the effects of total or selective amygdaloid lesions on emotionality, ge- neral activity and reactiveness to various stimuli had been investigated in increasing ...
ACTA NEUROBIOL. EXP. 1980. 40 : 911-932
EMOTIONALITY AND EXPLORATORY BEHAVIOR FOLLOWING CORTICO-BASOMEDIAL AMYGDALA LESION IN RAT I. EUKASZEWSKA, R. KORCZYRSKI. A. MARKOWSKA and E. KOSTARCZYK
Department of Neurophysiology, Nencki Institute of Experimental Biology Warsaw. Poland
Abstract. Twelve rats with amygdaloid lesion (CBM group) and 20 sham operated (Control group) were tested. Emotional reactivity evaluated by six category scale showed transient decrease in CBM group with respect to Control one. The CBM lesion enhanced the locomotor activity in electromagnetic activity meter in the initial phase of confinement in the apparatus. Open-field ambulation was higher in CBM rats than in controls on the first day of examination, while on the second day both groups showed similar locomotor activity. No between group differences were found in response to introduction of novel object. Both groups prefered to stay in box placed in open-field than move around the field. However, CBM rats were engaged in box exploration, whereas controls spent major proportion of time sitting motionless. These results indicate that CBM lesion increases some components of response to novelty. The pattern of changes and their transitory character suggest that cortico-basomedial region of the amygdala is involved in the control of processes underlying the initial phase of responding to novel environment. INTRODUCTION
Numerous experiments have established that in rat damage to the amygdaloid complex is followed by alterations in a variety of tasks, similarly as in other mammalian species. Since all of these tasks are to a considerable extent influenced by changes in untrained behavior,
the effects of total or selective amygdaloid lesions on emotionality, general activity and reactiveness to various stimuli had been investigated in increasing number of studies. However, the results do not provide the consistent picture. One of the reason is that different tests used in various investigations assess different aspects of behavioral alterations, which in addition to variability of amygdaloid lesions, may lead to discrepant interpretations. Thus, our aim was to perform a series of experiments with several test situations on the same animals, in order to obtain more complete information on lesion effects. Further advantage of this procedure results from the considerably long surgery - test intervals. This provides some indications on the durability of effects evoked by amygdaloid lesion. Our choice of the lesion site was determined by previous research (18, 19) dealing with similar region. The present paper reports data related to emotionality and different forms of locomotor activity. The next one (in preparation) concerns social and food-motivated behavior. GENERAL METHOD
Subjects. Forty male Wistar rats sup'plied by Animal Farm in Eomna were used. Two weeks after arrival animals attained 3 mo of age and were randomly assigned to amygdala (20 Ss) or sham operated (20 Ss) group. Throughout the experiments rats were housed in groups of 10 and had free access to food and water, except for a period of deprivation the day before surgery. Testing procedure. After surgery, brain damaged and sham operated rats were employed in four experiments testing (i) emotional reactivity (ii) activity in electromagnetic cage (iii) activity in open-field and (iv) preference for small box vs open-fic-ld. Surgery. Operation was performed under clean but not aseptic conditions. The animals were anesthetized with Nembutal (50 mg/kg i.m.) and positioned in Kopf stereotaxic instrument. Bilateral electrolytic lesions oriented to cortico-medial part of amygdala were produced by passing 2 mA of anodal current for 10 s with rectal cathode completing the circuit. Unipolar wolfram electrodes 0.5 mm in diameter, insulated by enamel except for 0.3 at the tip were placed using Konig and Klippel atlas (20) coordinates: AP + 4 . 5 ; L i 3 . 2 ; H -3.0. The animals in the sham operated group received and incision of skin and holes were drilled in their skulls, but no lesion was made. Histology. After completion of tests designed for the present paper and also the other one (in preparation) brain damaged rats were deeply anesthetized and perfused intracardially with isotonic saline followed
b y 10°/o formal-saline solution. The brains were removed and fixed in 1O0/o formalin for several d ~ y s .Transverse 50 pm sections were subsequently made with cryostat. The sections were stained by either Kliiver or the Nissl method. Neuroanatomical verification was made microscopically examining the damaged tissue surrounding each lesion. At the conclusion of anatomical verification 8 rats were discarded because lesions were not placed as intended and their records removed from analysis.
Localization of lesions in particular nuclei of the amygdala in individual rats AMe, medial n.; Aco, cortical n.; ABm, basomedial n., ABl, basolateral n.; AL, lateral n., AC, central n.; m. intercal., massa intercalata; V, ventral part; D, dorsal part; M, medial part; L, lateral part; Ant., rostra1 part; Interm., intermediate part; Post., caudal part.
Fig. 1. Drawings of sections with maximal lesions in individual animals.
ANATOMICAL FINDINGS
The extent and localization of amygdala damage in individual rats are summarized in Fig. 1 and Table I. In all 12 rats with satisfactory lesions the rostra1 portion of amygdala was destroyed. Bilateral destructions consistently involved cortical nuclei and in most instances basomedial nuclei. The medial nuclei were in general spared, although in some cases they were touched unilaterally. Since the damage comprised the cortico-basomedial amygdaloid region, the operated rats will be denoted as CBM group. EXPERIMENT I: EMOTIONALITY
Emotional reactivity of rats with amygdala damage was frequently evaluated by six category scale of King (15). However, the results of these studies were inconsistent. King (15), King and Meyer (16), Schwartzbaum and Gay (28) and Corman et al. (5) have found no change in emotional reactivity, while Kleiner et al. (17), Jonason and Enloe (11) and Jonason et al. (12) reported substantial increase. Increase in responses to capture and to tail tap in rats with induced epileptic foci in amygdala was found by Pine1 et al. (26). This discrepancy of results is difficult to explain since there is no discernable relationship between lesion size or placement and the effect on emotionality level. In most of the studies using multiple-category rating system data related to separate categories are not reported. Since important information may be lost when exclusive reliance is placed on the composite score, the present experiment attempts to evaluate also the contribution of particular categories. Method. Rats were tested for five consecutive days beginning on the 5th postsurgical day. Each rat was removed from home cage and placed singly on the table. Two observers independently rated the animal's reactions using slightly modified King's scale. Following categories were evaluated: (i) resistance to removal from the home cage (ii) resistance to capture and handling, (iii) vocalization during the testing session, (iv) reaction to a pencil presented close to the snout, (v) reaction to a light tap on the back, and (vi) urination and defecation throughout the testing session. Reaction were scored from 0 to 5 points on each category, except the last one, which ranged from 0 to 3. The sum of scores for all categories represented rat's emotionality rating for a particular day. Results and discussion. Mean emotionality ratings (sum of scores of six categories) across testing days are presented in Fig. 2. Ana-
lysis of variance (Groups X Days) yielded significant effect of Days ( F = 24.18, df = 41120, P < 0.001) but not of Groups. However, significant interaction between Groups and Days (F = 6.28, df = 41120, P < 0.01) indicated differential emotionality decrease in CBM group and C group. Control rats showed lowering of emotionality scores
Fig. 2. Emotionality ratings. Open circles, control group; filled circles, CBM group.
I
2
3
4
5 Days
already on Day 2 (P < 0.05, Duncan test) whereas in CBM group (cortico-basomedial) the scores remained the same through Day 1-3, and significant drop appeared between Day 3 and Day 4. Comparison between emotionality scores of CBM group and C group in successive days revealed that the only difference which attained the level of significance was that observed on Day 1 ( P < 0.01, Duncan test). This means that CBM rats showed lower emotionality scores than controls only on the very beginning of testing. Analysis of variance and Duncan tests performed separately for each of six categories indicated that lower emotionality scores of CBM group than C group on Day 1 were due to defecationlurination ( P < 0.05), vocalization ( P < 0.01), and reaction to tapping the back ( P < 0.01). Resistance to capture in home cage and to handling did not differ reliably between groups. Visual presentation of pencil was almost completely ignored by brain lesioned as well as control rats. In particular categories (apart from pencil presentation) scores for CBM group and C group tended to decrease during testing as shown by significant effect of Days (Ps (0.01 < 0.001, depending on the category). The only exception was resistance to removal from the home cage. Scores in this category remained on the same level throughout the testing period in both, CBM and C group (Days effect, P > 0.2 and interaction between Groups and Days, F < 1). The testing system employed in the present experiment involved several fearful conditions, mainly with tactual components, which might elicit various specific responses like muscle tension, startle
reaction, flight or attack. Vocalization, defecation and urination appear as "unspecific" reactions, since they may accompany the responses to other testing categories. It is important to stress, that except startle reaction to back tapping CBM rats received lower scores only for those "unspecific" reactions. In the previous studies using multiple category rating this finding could be overlooked due to focussing attention on overall emotionality scores. It should be mentioned that lowered defecation and urination as indicators of lowered emotionality of animals with amygdaloid lesions should be taken with caution in view of amygdala control of various autonomic responses. Less frequent defecation might be attributed to decreased intestinal movements (see review 8). EXPERIMENT 11. ELECTROMAGNETIC ACTIVITY METER
Different ways of assessing the locomotor activity in normal rats do not correlate well with each other (29) suggesting that locomotor activity is not a unitary dimension. It might be supposed that activity measured in small devices would primarily reflect the animal's mobility due to reduced opportunity for ambulation, while large apparatus, like the open-field would be more related to locomotion. Accordingly, the size of the test apparatus may be a relevant variable differentiating the influence of factors, which affect various aspects of activity. Previous study (19) has shown that lesion restricted closely to medial amygdala increased the rat's activity in electromagnetic meter (30). Since in the present experiment the most medial part of the amygdala was spared it seemed reasonable to compare the effects of these damages, differing somewhat in localization, on rat's activity registered in the same apparatus. Method. Apparatus. Electromagnetic activity meters of the Am-1 type had six independent solenoids. All movements to - or from any coil unsettled a resonance circuit, the output of which was standardized to square waves and registered on a cumulative recorder. Each activity meter was equipped with detachable box with 2 mm thick bottom and walls made of plastic. The bottom of the box was 2 2 x 4 0 cm, the walls were 18 cm in height. The box was covered with wire grid. Procedure. Rats were tested twice, on the 5th and 15th day after operation. First test was performed 1 h after emotionality rating. Each rat was put singly into the box for a 1 h session. Four rats were
tested simultaneously (2 cortico-basomedial and 2 controls) in four activity meters. Records were taken every 10 min period. Results and discussion. Three dimensional ANOVA (Groups X Days X Periods) performed on data presented in Fig. 3 indicated not significant effect of Groups ( P > 0.1), while effects of Days and Periods
Fig. 3. Locomotor activity in electromagnetic activity meter. Left panel, test performed on 5th postsurgical day; right panel, test on 15th day. Denotations as in Fig. 2.
were highly significant ( F = 18.92, df = 1/30, P < 0.001 and F = 218.66, df = 51150, P < 0.001, respectively). The only significant interaction was that between Days and Periods (F = 3.51, df = 51150, P < 0.005). This provided suggestion for the separate analyses of variance on data for 5th and 15th postsurgical days.
Analysis of variance for 5th day revealed not significant effect of Groups (F < 1) but highly significant effect of Periods ( F = 112.45, df = 51150, P < 0.001). Interaction between Groups and Periods was insignificant ((F< 1). Both groups showed similar level of activity at the beginning of test and similar intrasession decline. In contrast, on 15th postoperative day CBM group was more active than C group ( F = 4.27, df = 1/30, P < 0.05). As shown by Duncan tests this difference was due to first 10 min period ( P 0.01). Highly significant effect of Periods ( F = 82.82, df = 51150, P 0.001) indicated decrease of activity in successive time intervals in both groups. Although the interaction Groups X Periods did not attain the level of significance ( P < 0.1), decrease of activity between 10th and 20th min was obviously faster in CBM rats since their first record was significantly higher than that of controls and the difference vanished in the next time interval. To compare the activity of CBM group and C group on 5th and 15th postsurgical days, analyses of variance (Days X Periods) were performed separately for operated and control group. Both groups were more active on 15th than on 5th day after operation ( F 27.87, df = 1/11, P 0.001 for CBM group and F = 4.56, df = 1/19, P < 0.05 for C group). Although all 6 records taken with 10 min intervals were higher on 15th than on 5th day in both groups, the significant differences found by Duncan tests were only for records 1 and 2 (Ps 0.01 in each group). Lower activity of both groups in the first test performed early after surgery might be ascribed to reconvalescence processes. However, on 15th postoperative day CBM rats showed higher activity not only with respect to the first test but also in comparison to concurrently tested control rats. This suggests the possibility of the time dependent changes in the effect of amygdala lesions. Data of the test performed on 15th postsurgery day are in line with findings of Korczynski and Fonberg (19) who demonstrated increased activity of rats following basomedial damage when tested in initial phase of introduction to the electromagnetic cage. Further confinement (for 12 h night period) resulted in the decreased activity in comparison to the preoperative level. Similarly, McIntyre and Stein (23) using stabilimeter cage found postoperative decrement after destruction of central and medial amygdaloid regions. Contrary to these reports, the study of Mabry and Campbell (21) indicated enhancement of activity recorded in stabilimeter cage in rats with lesion of stria terminalis, as well as in those, lesioned in central and medial nuclei with additional interruption of stria terminalis. Records related to 22 h
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