aggressive behavior induced by electrical stimulation in the midbrain central gray of male rats

AGGRESSIVE BEHAVIOR Volume 8, pages 261-284

Aggressive Behavior Induced by Electrical Stimulation in the Midbrain Central Gray of Male Rats J. Mos, M.R. Kruk, A.M. van der Poel, a n d W. Meelis

Leiden University, Medical Centre, Department of Pharmacology, Sylvius Laboratories, Leiden, The Netherlands

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Electrical stimulation via electrodes implanted in the lateral hypothalamus may induce intraspecific aggressive behavior. Small electrolytic lesions placed via these electrodes resulted in a five- to tenfold increase in the current threshold for aggression. Degenerating fibers were stained by means of the Fink-Heimer method and could be followed caudally to the dorsal midbrain central gray and to the mammillary bodies. A few 8x011s could be traced rostrally to the medial septum. Aggression could be induced from 10 of 112 electrodes implanted in the central gray; the other electrodes elicited either locomotion, vocalization, jump, or “a~arm-like reactions.” The morphology of the induced aggression was similar to the morphology of the hypotha~amically induced aggression, though it was often accompanied with motor disturbances and was less intense. Hypothalamic stimulation was combined with simultaneous central gray stimulation in rats with electrodes both in the hypothalamus and in the central gray. Hypothalamic thresholds for aggression could be lowered by this stimulation of the central gray, even when no aggressive responses were observed during central gray stimulation alone. This suggests that, although aggression is not manifest, electrical stimulation may activate neural tissue involved in aggressive behavior. It is concluded that in rats central gray and hypothalamus are part of the same neural network mediating intraspecific aggression.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Key words: rat, aggression, hypothalamus, central gray, electrical brain stimulation,

neuroanatomy

INTRODUCTION

In recent years we have been working on the characterization of the neural structures involved in aggression in rats. Electrical stimulation in the hypothalamus of male rats may induce aggressive behavior towards conspecifics

Received for publication September 29, 1981; accepted January 18, 1982. Address reprint requests to J. Mos, Sylvius Laboratories, Dept of Pharmacology, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands.

0096-140X/82/0803-0261$06.50 0 1982 Alan R. Liss, Inc.

262 Mos et a1

[Koolhaas, 1978; Kruk et al., 1979; Panksepp, 1971; Woodworth, 19711. Stim- ulatioLl in a confined region of the hypothalamus results in attacks that, according to their morphology, can be classified as attack-jumps or bite-attacks. Apart from the initial decline, current thresholds that induce aggression remain stable for months [Kruk et al., 19791. Although several arguments favor the idea that a specific neural network subserving aggression is present in the hypothalamus, no definite statements can be made at present [Kruk and Van der Poel, 19801. To answer questions about the specificity of the effects induced by stimulation and whether or not one may speak of a neural correlate for aggression, more information about hypothalamically-induced attacks is needed. The purpose of the present investigation was to study which other regions may be involved in a neural circuit subserving aggression in rats.

The approach was essentially the same as the analysis that fly^ and coworkers [1979] adopted for the induced attacks upon stimulation in the cat brain. If the anatomical connections of the aggressive area in the hypothalamus with other parts of the brain can be shown to be relevant for aggression, it would support the hypothesis for a specific neural network for aggression. An initial step was to make small lesions via the aggression-inducing electrodes in the hypothalamus and to study the projections of degenerating axons with the Fink-Heimer technique (experiment 1). The next step was stimulation of one of these projection sites - the midbrain central gray- to find out whether aggression can be induced (experiment 2). The last step in this study was the simultaneous stimulation of the hypothalamus and the central gray area to answer the question of whether concomitant stimulation of the latter area could facilitate hypothalamic aggression (experiment 3).

GENERAL METHODS

For details on surgery, test cage, and stimulation techniques see Kruk et al. [1979, 19801.

Experimental subjects were brown-eyed, beige-colored, male CPB-WE-zob rats, originally supplied by the Central Institute of Laboratory Animals (CPB- TNO, Zeist, The Netherlands). Before the operation the rats were housed in groups of six to eight in large Macrolon@ cages in low-noise rooms at 22°C and 75% relative humidity. Food and water were available ad libitum and an inverted 14-hour day and 10-hour night cycle was installed. Prior to the operation the rats were handled once daily for one week. Male albino rats (CPB-WU Wistar random), weighing between 180 and 200 gm, were used as test partners.

Upon completion of the experiments, rats were anesthetized and perfused with physiological saline and 4% formalin. After at least a week of storage in situ in formalin the brains were removed from the skulls. Frozen sections were stained with the Kliiwer-Barrera method (except in experiment 1).

Central Gray and Aggression 263

For convenience the description of the procedure used in threshold determinations is repeated here. Threshold current intensities for aggression and vocalizations were determined according to the up-and-down method of Dix- on and Mood [ 19481 as modified by Wetherill [ 19661. The method consists of increasing the current intensity in fixed steps until a behavioral response is induced, then decreasing it until the response is lost, increasing the current until the response appears again. A response change is thus defined as the mean current intensity of a pair of successive positive and negative trials. The threshold of a behavior can be calculated from six subsequent response changes; it is the current intensity inducing that behavior in 50% of the stimulation periods. The up-and-down method reduces the number of stimulation trials needed to determine a threshold as compared to the method of limits. In threshold determinations the current is on for 10 seconds and off for 50 seconds periodically. The 50-second intertrial interval allows complete recovery from the afteref- fects from the previous 10-second stimulation train.

EXPERIMENT 1 Introduction

Flynn and coworkers [1979] were the first to study extensively the neural pathways related to aggression in cats. Principles and strategy have lately been summarized [Flynn et al., 19791. Briefly, an electrode is implanted in the brain and the effects of electrical stimulation by this electrode are studied. If a cer- tain behavior (e.g., aggression) can be reliably induced, a small lesion is made by passing a direct current through this electrode. The basic idea is that by this procedure a small amount of tissue involved in aggression is destroyed. The resulting pattern of degenerating axons is then studied. These studies differ from anatomical experiments in that the lesions are not necessarily placed in anatomically well-defined regions. Early behavioral studies included lesioning of a specific neural structure, after which impairments in performance of various behaviors were studied. Electrodes that yield a specific behavior upon stimulation are usually not placed in well-defined structures and therefore the neural pathways for such behavior cannot be directly inferred from data obtained in conventional neuroanatomical studies. We therefore operated on rats with chronically implanted electrodes and studied the behavior of these animals under stimulation before a small lesion was placed by this electrode. The degeneration resulting from the lesion was then investigated.

Methods Animals. Ten rats were used, each implanted bilaterally with a Pt-1OVoIr

electrode. The electrodes were aimed at the perifornical region at the coordinates - 2.5 mm dorsoventral, 1.5 mm mediolateral, and 5.5 mm anteropos-

264 Mos et al

terior of the stereotaxic atlas of Konig and Klippel[1963]. Aggression could be induced upon stimulation by at least one of the two electrodes in each rat. On- ly electrode implantations with stable thresholds for aggression were used in the lesion experiment, Most implantations had been used previously in other experiments.

Lesions. Upon completion of the experiments, electrolytic lesions were placed via one of the electrodes by passing a direct current of 30-50 p4 for 30 seconds. Immediately after the lesion, each rat was stimulated through the same electrode. If attacks could be induced at current intensities lower than 1 mA, a second lesion was made with the same parameters. During the survival time four electrodes in four rats were tested repeatedly for stimulation-induced aggression.

Histological techniques. Three to five days after placing the lesion, the rats, under deep ether anesthesia, were perfused transcardially with physiological saline and 4% formalin. After the brains had hardened in formalin for at least one week, they were placed in a solution of 40% sucrose and 4% formalin for several days to reduce the formation of ice crystal artifacts during sectioning. The brains were cut on a freezing microtome at 20 pm. At regular intervals sections were stained according to the Fink-Heimer method for degenerating fibers. Adjacent sections were stained by the Kluwer-Barrera method to identify cytological boundaries of structures. Photographic en- largements were made from stained sections. The degenerating fibers were indicated on pictures that were used to make the definite drawings for Figure 3. The anterior-posterior coordinates of the electrode tips were assessed by com- paring the relevant sections with the standard section of our own unpublished atlas (see Table I). In this atlas sections were made at 75-pm intervals, which yields a better resolution than is possible with the atlas of Konig and Klippel.

Control procedures. The electrodes in the hypothalamus passed through most of the dorsoventral extent of the brain. The following precautions were taken to control for degeneration resulting from the electrode track. Damage was minimized by using electrodes with a small diameter (150 pm). The electrodes were implanted 1-10 months before the lesions were made. The amount of degenerating fragments produced by the electrode track is considerably reduced in this period and morphologically distinguishable from the acute degeneration caused by the lesion. Since all animals had electrodes in the left and in the right hypothalamus, which had passed through the same overlying structures, it was the difference in degeneration between the left and the right side that was taken into account. In one rat (198L) the electrode was lowered at a different angle (45" to the coronal and midsagittal plane), so that the electrode penetrated different regions.

Behavior. Upon stimulation in the hypothalamus the rats showed pilo- erection and tooth-chattering. They approached the partner, often accom-


TABLE I. Relative Quantity of Degeneration in Selected Areas Following Lesions of Attack Sites in the Hvoothalamus"

Rat

54 R 72 R 79 L 80 L 92 R 99 L

103 L 138 L 140 L 198 L

Central gray

+ + + - + - + + + +

+ + + + + + + +

Corpus mammillary Septum

+ + + - + +

- ? + - ? + - + +

- + + - + + - + + + + + + + +

-

Fornix

Survival VMH time Tip lesion (days) localization

Maximum diameter

h m )

+ + +

+ + + + -

+ + + + + + + -

5 3 5 5 3 4 4

+ 4 + - 4 + 4

- - - - - - -

92 95

104 104 93 93 94

105 105 104

530 570 410 330 410 370 370 530 490 250

*The presence of degeneration in the fornix is indicated, and so is the extension of the lesion into the ventromedial nucleus. Symbols: ( - ) no degeneration present; (?) degeneration doubtful; (+ -, +, + +) denote increasing density of degeneration. In a separate column the anterior-posterior coordinate of the electrode localization is given in numbers of our own atlas. Plate 90 corresponds approximately to AP 5780 pm in the atlas of Konig and Klippel. Plate 104 corresponds to AP 4890 pm.

panied with lateral threat. They either jumped towards the partner, meanwhile trying to kick its belly and bite its head (jump-attack), or bit directly the head and neck region (bite-attack). This could culminate in a ferocious clinch fight. Fighting could end by a submissive posture of the partner or by retreat of the attacking rat. More than one attack could occur during the stimulation time, but they did not outlast this period (see Kruk et al., 1979 for a detailed description of stimulation techniques, apparatus, and behavior).

Results The small lesion elevated thresholds for aggression five- to tenfold. Postle-

sion thresholds were mostly higher than 1-1.2 mA, which was the current limit we employed. On subsequent days the thresholds decreased and reached a stable level after two days (Fig. 1). Thresholds on days 3 and 4 are 2.5- to 6-fold higher as compared to prelesion values, which indicates the destruction of a part of the tissue implied in the induction of the observed aggressive behavior. The diameter of the lesions was two to four times the size of the electrode tip (300-600 pm).

All lesions lie in what Saper et al. [1979] have defined as the tuberal part of the lateral hypothalamus. Some lesions extend partly into the ventromedial nucleus (VMH). An overview of the localizations of the electrodes is given in Figure 2. All electrodes lie well within the region where aggression can be easily

266 Moset al

Threshold I t 1200

1100

1000

900

800

700

600

500

400

3 00

200

100 ,

I

Rat 198 L

2 3 postlt 3n day

Fig. 1. Current thresholds for aggression in rat 198L before the lesion and after the lesion on four subsequent days. Immediately after the first lesion the threshold was lower than loo0 pA so a second lesion was placed.

induced [Kruk et al., 19821. The results of degeneration studies for all animals are summarized in Table I. Not all brains were equally well stained, which may be why less dense degeneration bundles sometimes are scarcely seen.

In general, after lesioning of hypothalamic attack sites, degenerating fibers could be traced from the lesion into the midbrain central gray, to the fibrous capsule of the mammillary bodies, to the mammillary bodies and to the medial septum.

Figure 3 shows the degeneration resulting from a lesion in rat 14OL, plotted schematically on frontal sections. This degeneration prevails to the midbrain central gray. It passes caudally through the periventricular bundle of Schutz into the central gray; initially it follows a dorsal route immediately next to the


ventricle. More caudally it turns more lateral and becomes more diffuse. The degeneration ends in the dorsal central gray at the level of the caudal ending of the red nucleus.

A rather thin rostra1 branch passes through the preoptic area into the diag- onal band of Broca and the medial septum, where it could not be traced any further.

Scattered degeneration in the mammillary bodies was often observed where degenerating fibers sometimes descended caudolaterally to the fibrous capsule of the mammillary bodies. No degeneration was seen to descend below the level of the mammillary bodies to the ventral tegmental area. Similar patterns

I 9 9 5 4

Fig. 2. Localization of the electrode tips plotted on two frontal sections of our own atlas of the hypothalamus. The black dots do not represent the actual size of the lesions. The maximal diameter of the lesions is given in Table I. A, caudal section; B, rostal section. Abbreviations: cair, capsula interna, pars retrolentricularis; f, columna fornicis; fmt, fasciculus mamillothalamicus; pv, nucleus paraventricularis; to, tractus opticus; vmh, nucleus ventromedialis; zi, zona incerta.

268 Moset al

Cenlral Gray and Aggression 269

of degeneration were observed in the rats with lesions extending into the VMH as in rats without damage in the VMH.

In some animals the electrode had not penetrated the fornix and no signs of degeneration were found in there. Since the degeneration pattern did not differ in these animals, it seems unlikely that the total pattern should be much influenced by damage to the fornix. The other control for damage by the electrode track is provided by rat 198L. In this rat the electrodes were implanted under a different angle damaging other overlying structures. However, no differences in degeneration were observed. We therefore feel that this pattern of degeneration was not caused by artifacts of the electrode implantation, but is due to destruction of a part of the tissue activated during stimulation-induced aggression.

Discussion In a similar study in cats, Chi and Flynn [1971a, b] described degenerating

axons after lesioning of hypothalamic attack sites. They discriminated between two types of aggression. Quiet-bite attack (QBA) could be induced in the more lateral and dorsal hypothalamus. This behavior is characterized by absence of vocalization, relative lack of autonomic signs, and by vicious biting of the head and neck region of the target animal (a deeply anesthetized rat). Affective attack (AA) is acccompanied by a marked display and intense autonomic arousal, and the attack is frequently made with unsheathed claws.

After lesioning of QBA sites degeneration was seen in the septum, the midbrain central gray, the dorsal hypothalamic area, the midline thalamus, and prevailed in the ventral tegmental area and reticular formation.

In contrast lesions of AA sites resulted in heavier degeneration in the midbrain central gray. With respect to the anatomical connections, the intra-

Fig. 3. Degeneration resulting from a lesion in rat 14OL plotted on drawings of frontal sections of our own atlas. 1, most frontal section; 6 , most caudal section. Corresponding sections in the atlas of Konig and Klippel (approximately): 1 = 8900 pm AP, 2 = 7600 pm AP, 3 = 4OOO pm AP, 4 = 3000 pm AP, 5 = 2400 pm AP, 6 = 1600 pm AP. The black region in section 3 is the caudal ending of the lesion. BST, nucleus interstitialis striae terminalis (bed nucleus); C, nucleus caudatus putamen; CA, commissura anterior; CAIR, capsula interna; CC, crus cerebri; CCS, commissura colliculorum superiorum; CP, commissura posterior; CO, chiasma opticum; DBB, nucleus tractus diagonalis (Broca); F, columna fornicis; FMT, fasciculus mamillothalamicus; FR, fasciculus retroflexus; GCC, genu corporis callosi; HIA, hippocampus pars anterior; ICM, insula Calleja magna; IP, nucleus interpeduncularis; LM, lemniscus medialis; MS, nucleus septi medialis; PCMA, pedunculus corporis medialis; R, nucleus ruber; SGC, substantia grisea centralis; SN, substantia nigra; TCC, truncus corporis callosi; TO, tractus opticus; TOL, tractus olfactorius lateralis; TULC, tuberculum olfactorium, pars corticalis, lamina pyramidalis.

270 Moset a1

specific aggressive behavior of the rat thus seems to show more relation to the AA of the cat, since both have dominant degeneration in the central gray.

The projections we found were also described in purely neuroanatomical studies on the efferents of the hypothalamus. Using the Nauta-Gygax method, Guillery [ 19571 found projections to the central gray, the mammillary bodies, and the septum. Since in that study the lesions were large, degeneration in other brain areas was also demonstrated, particularly the tegmental and in- trahypothalamic projections.

Conrad and Pfaff [1976] and Saper et al. [1979] have injected tritiated amino acids in the lateral hypothalamic area of the rat to study the efferent connections. Among projections to the central gray, the septum, and the mammillary region, they found projections to the ventromedial and dorsome- dial hypothalamic nucleus, the anterior hypothalamic area, the nucleus paraventricularis, and the ventral tegmental area.

Two major points deserve further attention. First we failed to trace degeneration into the ventral tegmental area and the reticular formation. As described above, the existence of this connection has been demonstrated by several investigators applying different anatomical techniques [Conrad and Pfaff, 1976; Saper et al., 1979; Guillery, 1957; Wolf and Sutin, 19661. We may have missed the projection because our method may not have been sensitive enough and the projection might be diffuse, or the fibers belonging to this projection may not be present in the vicinity of the aggression-inducing electrodes. Our results need not be surprising however, since we place our lesions based on the behavioral effect of electrical stimulation and are not guided by neuroanatomical considerations. Therefore our results will not always match the findings of studies where the lesions were placed in anatomically well-defined regions.

The second point concerns the origin of the observed degenerated fibers. Electrolytic lesions destroy cell bodies as well as fibers in the vicinity of the electrode tip. It may be that part of the degeneration is caused by passing fibers unrelated to the aggressive response. We cannot exclude this possibility by the present experiments. However, the projections we found were also observed in autoradiographic studies, in which only cell bodies are labelled [Conrad and Pfaff, 1976; Saper et al., 19791. Thus, to account for the observed pattern of degeneration it is not necessary to assume an important contribution of passing axons.

In accordance with the results of Chi and Flynn [1971a,b] we found that immediately after small lesions the current thresholds for aggression are substan- tially increased. Unlike other investigators we have repeated the threshold determinations and it appeared that the first two days after the lesion, the initial post-lesion thresholds decreased to some extent, though they remained much higher than the prelesion values. We checked one possible cause for this decline, namely the electrical properties of the electrode after the lesion. A


change in resistance and capacitance of the electode might result in less effective stimulation. Unpublished work of Kruk and van Strien in our laboratory showed that these electrical properties are only temporarily disturbed and re- turn to their normal prelesion values within 6 hours following a DC lesion. Therefore changed electrode properties cannot account for the initial decrease in postlesion thresholds during the first two days after the lesion. Neither do the results explain the lasting increase in current thresholds for aggression. It is thus reasonable to assume that the increased current thresholds do reflect a destruction of part of the tissue responsible for the observed aggressive behavior.


In the previous experiment an anatomical connection from the hypothalamus to the midbrain central gray was demonstrated. A second step in unravelling a neural network mediating aggression is to study the behavior resulting from local electrical stimulation in the central gray. If the projection to the central gray is indeed part of a neural network subserving aggression, then electrodes implanted in the degeneration region (see experiment 1, Fig. 3) should likely induce aggression, whereas from electrodes outside this specific region this would not be expected.

Therefore male WE-zob rats were implanted with electrodes in the central gray and the behavior induced by electrical stimulation was studied. In a number of these rats electrodes were implanted both in the hypothalamus and in the central gray, to permit studies on the interaction of these regions.

Methods Fifty-seven male CPBWE-zob rats weighing 350-500 gm, were each im-

planted with two bipolar Pt-1OVoIr electrodes. Electrodes were made by twist- ing two teflon-coated wires of 0.076-mm diameter (Medwire PtIr 3T); only the cross-sections at the tips were bare. The electrodes were implanted under an angle of 10" or 20" to the midsagittal plane and aimed at 1.8-3.0 mm antero- posterior, 0-0.5 mm mediolateral, and - 0.2- + 0.4 mm dorsoventral to the atlas of Konig and Klippel. Two electrodes were discarded because of poor electrical conductance; the remaining 112 electrodes were used for testing. Of the 57 rats, 24 also had two electrodes in the lateral hypothalamus (see experiment 3). No stimulation of these hypothalamic electrodes was performed in the present experiments.

When the rats had recovered from surgery (> 7 days), they were placed in the test cage with a partner weighing 180-200 gm. Stimulation was usually started at 50 PA, using biphasic pulses of 0.2-msec phase duration, with a fre- quency of 40 hz and a phase interval between the positive and the negative

272 Mos et al

phase of 12.5 msec. Stimulation lasted 2-3 minutes unless the response was too vigorous. An interval of at least 1 minute separated successive tests. Depend- ing on the result of the stimulation the current was increased or decreased. The step size of the current intensity was 20-100 PA; any current higher than 600 pA was not used. If a clear response occurred, testing was usually repeated with the same current intensity. If the behavior could be induced repeatedly, an attempt was made to determine a current threshold. Thresholds were determined with a train duration of 10 seconds and an intertrain interval of 50 seconds. For further details see General Methods.

Results Description of responses. The responses elicited upon stimulation can be

classified in two categories. In the first group, stimulation results in one dominant behavior. Although other responses may occur at much higher current levels the particular response was dominant at the lower current and consequently this behavior was used to classify the electrode. The second category is formed by a combination of responses. Several elements of behavior were taken together for reasons explained below.

Dominant responses. Aggression: Intraspecific aggression could be induced by stimulation via 10 electrodes in the central gray. The induced aggression consisted of approach, pilo-erection, lateral threat, and biting the head and neck region of the partner. The morphology of the behavior differed from hypothalamic stimulation in that no attack-jumps were observed. The behavior preceding the actual bite, however, was very similar to the hypothalamically-induced aggression [Kruk et al., 19791. Unlike hypothalamic attack the behavior induced by central gray stimulation is often accompanied by disturbed motor performance. These animals lean to the ipsilateral side, which could seriously interfere with the attack, especially if the current was increased. Most electrode placements did not permit threshold determination, because the attack occurred irregularly due to motor disturbance.

Locomotion: Stimulation by 23 electrodes resulted in locomotion. The locomotion increased with current intensity and could eventually pass into run- ning. Locomotion could be ipsilateral and contralateral in direction.

Vocalization: Scream-like sounds could be induced at 15 sites. The vocalizations were easily induced and current thresholds were stable from the first one onwards. They were independent of the simultaneous presence of partner, so it is unlikely that social interactions could underlie these vocalizations. Chronax- ies were estimated from strength-duration curves to be 0.53 k 0.05 msec (for details on chronaxie determinations see Kruk and Van der Poel, 1980).

Jump: The dominant response upon stimulation by seven electrodes was jumping. These were high enough to jump out of the 60-cm high cage, so the walls were heightened to prevent escape. Thresholds with one jump in 10 sec-


onds as criterion increased during the successive trials, until eventually some could no longer be measured. In one experiment the current was switched off once the animal had jumped at the ridge of the lower wall. This led to a decrease of the current necessary to induce jumping.

Other responses: Stimulation by seven electrodes seemed to inhibit normal behavior, because the animals remained sitting in one corner of the cage. No other noticeable change in behavior of these animals was observed after stimulation of these points. In three other cases the disturbance of motor performance prevented the animal from behaving normally. No further experiments were done with these electrodes.

Combined responses. Stimulation did not always elicit a dominant response. Locomotion, vocalization, and jumping from the cage seemed to be associated. The locomotion closely resembled Woodworth’s [1971] description of “rapid intermittent locomotion.” The rat sits in a corner, often in a crouched position, then runs to another corner, sits there for a while, and runs to the next corner of the test cage. Sometimes the animal vocalizes or tries to jump from the cage. With increased current, more vocalizations and jumps are elicited. Jumping is most often seen at high current intensities. For this behavior we have adopted the term “alarm” from Woodworth [1971], who described a similar pattern of responses following hypothalamic stimulation.

Histology. The localization of the electrode tips is shown in Figure 4. The majority of the electrodes were located in the central gray and the overlying posterior commissure. Visual inspection of the distribution of the aggressive points makes clear that these points cluster in and around coronal plane at 4500 pm. Vocalization points seem to be located more medially in the vicinity of the ventricle. No clear distribution pattern is seen in the points for locomotion alarm and jump.

In the first experiment a region was delimited in the central gray, where degenerating axons were found following small lesions from hypothalamic aggression electrodes (Fig. 3). An attempt was made for all electrode positions - both aggressive and nonaggressive - to assess whether or not they lay in this region. Table IIa is a chi-square table of these results. Significantly more

TABLE 118. Chi-square Table of the Distribution of Aggressive and Nonaggressive Electrode Localizations in the Central Gray*

Aggresive Nonaggressive electrodes electrodes Total

Inside degeneration area 8 Outside degeneration area 2

45 57

53 59

Total 10 102 112

*In the first experiment a degeneration area was delimited within the central gray, in which degenerating axons were found.

274 Moset al

276 Mos el ul

aggressive electrodes were located in the degeneration area than outside this area (p = 0.03, df = 1 , x2 = 4.7).

Discussion In this experiment it was shown that stimulation in the midbrain central gray

in rats may induce aggression. The morphology of this aggression is to a very large extent comparable to the bite-attack induced by hypothalamic stimulation. The fact that no attack-jumps were seen during central gray stimulation may be explained by the concomitant disturbances in locomotion, often seen as a result of stimulation by these electrodes. The similarity in morphology, combined with the already demonstrated anatomical connection, makes it plausible that the central gray and the hypothalamus are part of the same neural network involved in aggression. How these two regions interact and influence each other remains to be studied.

A feature shared with cats is the low percentage of positive electrode points for aggression. Nakao et al. [1968] found only a few aggressive sites in the central gray. Bandler’s [I 9771 description illustrates this. He stimulated the central gray in cats through moveable electrodes and sometimes obtained a weak response. If the electrode was moved just a bit deeper to get a better response, the aggression could completely vanish. This suggests that the target area in the central gray is very small. In contrast, our (unpublished) results indicate that the target area in the hypothalamus is several times larger than the diameter of the electrode (150 pm).

The same problem regarding the small target area probably applies to vocalization, jumping, and alarm. Although for some electrodes only one behavioral aspect was prominent, many electrodes supported more than one effect. Therefore it is difficult to assess the function of these effects in more natural situations. The stimulation may have aversive properties, as has been demonstrated in the rat [Schmitt et al., 1974, 19771 and in the cat [Nakao et al., 19681. Vocalizations have been induced in monkeys by stimulation in the rostral and caudal central gray [Jurgens, 1976, 19791, but only rostral sites supported self-stimulation. It seems unlikely that vocalizations are related to aggression since the localization of these points differ from the aggression points. Moreover, the occurrence of vocalizations is not dependent upon the presence of a partner.


In experiment 2 we found that although a considerable number of aggressive electrode positions were located in the degeneration area, quite a few elec-


trodes in this region did not support aggression. Bandler [1977] found that there are strong indications that the target areas in the central gray are small. We found differences in thresholds for vocalizations when each of the wires of the electrode served as a monopolar electrode (unpublished observations). This can be interpreted as evidence for small target areas that lie asymmetrical- ly with regard to the electrode tip. If indeed the target area for aggression is small, it is possible that the electrical stimulation does activate the aggressive substrate, but that the aggression does not become manifest. The simultaneous activation of other neural networks in the central gray may suppress overt aggression. This means that even though central gray stimulation alone does not induce aggression, it may facilitate the aggression induced by hypothalamic stimulation; i.e., if the central gray stimulation activates the aggressive network, thresholds for aggression in the hypothalamus should be lower.

Thus rats were supplied with electrodes in the midbrain central gray and in the hypothalamus. All electrodes were tested separately and the animals with aggressive electrodes in the hypothalamus were selected. The central gray was stimulated at several preset and fixed current intensities and the current threshold for hypothalamic aggression was determined, with or without simultaneous central gray stimulation.

Methods Male WE-zob rats were implanted with two electrodes in the hypothalamus

and two electrodes in the central gray. For a detailed description of the operation and electrodes see experiment 2 and Kruk et al. [1979]. All electrodes were tested separately and current thresholds for aggression were determined. When the thresholds for aggression in the hypothalamus were stable, the experiments were started.

Figure 5A shows the experimental set-up. The current in the central gray was fixed during one experiment, while the current in the hypothalamus was varied in an up-and-down fashion in order to determine a threshold for aggression (see General Methods). To prevent current flow from hypothalamic to central gray electrodes, pulses to the central gray were delayed 6.25 msec with respect to the hypothalamus. Both trains derive from separate stimulators and pairs of PSIU6 constant current units. Stimulation trains to the hypothalamus and the central gray were alternated with trains to the hypothalamus alone. Thus two thresholds were determined simultaneously. To test effects of central gray stimulation on hypothalamic aggression, the six response changes of both thresholds were tested against each other with a Student t-test.

Current intensities for stimulation in the central gray were selected based on previous testing sessions. We deemed it unlikely that the behavior at these current intensities could be incompatible with the hypothalamic aggression.

278 Mos et al

2 L

x a

Y - 8 " I

Central Gray und Aggression 279

Results Sixteen animals were tested on facilitation or inhibition. In ten cases the

concomitant central gray stimulation only inhibited aggression; i.e., raised the current thresholds for hypothalamic aggression. An example of this is given in Figure 5B. At the current intensities used, the central gray stimulation alone did not induce any particular change in the animals’ behavior. Nevertheless the hypothalamic aggression thresholds were increased.

In six animals the central gray stimulation facilitated lower aggression thresholds. Figure 5C shows an example of an electrode where the facilitation is clearly current-dependent. A biphasic effect is seen in Figure 5D with facilitation at low currents and inhibition at higher currents. Here the highest current intensity used for central gray stimulation was the threshold value for vocalization. These data do support the idea that the target area for aggression is small and close to other systems. At lower current intensities neither aggression nor vocalization are manifest. The lower current facilitates hypothalamic aggression, while at higher current vocalization is dominant over the facilitation effect.

Histology. The same procedure described in experiment 2 was applied in localizing the facilitation and the inhibition electrodes. Significantly more facilitation electrodes were located inside the degeneration area and more inhibition electrodes were situated outside the degeneration area (experiment 1) than might be expected with a random distribution (x2=4.26, p=O.O38, df = 1; Table IIb). Electrodes that were located at the border of the degeneration area were considered as lying in this area because the stimulation field of

Fig. 5. (A) Schematic representation of combined stimulation experiments. Thresholds are determined using pulse trains of 10 seconds, separated by 50-second intertrain intervals. During the 10-second pulse train both the hypothalamus and the central gray are stimulated, though the pulses to the two structures are spaced in time. The current in the central gray remains the same during all trains. The current intensity used for hypothalamic stimulation was varied in the intertrain interval, in order to determine current thresholds. (B) Inhibition of hypothalamic aggression by simultaneous central gray stimulation in rat 26813. Vertical axis: threshold values and standard deviation in FA. Horizontal axis: three pairs of current thresholds of hypothalamus stimulation alone (LH) or hypothalamus and central gray stimulation (20, 40, 60 pA CG). (*) p< 0.01, Student t-test. (C) Facilitation of hypothalamus aggression by simultaneous central gray stimulation in rat 266 L. Horizontal axis: hypothalamus stimulation alone (LH) or hypothalamus and central gray stimulation(50, 100, and 150pA CG, respectively). * p< O.Ol;* * p < O.ooO1 Student t-test. (D) Facilitation and inhibition of hypothalamic aggression by simultaneous central gray stimulation in rat 272L. Horizontal axis: hypothalamus stimulation alone (LH) or hypothalamus and central gray stimulation (300,200, 100 f i CG, respectively). Stimu- lation with 300 f i and 200 pA, at and below the vocalization threshold, inhibited hypothalamic aggression, whereas the effect of 100 pA CG stimulation facilitated the aggression. * p < 0.01 Student t-test.

280 Moset a1

TABLE IIb. Chi-square Table of the Distribution of Facilitatory and Inhibitory Electrode Localization in the Central Grav

~ ~

Facilitatory Inhibitory electrodes electrodes Total

Inside degeneration area 5 3 8 Outside degeneration area 1 I 8

Total 6 10 16

such electrodes at least partly activates this region. This was relevant to one facilitation and two inhibition electrodes.

Discussion The results show that more facilitation points lie inside the central gray de-

generation area (experiment 1) than outside this region. Stimulation at most sites outside the degeneration zone inhibits hypothalamic aggression. A p parently stimulation of some sites in the central gray is incompatible with the hypothalamically-induced attacks, even when stimulation of these central gray sites alone does not result in overt behavior. We have no explanation for this, but it implies that the total central gray area is not involved in aggression. It seems that the substrate for aggression is mainly present in the degeneration area. If this is the case, it also excludes the possibility that the degeneration from the hypothalamus to the central gray is caused by destruction of passing fibers irrelevant for the aggressive response.

Another point deserves special attention. This experiment may offer an explanation as to why so many nonaggressive electrodes lie in the degeneration region. Since we find facilitation of aggression, the neural substrate for aggression is apparently activated during stimulation, but for some reason or other the stimulation of these sites alone does not result in overt aggressive behavior. The most parsimonious explanation is that the simultaneous activation of other neurons produces effects that dominate and suppress the aggressive response.

In a study in cats Sierra et al. [1973] stimulated electrodes in the hypothalamus and the central gray, which both supported aggressive behavior. No dominance of one structure over the other was found, but the simultaneous stimulation resulted in a more direct attack than was observed after stimulation of either structure alone. They did not combine hypothalamic stimulation with stimulation of nonaggressive points in the central gray, therefore information about facilitatory central gray stimulation in cats is lacking. Sheard and Flynn [1967] used the combined stimulation technique to show facilitatory effects of the midbrain reticular formation on hypothalamic


aggression. They too, found facilitation by otherwise nonaggressive points in the midbrain. These studies suggest that the combined stimulation technique can be used fruitfully in unravelling the neural circuit of a behavior.

GENERAL DISCUSSION

Our results demonstrate the involvement of the central gray in intraspecific aggression. The central gray receives a direct projection from the hypothalamic site where aggression can be induced. Intraspecific aggression can be elicited, especially in the region where degeneration is observed. Sites that do not show overt aggression upon direct stimulation, however, may still facilitate hypothalamic aggression. These facilitation sites too, lie predominantly in the degeneration area in the central gray. These findings are in line with the results obtained by Flynn and coworkers 119791 in the cat, and Chaurand et al. [1972] in the rat. However, these studies deal with interspecific or predatory aggression. In earlier studies by Hunsperger [1956] and Skultety [1963] a neural circuit of hypothalamus and central gray was demonstrated to induce rage-like or defensive reactions.

Recent literature on aggression in rats and cats deals with the subdivision of aggression into offensive and defensive behavior [Blanchard and Blanchard, 1977; Adams, 1976; Lehman and Adams, 19771. Offense and defense form the extremes of a continuum of all aggressive responses shown by animals. Offen- sive behavior is typically seen as the behavior of a resident male against an intruder. This consists of full aggressive posture, sideways threat, pilo-erection, teeth-chattering, biting, and chasing the partner. Bites are mainly directed at the back of the intruder, whereas the bites of a defensive animal are directed at the snout of the opponent. Freezing, full submissive posture, etc. are charac- teristic of the defensive animal. Although there is no complete consensus about this terminology, it is widely used to evaluate several animal models for aggression. Electroshock-induced aggression for instance is now generally seen as defensive in nature.

More difficulties are met when one deals with the behavioral effects of electrical or chemical brain stimulation. Poor descriptions of the induced behavior and a restricted testing environment usually make it impossible to interpret older findings in terms of defensive or offensive aggression. The capability of an animal to “launch a well directed attack at a glove” [Skultety, 19631 says hardly anything about the cat’s behavior in a social context. in part the very distinction between offensive and defensive aggression is a source of problems in the study of natural and induced aggression. Since they are extremes of a continuous range of aggressive behavioral elements, the occurrence of pure offense or pure defense can hardly ever be expected. Consequently the classifica- tion of the displayed behavior in one or the other category will be complicated.

282 Moset a/

Stimulation of the hypothalamus or the central gray in cats may induce an affective attack, with much display and great autonomic arousal, or a more quiet form of attack. The latter is often called predatory aggression and is very effective in killing an anesthetized rat. The affective attack is generally seen as defensive aggression. Elaborate descriptive studies on the electrically-induced behavior of cats in a social context are lacking, so one must be cautious in in- terpreting older literature.

In our studies on rats we have reason to believe that the hypothalamically- induced aggression is on the offensive part of the continuum. However, it is important to realize that the test situation may be of great influence on the observed results. We always used a light, weaker rat as partner. The following arguments support the offensive nature of aggression: (1) pilo-erection is very often seen upon stimulation [Blanchard and Blanchard, 1977; Lehman and Adams, 19771; (2) all aggressive electrode positions also support teeth-chattering which is seen as an offensive component [Blanchard and Blanchard, 19771; (3) the attacking animal may show sideways threat, while actively approaching the partner; (4) no signs of flight are present in the stimulated animal, so there is no obvious reason for the rat to be defensive.

Moreover, the induced aggression as described by Koolhaas [1978] is considered to be offensive by Adams [1979]. We stimulate at the same hypothalamic site as Koolhaas [1978], although the test situation is slightly different. The wound pattern is puzzling. Blanchard et al. [1978] describe wounds caused by defensive rats as localized to the snout, whereas bites of territorial males are directed more at the back and neck region of an intruder. We find a mixed pattern with wounds on the snout and the back of the partner.

We believe that hypothalamic stimulation induces aggression of an offensive type. The projection to the central gray is part of this neural circuit and may therefore also be involved in offense. This view is contrary to the hypothesis of Adams [ 19791, who attributes a major role in defense and submission to the midbrain central gray. However, little work has been done at present on the neural circuitry underlying offense. This model may therefore have to be extended with a role of the central gray in offense.

As we demonstrated in experiment 3, one should be very careful in the inter- pretation of stimulation-induced behavior, since, especially in the central gray, overlapping substrates (perhaps for offense and defense?) may be activated simultaneously. Such an activation of several substrates in the central gray was most clearly demonstrated by Bandler [ 19771 ~ When he lowered an electrode into the central gray of cats over a small distance, he was able to induce affective escape, affective defense, and affective attack, respectively.

Many questions regarding this electrically-induced aggression have not yet been solved. What is the function of this neural substrate during naturally oc- curring aggression [see Kruk and Van der Poel, 1980]? Is it hierarchically or-


ganized and in what way? How is this substrate influenced by hormones and other substances? Some of these questions will be dealt with in a following paper, in which the effect of lesions in the central gray will be studied on hypothalamic and territorial intermale aggression. Although the concept of a specific substrate for aggression may prove to be an oversimplification of reality, clear-cut experiments can be derived from it. The results of the present experiments give no cause to reject the hypothesis of a specific neural network for aggression.

ACKNOWLEDGMENTS

We wish to thank Dr. A.H.M. Lohman for his help in the degeneration studies, Dr. E.M. Cohen and Dr. E.L. Noach for their critical reading and Miss R.H. F. de Ru for correcting the manuscript.

This study was supported in part by a grant from the Foundation for Biological Research (BION), which is subsidized by the Netherlands Organiza- tion for the Advancement of Pure Research.

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