however, about the neurochemical factors involved in the facilitation and inhibition of predatory aggression. Earlier research by Bandler 1 found that direct cholinergic (carbachoi) stimulation of the lateral hypothalamus facilitated predatory aggression in natural killer rats. Controls for osmotic effects, Brain Research, 20 (1970) 409-424
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R.J. BANI)LER
non-specific neural effects, and changes in general activity level failed to duplicate the carbachol effect, suggesting that the carbachol-induced facilitation of predator) aggression might be due to the cholincrgic action of the drug in the hypothalamus. This suggested, that the regulation of predator)' aggression at the lateral hypothalamus was cholinergic: acetylcholine functioned to transmit nervous impulses across the relevant synapses and was then promptly destroyed by cholincsterase. To test this hypothesis a number of predictions were made concerning the effect of central stimulation with acetylcholine, and cholinesterase inhibitors, and central and peripheral stimulation with cholinergic blocking agents: (I) direct. central stimulation of positive lateral hypothalamic sites, those sites at which carbachol facilitates predatory aggression, with acetylcholine should facilitate predatory aggression; (2) direct, central stimulation of positive lateral hypothalamic sites with cholinesterase inhibitors should potentiate a facilitation brought about by stimulation with acetylcholine; (3) given that endogenous acetylcholine is always being released at cholinergic synapses, central stimulation of positive lateral hypothalamic sites with cholinesterase inhibitors might allow enough of an accumulation of acetylcholine to facilitate predatory aggression; (4) peritoneal injections of atropine sulfate (a cholinergic blocking agent which readily crosses the blood brain barrier) 60-75 min before testing should inhibit 'natural" predatory aggression or block a cholinergically-induced facilitation of predatory aggression; (5) peritoneal injections of atropine methyl-nitrate (a cholinergic blocking agent which does not readily cross the blood brain barrier) should result in less interference with 'natural" predator S' aggression and cholinergically-facilitated predatory aggression than atropine sulfate. That is, the blocking effects of atropine sulfate would reflect both its action in the brain and on peripheral structures. The blocking action of atropine methyl-nitrate would reflect only its action on peripheral structures; (6) direct, central stimulation of positive lateral hypothalamic sites with either atropine sulfate or atropine meth)lnitrate should suppress 'natural" predatory aggression. By testing these hypotheses then, this study asked questions concerning, (I) the mechanism responsible for the carbachol-induced facilitation of aggression; (2) the identity of the neurotransmitter which regulates predatory aggression at the lateral hypothalamus. METHODS
Twenty-eight rats selected from a population of Long Evans derived male hooded rats weighing between 275 and 400 g at the start of testing were used in this experiment. All of the selected rats were natural frog-killers. Six of the rats were, as well, natural mouse-killers. The 28 rats were stereotaxically implanted with chemical stimulation cannulas while under pentobarbital anesthesia. Each cannula, after Grossman 4, consisted of a 23 gauge outer guide cannula which was permanently implanted and a 30 gauge inner cannula which could be raised and lowered within the guide cannula, allowing for repeated stimulation of the same brain site. Each of the rats received 2 cannulas Brain Research, 20 (1970)409--424
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CONTROL OF PREDATORY AGGRESSION
bilaterally aimed at the lateral hypothalamus. The coordinates were anterior-posterior 6.3-6.7, lateral 1.5-1.7, dorsal-ventral 7.3-7.7, from dura, with the incisor bar set 2.4 mm below the ear bar. Each rat was to be used in a later experiment and had 1-3 additional cannulas implanted at this time. Following a 3-5 day recovery period the subjects were retested for frog-killing and mouse-killing. A training period designed to stabilize attack and kill latencies was then initiated. All of the testing was conducted in each subject's home cage, a 10 in. x 7 in. x 7 in. metal cage with a plexiglass back which allowed for unobstructed observation of the subject. All observations were made from behind a oneway mirror. All subjects were maintained on an ad libitum food and water schedule throughout the experiment. The training procedure was as follows. On successive days each of the subjects was presented with 5 successive prey objects, frogs for the nonmouse-killers, mice for the mouse-killers. For each trial a metal barrier was inserted into each subject's home cage. This barrier isolated the subject in the back of the cage. The prey was then placed on the opposite side of the barrier. The removal of the barrier signaled the start of the trial. For each trial the latencies of attack, i.c., a bite on the body, and kill were measured with stopwatches and recorded. Either a kill or the passage of 6 min terminated the trial. The dead prey was removed immediately after the kill, the subject never being allowed to eat the prey. Each trial was separated by a 1 min inter-trial interval. For each nonmousekiller a final 6 min trial with a mouse was included. After 3-9 days of such training all subjects reached a stable level of performance. That is, the latencies of kill were not statistically different from one another for the last 3 days. Chemical stimulation treatments were then begun.
Cholinergic, anticholinergic and noradrenergic treatments Three different chemical stimulation treatments were administered twice each to each of the 28 subjects: (a) carbachol stimulation, (b) norepinephrine stimulation, (c) atropine application. For carbachol and norepinephrine stimulation, the inner cannula was removed from the outer guide cannula, packed with a 3 tap dose of the drug, and reinserted 7 min before the start of testing. For atropine application, the inner cannula packed with a 3 tap dose was inserted 12 min before the start of testing, removed 5 min before the start of testing, repacked with a 3 tap dose and reinserted 2 min before the start of testing. Microscopic analysis of cannulas had ascertained that 5-7 min was enough time for the drug to diffuse from the cannula. Thus the drug dosage, and hence the magnitude of the atropine effect could be gradually increased. This double stimulation method has been successfully used by Margules and Stein 1~. Atropine methylnitrate was used as preliminary work had indicated that it had more potent blocking effects than atropine sulfate. The drug dose was crudely quantified by the number of taps. The amount of drug injected per number of taps was determined in the following way. Ten empty cannulas were weighed twice each on a Mettler M5 microbalance. Each cannula was
Brain Research, 20 (1970) 409--424
412
R.J. BANDLER
then packed, tapped once into a glass mortar in which carbachol had been finely ground, and reweighed. This procedure was repeated for 2 and 3 tap dosages. The dosages for one tap varied from I to 5 fzg, mean of 3.2 /~g; for 2 taps. 3 to 7 !~g, mean of 5.7 ug; for 3 taps. 3 to 10 ug, mean of 7.2 t~g. The rats, since they could be stimulated via two cannulas received one stimulation via one cannula first, the next chemical stimulation via the other cannula, and so on in an alternating fashion. The order of chemical stimulations was randomized with the condition that the same site should not be stimulated with the same drug on two successive chemical stimulation days. Control days intervened between successive chemical stimulation days. On control days the subjects were stimulated with sodium chloride or no chemical (sham). Thus. 48 h intervened between successive chemical stimulation treatments unless a drug effect persisted. If a drug effect persisted, the latencies of kill for the control days preceding and following the chemical day were statistically different, the rat received only control stimulation until performance stabilized. That is, there had to be no statistical difference between latencies of kill for two additional successive control days before chemical stimulation was resumed. The amount of food and water consumed was recorded for the 1 h period following each stimulation. The subjects were closely observed throughout the 1 h test period and the data from any day on which a subject displayed abnormal behavior (e.g. convulsions) were discarded.
Stimulation with acetylcholine and cholinesterase inhibitors Six subjects with positive hypothalamic placements were selected for this experiment. For those subjects with positive bilateral placements all of the testing was done via the 'best" implant, the one with the most significant carbachol-induced decrease in latency of kill. Four different chemical stimulation treatments were administered twice each to each subject, (a) stimulation with acetylcholine, (b) stimulation with acetylcholine mixed with physostigmine sulfate. (c) stimulation with physostigmine sulfate, (d) stimulation with neostigmine bromide. Again, in order to increase the magnitude of the drug effect, two doses were administered before the presentation of the first prey object. For the acetylcholine and acetylcholine mixed with physostigmine treatments, 3 tap doses were administered 12 rain and 2 min before the start of testing. For the physostigmine and neostigmine treatments the 2 doses were administered 30 min and 7 min before the start of testing. All of the chemical stimulation treatments were administered in a randomized order. Control stimulation days intervened between chemical stimulation days. Food and water consumptiol, monitoring of each subject's test behavior and control for the persistence of the drug effects were the same as above.
Cholinergic stimulation in combination with systemically administered blocking agents Six subjects with positive hypothalamic placements were selected for this
Brain Research, 20 (1970) 409-424
CONTROL OF PREDATORY AGGRESSION
413
experiment. A total of 8 stimulation treatments were administered twice each to each of the subjects: (al, a2) peritoneal injections of atropine sulfate, I0 and 25 mg/kg, (bb b2) peritoneal injections of atropine methyl-nitrate, 10 and 25 mg/kg; administered 60-75 min before, (c) carbachol stimulation and (d) control stimulation. As preliminary testing confirmed Grossman's 5 report that high doses of atropine sulfate (50 mg/kg) produce extremely irritable and hyper-reactive subjects, 10 and 25 mg/kg doses were employed. The I0 mg/kg dose produced neither irritability nor hyper-reactivity, while the 25 mg/kg dose produced some irritability. Atropine sulfate and atropine methyl-nitrate were dissolved in distilled water and injected in solutions of both 10 and 25 mg/ml. That is, th.e amount of solution injected was constant for each subject regardless of the dose. Carbachol and control stimulation procedures were the same as before. All treatments were administered in a randomized order with the condition that the same treatment not be administered on 2 successive experimental days. Two control days (72 h) intervened between successive experimental treatments. On half of the control days, the subjects received peritoneal injections of appropriate amounts of distilled water to determine if any of the behavioral effects were due solely to the injection procedure. At the end of testing each subject was stimulated with carbachol on a subsequent chemical stimulation day to assure that predatory aggression was still facilitated.
Histology At the end of the experimental testing the subjects were sacrificed and perfused. The brains were removed, sectioned on a freezing microtome at 50/,m and stained with cresyl violet. Microscopic inspection determined the location of each implant. RESULTS
Cholinergic and noradrenergic stimulation Effects on predatory aggression. Complete test results were obtained from 16 of the 28 subjects. The attrition represents a loss of 5 rats following surgery, 2 rats with inner-ear disease, 3 rats that became 'sporadic' frog-killers during training, and 2 rats that became convulsive and then weak during testing and were sacrificed. Evaluation of the data was based on correlated t tests, with the data of each trial of each chemical day compared with the same trial of the preceding and following control day. It is seen in Table I, that of the 16 subjects from which complete test results were obtained, carbachol stimulation of 13 hypothalamic sites (Nos. 20-46 in Table 1) produced a consistent and reliable facilitation of predatory aggression. Data from the 19 sites at which carbachol stimulation failed to facilitate predatory aggression are not presented. Table I also includes complete data from 10 hypothalamic sites (Nos. I-18 in Table 1) at which it was previously reported 1 that carbachol stimulation facilitated predatory aggression. Data from 8 additional sites at which carbachol stimulation failed to facilitate predatory aggression are not presented. Brain Research, 20 (1970) 409-~124
414
R.J.
BANDLER
TABLE 1 MEAN LATENCIES OF ATTACK AND KILL (IN SECONDS) FOR CARBACHOL-INDUCED FACILITATION OF PREDATORY AGGRESSION AT HYPOTHALAMUS
All t tests on 4 df, * P < 0.05, t = 2.78; ** P < 0.01, t ~ 4.60; *** P < 0.001, t = 8.61. Sites at which carbachol induced significant increases in water consumption are printed in italics. R and L refer to stimulation at the right or left implant. Abbreviations: Ca, carbachol stimulation; Ct, control stimulation. S
Attack latency Ca
Killlatency Ct
t
37.0 6.3 71.5 84.8 25.8
103.3 139.7 224.8 181.4 263.9
2.20 7.22** 4.15" 3.52* 5.53**
4.21 * 3.14" 6.52** 1.88 I 1.10"**
5.5 9.5 67.0 131.0 149.0
92.3 106.0 201.4 216.8 253.2
3.73* 2.40 7.75** 2.82* 5.15"*
193.5 262.5 253.0 114.0 111.0
2.52 6.51 ** 10.92":'* 3.10" 2.51
40.0 45.0 83.0 56.0 43.0
266.0 300.0 274.0 155.5 134.5
4.02* 6.64** 5.25** 2.73 2.71
37R 37L
164.0 11.0 13.0 46.0 35.0
360.0 41.0 31.0 281.0 244.0
2.85* 5.08** 2.78* 7.38** 5.15 * *
! 78.0 22.0 28.0 55.0 40.0
360.0 47.5 51.5 305.0 262.0
2.88* 3.22* 2.58 7.10"* 6.74" *
41L 46R 46L
24.0 29.5 34.0
71.0 72.3 52.0
2.81" 3.81 * 2.66
32.0 84.7 56.0
171.0 137.5 87.5
2.83* 3.84* 2.77
1R
2R 7R 9R 12R 15R
15L 17R
18R 18L
20L 26R 26L
27R 27L 30R
35R 35L
Ct
t
4.7 3.3 62.0 74.0 9.0
29.2 82.2 167.8 154.3 146.8
2.61 3.98* 8.13"* 3.38' 8.47**
1.0 5.0 48.5 108.0 67.7
76.8 74.5 168.0 137.3 197.2
7.0 34.0 31.0 17.0 32.0
Ca
F i v e o f the s u b j e c t s w i t h p o s i t i v e h y p o t h a l a m i c p l a c e m e n t s ( N o s .
I, 2, 20,
30 a n d 35) w e r e m o u s e - k i l l e r s . T h e r e m a i n i n g 11 s u b j e c t s w i t h p o s i t i v e h y p o t h a l a m i c p l a c e m e n t s were n o n m o u s e - k i l l e r s . S e v e n o f these 16 subjects, I m o u s e - k i l l e r and 6 nonmouse-killers, had positive bilateral hypothalamic placements. That
the c a r b a c h o l - i n d u c e d
f a c i l i t a t i o n o f a g g r e s s i o n was a f a c i l i t a t i o n o f
p r e d a t o r y a g g r e s s i o n was s u g g e s t e d by the s t e r e o t y p e d killing r e s p o n s e (the d i r e c t e d biting in the neck r e g i o n o f the prey), a n d the n o n - i r r i t a b i l i t y o f the subjects. A f t e r c h o l i n e r g i c s t i m u l a t i o n , as b e f o r e , the subjects w e r e easily h a n d l e d : o n e c o u l d p l a c e a finger in m o s t s u b j e c t s ' m o u t h s w i t h o u t f e a r o f b e i n g bitten. T h e c a r b a c h o l - i n d u c e d f a c i l i t a t i o n o f p r e d a t o r y a g g r e s s i o n was r e p l i c a t e d on, at least, 2 successive c h o l i n e r g i c s t i m u l a t i o n days at e a c h p o s i t i v e site. T h e r e v e r s i b i l i t y o f the d r u g effect was e v i d e n c e d by the less t h a n I0°//o o f the t i m e the d r u g effect persisted. Brain Research, 20 (1970) 409-424
CONTROL OF PREDATORY AGGRESSION
415
The same unilateral cholinergic stimulation which facilitated frog-killing, however, failed to elicit mouse-killing in 10 of the I I nonmouse-killers. The one exception, No. 17, attacked and killed mice on each of 3 carbachol stimulation days and failed to kill on all control days. Bilateral cholinergic stimulation of the 6 nonmousekillers with two positive hypothalamic implants failed as well to elicit mouse-killing. The fact that mouse-killing could be elicited in only one nonmouse-killer, when the facilitation of 'naturally occurring' predatory aggression was easily obtained, deserves special consideration. Whereas King and Hoebe110 report that the electrical stimulation of the lateral hypothalamus elicits mouse-killing in nonmousekillers, Karli et alp report that while electrical stimulation of the lateral hypothalamus elicits an immediate killing response in the natural mouse-killer, it 'never elicits any attack against a nearby mouse' in the nonmouse-killer. The failure to elicit mousekilling in the nonmouse-killer while easily facilitating the killing of prey previously killed is not peculiar, then, to direct chemical stimulation. The elicitation of the killing of prey not previously killed may be dependent on variables such as the strain of rat selected, the parameters of stimulation, the kind of aggression elicited, or the criterion used to classify the rat as a natural non-killer of certain prey. (See Addendum.) Histological examination, as seen in Fig. 1, showed the cannula tip locations for the sites at which predatory aggression was facilitated to be between the mammillothalamic tract and the optic tract, lateral to the fornix at the level of the ventromedial hypothalamus. In addition, the histology indicated that the 'best' predatory aggression points were consistently ventral to the fornix. Compare (in Table I) the carbachol-induced facilitation following stimulation of points ventral to the fornix, for example 2R, 18L, 26R, 26L, 35R, with points dorsal to the fornix, for example, IR, 18R, 27L, 30R and 41L. Anatomically, the positive hypothalamic sites of this study correspond well to those sites at which King and Hoebel l° elicited mouse-killing by electrical stimulation. Fig. 2 is a representative photomicrograph of a hypothalamic section showing the sites of the cannula tips for a subject with two positive hypothalamic placements. As previously reported 1, norepinephrine stimulation at only 2 of 10 positive (carbachol-sensitive) sites, I R and 17R, produced a statistically significant suppression of predatory aggression, increase in attack and/or kill latencies. Similarly, norepinephrine stimulation at only 2 of the 13 new positive placements produced a significant increase in latencies of attack (46R, P < 0.05, two-tailed) and kill (35R, P < 0.05, two-tailed; 46R, P < 0.05, two-tailed). Bilateral norepinephrine stimulation for 3 subjects (Nos. 26, 27 and 37) failed to suppress predatory aggression. Norepinephrine stimulation at each of the 27 negative (carbachol-insensitive) sites failed to suppress predatory aggression. Effects on consummatory behavior. It is seen in Table i that carbachol stimulation at I I of the 23 placements at which carbachol facilitated predatory aggression produced significant increases in water consumption, 3.5-7.0 ml over control levels. While not seen in Table I, carbachoi stimulation at 9 of the 27 placements at which carbachol failed to facilitate predatory aggression yielded carbachol-induced drinking. Brain Research, 20 (1970) 409-424
4t6
R . J . BANDLER
j,
Fig. 1. Sections A - H are from K6nig and KlippeP 1 (Figures 29b, 31-36b, 38b). Cannula tip locations for cholinergic facilitation of predatory aggression (dot), drinking (asterisk), predatory aggression and drinking (dot with asterisk), and no effect (triangle). The number beside each symbol refers to the subject.
is
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0
,-.] 0 ,<
0
0
0 -I
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•
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.~
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, •
Fig. 2. Representative photomicrograph of hypothalamic section showing sites of cannula tips f~r subject No. 46.
Carbachol stimulation at some hypoth.alamic sites, then, can both elicit drinking and facilitate predatory aggression. However, this is not necessarily the case, as 12 of the 23 hypothalamic aggression sites were specific to only aggression, and 9 of the 20 hypothalamic drinking sites yielded only to drinking• In accordance with these behavioral data, the histology (as seen in Fig. It indicated some anatomical separation of the hypothalamic neural elements which regulate drinking and predatory aggression. It is seen that those points which proved positive for only drinking are posterior to most of th.e predatory aggression points• Further, while most of the drinking points are dorsal to the fornix, it will be recalled that the 'best" aggression points were consistently ventral to the fornix. The behavioral and histological data suggest, then, that the lateral hypothalamic neural elements which regulate predatory aggression are distinct from those which regulate drinking. No other effects on consummatory behavior were elicited by any stimulation treatment. Stimulation with acet.vlcholine and cholinesterase inhibitors As is seen in the first row of Table II acetylcholine stimulation at the 6 positive hypothalamic sites (Nos. 20L, 26R, 27R, 35R. 37R and 46R) failed to elicit a reliable Brain Research, 20 (1970) 409~,24
CONTROL OF PREDATORYAGGRESSION
419
TABLE II MEAN LA'FENCIES OF ATTACK AND KILL (IN SECONDS) FOLLOWING EACH STIMULATION TREATMENT AT HYPOTHALAMUS
All t tests on 5 df; * P < 0.05; ** P < 0.01. Abbreviations: ACh, acetylcholine stimulation ; Phy, physostigmine stimulation; Neo, neostigmine stimulation; Ct, control stimulation; Ex, experimental condition. Condition (N = 6)
Attack latency
Kill latency
Ex
Ct
t
Ex
Ct
t
ACh ACh/Phy Phy Neo
101.7 72.3 80.7 83.7
130.0 126.1 129.5 125.3
0.69 2.84* 1.93 2.86*
147.0 104.5 123.2 117.8
182.5 185.7 185.8 171.5
1.96 5.60"* 2.10 2.97*
facilitation of predatory aggression. The elicited behavior could be described as sporadic, with a decrease in attack and kill latencies for only 2 or 3 of the presented prey. It is seen in row 2, however, that stimulation with acetylcholine when mixed with physostigmine did effect a reliable decrease of both latencies of attack (P < 0.05, two-tailed) and kill (P < 0.01, two-tailed). This decrease was neither as pronounced nor as dramatic as the decrease induced by carbachol stimulation. Rows 3 and 4 of Table I! indicate that direct, central stimulation with the cholinesterase inhibitors had mixed results. Physostigmine sulfate stimulation failed to elicit a significant facilitation of predatory aggression, but stimulation with neostigmine bromide significantly decreased both latencies of attack (P < 0.05, two-tailed) and kill (P < 0.05, two-tailed). A recent study by Margules and Stein la has reported effects following stimulation with neostigmine to be significantly greater than those following physostigmine stimulation. These results concur with theirs. That acetylcholine mixed with physostigmine facilitated predatory aggression, suggests that the failure of acetylcholine alone was due to the rapid rate of destruction of acetylcholine by cholinesterase. Both this finding and the facilitation of predatory aggression subsequent to direct stimulation with neostigmine bromide is strong evidence for the participation of acetylcholine in the regulation of predatory aggression at the lateral hypothalamus. Since 5 of the 6 placements tested were negative for carbachol-induced drinking, it was to be expected that none of the 4 stimulation treatments produced significant increases in drinking. There were, as well, no changes in food consumption. Cholinergic stimulation in combination with systemically administered blocking agents
It is seen in the first and third columns of Table III that peritoneal injections of either atropine sulfate (10 and 25 mg/kg) or atropine methyl-nitrate (10 and 25 Brain Research, 20 (1970) 409-424
420
R.J. BANDLER
TABLE llI MEAN LATENCIESO1- ATTACK AND KILL (IN SECONDS) FOLLOWINGEACH COMBINED CENTRAL AND PERII'HERALSTIMULATIONTREATMEhT All t tests on 5 dr; * P < 0.05; * * P < 0.01 ; ** * P < 0.001. Significant differences between the placebo condition and the 4 experimental conditions are noted in the table. Abbreviations: Ca, carbachol stimulation; Ct, control stimulation; AS-10, atropine sulfate 10 mg/kg; AS-25, atropine sulfate 25 mg/kg; AMN-10, atropine methyl-nitrate 10 mg/kg; AMN-25, atropine methyl-nitrate 25 mg/kg. Co;Iditioa ( N -- 6)
Placebo AS-10 AMN-10 AS-25 AMN-25
Attack lateney
(A) (B) (C) (D)
Kill latency
Ct
Ca
Ct
Ca
48.8 224.2*** 123.6" 187.6"* 158.5"*
-104.1" 21.8 146.3"* 75.6
87.8 305.8*** 178.5' 263.9*** 192.3*
-196.5"* 58.5 261.5'* 88.7
3.01 * 2.51 *
4.09** 2.62*
!
A vs. B C vs. D
3.24* 0.78
6.93*** 4.33**
mg/kg) resulted in a t t a c k and kill latencies significantly greater than those in the placebo condition. It is also seen in the first a n d third c o l u m n s t h a t at the l0 m g ] k g dose, a t r o p i n e methyl-nitrate interfered less with the latencies o f a t t a c k ( P < 0.05, two-tailed) and kill ( P < 0.01, two-tailed) than the same dose o f a t r o p i n e sulfate; while at the 25 mg/kg dose, a t r o p i n e m e t h y l - n i t r a t e p r o v e d a less effective suppressant than atropine sulfate for latencies o f kill ( P < 0.05, two-tailed) but not a t t a c k (t = 0.78, N.S.). Since the injected a t r o p i n e m e t h y l - n i t r a t e w o u l d n o t be expected to pass the b l o o d brain barrier in any a p p r e c i a b l e a m o u n t s , the differential suppression o f ' n a t u r a l ' p r e d a t o r y aggression is a t t r i b u t a b l e to the central action o f the injected a t r o p i n e sulfate and is viewed as evidence f a v o r i n g the hypothesis o f the cholinergic regulation o f p r e d a t o r y aggression. That a t r o p i n e sulfate was a m o r e effective s u p p r e s s a n t o f p r e d a t o r y aggression at the 10 mg/kg dose than at the 25 mg/kg dose was a surprising result. A f t e r the 25 m g / k g i n j e c t i o n the subjects seemed s o m e w h a t i r r i t a b l e and were difficult to handle. S u c h an increased irritability m a y have f a c i l i t a t e d their aggressiveness t o w a r d the p r e y a n d thus lessened the suppressive effects o f the a t r o p i n e sulfate on a t t a c k and kill latencies. W i t h respect to the c a r b a c h o l - i n d u c e d facilitation o f p r e d a t o r y aggression, c o l u m n s 2 a n d 4 reveal t h a t the peritoneal injections o f a t r o p i n e sulfate (10 a n d 25 m g / k g ) not only blocked the c a r b a c h o l - i n d u c e d facilitation, b u t signific a n t l y increased the a t t a c k and kill latencies. The p e r i t o n e a l injections o f a t r o p i n e methyl-nitrate, while they blocked the c a r b a c h o l - i n d u c e d facilitation, did not signifiBrain Research, 20 (1970) 409-424
CONTROL OF PREDATORY A G G R E ~ I O N
421
cantly increase either attack or kill latencies. The relevant comparisons, as seen in columns 2 and 4, show that the 10 mg/kg dose of atropine sulfate more effectively interfered with the carbachol-induced facilitation of attack (P < 0.05, two-tailed) and kill latencies (P < 0.001, two-tailed) than the 10 mg/kg dose of atropine methyl-nitrate. Similarly, at the 25 mg/kg dose, the carbachol-induced decrease in latencies of attack (P < 0.05, two-tailed) and kill (P < 0.01, two-tailed) are more interfered with by atropine sulfate injection than by atropine methyl-nitrate injection. Since the differential effects are thought to reflect the unique action of atropine sulfate on the brain, the differential blockage of the hypothalamic, carbacholinduced facilitation of predatory aggression suggests that the carbachol-induced facilitation is due to the cholinergic action of the drug in the brain. Injections of both atropine sulfate (10 and 25 mg/kg) and atropine methylnitrate (10 and 25 mg/kg) blocked all carbachol-induced drinking. Atropine treatment
Direct, central atropine application produced a consistent and reliable suppression of attack ($35R, P < 0.01, two-tailed; $46R, P < 0.05, two-tailed) and kill latencies ($35R, P < 0.05, two-tailed ; $46R, P < 0.001, two-tailed) at only 2 of the 13 positive placements tested and none of the 19 negative placements tested. Bilateral atropine application for 3 subjects (Nos. 26, 27 and 37) also failed to suppress predatory aggression. It is probably not coincidental that both atropine and norepinephrine suppressed predatory aggression at the same two sites (35R and 46R). It is clear, however, from the lack of inhibition at all but these two sites that both the atropine and norepinephrine-induced inhibitions reflect more than a direct inhibitory influence on lateral hypothalamic synapses. Further, it is unlikely that the inhibitions are due only to spread of the chemicals to distal sites, as both atropine and norepinephrine application failed to suppress predatory aggression at all negative sites tested. The fact that neither atropine nor norepinephrine administration at the positive contralateral hypothalamic placement for each of the subjects (35L and 46L) suppressed predatory aggression suggests that the inhibitions do not reflect individual differences. One remaining possibility is that the inhibitions reflect a simultaneous drug effect at the lateral hypothalamus and by unique diffusion to other distal sites. No conjectures are offered concerning other possible sites. Atropine application had no effect on consummatory behavior. DISCUSSION
It is clear from the results of the study that the lateral hypothalamic regulation of predatory aggression is sensitive to cholinergic stimulation. The principle question which arises is what the data can tell us about the neurotransmitter which regulates predatory aggression at lateral hypothalamic synapses. The data of this study, in particular the differential inhibition of the carbacholBrain Research, 20 (1970)409-424
422
R.J. RANI)I+t R
induced facilitation of predatory aggression by the two systemically administercd cholinolytic agents, atropine sulfate and atropine methyl-nitrate, leave little doubt that the carbachol effect is due to the cholinergic action of the drug ill the brain. Further, the data favor the view that the action of the drug is a local one and does not reflect spread of the drug to other brain sites as has been suggested for dircct cholinergic and anticholinergic stimulation effects on water consumption in the rat 1G and direct cholinergic stimulation effects on affective aggression in the cat 3. That the facilitation is not the result of spread of the drug to the third ventricle and then by diffusion to other sites is suggested by the fact that direct cholinergic stimulation of brain sites medial to the fornix, but in the same anterior-posterior and dorsalventral planes as the positive aggression sites, has proved negative for predatory aggression. That the facilitation is not due to spread of the drug upward along the cannula shaft to dorsal sites is suggested by the fact that cholinergic and electrical stimulation t° of sites dorsal to the positivc sites have proved negative for aggression. In addition, it will be recalled that the 'better" aggression sites were ventral to the fornix, not dorsal to it. That the effects are not the result of extensive spread even within the hypothalamus is suggested by the fact that sites negative for predatory aggression or specific to drinking are often in close proximity to positive aggression sites. Finally, the fact that predatory mouse-killing has been elicited by electrical stimulation 10 of the +same' lateral hypothalamic area, which has proved positive for the cholinergic facilitation of predatory aggression, suggests that there is no need to postulate extensive drug diffusion in order to explain the facilitation of predatory aggression following cholinergic stimulation. The data presented, then, are certainly compatible with the notion that acetylcholine is involved in the regulation of predatory aggression at lateral hypothalamic synapses. In particular, the finding that predatory aggression is facilitated by direct central stimulation of positive hypothalamic sites with either neostigmine bromide or acetylcholine mixed with physostigmine sulfate rules in favor of acetylcholine involvement. The finding that the central application of atropine to positive hypothalamic sites failed to suppress predatory aggression is, however, inconsistent with this conclusion. Two explanations, the blocking action of the injected atropine being too temporary, or the diffusion of the atropine within the anterior-lateral h.ypothalamus being insufficient to block enough relevant neural elements, are unlikely explanations considering the amount of drug injected by the double-stimulation technique, and the failure of bilateral as well as unilateral atropine application. The data are compatible, however, with at least one alternative explanation: there might be such a redundancy of predation related neural circuitry in the brain that a functional lesion (cholinergic blockade) of the anterior-lateral hypothalamus is not disruptive enough to suppress predatory aggression. Karli and Vergnes's 8 report that only extensive, rostral to caudal, bilateral hypothalamic lesions block predatory mouse-killing is supportive of this conjecture. That is, it suggests that more extensive hypothalamic interference than a blockade of only anterior-lateral hypothalamic synapses is necessary to inhibit predatory aggression. This suggests the Brain Research,
20 (1970) ~09-424
CONTROL OF PREDATORY AGGRESSION
423
involvement of an additional lateral hypothalamic area in predatory aggression, possibly posterior to the level of the ventromedial hypothalamus (an area not extensively explored in this study). The involvement of just such an area in the regulation of predatory aggression is supported by the finding that electrical stimulation of posterior-lateral hypothalamus facilitates mouse-killing in natural mouse-killers (DeSisto, personal communication). It should be noted, however, that the failure of atropine to suppress predatory aggression at those hypothalamic sites at which carbachol facilitated predatory aggression suggests that the unique blocking effects of the systemically administered atropine sulfate must reflect anticholinergic action at sites other than, or in addition to, the anterior-lateral hypothalamus. This suggests, of course, the hypothesis of the cholinergic regulation of predatory aggression in other parts of the central nervous system. The results of this study, then, are interpreted to mean that acetyicholine is involved in the regulation of predatory aggression at, no less than, the anteriorlateral hypothalamus. ADDENDUM
Since the acceptance of this paper for publication, Smith, King and Hoebel (Science, 167 (1970) 900-901) reported that cholinergic stimulation (carbachol,
neostigmine) of the lateral hypothalamus elicited mouse-killing in nonmouse-killers. However, in contrast to the 3-10/zg doses used in this study, Smith et al. used doses of 50/zg 4- 25/~g. This suggests that their results are due to stimulation effects at both the lateral hypothalamus, and by spread of the drug, because of the high doses, to other brain structures. That such spread is in fact critical to their results is indicated by the long latencies for the reported drug effects: the mean elicitation of kill latency for carbachol stimulation was 45 min, for neostigmine stimulation 2.75 h. These latencies contrast with the 7 rain latency for the carbachol-induced facilitation of aggression and the 30 min latency for the neostigmine-induced facilitation of aggression reported in this paper. SUMMARY
Direct central stimulation, via chronically implanted cannulas, of sites in the lateral hypothalamus with either carbachol, acetylcholine mixed with physostigmine, or neostigmine, facilitated frog-killing and mouse-killing in natural killer rats. The cholinergic stimulation was believed to have affected predatory aggression because of (a) the stereotyped nature of the killing response, (b) the lack of increased irritability in nearly all subjects, (c) the neuroanatomical correspondence to the hypothalamic areas in rat and cat which, when stimulated electrically, elicit predatory aggression. These results, as well as the effects of systemically administered cholinergic blocking agents, were found to be compatible with the hypothesis that acetylcholine is involved in the control of predatory aggression at the lateral hypothalamus. Brain Research, 20 (1970)409--424
424
R. J. BANDLER The study also indicated that the lateral h y p o t h a l a m i c neural elements which
regulate predatory aggression are distinct from those which regulate drinking, although both are sensitive to cholinergic stimulation. ACKNOWLEDGEMENTS I am indebted to Dr. K. E. M o y e r for his guidance, s u p p o r t and e n c o u r a g e m e n t of this research. Dr. L. E. Jarrard c o n t r i b u t e d helpful advice and criticism. Dr. C. C. Chi and Dr. J. P. F l y n n reviewed the manuscript. 1 wish to t h a n k Fay G o m e s and Mildred Groves for p r e p a r a t i o n of the microscope slides. This research was supported by N a t i o n a l Science F o u n d a t i o n G r a n t GB 6652 awarded to Dr. K. E. Moyer, and was completed while R.J.B. was a predoctoral fellow in Psychology of C a r n e g i e - M e l l o n University, Pittsburgh, Pa.. U.S.A.
REFERENCES 1 BANDLER, R. J.. Facilitation of aggressive behavior in rat by direct cholinergic stimulation of the hypothalamus, Nature (Lond.), 224 0969) I035-1036. 2 BANDLER, R. J., AND MOYER, K. E., The objects naturally attacked by the rat, in preparation. 3 BAXTER, B. L., Comparison of behavioral effects of electrical or chemical stimulation applied at the same brain loci, Exp. Neurol., 19 (1967) 412-430. 4 GROSSMAN,S. P., Direct adrenergic and cholinergic stimulation of hypothalamic mechanisms, Amer. J. Physiol., 202 (1962) 872-882. 5 GROSSMAN, S. P., Effects of adrenergic and cholinergic blocking agents on hypothalamic mechanisms, Amer. J. Physiol., 202 0962) 1230-1236. 6 HUTCHINSON,R. R., AND RENFREW,J. W., Stalking attack and eating behavior elicited from the same sites in the hypothalamus, J. comp. physiol. Psycho/., 61 (1966) 360-367. 7 KARL1, P., The Norway rat's killing response to the white mouse, Behaviour, I0 (1956) 81-103. 8 KARLI, P., ET VERGNES, M., Dissociation exp~rimentale du comportement d'agression intersp6cifique rat-souris et du comportement alimentaire, C. R. Soc. Biol. (Paris), 158 (1964) 650-653. 9 KARLI, P., VERGNES, M., AND DIDIERGEORGES,F., Rat-mouse interspecific aggressive behavior and its manipulation by brain ablation and brain stimulation. In E. B. SIc~ AND S. GARATrIM (Eds.), Biology of Aggressive Behavior, Excerpta Medica Foundation, Amsterdam, 1969, pp. 47-55. 10 KING, M. B., AND HOEaEL, B. G., Killing elicited by brain stimulation in rat, Commun. Behav. Biol., 2 (1968) 173-177. 11 K/3NIG,J. F. R., AND KLIPPEL, R. A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, Md., 1963. 12 LEVINSON,P. K., AND FLVr~N,J. P., The objects attacked by cats during stimulation of the hypothalamus, Anita. Behav., 13 (1965) 217-220. 13 MARGULES,D. L., AND STEIN, L., Cholinergic synapses in the ventromedial hypothalamus for the suppression of operant behavior by punishment and satiety, J. eomp. physiol. Psychol., 67 (1969) 327-335. 14 MoveR, K. E., Kinds of aggression and their physiological basis, Commun. Behav. Biol., 2 (1968) 65-87. 15 ROaER'rS,W. W., Ar,~DKIESS, H. O., Motivational properties of hypothalamic aggression in cats, J. comp. physiol. Psychol., 57 (1964) 187-193. 16 RotrrrESBERG, A., Drinking induced by carbachol: thirst or ventricular modification, Science, 157 (1967) 838-839. 17 WASMAN, M., A~D FLVNN, J. P., Directed attack elicited from hypothalamus, Arch. Neurol. (Chic.), 6 (1962) 220-227. Brain Research, 20 (1970) 409-424