amygdaloid function in the central cholinergic mediation of shock-induced aggression in the rat

AGGRESSIVE BEHAVIOR Volume 2, pages 131-152 @ 1976, Alan R. Liss, Inc.

Amygdaloid Function in the Central Cholinergic Mediation of Shock-Induced Aggression in the Rat

R. J. Rodgers and K. Brown, Department of Psychology, The Queen; University of Belfast, Belfast, N. Ireland

................................................................................. ................................................................................. A series of experiments was designed to examine the role of central cholinergic mechanisms in shock-induced aggression. Cholinergic blockade in the basolateral amygdala, ventral hippocampus, or dorsal hippocampus resulted in greatly reduced levels of fighting in response to footshock. However, while pain sensitivity remained unaltered in the amygdala group, both of the hippocampal groups exhibited decreased shock sensitivity. Further investigation of the amygdala revealed ( 1) increased fighting in response to increased cholinergic levels, (2) neuroanatomical specificity to the basolateral division of this complex, (3) that an intact basolateral amygdala is essential to the normal manifestation of shock-induced aggression, and (4) that social attraction remains unaltered by cholinergic blockade of the basolateral amygdala. Motor coordination and motor activity were not significantly affected in any treatment condition.

................................................................................ ................................................................................ Key words: rat, shock-induced aggression, pain sensitivity, amygdala, hippocampus,

cholinergic mechanisms

I NTRO DUCT ION

Many recent investigations have studied the effects of peripheral drug administration upon shock-induced aggression (SIA) in both mice and rats. Most of these studies have manipulated levels of brain monoamines or circulatory hormones (Sofia, 1969; Conner et al., 1969; Powell et al., 1971; Crowley, 1972; Powell,

Manuscript received November 4, 1975; accepted for publication November 14, 1975

R. J. Rodgers is now at Undergraduate School of Studies in Psychology, University of Bradford, Bradford BD7 IDP, Yorkshire, England. Please send reprint requests to this address.

131

132 Rodgers and Brown

Walters et al., 1973). However, in view of the positive evidence for cholinergic involvement in other forms of aggressive behavior (McCarthy, 1966; Vogel and Leaf, 1972; Berntson and Leibowitz, 1973; Katz and Thomas, 1975) it is surprising that, to date, very few studies have investigated possible cholinergic mediation of SIA. On the one hand, Powell, Milligan el al. (1973) and Rodgers and Brown (1973a) found that peripheral injection of scopolamine, an anticholinergic agent, resulted in decreased fighting, whereas it’s quarternary methyl derivative (which does not readily penetrate the brain) did not have this effect. On the other hand, injection of physostigmine, an anticholinesterase agent, led to increased fighting, whereas neostigmine had no effect (Rodgers and Brown, 1973b).

Direct brain injections of various drugs have also implicated cholinergic mechanisms in different forms of aggression (muricide: Bandler, 1969; 1970; 1971a. b; Smith et al., 1970; IgiC et al., 1970; affective rage: Hull et al., 1967; Allikmets et al., 1969; Allikmets, 1974). These studies, together with various lesion experiments, have implicated the limbic system and associated regions in the central cholinergic control of aggressive behavior. The purpose of the present experiments was to investigate the role that limbic structures (amygdala and hippocampus) might play in the cholinergic mediation of shock-induced aggression.

A crucial behavioral control, too infrequently employed in studies on SIA, is that concerning treatment-induced changes in animals’ sensitivity to electric shock. Thus, the “jump-flinch’’ test for pain sensitivity (Lints and Harvey, 1969; as modified in our laboratory) was considered an essential control procedure in the present experiments.

MATERIALS AND METHODS

Subjects

Adult male Sprague-Dawley rats obtained from a local supplier (Olac, Ltd.) were used as subjects in this study. At the time of surgery, the animals were approximately 300 gm weight. All subjects were individually housed with food and water available ad libitum. Subjects were maintained on a 12 hr light/dark cycle (6 a.m.-6 p.m.), and all testing was performed under red light during the dark phase of this cycle, i.e. 6 p.m. onwards.

Surgery

Cannulation. Experimental subjects were bilaterally implanted with guide cannulas under Equi-thesin (Jensen-Salabury Lab., Inc.) anesthesia. These cannulas consist of a 0.6 mm 0.d. stainless-steel guide fitted with a 0.3 mm 0.d. obturator. The latter is removed and replaced by a 0.3 mm injection cannula prior to injection. The length of the injection cannula is such that when inserted into the guide, the tip of the cannula is flush with that of the guide.

Central Cholinergic Systems and Aggression 133

Lesions. Bilateral electrolytic lesions were made using a stainless-steel insect pin (diameter: 0.672 mm) insulated to within 1 mm of the tip. Under surgical conditions identical to above, this electrode was lowered into the amygdala and an anodal direct current of 2 mA was passed for 10 sec. An indifferent electrode was attached to the rat’s ear, with a saline connection.

Stereotaxic coordinates. Implantation coordinates for the ventral hippocampus were 5.6 mm posterior to bregma, 4.0 mm lateral to the sagittal sinus, and 6.0 mm below the surface of the brain (-5.6; ? 4.0; 6.0 down). Coordinates for the other areas were dorsal hippocampus (-5.6; f 2.8; 3.0 down), basolateral amygdala (-3.0; k 4.0; 7.0 down), and corticomedial amygdala (-4.7; * 3.4; 7.4 down). These values were calculated from the atlas of Konig and Klippel (1963). Ten days postoperative recovery was allowed before testing commenced.

Apparatus

Shock-induced aggression. A Grason-Stadler rat station (E3125A . . . IOO), measuring 24 X 20 X 29 cm served as the test chamber. The chamber was opaque apart from the perspex door, which also served as an observation window. A Grason-Stadler shock generator (E 1064) supplied scrambled shock of specified duration and intensity to the grid floor of the test chamber. The total number of shocks delivered in any series and the frequency of these shocks were con- trolled by relayed programing equipment (Grason-Stadler and Aim Biosciences).

Jump-flinch test. The same apparatus as that used for the SIA testing was employed in the screening of the animals for drug-induced changes in pain sensitivity.

Rota-rod. This apparatus consisted of a kymograph recorder (C. F. Palmer), with drum removed and placed in a wooden cradle so that the center spindle (rod) was horizontal. A thick wire mesh covering the rod provided a gripping surface for the animals. The apparatus was positioned so that the rod (length = 31 cm; diameter = 2.2 cm) was 65 cm above a peat-filled plastic arena. Rotation speed of this rota-rod was fixed at 4 rpm.

Activity box. The apparatus for this test consisted of a rat chamber, with the dimensions of that described above. The walls of this chamber were opaque to exclude external distractions. Four photocells were aligned across the width of the chamber in 2 rows of 2, 13 mm apart laterally and 8 mm apart vertically. Each photocell was connected to a separate counter, and a summation of these readings gave a measure of activity during the test.

Latane test. This consisted of an open field, 1.4 m in diameter with a 42 cm high wall. The floor and walls were painted flat white with the former marked off into 49 equal areas by a series of concentric circles and radii. Each area was labeled with a code number for ease of recording. An electronic digital timer (Forth Instruments) was used to record total contact time to the nearest l/lOO sec.


Procedures

SIA. Operated animals and unoperated stimulus animals were matched as far as possible for weight, and fighting pairs were randomly assigned to the various treatment conditions where appropriate. Each pair was placed in the test chamber and allowed 5 min exploration before testing began. Sixty preinjection shocks were delivered in 2 series of 30 shocks with an interseries interval of 60 sec. Shocks were either 1 mA or 2 mA intensity (depending on the particular experiment), 0.5 sec duration, and occurred every 10.5 sec.

The responses of both animals were noted during testing and scored as: (a) no response, (b) upright threat posture, and (c) a fight response/attack. The latter was recorded when one animal made a directed movement towards the other either by lunging forward or by making a striking action with the forepaws. In cases where both animals responded, that animal responding first was scored with an attack, so that only one fight was recorded per shock. Attack frequency of the operated animal was calculated and served as the preinjection baseline. Both animals were then removed, the cannulated animal injected, and both replaced in the chamber, where 2 further shock series were run to determine the postinjection response. A similar procedure was adopted for the lesion animals, except that these animals were not tested until 10 min after the drug injection (i.p.).

ber and received 6 series of 10 shocks (0.5 sec duration), delivered at 15 sec in- tervals to the grid floor. Shock series were administered in alternating ascending and descending order with intensities ranging between 0.16 and 1.3 mA in 10 steps. Responses were recorded as (a) no response, (b) flinch, and (c) jump. A jump threshold (the intensity at which the animal’s hind feet leave the grid floor) was determined for each series, and an overall mean value was calculated to give a preinjection baseline. The animal was then injected, replaced in the chamber, and immediately retested to yield a postinjection measure of pain sensitivity.

criterion of 4/5 trials (intertrial interval, 10 sec) after which they were tested under the various treatment conditions.

Activity test. Animals were individually placed in the test box and given a 10-min period to habituate to the new environment. The photocells were then activated, and activity of the animal was recorded over a 5-min period. This procedure was carried out under the various treatments.

Latank test. Pairs of rats (one operated, one unoperated) were placed in the open field for a 5-min period. Every 10 sec a note was taken of the position of each animal by recording the area occupied by the animal at that time. Total contact time between the pair was also noted over the test session. A computer program was used to calculate the distance traveled by each animal and the mean distance maintained between the pair over the session. The effects of various treatments were again studied on this test.

Jump-flinch test. Operated animals were placed individually in the test cham-

Rota-rod. Animals were trained to remain on the rotating rod for 60 sec to a


Drugs

Scopolamine hydrobromide (Sigma Chemical Co.) was used in a solution of 10 mg/ml. Physostigmine salicylate (Sigma Chemical Co.) was used in a solution of 5 mg/ml. Normal physiological saline served as vehicle for each drug and as an injection control.

Histology

Cannulated animals received an overdose of Nembutal and were perfused with normal physiological saline followed by 10% formal saline. Brains were removed, embedded in wax and serial sections cut at 10 p. Sections were stained for myelin (Lux01 Fast Blue), counter-stained for cell bodies (Pyronin Y), mounted, and examined for cannula tracts with reference to the atlas of Konig and Klippel (1963).

EXPERIMENT I

Electrolytic lesions of the amygdala (Eichelman, 1971 ; Kolb and Nonneman, 1974) or nonspecific neural blockade of this region produced by intracranial procaine injection (Rodgers, unpublished data) result in decreased SIA, without pro- ducing alterations in pain sensitivity. Also, electrolytic lesions of the hippocampus (Eichelman, 1971) or procaine anesthesia of the ventral hippocampus (Rodgers, unpublished data) produce a blockade of SIA. However, these effects are accompanied by changes in pain sensitivity.

The anticholinergic agent scopolamine hydrobromide ( 1 p1 bilateral injections; 10 pg/pl) was used to examine whether cholinergic mechanisms might be involved in these behavioral effects of amygdaloid and hippocampal blockade. The basolateral amygdala receives the strongest cholinergic innervation of this structure (Shute and Lewis, 1967; Girgis, 1972; Palkovits et al., 1974), while both the dorsal and ventral hippocampus receive strong cholinergic contributions (Lewis and Shute, 1967).

Thirty-eight rats were randomly divided into 2 groups of 19 rats. One group was used as unoperated opponents, while the other group was further subdivided into 3 operation groups. Group I (n = 7) was bilaterally implanted with guide cannulas aimed at the basolateral amygdala (BLA); group I1 (n = 6 ) was bilaterally implanted in the ventral hippocampus (VH); Group I11 (n = 6) was bilaterally implanted in the dorsal hippocampus (DH). A shock intensity of 2 mA was used in the aggression tests.

Results

representation of results the abscissa refers to 6 series of 10 shocks predrug and 6 series postdrug.

Two-tailed correlated t tests were used to analyze the data. In the graphic

I 3 6 Rodgers and Brown

SIA. The upper graphs in Figs. 1-3 indicate the effects of saline and scopolamine upon SIA, when injected into the basolateral amygdala, ventral hippocampus, and dorsal hippocampus. Scopolamine produced a significant reduction in all3groups(BLA: t=6 .71 ,d f=6 ,p<O.O01 ;VH: t = 4 . 2 8 , d f = S , p < 0 . 0 1 ; DH: t = 4.17, df = 5, p < 0.01) while saline was without effect (BLA: t = 0.67; VH: t = 0.75; DH: t = 0.25).

Pain sensitivity. The lower graphs in Figs. 1-3 illustrate the effects of scopolamine and saline injections upon jump thresholds (mA). Pain sensitivity was decreased in both VH(t = 2.59, df = 5, p < 0.05) and DH(t = 4.47, df = 5, p < 0.01) groups but remained unaffected in the BLA group (t = 0.56, df = 6, N.S.). Again, saline was ineffective in all groups (BLA: t = 0.57; VH: t = 0.65; DH: t = 1.00).

Motor coordination. Neither scopolamine nor saline produced any signs of motor impairment in any of the groups. Statistics were not required to show this lack of effect. BLA group (criterion: 60.0; saline: 60.0; drug: 59.3 f 3.6), VH group (criterion: 60.0; saline: 60.0; drug: 58.2 f 6.4), and DH group (criterion: 60.0; saline: 60.0; drug: 55.9 k 14.0).

1)’lbilateral Basolateral ~n iection AMYGDAL A

I I I I

I I

I I I

I 9

/

100s

1.01 1 I

-;+/- ‘./ I

I

I

- Saline &-A ScopoLarni ne(1W

I H 1809

I

PRE - DRUG f POST-DRUG

Fig. 1. The effects on SIA and pain sensitivity of bilateral injections of scopolamine (10 pg/ pl) into the basolateral amygdala.


Histology. Figures 4-6 give diagrammatic summaries of the histological verification of cannula tip placements in the 3 operated groups. The majority of cannulas were correctly placed with the exception of two amygdaloid cannulas which were positioned more medially, toward the central amygdaloid nucleus.

The results of this experiment suggest that the basolateral amygdala is specifically implicated in the cholinergic (muscarinic) control of SIA, whereas hippocampal involvement is relatively nonspecific. However, the finding that scopolamine injection into the hippocampus attenuates responses to aversive stimulation support Olds and Olds (1963) who reported that electrical stimulation of the hippocampus can be aversive in itself. This suggests that the hippocampus may be involved in a central system responsible for the mediation of responses to aversive stimulation.

EXPERIMENT 2

Rodgers et al. (1973b) found that SIA was increased with peripheral injections of physostigmine salicylate (which indirectly leads to increased synaptic acetyl choline [ACh]); Bandler (1970) and Smith et al. (1970) reported that injections

14 bilateral inje tion f VENTRAL

HI PPOCAM PUS 0-6)

I H 1805

I PRE-DRUG I POST-DRUG

Fig. 2. The effects on SIA and pain sensitivity of bilateral injections of scopolamine (10 p g / pl) into the ventral hippocampus.


19bilateral inject ion

I DORSAL HIPPOCAMPUS

(17.6) I I I

I ' d' I

I D O S I I

PRE- DRUG I POST-DRUG

Fig. 3. The effects on SIA and pain sensitivity of bilateral injections of scopolamine (10 p g / MI) into the dorsal hippocampus.

of physostigmine into the lateral hypothalamus resulted in increased muricide; IgiC et al. (1970) injected amitone into the basolateral amygdala and found increased muricide; HernandCz-Peon et al. (1963) produced the rage reaction in cats by physostigmine injection into limbic forebrain areas. Since a blockade of cholinergic receptors in the basolateral amygdala produced decreased SIA, it was postulated that a potentiation of synaptic ACh, by bilateral physostigmine injection (1 PI, 5 pg/pl), would have the opposite effect. Five rats of the BLA group used in Experiment 1 served as subjects in this experiment (for histology see Fig. 4). Their original unoperated opponents (n = 5) were also used. Shock intensity of 1 mA was employed to prevent the occurrence of any ceiling effect.

Results

One-tailed correlated t tests were used to analyze the data. SIA. Figure 7 (upper) shows the effect of saline and physostigmine upon

SIA, when injected into the basolateral amygdala. Physostigmine produced a


group. Fig. 4. Diagrammatic summary of cannula placements in the basolateral amygdala

A2970p

A2580p

A 2 1 8 0 ~

Fig. 5. Diagrammatic summary of cannula placements in the ventral hippocampal group.

I40 Rodgers and Brown

A3750,-

A 297Qr

A 2 5 8 0 r

Fig. 6. Diagrammatic summary of cannula placements in the dorsal hippocampal group.

significant increase (t = 2.95, df = 4, p < 0.025), while saline was without effect (t = 1.18, df = 4,N.S.).

Pain sensitivity. The lower graph in Fig. 7 shows the effect of physostigmine upon jump thresholds. Neither physostigmine (t = 0.67, df = 4, N.S.) nor saline (t = 1.63, df = 4, N.S.) had any effect on pain sensitivity. This experiment provided further support for the existence of a central cholinergic mechanism in- volving the basolateral amygdala which normally functions to facilitate shock- induced aggression.

EXPERIMENT 3

Although the amygdaloid complex is comprised of at least 5 major distinct nuclei, it has been traditionally considered as having 2 main divisions. The present study examined the possibility that the cholinergic mediation of SIA was a neuroanatomically specific effect within the amygdala, peculiar to the basolateral division. The corticomedial amygdala has been implicated in the control of the muricide reaction in both lesion (Karli et al., 1969) and pharmacological (Leaf et al., 1969) experiments. Since the corticomedial amygdala receives comparative- ly little cholinergic contribution but is strongly innervated by catecholamine systems (Ungerstedt, 1971; Lindvall and Bjorklund, 1974), it was predicted that 1 pl bilateral injections of either scopolamine (10 pg/pl) or physostigmine (5 pg/ pl ) would be ineffective in altering SIA.


7

PA€ - DRUG I POST-DRUG I

Fig. 7. The effects on SIA and pain sensitivity of bilateral injections of physostigmine (5 pg/pl) into the basokdteral amygdala.

Ten rats were randomly assigned to 2 groups; group I (n = 5) was bilaterally implanted with cannulas aimed at the corticomedial amygdala, while group I1 (n = 5) was unoperated and served as fight opponents. Shock intensity of 2 mA was used for scopolamine tests, 1 mA for the physostigmine tests.

Results

One-tailed correlated t tests were used in the data analysis. SIA. Figure 8 (upper) shows the effect, on SIA, of scopolamine and saline

injection into the corticomedial amygdala. Neither saline (t = 1.09, df = 4, N.S.) nor scopolamine (t = 0.57, df = 4, N.S.) had any effect on levels of fighting. Figure 9 (upper) illustrates the effects of physostigmine and saline on SIA. Again, neither saline (t = 1.00, df = 4, N.S.) nor physostigmine (t = 0.34, df = 4, N.S.) had any effect on aggression.

of saline and scopolamine injections into the corticomedial amygdala. No effect was obtained with either saline (t = 0.18, df = 4, N.S.) or scopolamine (t = 0.1 1,

Pain sensitivity. The lower graph in Fig. 8 shows the effect on pain thresholds


40 I

lPl bilat. Cort ico-medial n. AMY GOAL A

I (n-5) scOqO' ami ne

I I

c U 3

0 1 .o

0.5 E

0

I I c( 100s 1

I I

Fig. 8. The effects on SIA and pain sensitivity of bilateral injections of scopolamine (10 pg/pl) into the corticomedial amygdala.

df = 4, N.S.). Neither saline (t = 0.18, df = 4, N.S.) nor physostigmine (t = 0.54, df = 4, N.S.) had any effect on pain sensitivity when injected into the corticomedial amygdala (Fig. 9 , lower).

Motor coordination. Animals were trained to a mean criterion score of 60.0 sec Saline, scopolamine and physostigmine, in the doses used, were completely ineffective in altering the criterion score and thus did not produce motor impairment.

Histology. Figure 10 gives a summary of the histological verification of cannula placements. All cannulas were accurately positioned in the region of the corticomedial amygdala. The results of this experiment suggest that the corticomedial amygdala is not involved in the cholinergic mediation of SIA (or pain sensitivity).

EXPERIMENT 4

The present series of experiments suggest that the reduction in SIA produced by destruction of the amygdala results from damage to the basolateral division of this limbic forebrain area. If this is the case, then it would be predicted that (1)


4c

Y W a

s 2c G

0 1 .c

0.:

0

I

I I

I I

I 1 I

- 100s.

I I - saline I LA Physostigminc (05'1.) I I 1

ME-DRUG I POST-DRUG I

- 180s.

I

Fig. 9. The effects on SIA and pain sensitivity of bilateral injections of physostigmine (5 pg/pI) into the corticomedial amygdala.

Fig. 10. Diagrammatic summary of cannula placements in the corticomedial amygdala group.


electrolytic lesions of the basolateral amygdala would produce a specific blockade of SIA and that (2) increasing cholinergic levels in the amygdaloid-lesioned animals, by peripheral physostigmine administration (0.2 mg/kg), would not result in the increased SIA seen with intact animals (Rodgers et al., 1973b). This latter premise is based on the assumption that by destroying the cholinergic basolateral amygdala, no substrate for the action of the cholinergic agent would exist in this region. Thirty rats were randomly divided into 2 groups of 15 rats. One group served as unoperated opponents in the aggression test, while the other was further subdivided into 3 groups. Group I (n = 5) received bilateral electrolytic lesions aimed at the basolateral amygdala (BLA): group 11 (n = 5 ) served as sham-operated controls (SO) and was subjected to the identical surgical procedure as group I, except that current was not passed through the lesion electrode; group I11 (n = 5) was used as unoperated controls (C). A shock intensity of 1 mA was used in the aggression tests.

Results

Group differences, both on baseline and injection response measures, were analyzed using a one factor ANOVA and where significance was obtained, it was followed up with a test for comparison between treatment means (Winer, 1971, pp. 65-70). Within groups, differences between pre- and postinjection measures were analyzed using one-tailed correlated t tests.

SIA. Between groups: Figure 11 shows the effect on SIA of the various surgical and injection treatments. The 3 groups differed significantly on the predrug baseline fighting levels (F = 12.97, df = 2, 12, p < 0.001). Further analysis revealed that the BLA group fought significantly less than both the SO group (F=53 .96 ,d f= l ,12 ,p<O.O0l )and theCgroup(F=47 .42 ,d f= I , 1 2 , p < .001). Groups SO and C did not differ significantly from each other (F = 0.21, df = 1, 12, N.S.). After drug treatment, these differences were still apparent between the BLA group and the two control groups. When presaline baselines and postsaline responses for the groups were analyzed, the same pattern emerged; the BLA group was consistently different from the controls.

Within groups: Significant elevations in fighting were produced, by physostigmine, in groups SO(t = 5.66, df = 4, p < 0.005) and C(t = 4.95, df = 4, p < O.OOS), but not in the BLA group (t = 1 .OO, df = 4, N.S.). Saline was without effect in all groups (BLA: t = 0.00, SO: t = 1.08; C: t = 0.58).

In SIA in response to peripheral physostigmine, group BLA remained unaffected. Saline was without effect in all groups.

Pain sensitivity. Between groups: Figure i 2 shows the effect of the various surgical and injection treatments on pain sensitivity. Since data had already been collecied on intact animals (Rodgers et al., 1973b), group C was omitted from this test. No differences existed between the groups on either predrug (F = 0.48,


a l I- &.-.. un-operated

Saline I I - amygdala lesion

\

e - -A sham operated +

,100s

I

0 Pn-injection ' Post-Injection

baseline response

Fig. 1 1 . Effects of peripheral injections of physostigmine (0.2 mg/kg) on SIA in the 3 groups.

df = 1,8, N.S.) or presaline (F = 0.20, df = 1,8,N.S.) baselines, or on postdrug (F = 0.82, df = 1,8,N.S.) or postsaline (F = 0, df = 1,8,N.S.) responses. Pain sensitivity was therefore unaltered by BLA lesions.

Within groups: Neither physostigmine (BLA: t = 0.96, df = 4, N.S.; SO: t = 0.82, df = 4, N.S.) nor saline (BLA: t = 1.00; SO: t = 0.51) produced any alterations in pain thresholds.

Motor coordination. The BLA group did not differ significantly from the SO group in attaining criterion rota-rod performance (t = 2.02, df = 8,N.S.). Physostigmine did not produce any sign of motor impairment in the BLA group (t = 1.37, df = 4, N.S.) or in the SO group (t = 1.63, df = 4, N.S.).

Histology. Figure 13 gives a diagrammatic histological summary indicating the maximum extent of basolateral amygdaloid lesions. The lesions were not entirely restricted to the basolateral amygdala, with some damage occurring t o the central amygdaloid nucleus. Slight encroachment upon the medial nucleus was observed in one instance and upon the cortical nucleus in another. However, the distinction between the two major divisions was in general maintained.


Physostlgmine I I

OT I

a E

1 - 0

0- 7

0 '

Saline -

I *--a sham-operated )--. amygdala lesion

I I I __ 180s I

Pre-injection I Post- injection baseline response

Fig. 12. Effects of peripheral injections of physostigmine (0.2 mg/kg) on pain sensitivity in the lesion and sham-operated groups.

A3750p

A3290,-

Fig. 13. Diagrammatic summary of maximum extent of basolateral amygdaloid lesions.


The findings of this experiment are taken to suggest that an intact, facilitatory cholinergic mechanism in the basolateral amygdala is essential t o the manifestation of SIA.

EXPERIMENT 5

It has been suggested by various workers (Bunnell, 1966; Kling, 1972) that amygdaloid lesions may disrupt general social responsiveness in animals. Bilateral amygdaloid lesions have been shown to decrease social dominance in monkeys (Rosvold et al., 1954), decrease social dominance in dogs (Fuller et al., 1957), and decrease aggressive behavior in male golden hamsters (Bunnell et al., 1970). Such findings have generally been explained in terms of reduced responsiveness to social stimuli. Such an explanation would be more parsimonious than the present interpretation of results.

Experiment 5 was designed to test the possibility that bilateral scopolamine injections (1 pl, 10 pg/pl) into the basolateral amygdala, may decrease general social responsiveness in rats. A test of social attraction, devised by Latan6 (1969), was used in which i t was possible to measure various components of social attraction between paired rats. As an additional measure, rats were also tested in an activity box. Sixteen rats were randomly assigned to 2 groups: Group I (n = 8) was bilaterally implanted with cannulas in the basolateral amygdala, while group I1 (n = 8) served as unoperated stimulus animals, similar to the SIA paradigm. In both tests, a counterbalanced order was adopted to prevent any habituation effects.

Results

Latank test. ANOVA revealed that there was no difference between the different treatments on mobility (F = 0.23, df = 2,14, N.S.) contact time (F = 0.30, df = 2,14, N.S.) or mean distance (F = 0.05, df = 2, 14, N.S.).

ity in the LatanC test (Exp. 5) the activity of animals in this activity test was unaffected by any of the experimental treatments (F = 1.27, df = 2, 12, N.S.).

Histology. Figure 14 represents cannula tip locations in the basolateral amygdaloid region. Two placements were slightly more medial than was required (ie, in the central amygdaloid nucleus). However, the distinction between basolateral and corticomedial amygdala remained intact.

These results indicate that social attraction between paired rats remained unaffected by bilateral scopolamine injections into the basolateral amygdala. Additionally, motor activity was found to be unaltered by this manipulation.

Activity test. As was expected from rota-rod performance (Exp. 1) and mobil-


A 4 9 0 0 1

A 43 00f

Fig. 14. Diagrammatic summary of cannula placements in the basolateral amygdala group.

GENERAL DISCUSSION

Selective cholinergic blockade in the basolateral amygdala resulted in a specific reduction in SIA, whereas the same treatment of either the dorsal or ventral hippocampus produced a concomitant reduction in both SIA and pain sensitivity (Exp. 1). Thus it appears unlikely that the hippocampus is specifically involved in the cholinergic mediation of SIA. This experiment supports the findings of (1) Eichelman (1971) who reported large decrements in SIA with both laterally placed amygdaloid and hippocampal lesions, with only the latter resulting in changes in pain sensitivity (2) Miczek et al. (1974) who found that lateral amygdaloid lesions produced large decrements in SIA.

Microinjections of physostigmine into the basolateral amygdala resulted in a significant increase in fighting without altering pain thresholds or motor coordination (Exp. 2). This finding is consistent with Rodgers et al. (1973b), who reported increased SIA with peripheral physostigmine, and with lgi6 et al. (1970) who found increased muricide with intraamygdaloid injections of the anticholinesterase agent, amitone.

Neuroanatomical specificity, within the amygdala, for the cholinergic mediation of SIA was demonstrated in Exp. 3; in this experiment both scopolamine and physostigmine failed to alter SIA when injected into the corticomedial amygdala. In view of the neurochemical innervation of this amygdaloid region (Girgis, 1972; Brownstein et al., 1974; Ben-Ari et al., 1975) and the fact that only manipulation of catecholamine levels in the medial amygdala have pre- viously been shown to alter aggressive behavior (Leaf et al., 1969), the present

Central Cholinergic Sys tems and Aggression 149

finding that cholinergic agents are ineffective in this region is not inconsistent with earlier work. The first three experiments specifically implicated the basolateral amygdala in the central mediation of SIA and also confirmed, behaviorally, the work of the histochemists (Shute et al., 1967; Girgis, 1972; Palkovits et al., 1974) who have indicated strong cholinergic innervation to the basolateral amygdaloid region but only relatively weak cholinergic innervation to the corticomedial amygdala.

The importance of the cholinergic amygdaloid influence on SIA was demonstrated in Exp. 4. Not only did bilateral electrolytic lesions of the basolateral amygdala result in a specific blockade of SIA (thus confirming Eichelman, 197 1) but peripheral injections of physostigmine, known to increase fighting in intact animals, failed to have a facilitatory effect on SIA. It therefore appears that an intact cholinergic basolateral amygdala is essential to the manifestation of this type of aggressive behavior.

a1 amygdala had little effect upon social attraction between paired rats in the LatanC test, suggesting that the decrement in SIA following this treatment was not due to a nonspecific reduction in social responsiveness. This finding is in disagreement with Jonason and Enloe (197 1) who reported that bilateral amygdaloid lesions produce decreased social attraction in this test. However, it must be understood that the amygdala consists of a complex of nuclear regions (Eleftheriou, 1972) and as such may produce different behavioral effects depending on the nature and extent of the manipulation. It should not, therefore, be surprising that specific chemical blockade of the basolateral region produces different results to those produced by general amygdaloid destruction.

Present results, along with the findings on the cholinergic mediation of predatory aggression and the rage reaction, support the concept of a central cholinergic facilitatory system, or “trigger,” for aggressive behavior (Allikmets, 1974). This cholinergic influence on aggression would appear to be specifically related to “muscarinic” receptors in the brain. Present results indicate that central muscarinic receptors are involved in the cholinergic mediation of SIA, while the results of others (Bandler, 1969-1971b; Vogel and Leaf, 1972; Berntson et al., 1973) support this finding in relation to predatory aggression in both rats and cats. However, the possibility that “nicotinic” receptors may be involved cannot be ruled out and must await further research.

tion of muricide, it might be postulated that other brain regions such as the lateral hypothalamus, thalamus, midbrain central gray, and ventral midbrain tegmentum may be involved in the central cholinergic control of SIA. This possibility is currently under investigation in our laboratory. In this context it is interesting to note that the central pathway, linking laterally situated amygdaloid nuclei with the lateral hypothalamic area has been found to contain a strong

Experiment 5 found that bilateral injections of scopolamine into the basolater-

Considering the findings of Bandler (1969-1971 b) on the cholinergic media-


cholinergic component (Girgis, 1972). This pathway may transmit the mediating influence of the amygdala t o hypothalamic effector mechanisms and subsequent- ly t o lower brain centers.

The conclusion that a central cholinergic facilitatory system mediates aggressive behavior is not consistent with Moyer’s (1968) suggestion that the different forms of aggression have different physiological bases. While there is little doubt that different environmental cues result in various aggressive behaviors, this does not necessarily imply that the central brain mechanisms involved are different for each aggression type. The differentiation between aggression types may, therefore, only reflect the different stimulus situations which produce them and the final stereotyped, situation-specific responses which result.

REFERENCES

Allikmets, L. H . ( 1 974). Cholinergic mechanisms in aggressive behaviour. Med.

Allikmets, L. H., Vahing, V. A., and Lapin, I. P. (1 969). Dissimilar influences of Biol. 52:19-30.

imipramine, benactyzine and promazine on the effects of microinjections of noradrenaline, acetyl choline and serotonin into the amygdala in the cat. Psychopharmacologia (Berl.) 15 :392-403.

Bandler, R. J. (1969). Facilitation of aggressive behaviour in the rat by direct cholinergic stimulation of the hypothalamus. Nature (Lond.) 224: 1035- 1036.

Bandler, R. J. (1970). Cholinergic synapses in the lateral hypothalamus for the control of predatory aggression in the rat. Brain Res. 20:409-424.

Bandler, R. J.( 1971a). Direct chemical stimulation of the thalamus: effects on aggressive behaviour in the rat. Brain Res. 26:81-93.

Bandler, R. J. (197 lb) . Chemical stimulation of the rat midbrain and aggressive behaviour. Nature (Lond.) 229:222-223.

Ben-Ari, Y . , Zigmond, R. E., and Moore, K . E. (1975). Regional distribution of tyrosine hydroxylase, norepinephrine and dopamine within the amygdaloid complex of the rat. Brain Res. 87:96-101.

Berntson, G. G., and Leibowitz, S. F. (1973). Biting attack in cats: evidence for central muscarinic mediation. Brain Res. 5 1 :366-370.

Brownstein, M., Saavedra, J . M., and Palkovits, M. (1974). Norepinephrine and dopamine in the limbic system of the rat. Brain Res. 79:43 1-436.

Bunnell, B. N. (1966). Amygdaloid lesions and social dominance in the hooded rat. Psychon. Sci. 6:93-94.

Bunnell, B. N., Sodetz, F. J . , and Shalloway, D. I . (1970). Amygdaloid lesions and social behaviour in the golden hamster. Physiol. Behav. 5 : 153- 1 6 1.

Conner, R. L., Levine, S., Wertheim, G. A., and Cummer, J. F. (1969). Hormonal determinants of aggressive behaviour. An. N. Y . Acad. Sci. 159:760-775.

Crowley, T. J. (1972). Dose-dependent facilitation or suppression of rat fighting by methamphetamine, phenobarbital or imipramine. Psychopharmacologia (Berl.) 27:2 13-222.

Eichelman, B. S. (1 97 1). Effect of subcortical lesions on shock- induced aggression in the rat. J . Comp. Physiol. Psychol. 74 :33 1-339.


Eleftheriou, B. D., ed. (1972). “The Neurobiology of the Amygdala.” Volume 2

Fuller, J. L., Rosvold, H. E., and Pribram, K. H. (1957). The effect on affective of “Advances in Behavioural Biology.” New York: Plenum Press.

and cognitive behaviour in the dog of lesions of the pyriform-amygdala- hippocampal complex. J . Comp. Physiol. Psychol. 50: 89-96.

la and its role in aggressive behaviour in “The Neurobiology of the Amygdala” Eleftheriou, V. E., ed.). New York: Plenum Press, p. 283-295.

HernandCz-Peon, R., Chavez-Ibarra, G., Morgane, P. J . , and Timo-Iaria, C. (1963). Limbic cholinergic pathways involved in sleep and emotional behaviour. Exp. Neurol. 8:93- 1 1 1.

Hull, C. D., Buchwald, N. A., and Ling, G. M. (1 967). Effects of direct cholinergic stimulation of forebrain structures. Brain Res. 6:22-35.

Igid, R., Stern, P., and Basagid, E. (1970). Changes in emotional behaviour after the application of cholinesterase inhibitor in the septal and amygdala regions. Neuropharmacology 9 :7 3-75.

Jonason, K. L., and Enloe, L. J. (1971). Alterations in social behaviour following septal and amygdaloid lesions in the rat. J. Comp. Physiol. Psychol. 75:286- 301.

Karli, P., Vergnes, M., and Didiergeorges, F. (1969). Rat-mouse aggressive behaviour and its manipulation by brain ablation and brain stimulation. In “Aggressive Behaviour” (Garattini, S., and Sigg, S. B., eds.). New York: Wiley, pp. 47-55.

Girgis, M. (1 972). The distribution of acetylcholinesterase enzyme in the amygda-

Katz, R. J., and Thomas, E. (1975). Effects of scopolamine and a-methylpara- tyrosine upon predatory attack in cats. Psychopharmacologia (Berl.) 42: 153- 157.

Kling, A. (1 972). Effects of amygdalectomy on social-affective behaviour in non- human primates. In “The Neurobiology of the Amygdala” (Eleftheriou, B. E., ed.). New York: Plenum Press, pp. 5 11-537,

Kolb, B., and Nonneman, A. J. (1 974). Frontolimbic lesions and social behaviour in the rat. Physiol. Behav. 13:637-643.

Konig, J. F. R., and Klippel, R. A. (1963). “The Rat Brain - A Stereotaxic Atlas.” Baltimore: Williams and Wilkins Co.

Latan6, B. (1 969). Gregariousness and fear in laboratory rats. J. Exp. SOC. Psychol. 5 :6 1-69.

Leaf, R. C. , Lerner, L., and Horovitz, Z. P. (1969). Role of the amygdala in the pharmacological and endocrinological manipulation of aggression. In “Aggres- sive Behaviour” (Garattini, S. and Sigg, S. B. , eds.). New York: Wiley, pp. 120-131.

Lewis, P. R., and Shute, C. C. D. (1967). The cholinergic limbic system: projections to hippocampal formation, medial cortex, nuclei of ascending cholinergic reticular system and the subfornical organ and supra-optic crest. Brain

Lindvall, O., and Bjorklund, A. (1 974). The organisation of the ascending catecholamine neuron systems in the rat brain. Acta Physiol. Scand. (Suppl.)

Lints, C. E., and Harvey, J. A. (1969). Altered sensitivity t o footshock and de-

90:52 1-540.

4 12 1 -48.

creased brain content of serotonin following brain lesions in the rat. J. Comp. Physiol. Psychol. 67:23-31.


McCarthy, D. (1966). Mouse-killing in rats treated with pilocarpine. Fed. Proc. 25:385.

Miczek, K. A., Brykczynski, T., and Grossman, S. P. (1974). Differential effects of lesions in the amygdala, periamygdaloid cortex, and stria terminalis on aggressive hehaviours in rats. J. Comp. Physiol. Psychol. 87:760-77 1.

Moyer, K . E. ( 1 968). Kinds of aggression and their physiological basis. Commun. Behav. Biol. 2:65-87.

Olds, M. E., and Olds, J. (1963). Approach-avoidance analysis of rat diencepha- lon. J. Comp. Neurol. 120:259-295.

Palkovits, M., Saavedra, J. M., Kobayashi, R. M., and Brownstein, M. (1974). Choline acetyltransferase content of limbic nuclei of the rat. Brain Res. 79:443-450.

Powell, D. A., Francis, J., and Schneiderman, N. (1971). The effects of castration, neonatal injections of testosterone and previous experience with fighting on shock-elicited aggression. Commun. Behav. Biol. 5 : 3 7 1-377.

Powell, D. A., Milligan, W. L., and Walters, K. (1973). The effects of muscarinic cholinergic blockade upon shock-elicited aggression. Pharmacol. Biochem. Behav. 1:389-394.

Powell, D. A,, Walters, K., Duncan, S., and Holley, J. R. (1973). The effects of chlorpromazine and d-amphetamine on shock-elicited aggression. Psycho- pharmacologia (Berl.) 30:303-314.

Rodgers, R. J., and Brown, K. ( 1 973a). The inhibition of shock-induced attack in rats by scopolamine. J. Int. Res. Commun. Sys. (IRCS) (73-5):36.

Rodgers, R. J., and Brown, K. (1973b). Increased shock-induced attack in rats by physostigmine. J. Int. Res. Commun. Sys. (IRCS) (73-9):38.

Rosvold, H. E., Mirsky, A. F., and Pribram, K. H. (1954). Influence of amygdalectomy on social behaviour in monkeys. J. Comp. Physiol. Psychol. 47: 173- 178.

Shute, C. C. D., and Lewis, P. R. (1967). The ascending cholinergic reticular system: neocortical, olfactory and subcortical projections. Brain 90:497- 520.

Smith, D. E., King, M. B., and Hoebel, B. G. (1970). Lateral hypothalamus control of killing - evidence for a cholinoceptive mechanism. Science 167:900- 901.

mentally-induced aggression in rodents. Life Sci. 8:705-7 16.

rat brain. Acta Physiol. Scand. (Suppl.) 367: 1-48.

by repeated pilocarpine treatment. Physiol. Behav. 8:42 1-424.

McGraw Hill.

Sofia, R. D. (1969). Effects of centrally-active drugs on four models of experi-

Ungerstedt, U. (1971). Stereotaxic mapping of the monoamine pathways in the

Vogel, J. R., and Leaf, R. C. (1972). Initiation of mouse-killing in non-killer rats

Winer, B. J. (1971). “Statistical Principles in Experimental Design.” New York:

amygdaloid function in the central cholinergic mediation of shock-induced aggression in the rat

Documents