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Avoidance behavior and aggression in rats with transections of the lateral connections of the medial or lateral hypothalamus
Physiology and Behavior. Vol. 5, pp. 1103-1108. Pergamon Press, 1970. Printed m Great Britain
Avoidance Behavior and Aggression in Rats with Transections of the Lateral Connections of the Medial or Lateral Hypothalamus' S E B A S T I A N P. G R O S S M A N
Department of Psychology, University of Chicago, Chicago, Illinois 60637, U.S.A. (Received 4 June 1970) GROSSMAN,S. P. Av•idancebehavi•randaggressi•ninratswithtransecti•ns•fthe•ateralc•nnecti•ns•fthemedial•r•ateral hypothalamus. PHYSIOL.BEHAV.5 (10) 1103-1108, 1970.--Parasagittal surgical cuts in the medial quadrant of the lateral hypothalamus which isolated the medial half of the hypothalamus from all lateral connections produced marked deficits in avoidance learning but no d~scernible change m performance on a test of intra-species aggression. The animals showed transient motor deficits as well as aphagia and adipsia but were normal on all measures before the avoidance training and aggression tests were begun. Similar parasagittal cuts lateral to the lateral border of the anterior and medial hypothalamus (including the region lateral to the ventromedial nuclei) facilitated the acquisition of a shuttle box avoidance response but failed to affect intra-species aggression. These animals were aphagic and adipsic for longer periods of time but had recovered normal body weights and motor abilities at the time of testing. Hypothalamus-aggression Aggression-hypothalamus Hypothalamus-avoidance Hypothalamus-transectlon of connections Knife cuts-hypothalamus
MANY experimental observations suggest that components of the hypothalamus significantly influence the organism's response to noxious stimulation. This reaction can take two directions--aggression or flight--and both appear to be related to hypothalamic functions. Lesions in the medial hypothalamus result in vicious attack reactions to normal handling [30]; lowered thresholds for apparently affective reactions to electric shock [28] or noxious tastes [21]; and facilitated conditioned avoidance behavior (CAR) [12]. A pharmacological blockade of this region produces similar effects on avoidance behavior [12, 24] and disinhibits punished responses [19]. Electrical stimulation of the ventromedial area interferes with any ongoing activity and appears to be aversive [16]. Lesions in the lateral hypothalmus have also been reported to increase the rat's sensitivity to footshock [18]. Electrical stimulation of lateral as well as medial hypothalamic sites elicits defense or attack reactions [15, 29]. Chemical stimulation of some sites in the lateral hypothalamus induces mouse killing [25] and facilitates avoidance behavior [24]. The hypothalamus also controls autonomic reactions, and it has been suggested that it may contain a primary center for the integration of affective responses (see [8] for a review of this hypothesis). Extrahypothalamic structures have also been implicated in the control of aggressive or avoidance behavior but it can be argued that they exert their influence on behavior by modulating hypothalamic functions (see [13] for a review of the relevant literature). However, little is known about the
Avoidance--hypothalamus
pathways which interconnect the extrahypothalamic and hypothalamic portions of the postulated circuit or about the connections of the hypothalamus which specifically mediate reactions to affective stimuli. The present series of experiments were undertaken as a first step towards a more complete understanding of the functional importance of various hypothalamic connections. The medial half of the anterior and medial hypothalamus, including the ventromedial nuclei, was isolated from all of its lateral connections by parasagittal knife-cuts just lateral to the fornix which extended from A P = 7.0 to A P = 4.2 and from the base of the brain to the zona incerta. In other animals, similar cuts were made along the lateral border of the lateral hypothalamus. These cuts completely isolated the anterior and medial portions of the hypothalamus from lateral connections, notably with the amygdala. The wire knife used in these experiments produces very little damage to cellular components (because it is only 0.014 m m dia.) but completely transects transverse fibers. It is therefore probable that the effects of such cuts can be attributed to the disruption of fiber connections.
METHOD
Subjects Eighteen adult female albino rats of the Sprague-Dawley strain (Holtzman, Madison, Wisconsin), housed singly, in a
1Supported by grant MH 10130-06 from USPHS. The assistance of Lore Grossman and Anne Wilson is gratefully acknowledged. 1103
1104 continually illuminated and air-conditioned room were used. The animals weighed 220-260 g at surgery.
Apparatus Two identical double grill boxes constructed of transparent Plexiglas were used for the avoidance experiment. Each measured 53 × 22 × 32 cm and was divided into two compartments of equal size by a partial partition which extended 5 cm from each side of the apparatus. Both compartments had grid floors consisting of 4 mm dia. stainless steel bars placed 1 cm apart. The CS was supplied by a 7 W bulb placed in the center of the hd of each compartment. The UCS consisted of pulses (0.3 sec o n / 0 . 3 off) of constant current shock, supplied by a high-voltage source modified after Brown, Reus and Webb [3]. The aggression/dominance apparatus consisted of a clear Plexiglas tube, 94 cm in length and 4.9 cm dia. At each end of this tube were identical compartments constructed of white opaque plastic and measuring 29 × 29 × 28 cm. The floor of these compartments was constructed of stainless steel rods, 4 mm dia. and spaced 1 cm apart. The entrances to the tube which interconnected the two compartments were closed by black opaque plastic guillotine doors which could be raised from the outside of the apparatus. Two circular plastic food cups were mounted on the wall of the compartment which was farthest from the entrance of the tube.
Procedure (a) Behavioral tests. Food- and water-intake and body weight were recorded daily throughout the experiment. The animals were maintained on a powdered diet (Rockland Meal) except for several weeks after surgery when a liquid diet or pellets (Purina Lab Chow) were available. (The animals required intragastric feeding for several days post-operatively.) All animals ate normally and re-established normal body weights before training in the avoidance experiment was begun. On the first or second post-surgical day and again one week later, a series of tests [1] were given to ascertain the animals' motor capabilities. (1) The paw test consisted of lifting one of the rat's forepaws and recording the latency of the withdrawal response; (2) the pedestal test consisted of placing the animal on a small platform and recording the time taken to step down from it; and (3) the horizontal stabilization test consisted of placing the rat upside down on the inside of the front panel of its home cage and recording the time taken for the animal's voluntary descent into the cage. A maximum of three minutes was permitted for each test. Approximately six weeks after surgery, all animals were trained to avoid painful grid shock by jumping into the dark compartment of a double-grill shuttle-box in response to the onset of a light mounted overhead. Fifteen trials per day were given for 21 days as follows: The animal was placed into the dark compartment of the shuttle box. Sixty-three sec later, the overhead light was illuminated and the illumination of the adjacent compartment turned off. Five sec later, the grid floor in the illuminated compartment was electrified. The light and shock remained on in that compartment for the next 58 see. The CS and CS-UCS combination then switched to the previously safe dark compartment. The animals could escape or avoid the shock by always jumping into the dark compartment. The duration of the subjective intertrial interval was determined within a 63 see limit by the rat since the cycling of the CS and UCS was unaffected by the animals' behavior.
GROSSMAN Following the completion of the avoidance experiment, all rats were placed on a restricted feeding schedule designed to maintain each rat at approximately 90 per cent of its normal body weight. Each rat was then trained to run through the tube of our aggression apparatus for rewards consisting of highly pallatable bits of Froot Loop cereal. (The somewhat unusual rewards were selected because some of the animals reacted poorly to the deprivation regimen and would not run reliably for standard reward pellets.) After some initial shaping, each rat received 10 trials per day until its running latencies had stabilized. In the following tests of aggressiveness and dominance, each animal continued to receive 10 rewarded trials per day. On one randomly selected trial each day, two rats (one experimental animal and one unoperated control) were started simultaneously from opposite ends of the tube. Since the response latencies of both members of each pair were comparable, the two animals met near the middle of the tube. Vigorous fighting ensued because the diameter of the tube was selected so that the animals could not pass each other. Eventually, one of them backed up all the way to its starting compartment (and missed out on its reward at the other end of the tube). The other rat followed and was reinforced upon arrival in the opponent's start compartment. This test was repeated with the same pair of animals until one of them had dearly established dominance by winning six consecutive fights. The fights in the tube were initially prolonged and vicious but tended to become shorter and less acrimonious with repeated testing. (b) Surgery. Surgical knife cuts were produced under Nembutal anesthesia by stereotaxically placing a guide made of 30 ga hypodermic needle stock at the following coordinates (from the de Groot [6] atlas of the rat brain); A P = 7.4; L = 1.5; H = 0 . 0 or A P = 7 . 4 ; L=2.5; H=0.0. A 0.014 mm dia. tungsten wire was then extended from the slightly bent tip of the guide cannula until the wire protruded (at an angle of approximately 90 ° to the guide) posteriorly for 2-3 mm. The entire assembly was then lowered from H = 0.0 to the base of the brain and raised again to H = 0.0 The wire was then retracted and the guide cannula withdrawn. The design of the cannula has been described in detail elsewhere [23]. (It should be noted that approximately one-third of the animals which are subjected to this operation do not survive, the most probable cause of death being the transection of major blood vessels.) (c) Histology. Following completion of the experiments, all animals were sacrified with an overdose of Nembutal and perfused intracranially with a 10 per cent formalin solution. The brains were removed from the calvarium and frozen to permit the cutting of 25 ~t frozen sections. Alternating sections were stained with cresyl violet (to bring out cellular components) and Sudan black (to emphasize fiber tracts). The description of the anatomical results (see below) is based on microscopic analysis of this material. RESULTS AND DISCUSSION
(a) Effects of Transections of the Lateral Connections of the Medial Hypothalmus Bilaterally symmetrical parasagittal cuts which transected the hypothalamus approximately 1.5 mm from the midline were made in six animals. The cuts extended from A P = 7.07.4 posteriorly to A P = 4.2-4.6 and from the base of the brain to the zona incerta, terminating just below the medial lemniseus (see Figs. 1 and 2).
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FIG. 1. Examples of parasagittal knife-cuts (a) through the medial quadrant of the lateral hypothalamus (which isolated the medial hypothalamus from all lateral connections) and (b) lateral to the lateral border of the hypothalamus (which isolated the anterior and medlal aspects of the hypothalamus from lateral connections). The top sections are stained with cresyl violet to permit visualization of cellular components; the lower sections are stained with Sudan black to permit visualization of fibers. The extent of these cuts ~s illustrated in Fig. 2. The avoidance behavior of animals with similar cuts is shown in Fig. 3.
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TRANSECTIONS OF CONNECTIONS OF HYPOTHALAMUS
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FIG. 2. Schematic representation (after Pellegrino and Cushman [22]) of parasagittal knife-cuts (a) through the medial quadrant of the lateral hypothalamus and (b) lateral to the lateral border of the hypothalamus. Examples of the histological data on which these tracings are based are shown in Fig. 1. The avoidance behavior of animals with similar cuts is illustrated in Fig. 3.
These animals were aphagic and adipsic for 2-8 days after surgery but recovered the ability to regulate food intake on a powdered food diet within two weeks. They were subsequently hyperphagic when placed on a preferred pellet diet and had completely recovered normal body weights when training in the avoidance apparatus was begun. These animals appeared catatonic on some of the motor tests given on the first or second day after surgery but had recovered on the second tests, given one week later. All animals performed well within normal limits on a third set of motor tests given just before the beginning of training in the shuttle boxes. A detailed report of the food and water intake and motor
impairments of these rats and others with similar cuts has been published elsewhere [14]. At no time did these animals display the vicious attack reactions to normal handling which are commonly seen in rats with ventromedial hypothalamic lesions, suggesting that the influence of this region on aggressive behavior may not be mediated by direct lateral projections. This conclusion is supported by the observation that our experimental rats which no longer had these projections were not discernibly better or worse on our test of aggression or dominance. Two of the six animals with mid-hypothalamic knife cuts won all of their fights with an unoperated control rat; throe
1106
GROSSMAN when relatively high shock levels or complex learning situations are employed (unpublished data), and McAdam and Kaelber [20] have reported inhibitory effects on C A R acquisition in cats in a high-shock situation. It appears unlikely, however, that the results of our knife-cuts could be due to an excessive reaction to shock in view of the absence of attack reactions to handling and the animals' normal pattern of intra-species aggression. These observations suggest, instead, that the inhibitory influences which the ventromedial region appears to exert on affective reactions to noxious stimuli may be mediated by pathways which are not directly affected by our cuts. The work of Stein [26] suggests that the perlventricular fiber system may be a prime candidate for this function. If this analysis is correct, the observed depression of avoidance behavior in animals with cuts lateral to the medial hypothalamus could be due to an interruption of afferents to the ventromedml region which mediate inhibitory influences. Sepinwall's [24] recent observation that pharmacological blockade of the lateral hypothalamus (which should not affect fibers of passage) inhibited avoidance behavior whereas chemical stimulation of that region enhanced it, suggests further that the cells which may exert this inhibitory influence on the ventromedial area may be located in the lateral hypothalarnus. This interpretation is supported by a recent report [5] of avoidance deficits in rats with lateral hypothalamic lesions.
operated rats lost all of them; and one eventually won six consecutive fights but lost many fights in the process and did not appear dearly dominant in any of them. (If a clearly dominant and a clearly submissive partner were paired, very little fighting occurred after the initial encounter and the submissive animal backed off within a few seconds. When the dominance/submission relationship was less dearly defined, vicious and prolonged fighting often occurred even after as many as 10 or 15 encounters.) The pattern of dominance/ submission seen in the rats with parasagittal cuts which bisected the hypothalamus was dearly within the normal range. In the avoidance experiment, these animals performed reliably poorer than the normal controls (p < 0.01) or the rats with cuts along the lateral border of the hypothalamus (p < 0.01) at all levels of training (see Fig. 3). Half of the animals m this group failed to reach the criterion of 9/10 consecutive avoidance responses on two successive days within the 21 days of the experiment; two more required a greater than average nurnber of trials to do so. Only one animal which could not be distinguished on the basis of gross histological data learned the task rapidly. There was no discernible relationship between the animals' performance in the avoidance situation and their aggressiveness or the severity of the experimentally produced changes in feeding behavior. The impaired avoidance behavior of these animals was surprising in view of the commonly reported observation that rats with ventromedial hypothalamic lesions learn avoidance responses better than normals [11, 12, 17]. This finding is confirmed by the effects of pharmacological blockade of the ventromedial region [12, 24] and is in accord with the lesioned animals' increased sensitivity to footshock [28]. We have observed that the general hyperexcitability and hyperreactivity of rats with such lesions interferes with avoidance learning
(b) Effects of Transections of the Lateral Connecttons of the Lateral Hypothalamus Bilaterally symmetrical parasaglttal cuts, lateral to the lateral border of the anterior and medial hypothalamus, were made in six rats. These cuts coursed through the internal
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FIG. 3. Avoidance behavior of rats with l~asagitt~l labile-cuts in
the medial quadrant of the hypothalamus or lateral to the lateral hypothalamus. The data are expressed in terms of the number of trials required to meet increasingly stringent criteria of avoidance proficiency (such as 1, 2, 3. . . . 9 avoidance responses in 10 consccutivc trials). The line labelled "max" indicates the performance of a hypothetical animal which reaches the criterion of 9 out of 10 avoidances without error.
TRANSECTIONS OF CONNECTIONS OF HYPOTHALAMUS capsule and the interpeduncular nucleus and did not involve tissue of the lateral hypothalamus at any point. The cuts extended from A P = 7.0-7.4 posteriorly to A P = 5.0-5.8 and transected all lateral connections of the anterior and medial hypothalamus. The lateral connections of the posterior hypothalamus were left intact. (Cuts involving the lateral projections of the posterior hypothalamus were attempted but frequently resulted in such severe motor impairments that behavioral testing was impossible.) (See Figs. 1 and 2.) All animals with parasagittal cuts along the lateral border of the lateral hypothalamus were aphagic and adipsic following surgery. Some (not included in this discussion) failed to recover normal food or water intake and were discarded 60 days after surgery. Others, including the animals used in the present experiment, went through the recovery cycle described by Teitelbaum and Epstein [27] for lateral hypothalamic lesions and eventually recovered the ability to regulate food and water intake (the latter being purely prandial). These animals showed severe impairments on all of our motor tests given on the first or second post-surgical day but recovered to normal levels within one week (see [14] for a more detailed account). All animals performed normally on a third set of motor tests given just before the beginning of training in the avoidance apparatus, and had attained normal body weights (X: = 276.1 g). Daily food and water intake were recorded throughout the subsequent aggression experiment to assure adequate food and water consumption at all times. Isolation of the anterior and medial hypothalamus from its lateral connections had no more of an effect on inter-species aggression than transection of its medial connections (see above). Two experimental animals of this group won all of their fights with unoperated controls and two lost all of them. The remaining two won 6 consecutive fights only after 14 and 15 encounters respectively and never displayed a clear dominance relationship with their unoperated partner. These observations indicate that the aggressive reactions which have been elicited by electrical [7, 29] and chemical [25] stimulation of the lateral hypothalamus may be mediated
1107 by pathways which project in a rostro-caudal direction and do not depend on direct lateral or medial connections. The avoidance behavior of rats with parasagittal cuts lateral to the hypothalamus was superior to that of the unoperated controls or of rats with cuts in the medial quarter of the lateral hypothalamus (see Fig. 3). These animals performed no better than the unoperated controls during the early stages of training but reached higher terminal levels of avoidance performance reliably (p < 0.05) faster. The experimental animals of this group were significantly (p < 0.01) better at all levels of performance than the rats with cuts through the medial quadrant of the lateral hypothalamus. These findings suggest that the modification of avoidance behavior which has been observed after chemical stimulation or blockade of the lateral hypothalamus [24] may be due to stimulation or blockade of cells which receive inhibitory afferents via lateral connections. The origin of these connections is, as yet, unknown. The amygdaloid complex would, at first glance, appear to be a likely suspect. However, damage to most portions of the amygdala produces an impairment in C A R acquisition (see [9] for a review of this literature). Moreover, lesions which interrupt the ansa lenticularis, one of the major amygdalo-hypothalamic connections, have been reported to produce no selective effects on C A R acquisition [4]. There are, however, some recent observations which indicate that the amygdala may be the source of inhibitory as well as excitatory influences on C A R acquisition. Small lesions or pharmacological blockade of restricted aspects of the ventral amygdala facilitate avoidance learning [2] and low level electrical stimulation has been reported to have inhibitory effects [10]. Our results confirm Caruther's suggestion that excitatory amygdaloid influences on C A R behavior do not appear to be mediated by the ansa lenticularis or other lateral connections of the anterior and medial hypothalamus [4]. They indicate further that fibers carrying inhibitory influences of amygdaloid or other origin appear to enter the hypothalamus at its lateral border.
REFERENCES
1. Balagura, S., R. H. Wilcox and D. V. Coscina. The effect of diencephalic lesions on food intake and motor activity. Physiol. Behav. 4: 629--633, 1969. 2. Belluzzi, J. and S. P. Grossman. Avoidance learning: Longlasting deficits after temporal lobe seizure. Science 166: 14351437, 1969. 3. Brown, C. C., J. F. Reus and G. A. Webb. A new constant current stimulation circuit. In: Digest of the International Conference on Medical Electronics, edited by P. L. Frommer. Princeton, N.J.: Conference Committee of the Internation Conference on Medical Electronics, 1961. 4. Caruthers, R. P. Ansa lenticularis area tractotomy and shuttle avoidance learning. J. comp. physiol. Psychol. 65: 295-302, 1968. 5. Coseina, D. V. and S. Balagura. Avoidance and escape behavior of rats with aphagia produced by basal diencephahc lesions. Physiol. Behav. 5: 651-658, 1970. 6. de Groot, J. The Rat Forebrain in Stereotaxic Coordinates. Amsterdam: N.V. Noord-Hollandsche Uitgevers Maatschappij, 52: 1-40, 1959. 7. Fonberg, Elzbieta. The motivational role of the hypothalamus in animal behaviour. Acta Biol. exp. (Warsaw) 27: 303-318, 1967. 8. G-ellhorn, E. and G. N. Loofburrow. Emotions and Emotional Disorders. New York: Harper & Row, 1963.
9. Goddard, G.V. Functions of the amygdala. Psychol. Bull. 62: 89-109, 1964a. 10. Goddard, G. V. Amygdaloid stimulation and learning in the rat. J. comp. physiol. Psychol. 58: 23-30, 1964b. 11. Green, P. C. Effects of early vs. late lesions in cognitive-affective behavior in rats: VMH. Psychonom. Sci. 7: 11-12, 1967. 12. Grossman, S. P. The VMH: A centre for affective reaction, satiety, or both ? Physiol. Behav. 1: 1-10, 1966. 13. Grossman, S. P. A Textbook of Physiological Psychology. New York: Wiley, 1967. 14. Grossman, S. P. and Lore Grossman. Food and water intake in rats with parasagittal knife-cuts medial or lateral to the lateral hypothalamus. Submitted to J. comp. physiol. Psychol. 15. Hess, W. R. Diencephalon--Autonomic and Extrapyramidal Functions. New York: Grune & Stratton, 1954. 16. Krasne, F. B. General disruption resulting from electrical stimulation of ventromedial hypothalamus. Science 138: 822-823, 1962. 17. Levine, S. and S. Sohday. The effects of hypothalamic lesions on conditioned avoidance learning. J. comp. physiol. Psychol. 53: 497-501, 1960. 18. Lints, C. E. and J. A. Harvey. Altered sensitivity to footshock and decreased brain content of serotonin following brain lesions in the rat. J. comp. physiol. Psychol. 67: 23-31, 1969.
1108 19. Margules, D. L. and L. Stein. Chohnergic synapses in the ventromedial hypothalamus for the suppression of operant behavior by punishment and satiety. J. comp. physiol. Psychol. 67: 327-335, 1969. 20. McAdam, D. W. and W. W. Kaelber. Differential impairment of avoidance learning in cats with ventromedial hypothalamic lesions. Expl Neurol. 15: 293-298, 1966. 21. Miller, N. E., C. J. Bailey and J. A. F. Stevenson. Decreased "hunger" but increased food intake resulting from hypothalamic lesions. Science 112: 256-259, 1950. 22. Pellegrino, L. J. and Anna J. Cushman. A Stereotaxic Atlas oJ the Rat Brain. New York: Appleton-Century-Crofts, 1967. 23. Selafani, A. and S. P. Grossman. Hyperphagia produced by knife cuts between the medial and lateral hypothalamus in the rat. Physiol. Behav. 4: 533-538, 1969. 24. Sepinwall, J. Enhancement and impairment of avoidance behavior by chemical stimulation of the hypothalamus, jr. comp. physioL Psychol. 68: 393-399, 1969.
GROSSMAN 25. Smith, D. E., M. B. Kmg and B. G. Hoebel. Lateral hypothalamic control of killing: Evidence for a cholinocept|ve mechanism. Science 167: 900-901, 1970. 26. Stein, L. Chemistry of reward and punishment. In: Psychopharmacology: A Review of Progress, 1957-1967, edited by D. H. Efron. Washington: U. S. Government Printing Office, 1968. 27. Teitelbaum, P. and A. N. Epstein. The lateral hypothalamic syndrome: Recovery of feeding and drinking after lateral hypothalamic lesions. Psychol. Rev. 69: 74-90, 1962. 28. Turner, S. G. Sensitivity to electric shock after VMH lesions. Expl Neurol. 19: 236-244, 1967. 29. Wasman, M. and J. P. Flynn. Directed attack elicited from the hypothalamus. Arch. Neurol. 6: 220-227, 1962. 30. Wheatley, M. D. The hypothalamus and affective behavior in rats. Arch. neurol. Psychiat. (Chicago) 52: 296-316, 1944.
Avoidance Behavior and Aggression in Rats with Transections of the Lateral Connections of the Medial or Lateral Hypothalamus' S E B A S T I A N P. G R O S S M A N
Department of Psychology, University of Chicago, Chicago, Illinois 60637, U.S.A. (Received 4 June 1970) GROSSMAN,S. P. Av•idancebehavi•randaggressi•ninratswithtransecti•ns•fthe•ateralc•nnecti•ns•fthemedial•r•ateral hypothalamus. PHYSIOL.BEHAV.5 (10) 1103-1108, 1970.--Parasagittal surgical cuts in the medial quadrant of the lateral hypothalamus which isolated the medial half of the hypothalamus from all lateral connections produced marked deficits in avoidance learning but no d~scernible change m performance on a test of intra-species aggression. The animals showed transient motor deficits as well as aphagia and adipsia but were normal on all measures before the avoidance training and aggression tests were begun. Similar parasagittal cuts lateral to the lateral border of the anterior and medial hypothalamus (including the region lateral to the ventromedial nuclei) facilitated the acquisition of a shuttle box avoidance response but failed to affect intra-species aggression. These animals were aphagic and adipsic for longer periods of time but had recovered normal body weights and motor abilities at the time of testing. Hypothalamus-aggression Aggression-hypothalamus Hypothalamus-avoidance Hypothalamus-transectlon of connections Knife cuts-hypothalamus
MANY experimental observations suggest that components of the hypothalamus significantly influence the organism's response to noxious stimulation. This reaction can take two directions--aggression or flight--and both appear to be related to hypothalamic functions. Lesions in the medial hypothalamus result in vicious attack reactions to normal handling [30]; lowered thresholds for apparently affective reactions to electric shock [28] or noxious tastes [21]; and facilitated conditioned avoidance behavior (CAR) [12]. A pharmacological blockade of this region produces similar effects on avoidance behavior [12, 24] and disinhibits punished responses [19]. Electrical stimulation of the ventromedial area interferes with any ongoing activity and appears to be aversive [16]. Lesions in the lateral hypothalmus have also been reported to increase the rat's sensitivity to footshock [18]. Electrical stimulation of lateral as well as medial hypothalamic sites elicits defense or attack reactions [15, 29]. Chemical stimulation of some sites in the lateral hypothalamus induces mouse killing [25] and facilitates avoidance behavior [24]. The hypothalamus also controls autonomic reactions, and it has been suggested that it may contain a primary center for the integration of affective responses (see [8] for a review of this hypothesis). Extrahypothalamic structures have also been implicated in the control of aggressive or avoidance behavior but it can be argued that they exert their influence on behavior by modulating hypothalamic functions (see [13] for a review of the relevant literature). However, little is known about the
Avoidance--hypothalamus
pathways which interconnect the extrahypothalamic and hypothalamic portions of the postulated circuit or about the connections of the hypothalamus which specifically mediate reactions to affective stimuli. The present series of experiments were undertaken as a first step towards a more complete understanding of the functional importance of various hypothalamic connections. The medial half of the anterior and medial hypothalamus, including the ventromedial nuclei, was isolated from all of its lateral connections by parasagittal knife-cuts just lateral to the fornix which extended from A P = 7.0 to A P = 4.2 and from the base of the brain to the zona incerta. In other animals, similar cuts were made along the lateral border of the lateral hypothalamus. These cuts completely isolated the anterior and medial portions of the hypothalamus from lateral connections, notably with the amygdala. The wire knife used in these experiments produces very little damage to cellular components (because it is only 0.014 m m dia.) but completely transects transverse fibers. It is therefore probable that the effects of such cuts can be attributed to the disruption of fiber connections.
METHOD
Subjects Eighteen adult female albino rats of the Sprague-Dawley strain (Holtzman, Madison, Wisconsin), housed singly, in a
1Supported by grant MH 10130-06 from USPHS. The assistance of Lore Grossman and Anne Wilson is gratefully acknowledged. 1103
1104 continually illuminated and air-conditioned room were used. The animals weighed 220-260 g at surgery.
Apparatus Two identical double grill boxes constructed of transparent Plexiglas were used for the avoidance experiment. Each measured 53 × 22 × 32 cm and was divided into two compartments of equal size by a partial partition which extended 5 cm from each side of the apparatus. Both compartments had grid floors consisting of 4 mm dia. stainless steel bars placed 1 cm apart. The CS was supplied by a 7 W bulb placed in the center of the hd of each compartment. The UCS consisted of pulses (0.3 sec o n / 0 . 3 off) of constant current shock, supplied by a high-voltage source modified after Brown, Reus and Webb [3]. The aggression/dominance apparatus consisted of a clear Plexiglas tube, 94 cm in length and 4.9 cm dia. At each end of this tube were identical compartments constructed of white opaque plastic and measuring 29 × 29 × 28 cm. The floor of these compartments was constructed of stainless steel rods, 4 mm dia. and spaced 1 cm apart. The entrances to the tube which interconnected the two compartments were closed by black opaque plastic guillotine doors which could be raised from the outside of the apparatus. Two circular plastic food cups were mounted on the wall of the compartment which was farthest from the entrance of the tube.
Procedure (a) Behavioral tests. Food- and water-intake and body weight were recorded daily throughout the experiment. The animals were maintained on a powdered diet (Rockland Meal) except for several weeks after surgery when a liquid diet or pellets (Purina Lab Chow) were available. (The animals required intragastric feeding for several days post-operatively.) All animals ate normally and re-established normal body weights before training in the avoidance experiment was begun. On the first or second post-surgical day and again one week later, a series of tests [1] were given to ascertain the animals' motor capabilities. (1) The paw test consisted of lifting one of the rat's forepaws and recording the latency of the withdrawal response; (2) the pedestal test consisted of placing the animal on a small platform and recording the time taken to step down from it; and (3) the horizontal stabilization test consisted of placing the rat upside down on the inside of the front panel of its home cage and recording the time taken for the animal's voluntary descent into the cage. A maximum of three minutes was permitted for each test. Approximately six weeks after surgery, all animals were trained to avoid painful grid shock by jumping into the dark compartment of a double-grill shuttle-box in response to the onset of a light mounted overhead. Fifteen trials per day were given for 21 days as follows: The animal was placed into the dark compartment of the shuttle box. Sixty-three sec later, the overhead light was illuminated and the illumination of the adjacent compartment turned off. Five sec later, the grid floor in the illuminated compartment was electrified. The light and shock remained on in that compartment for the next 58 see. The CS and CS-UCS combination then switched to the previously safe dark compartment. The animals could escape or avoid the shock by always jumping into the dark compartment. The duration of the subjective intertrial interval was determined within a 63 see limit by the rat since the cycling of the CS and UCS was unaffected by the animals' behavior.
GROSSMAN Following the completion of the avoidance experiment, all rats were placed on a restricted feeding schedule designed to maintain each rat at approximately 90 per cent of its normal body weight. Each rat was then trained to run through the tube of our aggression apparatus for rewards consisting of highly pallatable bits of Froot Loop cereal. (The somewhat unusual rewards were selected because some of the animals reacted poorly to the deprivation regimen and would not run reliably for standard reward pellets.) After some initial shaping, each rat received 10 trials per day until its running latencies had stabilized. In the following tests of aggressiveness and dominance, each animal continued to receive 10 rewarded trials per day. On one randomly selected trial each day, two rats (one experimental animal and one unoperated control) were started simultaneously from opposite ends of the tube. Since the response latencies of both members of each pair were comparable, the two animals met near the middle of the tube. Vigorous fighting ensued because the diameter of the tube was selected so that the animals could not pass each other. Eventually, one of them backed up all the way to its starting compartment (and missed out on its reward at the other end of the tube). The other rat followed and was reinforced upon arrival in the opponent's start compartment. This test was repeated with the same pair of animals until one of them had dearly established dominance by winning six consecutive fights. The fights in the tube were initially prolonged and vicious but tended to become shorter and less acrimonious with repeated testing. (b) Surgery. Surgical knife cuts were produced under Nembutal anesthesia by stereotaxically placing a guide made of 30 ga hypodermic needle stock at the following coordinates (from the de Groot [6] atlas of the rat brain); A P = 7.4; L = 1.5; H = 0 . 0 or A P = 7 . 4 ; L=2.5; H=0.0. A 0.014 mm dia. tungsten wire was then extended from the slightly bent tip of the guide cannula until the wire protruded (at an angle of approximately 90 ° to the guide) posteriorly for 2-3 mm. The entire assembly was then lowered from H = 0.0 to the base of the brain and raised again to H = 0.0 The wire was then retracted and the guide cannula withdrawn. The design of the cannula has been described in detail elsewhere [23]. (It should be noted that approximately one-third of the animals which are subjected to this operation do not survive, the most probable cause of death being the transection of major blood vessels.) (c) Histology. Following completion of the experiments, all animals were sacrified with an overdose of Nembutal and perfused intracranially with a 10 per cent formalin solution. The brains were removed from the calvarium and frozen to permit the cutting of 25 ~t frozen sections. Alternating sections were stained with cresyl violet (to bring out cellular components) and Sudan black (to emphasize fiber tracts). The description of the anatomical results (see below) is based on microscopic analysis of this material. RESULTS AND DISCUSSION
(a) Effects of Transections of the Lateral Connections of the Medial Hypothalmus Bilaterally symmetrical parasagittal cuts which transected the hypothalamus approximately 1.5 mm from the midline were made in six animals. The cuts extended from A P = 7.07.4 posteriorly to A P = 4.2-4.6 and from the base of the brain to the zona incerta, terminating just below the medial lemniseus (see Figs. 1 and 2).
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(facing page
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TRANSECTIONS OF CONNECTIONS OF HYPOTHALAMUS
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These animals were aphagic and adipsic for 2-8 days after surgery but recovered the ability to regulate food intake on a powdered food diet within two weeks. They were subsequently hyperphagic when placed on a preferred pellet diet and had completely recovered normal body weights when training in the avoidance apparatus was begun. These animals appeared catatonic on some of the motor tests given on the first or second day after surgery but had recovered on the second tests, given one week later. All animals performed well within normal limits on a third set of motor tests given just before the beginning of training in the shuttle boxes. A detailed report of the food and water intake and motor
impairments of these rats and others with similar cuts has been published elsewhere [14]. At no time did these animals display the vicious attack reactions to normal handling which are commonly seen in rats with ventromedial hypothalamic lesions, suggesting that the influence of this region on aggressive behavior may not be mediated by direct lateral projections. This conclusion is supported by the observation that our experimental rats which no longer had these projections were not discernibly better or worse on our test of aggression or dominance. Two of the six animals with mid-hypothalamic knife cuts won all of their fights with an unoperated control rat; throe
1106
GROSSMAN when relatively high shock levels or complex learning situations are employed (unpublished data), and McAdam and Kaelber [20] have reported inhibitory effects on C A R acquisition in cats in a high-shock situation. It appears unlikely, however, that the results of our knife-cuts could be due to an excessive reaction to shock in view of the absence of attack reactions to handling and the animals' normal pattern of intra-species aggression. These observations suggest, instead, that the inhibitory influences which the ventromedial region appears to exert on affective reactions to noxious stimuli may be mediated by pathways which are not directly affected by our cuts. The work of Stein [26] suggests that the perlventricular fiber system may be a prime candidate for this function. If this analysis is correct, the observed depression of avoidance behavior in animals with cuts lateral to the medial hypothalamus could be due to an interruption of afferents to the ventromedml region which mediate inhibitory influences. Sepinwall's [24] recent observation that pharmacological blockade of the lateral hypothalamus (which should not affect fibers of passage) inhibited avoidance behavior whereas chemical stimulation of that region enhanced it, suggests further that the cells which may exert this inhibitory influence on the ventromedial area may be located in the lateral hypothalarnus. This interpretation is supported by a recent report [5] of avoidance deficits in rats with lateral hypothalamic lesions.
operated rats lost all of them; and one eventually won six consecutive fights but lost many fights in the process and did not appear dearly dominant in any of them. (If a clearly dominant and a clearly submissive partner were paired, very little fighting occurred after the initial encounter and the submissive animal backed off within a few seconds. When the dominance/submission relationship was less dearly defined, vicious and prolonged fighting often occurred even after as many as 10 or 15 encounters.) The pattern of dominance/ submission seen in the rats with parasagittal cuts which bisected the hypothalamus was dearly within the normal range. In the avoidance experiment, these animals performed reliably poorer than the normal controls (p < 0.01) or the rats with cuts along the lateral border of the hypothalamus (p < 0.01) at all levels of training (see Fig. 3). Half of the animals m this group failed to reach the criterion of 9/10 consecutive avoidance responses on two successive days within the 21 days of the experiment; two more required a greater than average nurnber of trials to do so. Only one animal which could not be distinguished on the basis of gross histological data learned the task rapidly. There was no discernible relationship between the animals' performance in the avoidance situation and their aggressiveness or the severity of the experimentally produced changes in feeding behavior. The impaired avoidance behavior of these animals was surprising in view of the commonly reported observation that rats with ventromedial hypothalamic lesions learn avoidance responses better than normals [11, 12, 17]. This finding is confirmed by the effects of pharmacological blockade of the ventromedial region [12, 24] and is in accord with the lesioned animals' increased sensitivity to footshock [28]. We have observed that the general hyperexcitability and hyperreactivity of rats with such lesions interferes with avoidance learning
(b) Effects of Transections of the Lateral Connecttons of the Lateral Hypothalamus Bilaterally symmetrical parasaglttal cuts, lateral to the lateral border of the anterior and medial hypothalamus, were made in six rats. These cuts coursed through the internal
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the medial quadrant of the hypothalamus or lateral to the lateral hypothalamus. The data are expressed in terms of the number of trials required to meet increasingly stringent criteria of avoidance proficiency (such as 1, 2, 3. . . . 9 avoidance responses in 10 consccutivc trials). The line labelled "max" indicates the performance of a hypothetical animal which reaches the criterion of 9 out of 10 avoidances without error.
TRANSECTIONS OF CONNECTIONS OF HYPOTHALAMUS capsule and the interpeduncular nucleus and did not involve tissue of the lateral hypothalamus at any point. The cuts extended from A P = 7.0-7.4 posteriorly to A P = 5.0-5.8 and transected all lateral connections of the anterior and medial hypothalamus. The lateral connections of the posterior hypothalamus were left intact. (Cuts involving the lateral projections of the posterior hypothalamus were attempted but frequently resulted in such severe motor impairments that behavioral testing was impossible.) (See Figs. 1 and 2.) All animals with parasagittal cuts along the lateral border of the lateral hypothalamus were aphagic and adipsic following surgery. Some (not included in this discussion) failed to recover normal food or water intake and were discarded 60 days after surgery. Others, including the animals used in the present experiment, went through the recovery cycle described by Teitelbaum and Epstein [27] for lateral hypothalamic lesions and eventually recovered the ability to regulate food and water intake (the latter being purely prandial). These animals showed severe impairments on all of our motor tests given on the first or second post-surgical day but recovered to normal levels within one week (see [14] for a more detailed account). All animals performed normally on a third set of motor tests given just before the beginning of training in the avoidance apparatus, and had attained normal body weights (X: = 276.1 g). Daily food and water intake were recorded throughout the subsequent aggression experiment to assure adequate food and water consumption at all times. Isolation of the anterior and medial hypothalamus from its lateral connections had no more of an effect on inter-species aggression than transection of its medial connections (see above). Two experimental animals of this group won all of their fights with unoperated controls and two lost all of them. The remaining two won 6 consecutive fights only after 14 and 15 encounters respectively and never displayed a clear dominance relationship with their unoperated partner. These observations indicate that the aggressive reactions which have been elicited by electrical [7, 29] and chemical [25] stimulation of the lateral hypothalamus may be mediated
1107 by pathways which project in a rostro-caudal direction and do not depend on direct lateral or medial connections. The avoidance behavior of rats with parasagittal cuts lateral to the hypothalamus was superior to that of the unoperated controls or of rats with cuts in the medial quarter of the lateral hypothalamus (see Fig. 3). These animals performed no better than the unoperated controls during the early stages of training but reached higher terminal levels of avoidance performance reliably (p < 0.05) faster. The experimental animals of this group were significantly (p < 0.01) better at all levels of performance than the rats with cuts through the medial quadrant of the lateral hypothalamus. These findings suggest that the modification of avoidance behavior which has been observed after chemical stimulation or blockade of the lateral hypothalamus [24] may be due to stimulation or blockade of cells which receive inhibitory afferents via lateral connections. The origin of these connections is, as yet, unknown. The amygdaloid complex would, at first glance, appear to be a likely suspect. However, damage to most portions of the amygdala produces an impairment in C A R acquisition (see [9] for a review of this literature). Moreover, lesions which interrupt the ansa lenticularis, one of the major amygdalo-hypothalamic connections, have been reported to produce no selective effects on C A R acquisition [4]. There are, however, some recent observations which indicate that the amygdala may be the source of inhibitory as well as excitatory influences on C A R acquisition. Small lesions or pharmacological blockade of restricted aspects of the ventral amygdala facilitate avoidance learning [2] and low level electrical stimulation has been reported to have inhibitory effects [10]. Our results confirm Caruther's suggestion that excitatory amygdaloid influences on C A R behavior do not appear to be mediated by the ansa lenticularis or other lateral connections of the anterior and medial hypothalamus [4]. They indicate further that fibers carrying inhibitory influences of amygdaloid or other origin appear to enter the hypothalamus at its lateral border.
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1108 19. Margules, D. L. and L. Stein. Chohnergic synapses in the ventromedial hypothalamus for the suppression of operant behavior by punishment and satiety. J. comp. physiol. Psychol. 67: 327-335, 1969. 20. McAdam, D. W. and W. W. Kaelber. Differential impairment of avoidance learning in cats with ventromedial hypothalamic lesions. Expl Neurol. 15: 293-298, 1966. 21. Miller, N. E., C. J. Bailey and J. A. F. Stevenson. Decreased "hunger" but increased food intake resulting from hypothalamic lesions. Science 112: 256-259, 1950. 22. Pellegrino, L. J. and Anna J. Cushman. A Stereotaxic Atlas oJ the Rat Brain. New York: Appleton-Century-Crofts, 1967. 23. Selafani, A. and S. P. Grossman. Hyperphagia produced by knife cuts between the medial and lateral hypothalamus in the rat. Physiol. Behav. 4: 533-538, 1969. 24. Sepinwall, J. Enhancement and impairment of avoidance behavior by chemical stimulation of the hypothalamus, jr. comp. physioL Psychol. 68: 393-399, 1969.
GROSSMAN 25. Smith, D. E., M. B. Kmg and B. G. Hoebel. Lateral hypothalamic control of killing: Evidence for a cholinocept|ve mechanism. Science 167: 900-901, 1970. 26. Stein, L. Chemistry of reward and punishment. In: Psychopharmacology: A Review of Progress, 1957-1967, edited by D. H. Efron. Washington: U. S. Government Printing Office, 1968. 27. Teitelbaum, P. and A. N. Epstein. The lateral hypothalamic syndrome: Recovery of feeding and drinking after lateral hypothalamic lesions. Psychol. Rev. 69: 74-90, 1962. 28. Turner, S. G. Sensitivity to electric shock after VMH lesions. Expl Neurol. 19: 236-244, 1967. 29. Wasman, M. and J. P. Flynn. Directed attack elicited from the hypothalamus. Arch. Neurol. 6: 220-227, 1962. 30. Wheatley, M. D. The hypothalamus and affective behavior in rats. Arch. neurol. Psychiat. (Chicago) 52: 296-316, 1944.